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This is to certify that the

dissertation entitled

An Investigation into the Clinical Manifestations and
Molecular Genetics of Bovine Hereditary Zinc Deficiency

presented by

Margo R . Machen

has been accepted towards fulfillment

of the requirements for
Large Animal

Ph.D. degree in Clinical Sciences

 

 

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Major professor

Date Dec /// /9?7

 

MSU is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

LIBRART

Michigan State
University

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PLACE IN RETURN BOX
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TO AVOID FINES return on or before date due.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1/98 cJClRC/DaleDue.p65—p.14

 

 

AN INVESTIGATION INTO THE CLINICAL MANIFESTATIONS AND
MOLECULAR GENETICS OF BOVINE HEREDITARY ZINC DEFICIENCY

By

Margo R. Machen

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Large Animal Clinical Sciences

1997

 

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ABSTRACT
AN INVESTIGATION INTO THE MOLECULAR GENETIC AND CLINICAL
MANIFESTATIONS OF BOVINE HEREDITARY ZINC DEFICIENCY

By

Margo R. Machen

Bovine hereditary zinc deficiency (BHZD), also referred to as Adema disease and
Lethal trait A46, is an autosomal recessive disorder which results in inadequate amounts
of zinc being absorbed from the gastrointestinal tract causing clinical zinc deficiency. A
pedigree was established at Michigan State University utilizing two heterozygous embryo
donors and semen from a homozygous, affected bull in artificial insemination and
embryo transfer studies. Five homozygous, affected calves, two of which were males,
and one heterozygous, unaffected heifer were obtained. Clinical observations,
biochemical assessment and histologic evaluation of skin biopsies allowed for the
chronological documentation of the onset of pathophysiologic changes in affected
animals. Affected BHZD calves had a decrease in plasma zinc concentrations below 0.5
ppm between two and four weeks of age. The animals then became lethargic and
developed diarrhea followed by parakeratosis at mucocutaneous junctions, a decreased

ability to sustain a proper suckling reflex and poliosis an alopecia around the orbits. A

 

 

 

 

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paralleled decrease in serum alkaline phosphatase and plasma zinc concentrations, which
correlated with the observance of pathophysiologic changes, was noted in the calves. The
oral administration of zinc in pharmacological amounts reversed all clinical, biochemical
and histologic alterations. In rodent intestinal cells a cysteine-rich intestinal protein

(CRIP) was identified, and believed to be responsible for the carrier-mediated transport of
zinc from the intestine. This dissertation hypothesizes that either a mutation at the CRIP
locus or a mutation in the 5’ promoter region and/or a trans acting factor suppresses CRIP
expression, and is responsible for BHZD in cattle. Molecular genetic techniques were
used to characterize the nucleotide and amino acid sequences of the bovine homolog of
CRIP in homozygous affected, heterozygous, unaffected and unaffected, unrelated cattle.
No mutations were detected in the coding region of the gene in affected animals. Four
polymorphic sites were detected in the nucleotide sequence, which resulted in three

amino acid substitutions between two unaffected, unrelated cows. The first two amino
acid substitutions occurred in the second zinc-binding finger at positions X51, tyrosine by
phenylalanine and X52, cysteine by glycine. The third amino acid substitution was in the
glycine rich third domain at position X63, glycine by serine. Studies with genomic DNA
detected the presence of an intron between the first and second zinc-binding fingers.
CRIP mRNA was detected in 24 tissues using RT-PCR. CRIP expression was not
altered by sex, zinc status, within affected BHZD animals, compared to controls and
Within the tissue distribution of individual animals. The results of the dissertation
indicate that a mutation of CRIP or its 5’ promoter region and/or a trans acting factor is

not responsible for the clinical manifestations of BHZD.

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ACKNOWLEDGEMENTS

Numerous people have motivated me and aided me in completing this dissertation and
my postdoctoral training in food animal medicine and surgery. To all of you, thank you.
There are several people who I owe special recognition to and without whose help, the
task at hand would not have been completed. First, I would like to acknowledge the holly
spirit that dwells within myself, that has been both patient and loving during my journey.
Second, the unyielding all encompassing support rendered by my family throughout my
transcendence from childhood to adulthood. Third, the members of my committee that
provided me with guidance and encouragement. Finally, I would like to recognize two
people who have helped to mentor and develop my professional and personal being. I
give special thanks to Patricia Lowrie, without whose support, intervention, inspiration
and patience, I would not have reached beyond; you have provided me with the bricks to
build my road to the future. I also give special thanks to Vilma Yuzbasiyan-Gurkan, who
provided me with a lab and the necessities to accomplish my research as well as taught
me how to envision and achieve, but even more so allowed me the latitude to grow.

Iwill complete the circle, as you have nourished and passed on what others have to

You, I will now go forth and do the same.

 

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TABLE OF CONTENTS
LIST OF TABLE ix
LIST OF FIGURES xj
LIST OF ABBREVIATIONS xiii
INTRODUCTION 1
CHAPTER 1
REVIEW OF THE LITERATURE 1
Metabolic Functions of 7 inc- 3
Distribution of Zinc in the Body' 9
Homeostatic Control of Zinc Absorption: 11
Hereditary Zinc Disorders 14
Acrodermatitis enteropathica 15
Lethal acrodermatitis in Bull Terriers 17
Inherited epidermal dysplasia 18
Bovine hereditary zinc deficiency 20
Zinc in the Diet: 29
Localization of Zinc Absorption in the Gastrointestinal Tract: ............................ 31
In vitro Qhu’lies 32
In vivo thdieq 33
Direct dosing procedure 38
Kinetics of Zinc Absorption: 42
Developmental Maturation of Zinc Absorptinn' 49
Zinc Absorption from the Gastrointestinal Lumen: 51
The role of intralumenal zinc binding ligands in intestinal zinc
absorption ' 51
Identification of intralumenal zinc binding ligands in the intestine ......... 53
Intracellular Mechanisms of Gastrointestinal Zinc Absorption: ........................... 68
The role of intracellular zinc binding ligands in intestinal zinc
absorptinn .. 68
Identification of prostaglandins as a zinc binding ligand, in rat intestinal
mucosal cells 70
Identification and characterization of metallothionein ............................. 71

vi

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Relationship between dietary zinc and metallothionein synthesis ............ 74
Developmental regulation of metallothionein expression in the
intestine 79
The function of metallothionein in the regulation of zinc absorption from
the gastrointestinal tract 80
The identification and characterization of CRIP ....................................... 94
Developmental regulation of CRIP expression in the intestine ............... 103
CRIP’s function in the regulation of zinc absorption from the
gastrointestinal tract 110
The CRIP locus as a candidate gene for bovine hereditary
zinc deficiency 118
CHAPTER 2
MATERIALS AND METHODS 120
Materials and Methods for the Clinical Investigation of Bovine Hereditary Zinc
Deficiency: 120
Producing the BHZD pedigree 120
Care and management of the calves 121
Trace element analysis 122
Clinical chemistry studies 123
Histopathologic examination of the skin 123
Treatment of affected calves 123
Discontinuing zinc treatment in adult ' ‘ 124
Molecular Investigation of the CRIP Locus: 1 2 5
Primer selection 125
Genomic DNA extraction from blood 126
Harvesting tissues 127
Total RNA extraction from tissues 127
Reverse transcription of total RNA preparations to produce cDNA ........ 128
Polymerase chain reactions 129
Extracting amplification products from agarose gels .............................. 130
DNA sequencing protocols 130
Agarose gels 131
Acrylamide gels 131
Determining the relative expression of CRIP in tissues .......................... 133
CHAPTER 3
RESULTS 135
Results of the Clinical Investigation of Bovine Hereditary Zinc Deficiency: ..... 135
Embryo transfer study .. 13 5
Clinical manifpctafinns 138
Histopathologic findings ,_ 143
Biochemical findings .146
Individual ' ' .149

 

 

Necropsy findings ..153

vii

Results of the Molecular Investigation of the CRIP Locus: ................................ 154

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Identifying the coding region of the bovine homologue of CRIP ........... 154
Gene structure of CRIP 163
CRIP tissue expression study 165
CHAPTER 4
DISCUSSION 171
Clinical Investigation of Bovine Hereditary Zinc Deficiency: ............................ 171
Molecular Investigation of the CRIP Locus: 179
Summary: 192
APPENDIX A 194
Possible Functions of CRIP: 194
APPENDIX B 70s
Protocols for the Molecular Investigation of CRIP: 70s
Purifying and preparing oligos for PCR 70s
Calculating a 20 1.1M concentration of primers for PCR .......................... 206
DNA extraction from blood using a Genomix kit 706
Phenol/Chloroform extraction protocol to remove proteins .................... 208
Isolation of total RNA from frozen tissues with TRIzol reagent ............. 208
DNAse treatment of total RNA preparations 709
Reverse transcriptase of total RNA preparations to produce cDNA ........ 210
Polymerase chain reactions 711
Extraction of DNA bands from agarose gels 712
Radiolabeling primers for DNA w. ' g 713
Sequencing PCR prndncts 714
Silver staining acrylarnide gels 718
Preparing SDS PAGE stacking gels 719
6% Acrylamide gels 771
Protocols for the Clinical Investigation of Bovine Hereditary
Zinc Deficiency‘ 773
Synchronization of donor and recipient cows 972
Embryo transfer protocol 773
APPENDIX C 774
Product and Manufacturers References: 994
BIBLIOGRAPHY 779

 

 

 

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LIST OF TABLES

 

 

Table 1- Six Classes of Zinc Containing 13-.., 6
Table 2 - Clinical Manifestations of BHZD 22
Table 3 - The Percent of the Administered Dose of 6SZn Absorbed from Different

34

 

Intestinal Regions in Two In Vivo Ligated Sac Studies

Table 4 - Kinetic Characteristics of Zinc Absorption in Three Different Sections of the GI
Tract Different Ages in the Rat 50

Table 5 - The Effects of Three Different Diets on Weight Gain, 6SZn Absorption by the

 

 

Kidney and Total Zinc Absorption 60
Table 6a - The Effects of Picolinic Acid on Intestinal Zinc Absorption in BHZD
Calves 65

Table 6b - The Effects of Bile and Pancreatic Duct Ligation on Intestinal Zinc Absorption
in Adult Rats 65

Table 6c - The Effects of F our Different Zinc-Binding Ligands on Zinc Absorption from
the Intestinal Lumen into the Vascular Space in Rats .................................... 65

Table 6d - The Effects of Picolinic Acid, Citrate and Diiodoquin on Net 65Zn Absorption,
Intestinal Mucosal Binding, Plasma Activity and Tissues Activity ............... 65

Table 7 - The Effects of Varying Dietary Zinc Concentrations on the Percentage of 65Zn
Absorbed and Mucosal Cell Distribution to Cytosolic Proteins in the Rat ....... 83

Table 8 - The Effects of Rats Fed Three Different Dietary Concentrations of Zinc on 65Zn
Uptake, Retention and Transfer to the Vascular Space by Intestinal Mucosal
91

Cells

Table 9 - The Location, Sequence and Types of Binding Sites Identified in the Promoter
Sequence of the 5’ Flanking Region of Rat CRIP 96

 

 

 

Table 10 - Six Classes of Zinc-finger Proteins, Amino Acid Sequences and Structural
100

Characteristics

Table 11 - The Percentage of 65Zn in Intestinal Mucosal Cells Cytosol Associated with

 

either Metallothionein or CRIP in Rats Fed Different Zinc Diets ................. 113
Table 12 - Primers Used for PCR Reactions, Identification Numbers, Nucleotide

Sequence and Origin 126
Table 13 - Primer Pairs and their Cycle Sequencing Times and Temperatures ............... 129

Table 14 - The Chronological Order of Development of Clinical Manifestations of Zinc
Deficiency in Effected BHZD Calves 138

 

Table 15 - Primers Used to Sequence the Coding Region of CRIP ................................. 155

Table 16 - Primer Pairs and Templates Used to Amplify and Sequence the Bovine CRIP
Gene 157

Table 17 - The Percentage of Homology that is Shared Between Species for the
Nucleotide Coding Region of CRIP and the Amino Acid Sequence ............. 160

Table 18 - PCR Products of Primers and Templates used to Characterize CRIP’s Gene
Stnicmre

163

Table 19 - The Ratios of the Counts Per Minute of PCR Products from Primer Pairs
158/159 and B—actin for twenty three Tissues Examined in Five Animals ....169

 

 

 

LIST OF FIGURES

Figure I - The Amount of Time in which Peak Concentrations of Serum, Cytosolic and
Metallothionein Bound Zinc and the 358 Incorporation into Metallothionein and

Metallothionein mRNA Levels 78
Figure 2 - Model of Zinc Absorption by an Enterocytes Involving CRIP, MT and

Nonspecific Zinc Binding Ligands, and a Paracellular Pathway © J Nutr, 1992

(122:89—95) 1 18
137

 

Figure 3 - Bovine Hereditary Zinc Deficiency Pedigree

Figure 4a - Cutaneous Lesions that Developed on the Cranium of Affected Calves with
BHZD 141

Figure 4b - Advanced Cutaneous Lesions Along the Stifle of Affected BHZD
141

 

 

 

 

 

 

Calves
Figure 4c - Advanced Cutaneous Lesions on the Rear F etlocks of the Affected BHZD
Calves 142
Figure 5a - Photomicrograph of a Characteristic Skin Lesion in Bovine Hereditary Zinc
Deficiency 145
Figure 5b - Photomicrograph of the Resolving Skin Lesion in Bovine Hereditary Zinc
Deficiency 145
Figure 6 - Longitudinal Observations on Plasma Zinc and Copper Concentrations and
Serum Alkaline Phosphatase levels 147
Figure 6 - (cont’d) 148
Figure 7 - Weekly Weight Gains of the Calves to 12 Weeks of Age .............................. 151

Figure 8 - Photograph of Two Affected BHZD Heifers and One Unaffected Heterozygote
Heifer l 52

Figure 9 - Nucleotide Sequence of the Coding Region of the Bovine CRIP Gene ......... 156

Figure 10 - A Comparison of the Nucleotide Sequences of CRIP in the Rat, Mouse,
Human and Bovine 159

Figure 11 - A Linear Model of the Bovine CRIP Protein with Two Zinc-binding
Fingers 162

Figure 12 - The Amino Acid Sequences of CRIP in the Rat/Mouse, Bovine
and Human 162

Figure 13 - Photograph of PCR Products of the Primer Sets Used to Characterize the
Gene Structure of CRIP 164

Figure 14 - Linear Range of Amplification of PCR Products for Primer Pairs 158/159 and
B-actin 167

Figure 15 - A Comparison of Amino Acid Sequences of LIM Containing Proteins in
Subfamily A 196

 

 

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ABBREVIATIONS

AAS Atomic Absorption Spectrophotometer

AE Acrodennatitis Enteropathica

a Adenine

ACD Acid Citrate Dextrose tubes

Alk Phos --- Alkaline Phosphatase

Amino Acid Residue Abbreviation --- [A-alanine; R-arginine; N-asparagine; D-

ASpartate; C-cysteine; E-glutamate; Q-glutamine; G-glycine; H-histidine; I-isoleucine; L-
leucine; K-lysine; M-methionine; F -phenylalanine; P-proline; S-serine; T-threonine; W-
tryptophan; Y-tyrosine; V-valine]

bp Base Pair

BHZD Bovine Hereditary Zinc Deficiency

Cytosine

3AT --- Chloramphenicol acetyltransferase
IRIP Cysteine-Rich Intestinal Protein

CRIP Bovine Cysteine-Rich Intestinal Protein

CRIP Human Cysteine-Rich Intestinal Protein
CRIP --- Mouse Cysteine-Rich Intestinal Protein
IRIP ..-

Rat Cysteine-Rich Intestinal Protein

 

cCRP --- Chicken Cysteine- Rich Protein

CPM Counts Per Minute

DNA Deoxyribonucleic Acid

DNCB --- Dinitrochlorobenzene

cDNA --- Complimentary Deoxyribonucleic Acid
gDNA --- Genomic Deoxyribonucleic Acid

DDW Double Distilled Water

DEPC Diethylpyrocarbonate

 

dATP 2’-Deoxyadenosine 5’triphosphate
dCTP --- 2’-Deoxycytidine 5’-triphosphate
dGTP 2’-Deoxyguanosine 5 ’-triphosphate
leP 2’-Deoxyinosine 5’-triphosphate

dTI'P 2’-Deoxythymidine 5 ’-tn'phosphate

iNTP Deoxyribonucleoside triphosphates
ldATP 2’3’-dideoxyadenosine 5’-triphosphate
[dCTP --- 2’3’-dideoxycytidine 5’-triphosphate
dGTP 2’3 ’-dideoxyguanosine 5 ’-triphosphate
dTTP --- 2’3’-dideoxythymidine 5 ’-triphosphate
iNTP 2’3’

-dideoxyribonucleoside 5 ’-triphosphat€
TT Dithiothreitol

)TA --- Disodium Ethylenediaminetetraacetate 0 H20

;P 1 --- Estradiol-Stimulated Rat Brain Protein

 

 

 

 

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ETOH --- Ethanol

'-FES --- Cellular Feline Sarcoma gene

'SH Follicle Stimulating Hormone

--- Gaunine

I Gastrointestinal

IRP Human Cysteine-Rich Protein

IRHP --- Human Cysteine-Rich Heart Protein

VIW High Molecular Weight

 

C 6 --- Intestinal Epithelial Cells

3 Inherited Epidermal Dysplasia
[W --- Low Molecular Weight

MLV --- Moloney Murine Leukemia Virus
E Metal Regulatory Elements

Metallothionein

Nuclear Magnetic Resonance
3-» Negative Regulatory Element
'E --- Polyacrylamide Gel Electrophoresis
Physiologic Saline Buffer

Polymerase Chain Reaction

- Prostaglandin

Ribonucleic Acid

A TOtal Ribonucleic Acid

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RNA --- Messenger Ribonucleic Acid
T Reverse Transcription

)S --- Sodium Dodecyl Sulfate

Single Stranded

.. Thyrnine

L-thyroxine

E Tris-Acetate EDTA

E --- Tris-Borate EDTA

-- Tris-EDTA

AED N,N,N’,N’-T~‘*

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Terminal Deoxynucleotidyl Transferase
-- Three Dimensional

Ultraviolet

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INTRODUCTION

Zinc is an essential trace element for a variety of metabolic functions. The
mechanisms responsible for its absorption and distribution have not been clearly defined.
Part of this study attempts to expand the understanding of the systems which may be
responsible for zinc utilization. It is clear that the failure to absorb zinc efficiently from
tonnal food sources is central to the basic defect in bovine hereditary zinc deficiency.

The existence of at least two biochemical pathways for the absorption of zinc from the

astrointestinal tract have been defined, mainly through the use of rodent studies. One of

lese pathways is a non-saturable system that probably involves the passive diffusion of

no between the enterocytes of the gastrointestinal tract. The second pathway is a
ulspOIt-dependent saturable system which is effective at low concentrations of lumenal
lC- A Cysteine-rich intestinal protein (CRIP) was discovered in the rodent, and
Eliminary experimental data suggested that it was involved in zinc transport in the
estinal tract. CRIP is an excellent candidate protein to facilitate the transport of line
at the intestinal lumen to the vascular space. By working in concert with

BliOIhionein, CRIP could regulate the absorption of zinc in the presence of eXCCSSive

tunts in the gastrointestinal tract.

 

 

2
If CRIP is responsible for the saturable component of zinc transport, then it also
:omes a candidate gene for a mutation at its locus being responsible for bovine
editary zinc deficiency and possibly it human homologue acrodermatitis enteropathica.
uedigree of cattle with hereditary zinc deficiency was established at Michigan State
versity to test the hypothesis that a mutation at the CRIP locus was responsible for
ine hereditary zinc deficiency. Observations of the affected animals in the pedigree
allowed for detailed documentation of the chronological onset and resolution, after
ment with large pharmacological amounts of zinc orally, of the clinical and
bemical manifestations of zinc deficiency in both calves and adults.
he molecular and clinical study of the affected calves has provided further insight
he biological role of zinc, as well as providing an animal model for the continued

tigation into the human homologue acroderrnatitis enteropathica.

 

Chapter 1

REVIEW OF THE LITERATURE

VIetabolic Functions of Zinc:

Zinc was first demonstrated in 1869 to be an essential trace element for the biological
motions of Aspergillus niger'. It has since been identified in over 300 metalloenzymes
id in over 100 proteins responsible for transcriptional regulation. Zinc is a transitional
ement that is stable and inert to oxireduction, it can exist in a hydrated and hydroxide
rm at a neutral pH, and provides structural coordination an activation of proteins,
aking it an optimal ion for forming stable complexes with organic compounds”.

Zinc binding proteins participate in numerous metabolic functions that range from cell
tmbrane stabilization to nuclear replication. The bulk of our knowledge about

.ctional zinc binding proteins is derived from observations of metabolic and

chemical abnormalities that occur in zinc deficient states both in vivo and in virro‘.

r stability of both cellular and lysosomal membranes is dependent on adequate zinc
:entrations being present in the tissues. Zinc appears to be important in membrane
ilization, however the exact mechanisms involved have not been determined. Some

6 possibilities in which zinc may stabilize membranes include; zinc forming

aptides with thiol groups of proteins which can link to phosphate moieties of

 

4
)spholipids; zinc interacting with the carboxyl groups of sialic acid; zinc directly
ding to proteins in plasma membraness. Zinc also appears to act as an antioxidant
venting free radical damage to cell membranes. Studies have demonstrated that zinc
an inhibitory effect on the activity of calcium-dependent adenosine triphosphates and
spholipase-AZ, both of which can affect the integrity of membraness.
Researchers have been able to demonstrate quite effectively that zinc deficiency
:ts gene expression, using both animals and cell cultures“. The expression of proteins
initiate and facilitate DNA synthesis and protein translation are dependent on the
ability of zinc to bind to metal-activated transcription factors and coordinate
idary structures responsible for DNA binding. A number of studies have shown that
leficiency impairs the incorporation of thymidine during DNA synthesis. The
arses in thymidine incorporation directly parallel decreases in thymidine kinase
y, which is a proposed zinc dependent enzyme responsible for thymidine
oration“. There have also been reports that DNA polymerase I in Escherichia coli
\IA polymerase and reverse transcriptase from avian myeloblastosis virus require
order to functions.
rently, there is some controversy as to whether zinc deficiency causes a decrease in
:pression or enzyme activation. In the case of thymidine kinase, fibroblast cells
with zinc chelators, to induce a deficiency, showed a corresponding decrease in
levels that paralleled the decrease in thymidine kinase activity“. More total RNA
NA was isolated from zinc-adequate rats than from zinc-depleted ratsé. These

uggest that zinc has a definitive role in the expression of genes at the

 

 

 

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translational stage, as opposed to the activation of the enzyme. There is also some
ication that even during zinc deficient states, certain cellular systems requiring zinc
protected. Zinc deficient cell systems have been able to maintain constitutive levels
roteins responsible for “house keeping chores”, such as S6 ribosomal protein and at
iame time the expression of proteins such as creatinine kinase and thymidine kinase
lepressed‘. Both of these enzymes require continuous regulation of their gene
ession. Physiological systems that have a high turnover rate of cells, such as the
tinal system and dermas require continual regulation of gene expression. Therefore,
0 deficiency affects genes that have varying expression rates and are responsible for
who functions that are not considered constitutive in nature, it stands to reason that
logical abnormalities would manifest themselves in these areas first, as observed
he development of diarrhea and parakeratosis in bovine hereditary zinc deficiency
urodermatitis enteropathica7.
merous enzymes, with critical functions in metabolism require zinc for optimal
V. To date there have been six structural classes of zinc enzymes determined3.
T contains the six classes of metalloenzymes with examples of enzymes in each
1eir fiinctions and structural characteristics. All of these enzymes are decreased in
eficient cell system or animal, but can be corrected with the supplementation of

left untreated, the decrease in enzyme activity can cause metabolic abnormalities.

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6

1- Six Classes of Zinc Containing Enzymes

 

 

 

 

 

 

 

Class and Enzyme Function Zn B‘inding Motif

' (Dehydrogenase)

:ohol dehydrogenase Catalyzes the oxidation of ethanol or the Tetrahedral coordination
reduction of acetaldehyde using of 2 cystiene, 1 histidine
diphosphopryidine nucleotide as a and NADH.

fl cofactor in the liver.

.7 (Transcarbamoylase)

mate Critical in the utilization of ammonia and Tetrahedral coordination
in the synthesis of urea. of 4 cystienes.

'bamoylase

RHydrolases)

line phosphatase Hydrolyzes a variety of Distorted tetrahedral
phosphomonoester as well as transfers coordination of 3
phosphate groups. histidines, 1 glutamate

and H20.
vxypeptidase A and B Hydrolyzes amino acids off the C-
terminus of proteins, peptides and esters
in the GI tract.
[Lyases)
tic anhydrase Catalyzes the reversible dehydration of Distorted tetrahedral
carbonic acid, which is critical in the coordination of 3
transport and elimination of C02, histidines and H20.
Aetalloenzyme)
)thionein Functions are not completely understood, Thiol clusters of

but the many isoforms that exist implies
it has a multifimctional role in
metabolism.

cystiene.

 

iene expression)

ption factor III A

Activates the transcription of SS RNA
gene in Xenopus.

 

 

Zinc finger motifs with
tetrahedral coordination
of 4 zinc binding amino
acids and thiol clusters.

 

 

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7

Metallothionein (MT) is a metal binding protein that has an intricate and diverse role

inc absorption, metabolism and excretion. It is widely distributed throughout tissues

 

occurs in different isoforms within and between speciess’g’w. Metallothionein
ably participates in the metabolism of essential rather than nonessential elements”.
characteristics of MT protein and its role in the absorption of zinc from the GI tract
e discussed in detail in the “Mechanisms of Zinc Absorption Section”. The
' g will focus on zinc’s interaction with MT and the metabolic functions it
tates.
etallothionein was first discovered in equine kidney cortex, in 1957, due to its
e ability to bind cadmium, a metal whose presence has no apparent nutritional
[canoe and is toxic in large concentrations" ‘. Metallothionein’s function in
fying heavy metals is probably a consequence of their similarities to zinc that
them to be bound, making it a secondary rather than a primary function of the
. The half-life of MT in humans and rats on average is about 2.5 days, which is
hort if it is to be considered avprimary detoxification mechanism”.
[zinc and copper bound to MT intracellularly may serve as a reservoir to insure
ady supply of both metal ions will be available for metabolic functions. The
r MT to provide storage for these elements also implies another function that it
e in metabolism, as a metal transfer protein”. The function of MT in such a
has been hard to determine since it has been difficult to follow the fate of the

ted metal. However, there has been in vitro evidence that shows the direct

 

r_i—* W
8

hange of zinc bound to MT to apofonns of zinc dependent enzymes such as alkaline

sphatase and superoxide dismutasew'”.

Zinc bound to MT is required for cellular differentiation in fetal livers”. It also

14

tars that there is an increased amount of zinc bound to MT in the nuclei of neonates .

1mans, several MT genes have been identified to regulate cellular differentiation.

 

MT III gene of the human brain is a brain-cell growth inhibitory factor, which is
iely down regulated in patients that have Alzheimer’s”. Metallothionein I F is a

tn developmental regulatory gene which is located on chromosome 16”.
etallothionein synthesis is stimulated by a wide variety of pathophysiological

s, implying that the protein has a multifunctional role. Acute phase induction of
ray allow cells to accommodate increases in intracellular metal concentrations that
in stressed animals. Increased MT levels may also increase the availability of zinc
bilizing cell membrane structures. A common feature of all pathophysiologic

us is to increase hepatic MT concentrations and therefore the cellular uptake of
'hich decreases serum zinc concentrations”. It is not clear which acute phase

:e mediators induce MT synthesis; however the various possibilities include:
trticoids, catecholamines and the second messengers analogs of cyclic-AMP,

rine and glucagon.

llothionein is also a free radical scavenger. Both neutrophils and macrophages
hydroxyl radicals in an effort to destroy bacterial or virally infected cells. These
cals can cause damage to DNA and cell membranes. Scavengers such as vitamin

irione, glutathione peroxidase and zinc bound to MT have been shown in vitro to

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ective effects. Metallothionein alone does not confer total protection against
; but, does show specific cellular loci protection of DNA. Zinc bound to MT
:1" directly protect DNA by displacing iron and copper from DNA bound sites,
y mediate oxidative damage or MT may scavenge these oxidative elements
.em damaging DNA".
also necessary in the development of cell-mediated immunity as well as the
:ic properties of T-lymphocytes and macrophages”. Zinc deficient animals
rophic thymus, tonsilar tissue, Peyer’s patches and lymphoid tissues. Thymulin
5 specific hormone that requires zinc for biological activations. Thymulin
affinity receptors on T-cells and induces T-cell markers. It also promotes
ytotoxicity, suppresser functions and interleukin-2 (IL-2) production5.
e activity of thymulin, there is an alteration in the subpopulations of T—cells
ed reduction in antibody mediated responses to T-cell-dependent and T-cell-
.t antigens. In lymphocytes evidence indicates that zinc directly stimulates
esis and/or RNA synthesis of enzymes that regulate the proliferation of the
all populations. Clinically zinc deficient animals have secondary bacterial,

'iral infections that often become fatal.

on of Zinc in the Body:
rity of zinc in the vascular system is bound to carbonic anhydrase in
Only 10 to 20% of zinc is bound loosely to albumin, and this fraction is

angeable with tissues“. The albumin binding site for zinc is distinct and does

 

 

 

10

ther trace elements. The remaining fraction of zinc not bound to albumin is
zinc-aZ-macroglobulin. The zinc-aZ-macroglobulin is a histidine-rich
ein that is present in low concentrations in the plasma“. The protein has a high
'ty for zinc, which makes it unlikely that it will donate zinc to tissues.

‘ s also contain zinc that is not exchangeable with plasma zinc and the

8,18

ion is not affected by dietary zinc levels
inc has crossed into the vascular space it is distributed via albumin to various
[genera], small fluctuations in dietary zinc levels do not have a pronounced

3‘19. This appears to be even more so in the

inc concentrations physiologically
rinants'g. The only obvious decrease in zinc concentration is in the feces,
sates an increase in the retention of zinc by the body. Severe dietary

or metabolic diseases that prevent sufficient zinc absorption for prolonged
irne will cause fluctuations in the zinc concentrations of various tissues and
:eins.

deficient state plasma concentrations of zinc that are loosely bound to

dily exchange with tissues that are metabolically active. The most responsive
than the intestine, to dietary zinc status is the livers. The liver absorbs zinc

a process. The first process, in which hepatic receptors that contain

roups bind zinc is carrier-mediated and energy dependent. The second

sents a slow, non-saturable uptake and is responsible for 90% of zinc

ther metabolically active organs which are responsive to dietary zinc levels

20,2]

as, kidney, lungs, heart, and testes Organs that are not as metabolically

 

 

 

 

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ve act as a storage pool for zinc and are not affected by deficiencies until extremely
concentrations are reached. These organs are bone, red and white blood cells,

cle, hair and skin”.

neostatic Control of Zinc Absorption:

his section will only discuss the physiologic factors that regulate zinc absorption, the
osed mechanisms responsible for regulation will be discussed in the “Mechanisms of
Absorption Section”. Homeostatic regulation of zinc absorption from the

ointestinal (GI) tract occurs at a variety of sites and is driven by the physiological

urds of the animal and dietary zinc concentrations. Abnormal regulation at one site

3e compensated for by another site to maintain the overall balance of endogenous
:oncentrationsz‘. There are four primary factors that regulate zinc homeostasis and

11 rrrinor factors that although influence zinc status, only do so after prolonged zinc
ency. The primary regulators are dietary zinc amounts, excretion into the GI tract,
ion into the urine and tissue demands. The minor factors are pools of zinc that have
rrates ofturnover, such as bone, muscle and red and White blood cells. These pools
tgenerally influenced by an animal’s zinc status until they experience severe

:ncy”.
tumin has been proven not to limit the absorption or excretion of zinc to and from
al cells. Experimental evidence has shown that zinc depleted rats with large

ts of intravenous zinc injected into them do not absorb less GI zinc than depleted

 

12
it are not injected, which would be expected if albumin binding sites were
ed”.
tary zinc content regulates the acquisition and retention of zinc in mucosal tissue.
)resence of adequate or excessive concentrations of zinc in the diet, the amount of
L1 zinc absorbed by animals of normal dietary zinc status is decreased and the
: of zinc retained in mucosal tissues is then increased“. Zinc deficient animals that
in oral doses of “Zn have an increase in the amount of zinc taken up by GI tract
y retain the 65Zn in their tissues longer. Thus, it appears that there are brush
nechanisms operating to up-regulate or down-regulate the absorption of zinc from
ract depending on the animals physiologic zinc statuss.
prption of dietary zinc from the GI tract is closely tied to the amount of zinc that is
into the tract via mucosal cells from the vascular space”. Zinc is primarily
in the feces of animals through three pathways; pancreatic ducts, bile ducts and
cells. Zinc secreted from the pancreas is primarily bound to high molecular
roteins and biliary zinc is bound primarily to a tripeptide, glutathiones. Just how
secreted from the pancreas and gall bladder affect the uptake of lumenal zinc by
cells is unknown, but the proteins are believed to decrease the amount of free
able for absorption. Mucosal cells containing zinc slough into the GI tract,
ng to the overall zinc content in the feces of animals. Rats that have their bile
eatic ducts ligated still have appreciable amounts of zinc detected in their feces,
of which has been determined to be mucosal cells“. Mucosal cells are also

) be able to secrete zinc into the GI tract, via a mechanism other than

 

 

 

 

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squamation. Albumin loosely binds zinc that is interchangeable with mucosal cell zinc
318 which are excreted into the GI lumen. An increase in endogenous mucosal cell zinc

)15 decreases the amount of zinc that can be absorbed from the GI tract, by down-

 

plating absorptive mechanisms at the brush borderu. Ruminants with adequate dietary
: concentrations have more zinc detected in their feces than animals fed zinc deficient
s. A study conducted in calves that were fed zinc deficient and zinc adequate diets for
lays and then given oral 65Zn resulted in the zinc deficient calves having an increase in
[the amount of orally absorbed and retained 65Zn and a decrease in fecal losses”. The
as responded to their zinc deficient state by decreasing fecal losses and increasing
rption of zinc”.

rinary excretion of zinc is usually minimal in animals unless they are diseased.

 

ased loss of zinc can occur from certain feed additives that enhances glomerular

ion and physiologic states causing increased nitrogen excretion, such as insulin

[dent diabetesw. However, in general, zinc that is bound to albumin is not filtered

: glomerulus and remains in the body“.

:sue demands also regulate the amount of zinc absorbed from the GI tract.

ing the growth rates of calves by restricting their energy and protein intakes has
hown to decrease the amount of orally administered “Zn absorbed from the

re when compared to controls”. Calves fed high, but non—toxic and restricted

of zinc for prolonged time periods have alteration in the absorption and distribution
l orally and intravenously administered 65Zn’9'2“. In the calves fed zinc restricted

Lssues with high metabolic rates, such as the pancreas, kidney, liver and intestine

 

 

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14

ially have sudden decreases in their zinc concentrations, which were followed by more
adual”. When these calves were given both oral and intravenous doses of 65Zn, the
bolically active tissues also had the greatest and fastest uptake of zinc”. The calves
'gh, but non-toxic, concentrations of zinc had an initial increase in the amount of
taken up by the metabolically active tissues, but then the uptake plateaued. The

'all conclusion is that homeostatic regulation of zinc uptake fiom the GI tract is also

an by the requirements of metabolically active tissues‘9’26.

he interaction of all of these factors helps to regulate zinc homeostasis in animals and
The tissue requirements and amount of zinc available in the diets of animals helps

tennine the amount of zinc that will be secreted into mucosal cells and subsequently

: GI tract, which influences the zinc uptake.

aditary Zinc Disorders:

rc deficiency can be caused by a lack of dietary intake, excessive secretion,

:titive inhibition of absorption, and hereditary disorders that prevent adequate

.ts of zinc from being absorbed by the GI tract into the vascular system. The first
ctors causing zinc deficiency, lack of dietary intake, excessive secretion and

itive inhibition of absorption can usually be corrected once the inciting cause of

ciency is identified. However, hereditary zinc deficiency once identified may or
be manageable by zinc treatment. There are a number of species that have

characteristics indicative of hereditary zinc deficiency. The causative defects for

cies and the different syndromes of zinc deficiency that occur within a species

tel-ii- " . 15",“

 

 

15

tknown. Hereditary zinc deficiency often results in the development of diseases
re life threatening, therefore they are often referred to as lethal traits. Thus far,
hereditary zinc deficiency syndromes include acroderrnatitis enteropathica (AB), in
us, lethal acroderrnatitis in Bull Terriers, bovine hereditary zinc deficiency (BHZD)
herited epidermal dysplasia (IED), in cattle.
lermatitis enteropathica
reditary zinc deficiency in humans was first described in 193 6, however at the time
referred to as “Acro continua with familial occurrence”. In 1942 the current
AE, was proposed for the disease”. It is an autosomal recessive disorder with a
milial recurrence and if left untreated, can be lethal.
clinical characteristics of the disease usually occur shortly after infants are
fiom human breast milk to cow’s milk‘7’28'29'”. Signs of the disease can occur in a
of combinations and have been known to manifest themselves periodically3‘32'33.
re a number clinical findings that include the following: symmetrical alopecia;
atous; vesicopustular dermatitis localized around mucocutaneous junctions;
:ivitis; stomatitis; glossitis; blepharitis; corneal opacities; photophobia;
sia; gonadal atrophy and growth retardation. Histologic evaluation of the skin
veal parakeratosis, hyperkeratosis and polymorphonuclear leukocytic infiltrates.
ically, AE patients have a decrease in their plasma zinc concentration that is
by a decrease in the zinc dependent enzyme, alkaline phosphatase. These
3 often have diarrhea and secondary, opportunistic fungal and bacterial

, due to impairment of their immune system.

 

 

 

 

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16
1e secondary infections are usually the cause of death in these patients. There have
a number of studies conducted to determine which components of the immune
n are affected. Autopsy results have found an atrophic thymus and nricroscopic
nation of the tissue reveals that it was primarily composed of epithelial cells with
3w thymocytes and Hassall’s corpuscles5'3‘. There also appears to be a lack of

29’3 l. The anatomic

:issue, lymph nodes and Peyer’s patches in the ileum
ralities result in antibody-mediated responses to both T—cell dependent and T-cell
rdent antigens being significantly reduced; along with a decrease in the cytosol T-
ponses, natural killer-cell activity and delayed-type-hypersensitivity reactionss.
:motactic activity of the cytotoxic immune cells is also depressed but can be

after the cells are incubated in zinc sulfate solutions, in vitro”.

reatment of patients with AE results in the reversal of clinical manifestations as
riochemical abnormalities. Historically the treatments have consisted of

g the infant to mother’s milk, administering halogenated derivatives of a 8-OH

:s (Diodoquin) and providing an additional exogenous source of zinc sulfate

. Originally the administration of Diodoquin was thought to act as a chelating
he gastrointestinal tract, enhancing the absorption of zinc by an unknown

[11, this is no longer used as a treatment protocol due to its toxic side effects.

.gh the etiology of the disease is unknown, it is evident that the cause is due to
y of the GI tract to absorb normal dietary levels of zinc in adequate amounts.
teresting to note is that there have been several reported cases in which the

l apparently resolve the clinical manifestations without treatment

l7

intervention”. There also seems to be a variation in severity of presentation of the
lisease, in which normal zinc supplementation protocols are ineffective in treating the
ratients and much higher doses of zinc need to be administereds. Whether the variations
II clinical presentations stated above reflect different hereditary mutation or variations in
enetrance, remains to be determined.
.ethal acrodermatitis in Bull Terriers

Lethal acroderrnatitis in Bull Terriers has been widely recognized by breeders for quite
few years. It is an autosomal recessive genetic disorder that was first reported in the
erature in 1986“. What makes it unique when compared it to both AB in humans and
TZD is that the disease does not appear to be entirely responsive to supplemental zinc
:rapy.
From birth, pups affected with lethal acroderrnatitis have clinical manifestations of the
case. These manifestations rapidly progress and eventually end in death by 7 months
tge. The clinical and histopathologic manifestations were similar to those listed above
AE, but there were a few that appear to be unique to the Bull Terriers. The affected
5 have less skin pigmentation at birth which continues to dilute with age; they also
am to have difficulty nursing and when weaned, have abnormal mastication of solid
is. The majority of the pups have protruding nictitating membranes and their feet
at to be splayed“. The skin lesions begin as small crusted sores between their digits
:0 10 weeks of age. Papular and pustular eruptions also became prominent at
tcutaneous junctions which quickly progressed to generalized pyoderma and

' 3 . . .
lyChla 4. Areas that are exposed to frrctron or excessrve wear, such as the footpads,

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e excessively keratinized and develop folliculitis. Another unique feature is the

cc of external otitis which progresses to purulent otitis with hyperplastic changes
'ng in the pinnae“. By 16 weeks of age the pups are lethargic and begin to develop
ary infections due to immune system compromises. Necropsy findings parallel

f AE patients, with the additional findings of a hypoplastic trachea, diffuse, red
ration of the small bowel and esophagus and lateral cerebral dilatation“.

unique features are the plasma zinc concentrations and the lack of responsiveness

therapy in affected puppies. Although the plasma zinc concentrations are
:antly lower than the controls by statistical analysis, their range of values overlap
'er range of the controls“. This not only prevents the use of plasma zinc as a
;tic indicator of lethal acroderrnatitis, but it also indicates that the defect may not
be one of absorption from the GI tract. This is further supported by the fact that
5 show very little improvement in their clinical condition when administered zinc
both orally and by intraperitoneal injection. Perhaps the defect involved

1 the utilization of zinc rather than its absorption from the GI tract. Some of the
:nts have been reported to have normal to increased plasma zinc concentrations,
rad clinical manifestation of zinc deficiency that were responsive to large doses
Jpplementation”. These patients along with the Bull Terriers may represent a
form of hereditary zinc deficiency that is the result of a unique defect".

l epidermal dysplasia

:ed epidermal dysplasia (IED) in cattle shares two clinical features that are

lethal acroderrnatitis in Bull Terriers and some of the patients that have been

 

19

ed to have AB. The similarities are the failure of zinc supplementation to reverse
symptoms and the finding that plasma zinc concentrations in the affected cattle
e low, normal or even high, indicating that the defect in zinc metabolism may be

g in metabolic abnormalities that are not confined to absorption from the G1

955, IED was first reported in calves of Holstein-Friesian decent, in Canada. It is
hereditary disorder and appears to be a single autosomal recessive trait. The
lmanifestations of the disease indicate that it is probably a disorder involving zinc
vlism. The clinical presentation of the calves at birth are normal, but by 6 to 12
hey begin to appear unthrifty with noted weight loss, despite a continued good
a. By 3 to 5 months of age, they have long slender legs and walk with a painful
gait. Their hooves are narrow and elongated with horizontal ridges on the hoof
face”. There is also a failure of horn bud development and the tips of their
pinnae’s curl backwards. The skin of the calves is scaly and there are areas of
mg with suppurative fissures over the joint surfaces, histologically there is no
: of parakeratosis. It also appears that the development of skin lesions does not
1 anatomical pattern. The calves mucocutaneous junctions do not always form
stular dermatitis, as with AE and lethal acroderrnatitis in Bull Terriers.
' findings reveal expected atrophy of the thymus grossly and histologically”.
ttological results are within normal limits, indicating an intact immune system.

ocherrristry results are also within normal limits, indicating that zinc dependent

 

20

es such as alkaline phosphatase, which is decreased in the two previously

bed conditions, is not affected in this particular disease.

6 basic defect that causes the clinical manifestations of this disease is not known,

gh some of the characteristics appeared to be related to a zinc deficiency syndrome.
'que features of this disease such as hematological and serum biochemical

es being within normal limits in addition to the failure of zinc supplementation to
clinical symptoms indicates that the defect is probably not related to the

tion of zinc from the GI tract, but in one of the pathways responsible for zinc

nlism.

: hereditary zinc deficiency

ine hereditary zinc deficiency (BHZD), also known as, Lethal Trait A46, Adema

hereditary parakeratosis, hereditary thymus hypoplasia and hereditary zinc

.cy, was first described in 1964, in Black Pied F riesian cattle37’38‘39'40’“. Black Pied
cattle descended from two breeds, Black Pied J utlands from Denmark and

s from Holland”. Genetic investigations into the pedigrees of affected cattle have

d one common ancestor, a bull named Egbert NRS 13110, born in 193239'42’43 .

ase, however, is most commonly associated with another bull named Adema 21

’oudhoeve; thus the name " Adema disease“3

. Initial investigations into the
: of affected cattle using a fixed sample size and chi square analysis determined

ic transmission to be caused by a “double dose of lethal factor”39'“. The current

‘gy for “double dose of lethal factor” is recessive inheritance. Since both males

 

 

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21
nd females were equally affected and all affected individuals died without treatment,
IHZD was also considered to be autosomal recessive with complete penetrance39'”.
The clinical manifestations of BHZD primarily appear between 4 to 6 weeks of age in
ffected calves and if the animals are lefi untreated they usually die by 4 months of age.
'able 2 contains a list of clinical manifestations reported in BHZD, in the approximate

38,42,43,

rder of occurrence “"5”“. Clinical signs appear more rapidly and severely in calves

; compared to adults that are allowed to become zinc deficient after being treated“.

 

22

[‘able 2 - Clinical Manifestations of BHZD

 

Clinical signs

2 Depression

2 Excessive salivation

2 Diarrhea

2 Rough dry hair coat with scaly debris diffusely throughout the body

2 Hair loss on the legs

2 Calves walk with a “stilted gait”

2 Symmetrical presentation of the skin cracking over joint surfaces, that
progresses to cover the body. The areas are characterized by surface exudate
and matted hair. Removal of the mats reveals inflamed, hemorrhagic skin.

- Carpus o Inguinal area
I Hocks o Axillary area
2 Crusting around mucocutaneous junctions:
o Muzzle 0 Eyes 0 Vulva
0 Base of the ears - Anus o Scrotum
2 Oral lesions may be present on the dental pad, which can cause inappetence.

Tistologic characterization of the skin lesions
2 Focal orthokeratotic hyperkeratosis of a parakeratotic nature

'hysiologic abnormalities
2 Acidosis caused by the diarrhea and excessive salivation
2 Secondary bacterial and viral infections due to abnormalities in the immune
system
2 Growth retardation
2 Reduced testicular development, causing a decrease in the concentration and
motility and increase in morbidity of sperm.

nst—mortem findings
2 Hypoplasia of the following organs:
0 Thymus o Spleen 0 Regional lymph nodes
0 Peyer’s patches
2 Parakeratosis in the:
a F orestomachs o Esophagus

 

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Blood chemistry comparisons between controls and BHZD calves are unremarkable at
irth, but by 3 to 4 weeks of age the plasma zinc and serum alkaline phosphatase
oncentrations begin to decrease. Other biochemical variables appear unremarkable
nless the affected calves have been debilitated by infections and dehydration.

The fact that BHZD calves were susceptible to secondary bacterial and viral infections
1d the hypoplasia observed postmortem in the thymus and lymphoid system, suggested
till the disease was primarily an immune deficiency, to early investigators". The white
ood cell differential is often unremarkable in the affected calves until they become
'stemically ill. However, prior to that they may have had a mild leukocytosis, but
ukopenia has not been observed. The circulating lymphocytes appear large and
unature microscopically, but whether this prevents them from fimctioning normally has
tbeen determined". The thymus of the affected BHZD calves is very hypoplastic and
average weighs 18 g, normal thymic weight should be 250 to 400 g”. Histologic
unination of lymphoid tissues reveals that there is a deficiency of small lymphocytes in
thymic cortical region and nodules are atrophic in the spleen and Peyer’s patches, but
lules in the regional lymph nodes appear normal“.

The clinical characteristics, immunological findings and hypoplastic lymphatic organs
vell as reduced resistance to bacterial infections in BHZD is very similar to AB in
tans“. In addition, both diseases can be treated with pharmaceutical amounts of zinc
ment to reverse all the clinical signs. This makes BHZD an excellent animal model
he investigation of AE. Most of the efforts of producers of animals with genetic

‘ders goes towards identification and eradication; however, the short coming of this

 

 

 

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24
r be realized in that BHZD was being treated with zinc almost 10 years prior to that
atment being instituted in humans”.
Two experiments have been conducted to further define the immunologic
normalities that were grossly observed in the affected BHZD calves. The first
eriment was conducted in vivo and evaluated the humoral and cell-mediated response
ffected BHZD calves as compared to controls. The humoral response was examined
noculating the calves with tetanus toxoid injected intramuscularly once a week for 7
ks. Blood samples were then drawn weekly to determine the anti-tetanus antibody
:entrations, by single radial diffusion technique". Cell-mediated immune responses
: judged by the ability of the calves to develop hypersensitivity reactions to
abacterium tuberculosis and dirritrochlorobenzene (DNCB)". All the calves tested
tive to tuberculin sensitivity prior to the injection of heat inactivated Mycobacterium
culosis. They were then tested again for sensitivity to the two antigens, 1 month
ving inoculation. Dinitrochlorobenzene testing is used in humans to determine cell-
Lted response ability. The injection site after sensitization histologically appears to
)erivascular cuffing in the corium and hypertrophy of endothelial cells". The
tmatory cells present in the cuffing are primarily lymphocytes, eosinophils,
phages and neutrophils. The total serum protein and immunoglobulin
:trations were also compared between the control and affected calves.
results indicated that were no differences in the serum protein levels between
sand BHZD calves. However, the affected BHZD calves did have elevated serum

gG 2 and IgM levels". The initial humoral response in affected calves was

 

 

 

 

 

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nparable to the control animals, but antibody levels began to decrease after 3 to 4

:ks in the affected calves, as compared to controls". The affected calves in this study
not live long enough to induce a secondary humoral response. The cell-mediated
onses in the affected calves were negative to weak compared to the controls for both
Mycobacterium tuberculosis and DNCB. The cell-mediated response of the BHZD
:5 indicated a dysfunction in thymus dependent lymphocytes. There was no direct
me to indicate a dysfunction in humoral responses, since the observed experimental
ts may have resulted from a failure of T- and B-cell interactions“.

1e second study attempted to further characterize lymphocyte alterations in BHZD
5 and examine primary and secondary humoral response to antigenic stimulation.
bility of the calves to mount a secondary humoral response to an antigen would

te that they can produce memory cells. The study used two affected calves with

l and nine controls, to examine lymphocyte responses to antigenic stimulation in

)y the analysis of isolated mononuclear cell types and antibody responses to

)n of bacteriophage @X1 74, in viva”. A lymphocyte blastogenesis assay was

ted by collecting the blood of the calves and isolating the mononuclear cells. The
ere then cultured and placed in six separate wells for 72 hours, that contained: no
1, two different concentrations of phytohemagglutinin-P, concanavalin A and poke
itogen”. Twenty four hours before the end of the incubation, 3H-thyrnidine was

) the cultures and its incorporation into the cells as determined by liquid

tion counting. Monoclonal antibodies were used to identify the lymphocyte,

te and neutrophil populations in the blood samples”. At 4 and 10 weeks of age

 

 

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he calves were injected intravenously with a phage preparation containing 9X] 74, then
heir serum was collected weekly to evaluate primary and secondary antibody production.
The lymphocyte responses of the affected calves compared to the controls varied with

re zinc status of the animals. The affected calves lymphocyte response was within

ormal range at birth then became subnormal with decreasing circulating plasma zinc
oncentrations and returned to that range after treatment”. Thus, the original

assification of BHZD as a primary immune disorder is incorrect, the immune disorder is
secondary complication to the primary hereditary defect of zinc absorption. The
imposition of the mononuclear cell population revealed that the affected calves had a
gnificant decrease in the number of B lymphocytes at 8 weeks of age. The CD4+ and

)8+ T lymphocytes were elevated in one of the affected calves at 2 and 4 weeks of age

:l slightly lower in the other affected calf, but by 12 weeks of age the CD4+ T

nphocytes had decreased in both calves”. The primary antibody response to the
nunization of 9X] 74 phage was within normal limits of the controls, but the

ondary response was significantly lower in both affected calves”. The reduction in B
lymphocytes may have contributed to a decreased secondary antibody response to the
teriophage. The lymphocyte alterations in the affected calves were significant and
ributed to the increased susceptibility to infections; however the observed

rhocyte defects in this experiment were not profound enough to be lethal by

selves”.

 

27
Clinical investigators recognized early on the similarities that existed between calves
vith BHZD and calves that had been fed zinc restricted diets. The onset of clinical signs
nd development of parakeratosis along with the decreases in serum alkaline phosphatase
nd plasma zinc concentrations were identical‘g’so’SI'”. latrogenically induced zinc
eficierrt calves also had a reversal of all clinical signs upon reconstitution of adequate
letary zinc levels”. The benefits of zinc treatment with dermatological diseases have

so been well documented in chickens, swine and cattle46

'53. It was therefore of interest
r researchers to study the affects of zinc treatment in calves that had BHZD. The first
My examined the affects of daily oral zinc oxide treatment on 2 affected calves \
;playing clinical signs, ages 1.5 and 2.5 months. Both calves had a resolution of all

nical signs within 1 to 2 months after treatment began“. The calves recovered quickly

m their symptoms, with improvement in attitude and appetite occurring 1 to 2 days

3r treatment began, most of their skin lesions healed by 7 weeks and new hair growth

sevident at 11 weeks‘3'“. At 5 months of age both calves were sacrificed.

trnortem examination of the thymus and Peyer’s patches appeared normal both

phologically and histologically. The conclusion drawn from this experiment was that

cted BHZD calves were unable to utilize the zinc that normally is available in the

lsupply‘w. Future studies revealed that discontinuing the zinc supplementation led to

recurrence of clinical signs”.

esearchers then focused their attentions on trying to localize the defect in zinc

bolism. Two separate experiments were able to confirm that the defect occurred at

vel of zinc absorption from the GI tract. The basic design of both experiments was

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ame. Control and BHZD affected calves were given radiolabeled zinc either orally
injection into the abomasum and vascular systemms. Then the uptake of the
labeled zinc from the intestinal tract and vascular supply were compared to the
ols. The results indicated that the BHZD calves absorbed zinc three times slower
the intestinal tract than the control animals”. However, the decline in the
labeled zinc activity of the vascular supply was either the same or greater in BHZD
; as compared to controls3s’”. The BHZD calves with extremely low zinc
ntrations absorbed greater amounts of zinc from the vascular supply into their
;. The results made it apparent that the defect in zinc metabolism was related to the
l mechanism responsible for zinc uptake from the GI tract into the vascular supply.
Ier, it has not been determined if the defect occured at the intestinal to cellular
or the transcellular movement of zinc into the vascular supply.
ability of zinc in high oral doses to reverse clinical manifestations of BHZD is
d to occur through a passive diffusion mechanism. The increase in zinc ion
ations in the intestinal lumen and the decrease in concentration in the vascular
oduces a gradient which may allow for sufficient quantities of zinc to diffuse into
als”. Since BHZD disorder can be treated with zinc, the pathway responsible for
t is assumed to be involved in a carrier—mediated cellular mechanisms
le for zinc absorption. Recently investigators identified a cysteine-rich
protein (CRIP) in large quantities in developing rat intestine”. A model for
rption was subsequently proposed that depicted the interaction of CRIP, MT and

binding ligands, along with a paracellular pathway”. The essential role that

 

29

plays in the model for zinc absorption, implies that it is necessary for the
rption of zinc from normal dietary sources.

e subject of this dissertation is to investigate the clinical and biochemical
festations of BHZD during neonatal calf development and the resolution of signs
treatment, as well as to characterize CRIP in bovids and test its role in BHZD. The
' used a pedigree of five affected calves and one obligate heterozygote produced by
rbryo transfer study using semen from an affected bull and two obligate
)zygote embryo donors. In both AE and BHZD, the basic defect is an inability to
:ntly absorb zinc from the gastrointestinal tract, this may not be the etiology of IED
lal acroderrnatitis in Bull Terriers. Studying affected calves will allow further
t into both the human and bovine diseases, thus providing valuable genetic systems
can be studied to elucidate the metabolic steps necessary for the absorption and
ition of zinc.
next sections will review what is currently known in the literature about the

and mechanisms involved in the absorption of zinc from the GI tract.

the Diet:

es have indicated that zinc absorptiOn by the GI tract is dependent on the
bility of the form of zinc, the presence or absence of other elements and

' g agents, the lumenal pH and the dietary zinc status of an anirna158‘59'w. The
hich zinc is transported across the brush border membrane is unknown;

zinc can be absorbed as an oxide, sulfate, acetate and carbonate. Zinc supplied

 

30
s an oxide is absorbed with 63% efficiency, when compared to a sulfate or acetate“.
igh levels of dietary proteins have been demonstrated to directly increase zinc
)sorption and protein restricted diets to limit zinc uptake“. This is thought to occur
rough the increased presence of zinc protein chelators contained in high protein diets.
re chelating proteins hypothesized mechanism of action is to bind lumenal zinc and
event it from being bound to other factors or being utilized by bacteria making the zinc

available for absorption by the mucosal cells”. Some studies have indicated that the

 

ditions of disodiurn etliy‘ 1' ' ‘ ‘ ‘ ‘ (EDTA), into animal feed enhances

 

ight gains and zinc plasma concentrations”. Disodium cthy' " ' ‘ r ‘ ‘ is
trong zinc chelating agent, it has been hypothesized to work through forming a

nplex that is more readily absorbed by the gut than zinc that is not complexed”. The
ial physiologic mechanisms responsible for EDTA’s affects on zinc absorption by the
rave not been determined.

Zinc absorption is decreased by a number of factors. Iron competitively depresses
absorption when present in excess amountsg. Grain based diets have a lot of fiber

:h contains phytates that complex with zinc making it unavailable for absorption in

il tracts”.

re dietary zinc status of an animal strongly influences the amount of zinc that is

bed from the GI tract; this has been demonstrated in rats, goats and calvess"9'58'63.
leficient ruminants and rats absorb a higher percentage of orally administered 652o

0 animals of adequate dietary zinc status'9'58’6“.

 

31
.ocalization of Zinc Absorption in the Gastrointestinal Tract:

Investigations into determining the site at which zinc is maximally absorbed by the

64,65,66,67,68,69,70,7 l ,72,73,74,75,76 There have been

.testinal tract, have yielded conflicting results
veral experimental techniques used, each one designed to account for as many

lysiologic factors as possible in an effort to generate more accurate data. In vitro studies
ve employed rat tissue bath preparations, resulting in data that indicated that the ileum
lS the primary site of zinc absorptiona’“. In vivo experiments used in situ ligated loops
rat bowel, to indicated that the duodenum was the site of maximal zinc
:orption"'72'73'74'75’7‘. Direct dosing techniques in live intact rats and calves yielded no
nificant regional differences in total zinc absorption capabilities in the gastrointestinal

16"“. One point in which all the studies seem to agree upon was that there was very

3 absorption of zinc from the stomach, cecum and colon of rats‘9'7l'75'76.
he discrepancies between experimental results can be accounted for by examining
rariety of experimental techniques utilized. The most obvious difference between
riques is whether the experiment was performed in vivo or in vitro. Comparing
rimental results is difficult since they have been recorded and reported in numerous
of measurement. Some of the experiments reported their results by comparing the
ntage of administered 65Zn dose absorbed by each intestinal region, and other studies
‘rcentage of uptake of 65Zn by various tissues upon administration of zinc at various
as. The units in which the data were collected and calculated could be in per unit

of intestinal segment, per unit weight of intestinal segment and per unit weight of

’. All of these studies were trying to determine which intestinal region, by

 

 

 

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32

omparison, absorbed the greatest quantity of zinc and the efficiency of absorption for the
:gions.
>1 vitro studies

The primary advantage of using in vitro techniques to study intestinal zinc absorption
that they are easily manipulated and controlled systems. In vitro experiments
:termined the ability of different intestinal regions to absorb zinc by utilizing both strips
.d everted sacs of tissue. The strips were incubated in precounted “Zn solutions for time
:ervals up to 2 hours“"7'“'". The everted sacs were placed in the same type of
:counted incubation solution and the solution as well as the fluid in the pouch of the
:s was counted after incubating. The decrease in activity of the “Zn incubating
ution was then quantified and their activities compared between intestinal segments“.
The results indicated that the distal portion of the isolated rat gut absorbed a greater
ntity of “Zn than the proximal or middle portions of the intestine66'67’68'“. This portion
‘esponds to the ileum. The duodenum absorbed 14.8% less zinc than the jejunum and
ejunum absorbed 27.2% less zinc than the ileum“. Data from several studies
:ated that the ileum absorbed more zinc in both the strip and everted sac experiments
'dless of whether the results were reported in 65Zn uptake/g of fresh tissue or 6SZn
:e/percentage of total length of intestine“.
[ere are many disadvantages in performing in vitro experiments, all of which were
:1 to the lack of normal physiological parameters. The lack of an intact blood supply
tissues could have a number of effects on the intestine’s ability to absorb zinc. Any

:nous regulation of zinc absorption that may occur as a result of plasma zinc levels

 

33
uld not be taken into accounted for. Although attempts were made to provide the
rues with oxygen and nutrients to sustain their metabolic functions, they would still be
me to degradation, which may alter the metabolic processes involved in zinc
orption. In vitro experiments have no way of accounting for peristaltic movement of
GI tract. An alteration in the normal peristalsis of the GI tract also changes the
runt of time that ingesta is in contact with various intestinal regions, which in turn
le determine how much zinc could be absorbed by an area in that time period. The
tion and length of the intestinal segment used to assess zinc absorption for that region
,e GI tract may not be representative of the organs total zinc absorption capacity. For
rple, the proximal duodenum and ileum have been reported to absorb zinc at a slower
han their distal counterparts“. The time intervals and the overall length of time that
periment runs influences the outcome of results, if one region of the GI tract has a
:r efficiency of zinc absorption than another. The dietary zinc status of the animals
to the tissues being harvested has also been shown to influence the net “Zn
)tion from the GI‘9'58'“.
9 studies
a in vivo studies were performed on anesthetized rats and involved isolating
ral segments of large and small bowel with ligatures. This procedure attempts to
.ze the competition that the presence of large amounts of non-radioactive zinc in
may have on the absorption of “Zn“. The sacs were made by placing a ligature at
ds of the intestinal segment, with care being taken not to disrupt the blood

”5'76. “Zinc was then introduced into the intestinal sac at the proximal end and

 

 

34
intestine was temporarily placed back into the abdominal cavity. The rats were
ificed at time intervals up to 2 hours post-injection and the intestinal sacs and body
us were removed to determine their “Zn activity. Intestinal absorption of “Zn was
essed both as a percentage of the administered dose in each segment and as a
:ntage of “Zn administered per gram of intestinal tissue“. The tissue retention was
:ssed as a percentage of administered “Zn per gram of total organ and the whole
I was expressed as a percentage of “Zn per ml of fluid75’76.
,ble 3 is a comparison of the percentages of the administered dose of “ Zn absorbed
ferent intestinal sections in two separate studies that utilized in vivo ligated sacs.
A measured absorption at 10 and 120 minutes in three different tissues”. Study B

red the absorption at 15, 30 and 120 rrrinutes in two different tissues”.

3 - The Percent of the Administered Dose of “Zn Absorbed from Different
Intestinal Regions in Two In Vivo Ligated Sac Studies

 

 

 

Intestinal Regions 10 min 15 min 30 min 120 min
A Duodenum 7% --- --- 45%
Jejunum 3% --- --- 21%
Ileum 3% --- --- 16%
B Duodenum —-- 9.2% 23.1% 32.1%
Jejunum --- 1.5% 8.3% 18.5%

 

 

 

 

 

 

esults of the two studies indicated that the duodenum absorbed the greatest
of “Zn in the shortest amount of time75'76‘77. The highest levels of uptake into

tans such as the liver, kidney and heart were observed when “Zn was injected

 

 

 

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35

the duodenal sac as compared to other GI regionsm‘.

There was a discrepancy

veen the two experiments as to which intestinal segment absorbed the second highest
ntity of “Zn. The results of one study clearly indicated that the ileum absorbed and
ibuted to the organs more zinc than the jejunum“. In the other investigation the

,um was found to absorb slightly more zinc than the ileum“. On average the stomach
:ecum absorbed 0.2% of the administered “Zn dose and the colon 2.2%". The

rined data of the studies indicated that the absorption of “Zn continued to increase

'ly for 2 hours post-administration.

.e aforementioned experiments have been designed to reflect the amount of

nous “Zn absorbed by intestinal segments or retained in body tissues75'76’77. They
take into account the amount of endogenous zinc present in the tissues of the

nal regions and its influences on the absorption of exogenous zinc. Endogenous
present in all regions of the intestine in the process of being excreted back into the
of the GI, as well as being stored by intracellular proteins. A previous study

d that only mucosal zinc content is representative of the absorptive capacity of an
al region, due to the fact that it also accounts for the endogenous intracellular zinc
rat may regulate additional zinc absorption". In turn, endogenous zinc levels are
in the dietary zinc status of an animal. Therefore, it was necessary to design a

rat compares the ability of intestinal regions to absorb zinc independent of the

es of endogenous zinc levels. The study required two groups of rats fed identical
ne group had tubes surgically placed in the ligatures of both ends of the in vivo

res and ZnSO4 solution circulated through them for 2 hours”. At the end of the

 

 

 

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36

ion, the fluid was collected and the intestinal segment removed from the rats. The
.d group served as a control, they too had their intestinal tissues harvested and their
lnal zinc concentrations served as a base line. Both groups had the mucosal surface
:d from their intestinal segments and ashed for zinc absorption analysis by atomic
rtion spectrophotometry (AAS)7°. By determining the zinc concentration of the
[131 regions of the control group and the perfused group results were expressed as
Ivement of zinc into the mucosa per gram of mucosal dry weight over 2 hours. The
solution that was collected after perfusion also had its zinc concentration
ined. The difference in concentrations pre— and post-perfusion were expressed as a
age of zinc absorbed by the intestinal sac.
he perfused intestinal segments had higher zinc concentrations than the controls.
3 control and perfused groups had higher zinc concentrations in the ileum than the
un and jejunum”. The ileum also had the highest percentage of zinc absorbed at
he jejunum was 20.2% and the duodenum 19.1%”. The combined data of the
iicated that the ileum had the greatest capacity for zinc absorption. These results
:t the previous in vivo ligated sac studies, that indicated that the duodenum
zinc in the greatest quantity compared to other intestinal regions. The
y in results between the experiments indicates the significance of maintaining
eristaltic activity of intestinal regions and proximal to distal flow of intestinal
’. Allowing the intestinal contents to flow through the region during the
it limits the possibilities of nonspecific binding of zinc to ileal contents and the

ty for utilization by bacterial organisms. In addition, the movement of the

 

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37

sate prevents the saturation of zinc absorption mechanisms allowing the segment to
nue to absorb zinc. These findings also collaborated with those previously reported
vitro experiments“'“'“. There is no dispute that the duodenum absorbs zinc with

:r efficiency than the other intestinal segments“. If the duodenum also transfers zinc
vascular space quickly, as indicated by the faster appearance of radioactivity in the
kidney and heart, then the endogenous mucosal zinc levels would be lower in the

num; which would allow for the absorption of additional “Zn from the lumen of the
fthe ileum transfers zinc to the vascular space at a slower rate, then there would be
5Zn present in the mucosal cells for a longer amount of time, which would decrease
litional absorption of “Zn from the GI lumen: Accounting for the differences in

nous levels of zinc in the mucosal cells of the two regions by the use of perfused
ligated sacs, the ileum appeared to absorb a greater quantity of zinc than the

urn.

variation in results between in vivo experiments can be attributed to many of the
)reviously stated as disadvantages of in vitro experiments, with the exception of

v0 experiments maintaining an intact blood supply to the tissues. One

ltage unique to in vivo ligated sac studies is the effects of anesthesia on

sm. The anesthetics used to perform surgical procedures may influence

:ntal results by affecting blood flow and nerve enervation to the intestine altering
alsis of the digestive tract and/or zinc absorption mechanisms-’0“.
>parent that prevention of normal peristalsis of ingesta and limitations of the

1 location of the intestinal region studied raises some doubts in regards to the

 

 

 

38

s of in vivo ligated sac studies. To examine the absorption abilities of intestinal
5 without these experimental limitations, a group of rats were administered “ZnCl2
on through gastric intubation and sacrificed at specific time intervals“. By
'stering “ZnCl2 through intubation and following its decent through the GI tract,
estigators were able to determine the ability of each intestinal region to absorb zinc
mpare their values. The results of the study indicated that “Zn was absorbed from
denum in the greatest quantity and efficiency when compared with other intestinal
s“. In addition, results indicated that the passage of “Zn along the intestinal tract
=ry rapid and the majority of it was excreted in the feces. Indeed these frndings
milar to those of the in vivo ligated sac studies that did not maintain continual
l perfusion.
dosing procedure
direct dosing procedure was another technique utilized to determine the site of
.l zinc absorption in the intestinal tract. The direct dosing protocol accounts for
:rent rates in which ingesta passes through intestinal regions and allows an
ent of the contribution of each region to the overall absorption of zinc“. Animals
diets that were zinc deficient or adequate for a designated period of time.
:ized animals then had “Zn directly injected into the proximal end of small
regions via a laparatomy or a paralurnbar incision“‘“. Various organ samples
were then collected and analyzed 24 hours after dosing to determine their “Zn
6 tissue “ Zn retention values were plotted against the site of injection, which

ssed as a percentage of intestinal length from the proximal to distal end“.

 

 

39
This protocol was originally employed in two studies in the early 1970’s by the same
:stigatorM'“. One study utilized this technique in adult male rats and the other in
stein bull calves approximately three and a half months in age. In both studies the net
absorption was found to be uniform in all regions of the small intestinal tract“’“.
duodenum was more efficient at absorbing zinc, compared to the jejunum and ileum.
fastest rate of ingesta passage in the rat occurred in the duodenum and decreased by a
r of almost twenty as it progressed to the distal end of the ileum““'“. Thus even
gh the ileum may not have absorbed zinc as fast as the duodenum, it had a longer
to do it“‘“’". The net effect of these factors, faster rate of absorption but shorter
me time in the duodenum and slower rate of absorption but higher residency time
ileum, is the finding of similar net zinc absorption throughout the small
ne““. Rats fed zinc deficient diets had more “Zn absorbed from the distal portions
r small intestines than the proximal and overall had higher “Zn activity levels in
rgans than zinc adequate rats.
) direct dosing studies with modified protocols involving rats and calves produced

64,65

ting results when compared with the aforementioned studies . Their results

69,7]

:d that the duodenum was the major site of net zinc absorption . Rats were fed
eficient diet for 2 days then their food was withheld for 18 hours prior to the

r of “Zn into the various intestinal regions. Restricting the rats’ feed intake was
decrease intestinal levels of endogenous zinc that could compete with the

an of injected “Zn. Their whole-body was assayed for “Zn for 5 days along with

s or urine they produced. The results for each region were expressed as a

 

—.————— ."5

40
:ntage of the “Zn absorbed by the entire intestine. The duodenum absorbed 57.9%,
:junum 8.4% and ileum 3%, these results were consistent with the gastric intubation
n vivo ligated sac studies previously described".
he study with the calves investigated “Zn absorption rates in zinc adequate and zinc
ent animals. The modifications in the direct dosing protocol included the surgical
nent of a catheter three weeks prior to the experiment being conducted“. The
nent of the catheter prior to conducting the experiment was done to prevent the
: of anesthesia and postoperative recuperation on the results. Absorption of “Zn in
periment was defined as the “Zn not recovered in the intestinal contents and the
. The results indicated that 46% to 58% of zinc was absorbed within the first hour
)sing, which corresponded to a greater quantity of zinc being absorbed more
fly from the duodenum relative to the jejunum and ileum, and little absorption
59. The zinc deficient calves had a higher percentage of “Zn absorbed than the
ed zinc adequate diets.
f the direct dosing studies concluded that zinc was most rapidly absorbed from
lenum“’“'“'". The modified protocol for direct dosing only measured the
)n of zinc by the intestine for 1 hour post-dosing. This helps to explain why they
ed the duodenum to be the major site of absorption. The jejunum and ileum
it have had enough time to absorb zinc from the lumen to their maximum
1nd the dosage would not have traversed the full lengths of both regions. Thus,

num would appear to absorb the greatest quantity of zinc.

 

41
heir findings also concurred that deficient calves and rats absorbed more zinc and
red it longer in their organs. There could be several explanations as to why the zinc
ient calves absorbed and retained more of the “Zn than the zinc adequate calves.
irst, is that the zinc deficient calves may have decreased peristaltic activity in their
ct, allowing for an increase in the amount of time that zinc can be absorbed from
gesta. The second, could be that the mechanisms responsible for zinc absorption in
tract are up-regulated and are more efficient in zinc depleted animals. Third, the
;e in “Zn absorption and tissue retention in zinc deficient calves could be the result
crease in endogenously produced zinc which dilutes the radiolabeled zinc and
res its apparent absorption in zinc adequate animals“. Forth, a zinc deficient
ofien has a decrease in appetite, therefore there would be less ingesta in the
'e tract that could contain competing ions, proteins to form zinc complexes and
ctors.
to the numerous experimental variables that can occur within a study and between
it is not surprising that there have been so many different conclusions drawn as to
ion of the GI tract absorbs zinc in the greatest quantity and most efficiently.
he obvious differences between in vitro and in vivo studies, the next most
t variable is the dietary zinc status of the animal. The impact of moderate
as in dietary zinc status on individual intestinal regions to absorb zinc is not
y clear, however the overall impact on zinc absorption is evident.
tended time over which direct dosing experiments measure zinc absorption may

I the discrepancy in results of these studies as compared to the in vivo and in

 

 

42

0 studies which only last 2 to 3 hours. The studies have demonstrated that the distal
turn has the capacity to absorb large quantities of zinc, however, this takes place over
tended periods of time and maximum zinc absorption occurs between 4 and 8 hours
er introduction”. The studies conducted in viva and in vitro do not extend past 2 to 3
rs, producing results that over—evaluate the total absorption of the proximal intestine
itive to the distal intestineég'".
Overall the results from direct dosing studies indicating that net zinc absorption is
ilar in the duodenum, jejunum and ileum are probably correct. Although the
denum absorbs zinc with the greatest efficiency, it also has the shortest amount of

in which the ingesta has contact with the mucosa. The jejunum and ileum are both
er in length than the duodenum and move ingesta slower. Therefore, although they
absorb zinc with less efficiency, they would have a longer time and a greater amount
:a to absorb it. The overall effect of these factors would be that the net absorption of
vould be similar for all the intestinal regions. An interesting note is the differences
orption rates between the three segments and what accounts for the differences in
:es. Whether absorptive proCess are more active or different in the duodenum than

rer two regions remains to be determined.

ics of Zinc Absorption:
re have been a number of studies performed with zinc radioisotopes to develop
models that represent the mechanisms of zinc absorption”. These studies were

i to measure the rate in which exogenously introduced “Zn was absorbed from

 

 

 

43
gastrointestinal tract and into the vascular space. The findings have led to the
rosition of a model in which two pathways are responsible for zinc absorption from
gastrointestinal tract, the first is a saturable pathway and the second a nonsaturable
vay68'7m'73'75’78. In vitro studies have measured the rate of zinc accumulation in the
:inal wall and the rate of transmural passage to the serosa“. In viva studies have
ured several factors overtime, some have determined the amount of “Zn activity in
usocal tissue, others the loss of “Zn activity from lumenal contents and appearance
a in the vascular space7”72’75’78. Additional studies in rats have examined the affects
ary zinc depletion on the rates of absorption of zinc from the lumen of the gut and
m to the vascular space.
transmural absorption of zinc from the lumen of the intestine to the vascular space
:s two stages. The first stage is the uptake of zinc from the lumen into the mucosal
d the second stage is the movement of zinc through the mucosal cells to the
r space22'68'73'78. The first stage of zinc absorption appears to occur by two
isms. To further define the characteristics of the mechanisms responsible, in
fed zinc adequate diets, studies have relied on the administration of doses of “Zn
intestinal lumen and the measurement of uptake into mucosal cells over
”5'79. When “Zn activity in mucosal cells was plotted as a fimction of time, two
relationships were apparent in the graphs: one was curvilinear, indicating a

mechanism and the other was linear, indicating a nonsaturable mechanism.

 

 

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Doses ranging from 5 to 50 pg of “Zn injected into in viva ligated loops of
)denum, had two distinct absorption curves". The first curve indicated that animals
:ed with 5 pg absorbed “Zn linearly within the first 15 minutes of dosing; however, as
dosages were increased to 50 pg and the time of measurement extended to 30
lutes, the absorption began to plateau . When results were plotted in the double-
.procal manner of Lineweaver-Burke, a relationship characteristic of a carrier-
1iated process was observed“. Gastric intubation of “ZnCl2 in rats indicated that the
:entage of “Zn absorbed by these animals was not dependent on lumenal zinc content
1e range of 0.25-1.0 pmol and saturation of the process occurred when lumenal zinc

lent reached 5 pmol22

. The daily intake of zinc in rats fed standard laboratory diets is
oximately 3 to 4 pmol/day, therefore there is probably less than ’1" pmol of zinc in the
"ointestinal tract at a time, meaning that most rats that are not zinc depleted absorb
within the linear range of the carrier-mediated process”.

[though there was a saturable carrier-mediated process that accounted for a portion
:zinc absorbed from the lumen, an additional mechanism was responsible for the
med absorption of zinc when lumenal zinc content increased above the capacity of
trier-mediated pathway. Several experiments have shown that when lumenal zinc
It exceeds 5 pmol for more than 30 minutes, the amounts of zinc absorbed over
'hen plotted against increasing zinc dosages produces a linear relationships’zz'".
rear relationship implies that the absorptive process is nonsaturable and occurs

Jmenal zinc concentrations exceed the carrier—mediated capacity. It still remains

=termined if the nonsaturable component is reliant on nonspecific binding of low

 

 

 

 

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olecular weight proteins, passive diffusion between enterocytes or leaky membranes
.owing diffusion into the cells.

The second stage of zinc absorption involves the mucosal to vascular flux of zinc.
1e kinetics of zinc absorption intracellularly have been determined, but the mechanisms
:ponsible for its movement have not been fully elucidated. There appears to be two
ols of zinc in mucosal cells. The first pool of zinc is rapidly transferred to the vascular
ICC and the second pool of zinc is slowly transferred to the body over time.
The rapid transfer of zinc intracellularly to the blood supply occurs without zinc being
Ind within the mucosa”. When zinc is first introduced into the lumen of the
trointestirral tract the mucosal uptake peaks 30 minutes after infirsionmm'". The
kmucosal “Zn activity corresponds to a peak in plasma “Zn activity after gastric
bation“. This indicates that the majority of the mucosal zinc absorbed in the initial
ninutes is transferred into the vascular space almost immediately. Studies utilizing in
ligated sacs have produced results that indicate that 30% of the “Zn introduced into
ac was absorbed into the vascular space within the first 30 minutes". The
ously described experiments determined that both a carrier-mediated process and a
rturable process accounted for the absorption of zinc from the lumen of the GI into
sal cells. The carrier-mediated process became saturated 30 minutes after the
lal zinc content exceeded 5 pmol”. The carrier-mediated pathway is the primary
ay in which zinc is absorbed in the first 30 minutes after introduction into the GI

and peak serum zinc concentrations are also attained within this time; thus it

 

 

46
rs that the carrier-mediated mechanism of zinc absorption from the lumen of the GI
LlSO account for the pool of zinc that was rapidly transferred to the vascular space.
re in vitro study indicated that zinc was absorbed very rapidly from the lumen of the
It subsequent transmural movement of zinc into the vascular space was undetectable
to 1 hour“. The contradiction in results can be accounted for in the variation in
mental techniques. The in vitro study involved the removal of the intestinal
nt from the rat, which eliminated vascular perfusion. The continual blood flow to
estine allows zinc to be removed quickly from the basolateral surface of mucosal
75. The GI lumen to mucosal flux was not affected by the lack of blood flow over
6 the experiment was conducted. However, the mucosal to vascular flux of zinc
occur within the first hour of measurement; therefore, it appeared the uptake of
‘zinc by the vascular supply maybe dependent on blood born components and/or
:ed by vascular perfusion.
slower transfer of zinc from the mucosal space to vascular flow involved it being
1 the mucosal cells. Two studies utilizing in viva ligated sacs, were conducted to
re first, if mucosal bound zinc transferred to the vascular supply and second, to
ize the mechanism responsible. In the first study, 5 pg doses of “Zn were
:d into the lumen of in viva ligated sacs of duodenum and the loops excised at
i 60 minutes post-dosing, then hourly up to 6 hours. The results indicated that
rtes approximately 30% less zinc was in the mucosal tissue than at 30 minutes,
vas recovered from the intestinal washing of the lumen". Therefore the zinc

ransferred into the blood stream and not secreted back into the intestinal lumen.

 

 

47

the second study, dosages of “Zn ranging from 1 to 50 pg were introduced into the
testinal lumen for 15 minutes, the amount of “Zn activity in the body was assayed
urly". By plotting the mean rates of loss of “Zn activity from the intestinal lumen

m l to 3 hours against the mean “Zn content of the body at 1 hour, a curvilinear graph

produced. Thus, the slower phase of zinc transfer from the mucosal cell to the
scular space was proposed to be a carrier-mediated process.
Investigators have attempted to determine whether the lumen to mucosal flux or
cosal to vascular flux of zinc is affected by changes in dietary zinc status. It is widely
:ed that the absorption and retention of zinc from the gastrointestinal tract is increased
inc deficient animals8'22'73'78. Two experiments conducted to examine the effects of
deficiency on zinc absorption employed the techniques of gastric intubation and
Iltaneous perfusion of the GI lumen and mesenteric artery to the portal vein. The
ic intubation study had groups of rats maintained on diets containing different zinc
entrations for two weeks prior to “ZnCl2 administration”. In the latter study, the rats
fed zinc deficient diets 3 days prior to the lumen of the small intestine being
sed with “ZnCl2 concentrations ranging from 5 to 200 pM for 30 minutes”.
re uptake of zinc from the GI lumen was significantly greater in the zinc depleted
)mpared to the zinc adequate rats, when “Zn lumenal concentrations were less than
I; however, above 50 pM the uptake of zinc was similar between the two groups".

'mal transport rate for the carrier-mediated pathway, was three times higher in

c depleted group, but the non-saturable pathway had the same transport rate for

cups”. Therefore if the lumenal zinc concentrations were within the linear range

v Vv-T“. ,7

 

48

rsorption for the carrier-mediated process the zinc depleted rats had an increase in
ability to absorb zinc, as compared to zinc adequate rats. At concentrations above
near range of absorption for the carrier-mediated mechanism, zinc was absorbed by
on-saturable mechanisms at the same rate for both groups. The gastric intubation
iment also came to a similar conclusions, finding that the total amount of “Zn that
be bound to the mucosa was independent of zinc dietary status”.
.6 increase in the ability of the carrier-mediated pathway to absorb zinc, in the
r to mucosa stage of absorption in zinc depleted rats, can be explained by two
misms. The first would be an increase in the number of mucosal binding sites, the
i would be an increase in the efficiency of the carrier-mediated pathway to absorb
Lnsfer zinc. Since the gastric intubation experiment indicated that the decrease in
NM to the mucosa, was at the same rate in zinc depleted and zinc adequate rats, it
5! that there were the same number of binding sites present in the mucosa between
. groups”. Therefore the increase in zinc absorption from the GI lumen has to be
in an alteration in the carrier-mediated pathway. If the carrier-mediated pathway
absorption from the lumen to mucosal flux represents the rapidly transferred pool
.n the mucosal to vascular flux, as previously stated, then in order for there to be
ase in zinc absorption in zinc depleted rats, there would have to be an increased
of carrier-mediated bound zinc to the vascular space”. Thus the lumen to

zinc flux would have to be a mechanism that was stimulated when lumen

zinc content fell below 0.24 pmol and would obligatorily absorb zinc into the

asorbed pool to be transferred to the vascular space”.

 

 

I...

s...

VV‘

‘1

\i

 

49
An additional simultaneous perfusion study also verified that the increase in the
0nd stage of zinc absorption, mucosal to vascular flux, in zinc-depleted rats was due to
ncrease in the turnover of zinc in the rapidly absorbed pool and not an increase in the
: of the rapidly absorbed pool. In this study, rapidly absorbed pool did not increase in
until the total “Zn mucosal content exceeded 200 nmol/g of tissue”. The zinc
:entration in the lumen was no longer in the linear range at this concentration and
ration of the carrier-mediated process would have occurred. The half-life of the zinc
)ver rate was examined in the rapidly absorbed pool and found to be 50% less in the
depleted group than the zinc-adequate group, indicating that the zinc-depleted rats

:zinc from the rapidly absorbed pool into the vascular space faster”.

 

appeared that the total amount of zinc that can be bound to the mucosa is not

need by dietary status. The increase in intestinal lumen to mucosal flux of zinc
ption in zinc depleted rats occurs by the stimulation of a carrier-mediated process,
also represents the rapid absorption pool in the mucosal to vascular flux. The

:al transfer of zinc to the vascular space is increased by increasing the turnover rate

rapidly absorbed pool, but not the number of binding sites available.

opmental Maturation of Zinc Absorption:

r1 effort to further characterize the mechanisms responsible for zinc absorption, a
'as conducted to examine the existence of a maturational pattern. Using an in viva
rass perfusion technique, the study examined net zinc absorption and lumen to

l flux of zinc fi‘om segments of small and large intestine of suckling (14-15 days),

a...

7..

\

 

\\.v... \Tm \ -.a..u\

 

50
mling (21-22 days) and adolescent rats (42-45 days)”. The intestine was divided into
:e segments proximal, distal and colon, to study the absorption characteristics of each
1e different age groups. The net zinc absorption and the lumen to mucosal flux was
rrnined by the disappearance of “Zn from the intestinal lumen. The results of the
y demonstrated that all segments of the gastrointestinal tract at all ages transported
out of the GI lumen”. Table 4 represents a description of the graphs produced by the
ionship of the rate of zinc absorption to the initial concentration of zinc in the
ion perfusing the intestinal segments. The term saturable refers to a graph that fits
aelis-Menten kinetics, of a carrier—mediated process. The term non-saturable refers
raph that fits a straight line when analyzed by the formula (Y = A+Bx) of the least

a fit.

4 - Kinetic Characteristics of Zinc Absorption in Three Different Sections of
the GI Tract at Different Ages in the Rat

 

above data indicated that rats are born with an operational carrier-mediated zinc
on mechanism in the proximal intestine, that develops progressively with age in
1 segments of the GI tract“. The affinity of the rats to absorb zinc by the carrier-

1 pathway for each the intestinal segments as they manned were calculated using

 

51

values for the Michaelis-Menten kinetics equation. The results of the study
onstrated that the KIn value of rats increased with age, therefore the carrier mediated
way in suckling and weanling rats had a higher affinity to absorb zinc during periods

ctive growth”.

rc Absorption from the Gastrointestinal Lumenal:
here have been various models proposed for zinc absorption from the intestinal
:n, some have generated controversy and some have been disproven. One area of
: debate has been the possible role of intralurnenal and/or intracellular zinc binding
ds (ZBL) in zinc absorption.
“ole of intralumenal zinc binding ligands in intestinal zinc absorption
re facilitation of zinc absorption by intralurnenal ZBLs can occur in several ways.
auld involve the binding of zinc to a specific or nonspecific ZBL in the intestinal
. Once zinc was bound in a complex with a ZBL, it could be transported into
a1 cells at the apical cell surface”. At the basal membrane, zinc could then be
rred to plasma albumin. This mechanism could account for either the saturable or
urable pathway of zinc absorption from the intestinal lumen into mucosal cells. In
nario if the concentration of lumenal ZBLs was limited, then they would become
d in the presence of high lumenal zinc concentrations. An alternative scenario
e that ZBLs would always be in excess and zinc could be transported into the
cells and to the vascular space, without the ligand being saturated, which may

mm for the intracellular rapidly absorbed pool.

 

52

Another mechanism would be the disassociation of zinc from the lumenal ZBL at the
rcosal cell surface and the reassociation of zinc to a cell surface ligand. Once the zinc

s bound to the cell surface it could be transported into the cytosol. There are pH

dients present along the brush border membrane of intestinal epithelial cells”. The
rssociation of zinc from a lumenal ZBL could be facilitated by the complex coming
contact with a different pH, as the zinc is released, it could become reassociated with
ll surface ZBL as it experiences a change in pH. If the cell surface ZBL had a higher
binding affinity, the lumenal ZBL would be obligated to give up its zinc. This
ranism would represent the saturable pathway of absorption from the intestinal

n and could be responsible for either the rapidly absorbed pool or slowly transferred
of zinc intracellularly. If the transfer of zinc to a cell surface ligand represented the
y transferred pool of zinc to the vascular space, then it could work in tandem with a
anism that would be responsible for the rapidly absorbed pool. The rapidly

:ed pool of zinc could utilize a paracellular pathway, or a lumenal zinc binding

ex could form in the presence of high zinc concentrations and be absorbed through
rsh border membrane in its entirety.

: final mechanism proposed would involve the transport of the complex into the

11 cells, where the zinc would disassociate and reassociate with intracellular

. The intracellular ZBLs could be designated for both storage and transport to the
r space. Although this model would satisfy the intracellular kinetics of zinc

ion, which require both a rapid and slow zinc transfer pool, it is hard to satisfy the

3 mucosal cell flux kinetics of absorption. This however leaves the mechanism

 

.1
ur'
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...1.. ..x... .1.. w. .I\

 

 

 

53

rat would account for the saturable, carrier-mediated portion of zinc absorption
nexplained. If the lumenal ZBLs were limiting and they became saturated with zinc
ren the non-saturable mechanism of zinc absorption would be unexplained.

Whatever mechanisms involved in intralumenal ZBLs assisting or facilitating zinc
rsorption from the GI tract, the system would probably be driven by the binding
finities of the proteins or other compounds for zinc along with varying pH gradients.
1e alternative would be an active transport mechanism. There has been some research
0 the possibility of an active transport mechanism operating during the absorption of
c from the GI, but it is inconclusive as to which area of transport it may occur at and if
ccurs at all”.
e identification of intralumenal zinc binding ligands in the intestine
?revious experimental data determined that the carrier-mediated mechanism
ronsible for zinc absorption from the intestinal lumen into mucosa cells was not fully
:loped until rats reached adolescence“. Therefore, in order for neonates to be able to
rb zinc in sufficient amounts from nutritional sources, there has to be a mechanism
:an facilitate zinc absorption until the maturation of the GI tract. Determining the
al developmental steps may define the components responsible for zinc absorption
r gut of mature animals and humans.
sorders of hereditary zinc deficiency such as AB, in humans and BHZD are
ent models for the study of intestinal zinc absorption. Patients with AB do not
11y display clinical symptoms until neonates are weaned from breast milk to cow’s

Human milk has been therapeutically administered to AE patients". Thus, there

 

 

54

ay be a special ligand present in human milk that is either lacking, or insufficient in
juantity, or inhibited in cow’s milk. This ligand might allow zinc to be absorbed in
rdequate amounts by infants. Although there was a distinct therapeutic effect of breast
rilk in AB affected individuals, there was no therapeutic effect evident with the
ontinued administration of dam’s milk from lactating cows whose calves were affect
ith BHZD. All the affected BHZD calves, whether they were maintained on maternal
ilk or milk replacer developed clinical manifestations of zinc deficiency between 3 to 7
eeks of age. Gel filtration and chromatography of bovine and human milk samples
ows for the identification and comparison of proteins that bind zinc.
Cow’s milk is very rich in proteins, 80% of which is the high molecular weight
MW), phosphorous-rich casein protein“. Human milk only contains about a fourth of
protein content of cow’s milk and the majority are whey proteins. Human milk also
rtains less zinc than bovine milk and the concentration decreases in both species as
ation progresses. In humans zinc primarily complexes with low molecular weight
IW) proteins”. The discovery that zinc was forming complexes with LMW proteins
urnan milk, that may not be present in cow’s milk, led to the proposal that one or
e of these complexes were responsible for the therapeutic value of human milk in the
ment of AE patients. These same LMW, ZBLs could also serve to enhance the
rption of zinc in neonates prior to their intestinal lumenal mechanisms being
rtional’”.
re zinc binding characteristics of rat milk ligands were examined to determine if they

be used as a model to study human milk zinc binding characteristics“. Rat milk

 

 

55

ands were isolated and identified by harvesting the milk from rats with litters that had
kled for 10 days. The defatted milk was then passed through two gel filtration
umns, the resulting fractions were assayed for zinc content by AAS“. The elution
em from the gel filtrates produced three peaks; one was similar to that observed in
an milk samples but was absent in bovine milk samples. It was concluded that the
ans and rats had a similar ZBL present in their milk and the rats were a suitable
el to study.
rttention was then focused on the mucosal scraping of neonatal rats ranging in age
1 7 to 28 days, to determine if the same ZBL that was present in the rat’s milk was
present in the intestinal mucosa of rat pups. The results indicated that prior to 14
the majority of zinc was associated with HMW proteins in the mucosal scrapings,
: 16 days of age a ZBL was eluted that had similar characteristics to the one found in
'. This is the same age at which the kinetic maturation study also determined that
oximal intestinal tract was beginning to exhibit saturation characteristics, indicative
urier-mediated process”. The presence of a ZBL in rat milk prior to the
pment of the intestinal ZBL supported the hypothesis that the ligand may enable or
:e the intestinal absorption of zinc during the neonatal period. The human
gue of the ZBL, when it was purified and administered to rat pups enhanced the
al absorption of zinc“. Low molecular weight, ZBLs have also previously been
d in the intestinal mucosal cells in adult rats and chickens““. However, their
'on with intestinal maturation and similarities to milk ZBLs have not been

ted.

 

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56
‘wo LMW, ZBLs that have been identified as unique by either their presence or
:entration in human milk as compared to cow’s milk were picolinic and citric

"'85'86'87'“. Each ligand was identified by two different investigators and their

 

>very generated a heated controversy as to which one was ultimately responsible for
bsorption in human infants and adults. Part of the conflict that arose between the

houps was centered around the suggestion that picolinic acid and zinc

ementation be used in concert to therapeutically treat AE patients. Picolinic acid is

nitant and if its administration was unwarranted as the citric acid group claimed,

is use as a therapeutic agent was undesirable.

rate is a component of milk in many species“. Cow’s milk contains almost twice

rate concentration of human’s milk, yet the human form has four times the amount

: bound to it. This was thought to occur because of the high percentage of casein

: in cow’s milk which complexes with zinc preferentially over LMW compounds
citrate“. Since human milk contains fewer HMW proteins, more zinc is available

)lex with LMW ligands. Citrate in human milk was determined to exhibit

:ristics of a LMW, ZBL by several experimental techniques“'“. Defatted human

ine milk was filtered by gel and ultrafiltration techniques. Polyacrylamide gels

vith borohydride to reduce charged groups and Sephadex columns equilibrated

M ammonium acetate, were used to prevent the disassociation of zinc from

igands . The fractions from the purification procedures produced two peaks

ssed through ion-exchange chromatography columns, one was a HMW, ZBL

ran 70,000 Dalton and the other was a single LMW, ZBL of 600~650 Dalton.

r s. “- ' -' ",v.;,-x._»: >71." ‘r
,.,. n... ".l‘ .1

 

uh.“ r..." .\.|\|m 1‘ -. a \rar lv d t 1.. r .r ibt ..&w\ .51

 

 

 

 

57

molecular weight and peak of the smaller protein coincided with synthetic zinc-

ate complexes, which have a weight of 572 Dalton“. Further confirmation was made
ared spectroscopy of pooled ion-exchange chromatography elution, which

.cated carboxylic groups were present. The carboxylic group’s nuclear magnetic
Inance (nmr) spectrum consisted of a pattern which was confirmed to be citrate. The
rte concentrations of the LMW, ZBL were also measured enzymatically using
ritase-isocitrate dehydrogenase method, which converted citrate into isocitrate.
trate was then converted into a-ketogluterate of which NADPH could be measured
ometrically.
Titrate was identified by the same investigator who determined the presence of a
’, ZBL in the developing mucosal cells of rat pups, which comigrated with the
ma] ligand identified in milk“. Although the intracellular ZBL was not
:terized as citrate, a hypothetical model was developed by combining the
mental results. An intestinal lumen LMW, ZBL originating from maternal milk
be responsible for facilitating or enhancing zinc absorption until an intracellular
nism matured, which could be the same ligand or have similar characteristics“.
'er, the model did not propose how the developing intracellular ZBL would interact
menal zinc to facilitate absorption once the animal had been weaned.
1e same time that citrate was being identified in human and rat’s milk as the
ZBL responsible for neonatal zinc absorption, another group of investigators
:d picolinc acid as the possible ligand“. Their experimental procedure was based

dified gel filtration chromatography technique. After each purification step

‘VP'I

 

58
ultrafiltration, cation and anion exchange) the fractions recovered were run through a
iephadex column equilibrated with Tris acetate and zinc, to confirm the presence of a

“'83. The modified technique prevented the disassociation of zinc from

ZBL complex
MW binding ligands, by equilibrating the columns with zinc prior to the samples being
111 through them. The eluted fractions from ion-exchange chromatography were then
:amined by AAS, which indicated that there was one LMW, ZBL peak, that coincided
th the peak that a synthetic picolinic acid-zinc complex peak“. The modified

thnique also was used to quantitate the amount of picolinic acid present in human and
111’s milk. The concentration of the picolinic acid in hurnan’s milk was found to be 38
'ml and in processed cow’s milk it was 20 pg/ml in one sample and undetected in
ther“.

Picolinic acid is known as a bidentate chelating ligand and is a by-product of

tophan metabolism”. Evidence has suggested that picolinic acid enhances the

anal absorption of zinc when given as a supplement to both neonates and adults.
:reatic secretions contain high concentration of picolinic acid”. Human infants with
hat have been therapeutically treated with pancreatic extracts and supplemental zinc
been reported to resolve their clinical manifestations earlier than patients

ristered zinc alone“.

or to the discovery of picolinic acid’s possible role in zinc absorption, a LMW,
vas identified in both the pancreas and intestinal mucosal cells of rats and the

83,91

tatic secretions of dogs . Homogenized rat pancreas and pancreatic secretions of

lere run through equilibrated columns to isolate proteins whose molecular weights

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59

were less than 1500 Dalton”. The proteins were then incubated with “Zn for 15 minutes.
After the proteins were incubated in the solution, less then 50% of the free “Zn was
recovered, indicating that both pancreatic preparations contained ZBLs. Two additional
in viva and in vitro experiments were also performed to confirm that the pancreatic ZBLs
were involved with intestinal zinc absorption. Rats surgically had their common bile duct
ligated, preventing pancreatic secretions from reaching the GI tact, then were given “Zn
3y GI intubation. The rats in this study had a decrease in whole body absorption of “Zn
when compared to those that were not ligated”. An additional in vitro study used rats
hat were fasted 40 hours prior to sacrifice. Inverted sacs of their small intestine were
ncubated in solutions containing “Zn and either canine pancreatic secretions or a buffer
olution for 1 hour, prior to assessment of the absorption of “Zn by the epithelial cells".
he epithelial cells that were incubated in the dogs pancreatic secretions had an increase
1 the absorption of “Zn compared to the segments that were incubated in control buffer.

The discovery of a LMW, ZBL in pancreatic secretions, that increased the uptake of
Zn in the above experiments, along with the finding of higher picolinc acid
)ncentrations in these secretions, suggested that picolinic acid may have a role in the
rsorption of zinc from the GI lumen of adults“. To determine the affects of endogenous
colinic acid on zinc absorption in adult rats three groups were fed the same basal diet
ntaining 5% vitamin-free casein. One group acted as a control and the other two had
ir basal diets supplemented with tryptophan and picolinic acid”. Tryptophan is
verted to picolinic acid through a series of enzymatic reactions. After the rats had

It on the diets for 7 days, they were given an intramuscular injection of “Zn, 9 days

itblr

 

 

 

 

60

later their feed intakes, weight gains, excretions and tissue absorption of “Zn were
assayed daily for 5 days. Table 5 below summarizes their experimental findings for the
three groups of rats fed basal diets, basal diets supplemented with tryptophan or picolinic

acid.

Table 5 - The Effects of Three Different Diets on Weight Gain, “Zn Absorption by
the Kidney and Total Zinc Absorption

   

+
.Measured Picolinic Acid

The results of the experiment indicated that rats fed the basal diet absorbed

gnificantly less zinc on a daily basis than rats fed the basal diets supplemented with

ther tryptophan or picolinic acid. The supplemented rats also had higher weight gains

rd kidney uptake of “Zn“. An interesting note to this experiment was that the basal diet
rntained a salt mixture in which zinc was supplied in the form of zinc—citrate; yet

iximal zinc absorption did not occur without the presence of picolinic acid or its
:tabolic precursor. The conclusions were that dietary zinc absorption in adult rats was
:ilitated by both endogenous and exogenous picolinic acid”.

This led to the hypothesis that rat and human milk contained picolinic acid which

ilitated the absorption of zinc in neonates until exocrine pancreatic functions were able

 

 

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61

) secrete adequate amounts. Since the investigators had previously identified a LMW,
BL in the mucosal cells of adult rats, they also proposed that the picolinic acid-zinc
)mplex in the GI lumen was transported across the brush border membrane into mucosal
:lls83’88'90. However, the investigator never confirmed the presence of a picolinic acid-
nc complex in mucosal cells, showing only that there was a LMW, ZBL present in the
ucosal scrapings of the rat intestine“. Thus the evidence for picolinic acid as the
itical ZBL for zinc absorption was mostly indirect.

The differences in ZBL that were identified by the two research groups could in part
due to the gel filtration and purification techniques of the LMW, ZBLs. The columns
: two experiments used to elute the zinc bound proteins were equilibrated using
ferent solutions with varying pH and salt concentrations. The variation in equilibration
hniques may have resulted in the disassociation or inappropriate association of
olinic or citric acid with zinc in each of the respective studies. The group that
ntified citrate as the LMW, ZBL, used the same equilibration techniques for
sequent experiments used to investigate picolinic acid’s role in binding zinc”.
:refore, they did not repeat the experimental procedure conducted by the other group
their results were similar to their original investigation. Because of the difference in
:rimental procedures, it is difficult to validate or invalidate the results of one group as
pared to the other. It would have been more convincing if the citric acid group had
roved picolinic acid’s role in zinc absorption using the identical protocol which was

nally employed.

'11

a

 

- up. . .

 

 

62

The concentration of picolinic acid present in human milk was determined to be 38
.g/ml, this was considered very high compared to previous studiesss‘“. The citric acid
roup measured the picolinic acid concentrations in milk, pancreatic secretions and
rtestinal mucosal cells of a humans and a rats, with high pressure liquid chromatography
iPLC). Their results indicated that picolinic acid concentrations in human milk were
:53 than 3.7 pM, which was insufficient to bind zinc as a primary transport mechanism“.
lthough the sample size was n=1 for the human and rat pancreatic secretions, there was
> trace of picolinic acid detected in either subject. Picolinic acid was also undetectable
adult and neonatal rat intestine as well as human infant jejunum.
The citric acid group dismissed the hypothesis that picolinic acid played a normal
ysiologic role in the absorptive process of zinc in the lumen of the G192. They
ributed the increased absorption of zinc in the previous investigations presented above,
the chelating properties that picolinic acid possessed.
The effects of both citrate and picolinic acid on zinc absorption has been extensively
'estigated in numerous studies. However, the sum total of the experimental results thus
indicate that neither of the two compounds are solely responsible for zinc absorption
reonates or adults. Table 6 is a summation of the experimental results of four studies
igned to examine the affects of the two ZBLs on zinc absorption in rats and calves.
1eriment A was conducted in two calves that had BHZD and one control calf". The
:riment consisted of two periods, one in which a large oral dose of “ZnCl, was
inistered, followed by assessment of “Zn in plasma daily for 7 days. During the

nd period the calves were given daily oral doses of either hydroxyquinoline or

 

 

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63
icolinic acid after GI intubation of “ZnClz. Plasma samples were obtained daily an
;sayed for absorption of “Zn. The activity levels of the plasma samples were then
)mpared between the two experimental periods to determine the differences in the
ansfer of zinc from the GI lumen to the vascular space, the results are displayed in Table
1.

Experiment B was conducted in adult rats, using in viva ligated sacs of the duodenum,
at were perfused with ZnSO4 solution for 2 hours”. Two groups of rats were used, one
cup had their bile and pancreatic ducts ligated. At the completion of the perfusion, the
testinal segments were removed from the rats and their mucosal cells were scraped and
llected for zinc analysis by AAS. The percentage of zinc absorbed for the two groups
is then determined, the results are displayed in Table 6b.

Experiment C used adult rats with a simultaneous GI lumenal and vascular perfusion

rtem to measure the mucosal uptake and transfer of zinc to the vascular space”. The

nenal perfusate solutions each contained one of the following ZBLs combined with

c and “Zn; citrate, picolinic acid, tryptophan or EDTA. There were two perfusion
rtions prepared for each of the ZBLs, at concentrations of 110 pM or 550 pM. The
run: of zinc that was transported to the vascular space was measured in nM per hour.
effect of each ligand on the absorption of zinc was evaluated. The results are

layed in Table 6c.

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64
Experiment D used rats that were given an oral emulsion of “ZnClz and either
diodoquin, citric or picolinic acid”. The influences of the ZBLs on net “Zn absorption,
the amount bound to the mucosa, plasma activity and absorption by the carcass were

compared to the control group to determine their effects. The results are displayed in

Table 6d.

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65

Table 6a - The Effects of Picolinic Acid on Intestinal Zinc Absorption in BHZD

 

 

 

Calves
Supplementation BHZD Calves Control Calves
Hydroxyquinoline T zinc absorption -----
Picolinic acid slight T zinc absorption No effect

 

 

 

 

Table 6b - The Effects of Bile and Pancreatic Duct Ligation on Intestinal Zinc

 

 

Absorption in Adult Rats
Obstructed bile and
pancreatic ducts Unobstructed ducts
% of zinc absorbed 32.0 19.1

 

 

 

 

Fable 6c - The Effects of Four Different ZBLs on Zinc Absorption from the
Intestinal Lumen into the Vascular Space in Rats

 

 

rble 6d - The Effects of Picolinic Acid, Citrate and Diiodoquin on “Zn Net
Absorption, Mucosal Binding, Plasma Activity and Tissues Activity

absorbed bound “Zn Plasma “Zn Tissues “Zn

 

 

66

The results of Experiment A indicated that picolinic acid supplementation in calves
ith BHZD had little to no effect on their ability to absorb zinc”. There have not been
1y reports confirming the presence of picolinic acid in the milk, pancreas or GI tract of
1ws. In fact, picolinic acid is easily degraded by enzymes in the GI tract of ruminants.
lthough AB in humans and BHZD in cattle appear to have similar clinical and genetic
.aracteristics, the possibility exists that there are different mechanisms responsible for
rc absorption and therefore picolinic acid could be a species specific ZBL. The slight
:rease in zinc absorption observed in the BHZD calf given picolinic acid was probably
e to its chelating effects. The results of Experiment B were the opposite of what would
expected, if the pancreas contained picolinic acid, that was responsible for zinc
:orption in the intestine”. The presence of the pancreatic secretions in the duodenum
:reased the amount of zinc absorbed. In Experiment C, only tryptophan and EDTA

sed significant changes in zinc transfer to the vascular space when compared to the
trols. Tryptophan caused a decrease in zinc absorption at both the concentrations
3d and EDTA caused an increase. However, neither picolinic nor citric acid had a
ificant influence on the absorption of zinc”. Net “Zn absorption by rats, in
eriment D, was not affected by the administration of picolinic or citric acid. Picolinic
did not effect any of the variables measured. Citric acid appeared to cause a slight
:ase in the amount of “Zn activity observed in the mucosal cells and in plasma, but
crease in tissue activity. Since citrate did not effect the net amount of zinc absorbed,
)lies that it may have altered the pool of zinc that binds to mucosal cells ligands”.

.oquin main effect was to increase net zinc absorption without appearing to effect

 

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67
nc binding in the mucosal cells; this is consistent with alterations in the rapidly
rsorbable pool of zinc intracellularly.

The inconsistencies between the experimental results obtained by the aforementioned
1dies and the two that originally identified picolinic and citric acid as the primary ZBL
rat and human milk may partially be explained by the assumptions that were made
ren these experiments were designed. Both research groups investigating picolinic and
ric acid, were trying to identify a LMW, ZBL that was exclusive to human and rat milk
compared to bovine milk. Although they acknowledged the differences in content and
tribution of HMW and LMW ligands between the species, they assumed that the
tribution of the ligands that bound zinc were fixed. This distribution has been
rerimentally proven not to be fixed, but easily altered both in milk and mucosal cells
increasing and decreasing protein and zinc content“. Since HMW proteins bind zinc
:"erentially over LMW proteins, they would have to become saturated prior to the
W proteins binding zinc. The observation that a LMW, ZBL is primarily found in
ran or rat’s milk as opposed to cow’s milk, may be explained by the fact that cow’s
: contains 75% more HMW proteins“. Therefore, both picolinic and citric acid may
re exclusive ligands in human and rat’s milk, but only have a increased opportunity
nd zinc due to the absence of substantial HMW protein concentrations.
xperiment C also tested the influences of histidine, cysteine, methionine and
thione, on intestinal zinc absorption and found that none of them increased uptake“.
will bind to all of these ligands. However, since their complexes did not appear to

we mucosal uptake, it appears that they form nonspecific complexes in the intestinal

”M
iv.

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68

umen. The complexes may only exist to maintain the bioavailability of zinc so that it
aybe absorbed by mucosal cell ligands. Therefore zinc absorption would depend on
tracellular ligand populations and not a specific lumenal ZBL . The characteristics of
tracellular ZBL and the intracellular pathways they could be involved with in

ansporting zinc to the vascular space will be discussed in the next section.

tracellular Mechanisms of Gastrointestinal Zinc Absorption:

he role of intracellular zinc binding ligands in intestinal zinc absorption

The presence of intracellular ZBLs has been confirmed in numerous studies. The size,
llular localization and function of the ligands have not been fully elucidated. An
racellular mucosal ZBL was first identified in 1971 in chickens and rats. The ZBL in
icken’s intestinal cells was only defined as having a molecular weight less than plasma
rumin“. The mucosal cells of the rat small intestine when homogenized and passed
)ugh columns, yielded two pools of zinc binding proteins, one contained eleven
teins, with molecular weights greater than 15 x 104, the other three, with molecular
ghts between 103 and 15 x 103 95. The experiment did not however confirm that all the
:eins identified bound zinc, only that one or more in the pool was capable of binding
. Neither of these studies were designed to elute LMW fractions“. In 1973, LMW,
.s were detected in mucosal scrapings of adult rats“. Since the discovery of the
N, ZBL in 1973, the findings have been repeated by subsequent studies and it is clear
there are a variety of intracellular ZBLs present. While the existence of various

in the intestinal mucosa has been established, their identity and physiologic

 

69
imctions are not clear“. Establishing the roles of ZBLs in metabolism has been an on
going process. Most of the methods used in investigations have tried to determine what
alters or induces expression of the proteins.

The fractionation of mucosal cell cytosol along with differential hybridization
techniques, have been utilized to determine the effects of developmental, hormonal or
other physiologic changes, on the expression of potential ZBLs. Proteins that appear to
be affected are then characterized and studied further to identify cellular localization and
hypothesized mechanisms of action. Mucosal cell homogenates labeled with “Zn were
fractionated by gel filtration and ion-exchange chromatography columns to elute proteins
by molecular weight and determine their amino acid content. The method of differential
hybridization, or “plus-minus” screening, is well suited for examining differences in gene
expression in different nutritional states or during developmental stagesé. The procedure
nvolves the harvesting of flesh tissue preparations from the organ to be studied in the
xperimental group and producing a cDNA library that can be screened by two different
ets of labeled cDNA probes. One probe is produced from the same tissue that the library

as made and the other totRN A isolated from a control group. Signals differentiation by
toradiography can then be used to identify the cDNA colonies that are unique to the
perimental group6. The inserts contained in these colonies can be sequenced and
Dmpared to known DNA and amino acid sequences of other proteins and inspected for
rnsensus regions to gain insight into protein function.

Examination of the affects of dietary zinc and intestinal maturation on the intracellular

pulation of proteins and ligands in epithelial cells has resulted in the identification of

it

m.
.3"
uh
..
. c
.u
.-m
..~.~.
-
1. u.
...
. c
1‘1
.-x
(TM
..1.-
r\\.\
F-
.\

 

 

 

7O

nee LMW, ZBLs that appear to be involved in intracellular mechanisms of zinc
bsorption from the GI tract.

,dentification of prostaglandin as a zinc binding ligand, in rat intestinal mucosal
:ells

One of the initial studies conducted to identify the chemical nature of LMW, ZBLs in
'at intestine found a ligand that separated with the cytosol fraction of mucosal cells that
round “Zn. Further experimentation revealed that the ligand had a molecular weight of
300 D and its identity was determined to be a prostaglandin (PG 96. In vitro studies
established that the PG readily formed complexes with zinc, which enhanced the ability
)f zinc to be absorbed by the GI tract. In addition to demonstrating that PGs bind zinc
he study also provided evidence that they were involved in the regulation of zinc
tbsorption. Experiments using ethyl acetate extracts of PG from rat small intestine were
njected into jejunal segments with “Zn“. The uptake of “Zn was three times greater in

e rats injected with the PGs than the controls. When the segments were pretreated with
domethacin, which inhibits PG synthesis, “Zn absorption was decreased by 60% of the
ntrol values, when the same segments had PG extract added to them their zinc
sorption increased by 70%“. Thus, the study appeared to present convincing evidence
at at least one of the species of LMW, ZBL involved in zinc transport in mucosal cells
f5 a PG.
The results of this experiment were not duplicated by other researchers. Although
rer experiments were able to isolate LMW, ZBLs from the mucosal tissue, none of
m were characterized as a PG“'“. In addition, the low levels of PGs present in the

k of lactating human’s would not facilitate the absorption of enough zinc to meet the

 

71

hysiologic requirements of neonates. Computerized models of zinc complexes with
gands also indicated that the divalent ion would be unlikely to bind to an oxygen rich
rolecule such as a PG“. Therefore, the findings of this experiment were dismissed by
e scientific community as inaccurate“.
lentification and characterization of metallothionein

A low molecular weight zinc-binding protein was identified in the mucosal cells of
ults and neonates of a variety of species including rats, cows, humans and
ickens97’98‘99"“"0”“. Analysis of this protein revealed that the amino acid content and
tribution was consistent with that of MT. Metallothionein has a single polypeptide
tin with a molecular weight of s 6000 Dalton. On average the protein has 61 amino
ds, 30% of these are cysteines, which are distributed in a fixed pattern. There are two
es of cysteine residues in the protein, those that bind one metal ion “terminal
teines” and those that bind two metal ions “bridging cysteines”. The bridging
:eines are important for protein stability. Overall the fold of the protein is determined
:ysteine arrangement and not by intervening amino acid residues”. The intervening
ues act as flexible spacers that connect the metal chelating cysteines.

etallothionein is present in a wide range of tissues in vertebrates, as well as in fungi,

and a number of single celled organisms. The amino acid sequence at the N-
'nus is specific for various classes of vertebrates; mammals have a (MDPN), the

sequence is (MDOQD), and the piscine sequence is (MDP)‘°3.

 

 

72

There are various isoforms of MT that exist within and between species;
haracterization of the genes has focused on their identification and determining their
ssue specific distribution. Each isoform has a slightly different amino acid content that
onfers metal binding affinity and regulatory sites that allow for differential tissue
xpression‘5'97. Rodents have only two functional MT genes, MT-l and MT-2, both are
xpressed in the intestine but MT-l is the predominant species presentl“.
ietallothionein—Z is predominant in bovines and ovids, but the animals also appear to
ave several minor MT species in their tissues that bind less zinc than MT—297. Of the
rurteen MT genes that have been identified in humans, six are expressed. Each human
me has a unique expression profile and responds to a number of different regulators“.
hus, the number, type, ionic strengths and tissue distribution of the MT isoforms vary
ithin and between species”.

Although MT has the ability to bind a number of heavy metals, only zinc and copper
3 of nutritional significance. Zinc is bound by a tetrahedral arrangement of four sulfur
is and one mole of protein can bind 7 g of zinc”. Copper is bound in a trigonal
angement of three sulfirr ions and one mole of protein can bind 12 g of copper”.
pper also has the ability to displace zinc from MT. Zinc preferentially binds to the
ha domain of the C-terminus and copper to the beta domain of the N-terminus.
Metallothionein is primarily localized to the cytosol; however, it has also been found
re nuclei, mitochondria and rrricrosomes of cells9"°3"°". Although MT is primarily
lcellular in origin, it does occur in the plasma, bile and urine. It is believed that the

'is the primary source for MT in all species.

"Wt.

I
a

W

 

is"

 

 

73
Metallothionein can be induced by metal ions, corticosteriods and stress. Recent
vestigations have determined that inducing agents regulate specific isoforms of MT

“"06””. The inducing agents that are involved in differential gene

pression in tissues1
pression alter the MT mRNA population and conversely protein expression. The
luction of MT synthesis may be initiated through DNA interactions with metal ions
'ectly or transcriptional regulators. There are four distinct metal regulatory elements
RE) and three promoter regulatory elements for glucocorticoids, interferon and
iotoxin-factors which have also been identified in the MT promoters“). The degree of
[thesis depends on the inductor and the isoform of the gene. After MT expression has
:n induced, it takes approximately 6 to 8 hours for maximum mRNA levels to be
:hedw.

There is not a lot of information available about the degradation of MT. It is clear that
isoform and the metal it is bound to determines the degradation rate. The rate limiting
in degradation is the removal of the metal, the easier it can be displaced, the faster
)rotein is degraded”. When the bound metal is lost fiom MT, the protein loses its
ormation and becomes susceptible to proteolytic enzymes. Decreases in pH can

3 loss of bound metals and rapid proteolytic degradations. Metallothionein

rating from desquamation of mucosal cells has been found to be excreted without
dation in urine and feces. But primarily, MT experiences turnover in the organism

:tabolisms.

 

74
elationship between dietary zinc and metallothionein synthesis
In order to determine the relationship between dietary zinc and MT synthesis several
chanisms need to be explored. First it has to be demonstrated that zinc induces the
lthCSlS of MT. If possible the transcriptional mechanism which zinc utilizes to initiate
thesis should be defined. Second, a relationship between dietary zinc levels and MT
thesis should be demonstrated. Third, dietary zinc fluctuations should be shown to
ICC or suppress the nuclear mechanism responsible for MT production.
Vietallothionein concentrations have been studied in rat and bovine liver and intestinal
;. Parenteral injections of 6SZn in rats have been shown to increase the incorporation
C-labeled cysteine and 65Zn into the MT of hepatic and mucosal cellsm’m". The
:ased incorporation of labeled cysteine was disrupted by the administration of
:omycin D prior to “Zn inj ectionsm. Actinomycin D inhibits DNA-dependent, RNA
lesis of proteins, which implies that zinc plays a role in inducing MT synthesis in
‘ystems'm. Just how zinc increases the expression of MT at the nuclear level is
what of a mystery. Experiments have demonstrated that varying the zinc content of

1103. However

does not affect the net amount of zinc located in the nuclear poo
listered of 65Zn through the portal vein has been shown to lead to rapid

rulation of zinc in hepatic and intestinal nuclei. Increases in the amount of 65Zn in
clear pool were proportional to the increased concentrations of 652m administered
creased MT synthesism. There is evidence that newly acquired zinc binds to

iption factors that bind known MREs on the MT promoter103 . The transcriptional

:ion of MT synthesis by zinc has been hypothesized to occur through small

 

 

til.

III:
mi

‘41

 

75
fluctuations in the intracellular concentrations of zinc that stimulate the nuclear uptake of
either free zinc, or zinc bound to a transcription factor in the cytosol. In the nucleus, the
zinc complex binds to an MRE site in the MT gene to induce synthesis. This led to the
)roposal that since MT synthesis appeared to be regulated by the presence of zinc, this
nducible ligand could be altered by dietary levels of zinc62"".
To determine the relationship between dietary zinc and MT accumulation in tissues,
xperiments have been conducted in cattle, sheep and rats. A bull and steer and two
roups of sheep, were fed two different diets, one supplemented with 2000 ppm of zinc,
1e other supplemented with zinc after being fed a basal diet for two weeksg. Liver
iopsies were taken at two week intervals for 70 days, then the animals were sacrificed
1d had their tissues collected. The half-life of MT in bovine and ovine hepatic cells was
:termined. Homogenates of the various cell fractions were gel filtered and analyzed for
ac content by AAS9. In the bulls and sheep fed the basal diets plus high levels of
plemental zinc, MT had the greatest increase in the liver and kidney. The
centration of zinc bound to MT was highest in the small intestine9. There was no zinc
d to MT detected in the rumen papillae and abomasum and there were no increases
e amount of zinc bound to MT in the heart and testes. Hepatic cellular accumulation
' c bound to MT was greater in the zinc supplemented animals and the lowest in zinc
leted animals as compared to controls fed the basal dietsg. The half~life of hepatic MT
determined to be 24.1 days and 22.6 days respectively for the cattle and sheep.
e above results indicated that an increase in dietary zinc caused an increase in MT

Is in the liver, kidney and small intestine of both species. The fact that MT did not

 

76
rease in the heart and testes indicated that MT was not regulated by dietary levels of
c in all tissues and another regulatory process was operational in these tissues. The
f-life of hepatic MT in cattle and sheep is considerably longer than that reported in rats

9"“. There were also differences

chickens which is 1.7 and 1.5 days respectively
:d in the maximum amounts of MT that could accumulate per gram of tissue in the
r between the species, with the ruminants having the ability to accumulate a hundred
more than ratsg.
t similar study conducted in rats examined the change in amounts of zinc bound to
in response to normal dietary fluctuations in zinc levels. The experiment was
gned to determine the relationship between serum zinc concentrations and zinc-
ing proteins intracellularly as well as the effects of actinomycin D on the proteins”°.
investigators found that brief and minor fluctuations in dietary zinc levels were
leled by changes in zinc concentrations of the serum, liver and intestinal cytosol”°.
olic zinc accounted for 50% of the total amount of zinc in the liver. In the rats that
fed lower zinc concentrations, the zinc bound to MT was 1% of the total amount of
the liver and in those fed higher zinc concentrations, zinc bound to MT
ented 25 to 30%”). Thus, quantitative changes in dietary zinc were also paralleled
titative changes in the amount of zinc bound to MT in hepatic cells. Rats treated
tinomycin D had no increase in the amount of zinc bound to MT in mucosal cells
cells, but they did have a 200% increase in their serum zinc concentrations‘ '0.

of the aforementioned studies support the concept that dietary zinc content had a

d influence on MT concentrations in tissues and the association of zinc bound to

 

.’ ‘
1‘ N

5.~

.
.
!

E
r

'-..l

‘Q
l.
‘i

N

77
‘ in intestinal and hepatic cells. The studies implied that zinc regulates its own
orption from the intestine by altering cellular MT concentrations.
A subsequent study in rats was able to directly evaluate the effects of dietary zinc
:tuations on the nuclear mechanism responsible for the expression of MT in mucosal
sand the influence that this had on the absorption of zinc from the GI tract. Several
ables were measured, serum and mucosal zinc content, the rate of MT synthesis post
ntubation with zinc, and alterations in the amount of mRNA coding for MT‘”. The
lts are presented in Figure 1. The end of the line indicates the time at which the peak
:entration were reached for the variable being measured, afterwards the
entrations declined, or were stable as indicated by the dashed line. Serum zinc
entrations were measured in microgram of zinc per deciliter of serum; zinc
entrations in the mucosal cell cytosol was measured in microgram of zinc per gram
[ucosa tissue; zinc bound to MT was measured in nanogram of zinc per milligram of
:01 protein; and the last two variables were measured as increases above the control

values1 ‘2.

 

78

Time post-administration of ZnSO,

 

 

 

 

 

 

Parameter Measured 0hr 3hr 6hr 9hr 12hr
Serum Zn concentration ____, 320 itng
Mucosal cell cytosol Zn ; 150 pg/g
concentration
Zinc bound to MT = 170 ng/mg
35S incorporation into MT ; 4x 4x
MT related mRNA ; 5x

Figure l - The Amount of Time in which Peak Concentrations of Serum, Cytosolic
and Metallothionein Bound Zinc and 35S Incorporation into
Metallothionein and Metallothionein mRNA Levels were Attained

The results indicated that intestinal MT could be directly induced by dietary zinc
:oncentrationsm. All five of the parameters measured exhibited a consistent time-related
esponse to changes in dietary zinc statusI 12. A probable sequence of events were
ypothesized from this data as follows’”. 1) Zinc enters the intestinal mucosal cells and
:cumulates in the cytosol until it peaks at 6 hours. 2) Once zinc enters the mucosal
:lls it is quickly transported to the vascular supply, where peak concentrations are
ached in 3 hours. 3) The increased zinc concentrations in the mucosal cells induces
her directly or via a transcriptional regulator the transcription of MT mRN A. 4)

anslation of the MT mRNA occurs and peak mRNA and MT cellular concentrations
treached at 6 hours. 5) Available intracellular zinc is bound to synthesized MT, peak

lcentrations of the complex are reached at 9 hours.

 

 

79

The regulation of MT synthesis by zinc entering the cells could be accomplished by
mpetition between thionein, the metal free form of MT and transcription regulators of
T synthesis”. Once thionein sites have been saturated, then zinc would bind to
mscriptional factors that in turn bind to the MRE sites on the MT gene, to induce
nthesis”. When the cellular zinc concentrations decrease the transcription factors no
tger bind zinc and the synthesis of MT is down-regulated, thus implying that dietary
[C can regulate the production of MT in mucosal cells and therefore regulate its own
:tabolism.

The expression of MT in the intestine, liver and spleen by various inducing agents, has

107

3 been investigated in adult rats, with Northern blot analysis . The results indicated

t MT synthesis in the intestine and spleen was induced only by zinc and was refractory

lexamethasone and interleukin lot107

. The liver had increases in MT synthesis in
Jonse to all three inducing agents. The evidence shows that there is tissue specific
ilation of MT synthesis, and zinc is one of the primary intestinal regulatorsm.
‘elopmental regulation of metallothionein expression in the intestine
)evelopmental regulation of the expression of MT in various organs from fetal and
fling rat has also been examined. Isolated totRNA from 17 and 22 day old fetuses
rrun on Northern blots and probed with MT cDNA, labeled with 32P.

Jlothionein was found to be expressed in fetal intestine starting at 20 days gestation
ncreased continuously up until birth, then the levels gradually decreased until the
were weaned“°. The induction of MT synthesis in the fetal intestine was stimulated

llO

ogenous levels of zinc but not corticosteriods . The increase in intestinal MT also

 

 

80

vincided with endogenous increases in zinc in the dams of rats prior to parturition, in
hich serum levels were five times higher than the serum level of rats at weaning (there
no other experimental data to support the high zinc serum levels in rats at parturition

id in fact, both humans and cows do not have increased serum zinc levels) ”°. It was

It clear if the increase in MT levels that occurred prior to parturition were to enable
onates to absorb adequate amounts of zinc during suckling or if it was a response to the
:reased circulatory levels of zinc in the dams. This emphasizes the differential

pression of MT in tissues and developmental variations that could account for

:reased alterations in zinc distribution during growth".

.e function of metallothionein in the regulation of zinc absorption from the
itrointestinal tract

Since, MT is primarily a cytosolic protein, it can be expected to influence the

:ntion and/or release of intracellular zinc, where as the uptake of zinc from the

:stinal lumen into mucosal cells would be mediated by another mechanism and may
directly be influenced by cytoplasmic factors'“. There are a number of theories
1rding the role MT plays during zinc absorption and distribution; however, the

)rity can be condensed into four concepts. The first is that MT functions as an

:tinal zinc buffer, that is turned on when serum zinc concentrations are elevated’m’l”.
MT binds to zinc and limits the amount that can be transferred to the vascular space.
second theory is that zinc absorption is independent of intestinal MT levels and that
=gulation of zinc absorption is controlled by intestinal excretion‘”. The third is that
ises in intestinal lumenal zinc induces the synthesis of MT, which facilitates the

tort of zinc to the vascular space1 '4. The fourth theory integrates both the concept of

 

 

81
MT acting as a buffer system for mucosal cell zinc, in the presence of elevated
endogenous zinc concentrations and MT as an facilitator for zinc absorption, in the
aresence of increased zinc content in the intestinal lumen’“.

The first theory, which hypothesized that intestinal MT acted as a zinc buffer when
lietary and endogenous zinc concentrations were elevated, evolved from the results of
wo studies from the same group of investigators. The first study examined the effects of
ndogenous zinc on the absorption and distribution of “Zn, as well as MT synthesis in

ssues ' 1 1

. The experiment involved the use of intraperitoneal injections of zinc to elevate
:rum zinc levels, then measured the zinc fluxes in the serum, intestine and hepatic cells.
lthe second study, the affects of fluctuating dietary zinc concentrations and actinomycin
administration on serum zinc concentrations, cellular MT levels and the ability of the
l tract to absorb and distribute orally administered “Zn were measured”).

The results of the first study indicated that serum zinc concentrations increased after
: administration of a zinc load by injection. The zinc loaded rats absorbed less “Zn
ough the GI tract than the control group, most of the “Zn was bound to MT is in the

osol of mucosal cellsl “

. However, in the control group most of the newly introduced
was associated with LMW, ZBLs in the mucosal cytosol‘”. The results also

'cated that the elevation in serum zinc concentrations caused an increase in the
esis of mucosal cell MT, which bound newly introduced “Zn, preventing it from

g transported to the serum. If the serum zinc concentrations were normal or

eased, then most of the newly introduced “Zn was associated with LMW, ZBLs in

osal cells, and was quickly transported to the vascular space. The “Zn was primarily

 

 

82

associated with LMW, ZBLs in the rats that were administered actinomycin D, which
nhibited the de novo synthesis of MT regardless of whether the animals were zinc loaded
r not. Most of the “Zn introduced into the GI tract was quickly transported to the

erum, where the zinc concentrations remained elevated until the effects of the

 

:tinomycin D subsided.
In the zinc loaded rats as the amounts of zinc bound to MT in hepatic cells increased,
rum zinc concentrations decreased. After a peak in serum zinc concentrations, 80% of
increased liver zinc, was bound to newly synthesized MT. The decrease in serum
c concentrations between 6 and 8 hours post-injection and the fact that administration
actinomycin D blocked the reduction in serum zinc concentrations, indicated that zinc
arance from the vascular space was directly related to the synthesis of hepatic MT'”.
The results of the second experiment reflected the influences of fluctuating dietary
3 content on the absorption and distribution of zinc. The results confirmed the findings
he zinc loading experiments. The rats fed the higher dietary zinc contents absorbed
65Zn than the rats fed the lower amounts of dietary zinc. Elevations in dietary zinc
ent induced MT synthesis in hepatic cells, causing an uptake of zinc fi'om the
ular supply and also in intestinal cells, preventing the transfer of zinc to the vascular
ly”°. Gel filtration chromatography allowed the assessment of the association of
in the mucosal cells with MT, HMW and LMW proteins as the dietary zinc contents
varied. The results indicated that the association of zinc to the protein population in
sal cells was also dependent on the concentration of zinc received in the diets of the

The percentage of “Zn absorbed from the GI tract and the distribution of zinc bound

in“
oi L

lal

 

 

83

to proteins in mucosal cell cytosol of rats fed the three different diets are present below in

Table 7’”.

Table 7 - The Effects of Varying Dietary Zinc Concentrations on the Percentage of
“Zn Absorbed and the Mucosal Cell Distribution to Cytosolic Proteins in
the Rat Intestine

“Zn absorbed/hour to

i to ,
ZBLs little LMW or MT

:i: to ,
very little to MT, increasing
amounts bound to LMW
to ,
HMW, ZBLs

 

The distribution of zinc in proteins in the two groups indicated that rats fed the higher
inc content diet had most of the “Zn bound to MT and a small amount bound to HMW,
SBLs in mucosal cells. In rats fed the lower zinc concentration diet, most of the zinc
was bound to LMW, ZBLs. The administration of actinomycin D, caused an increase in
re amount of “Zn bound to LMW, ZBLs and transfer to the vascular space. The binding
forally introduced “Zn to mucosal MT was inversely proportional to the amount of “Zn
)sorbed‘w. This further supports that MT is binding zinc in intestinal cells.

Most of the “Zn was bound to HMW, ZBLs and MT in liver cytosol”. As dietary
1c content increased, serum zinc concentrations increased and less “Zn was found

sociated with all liver cytosol fractions. This is consistent with the observation that less

 

 

?,

NJ

{'1'

“U
K“

 

84

In was transferred to the vascular space due to increases in MT synthesis in mucosal
lls.

Dietary and serum zinc concentrations modulate the synthesis of MT and its
sociation with zinc in intestinal and hepatic cells. Intestinal and hepatic MT synthesis
: both stimulated by increased serum zinc concentrations, suggesting that serum zinc

:diates a homeostatic response via effects initiated in both tissues”°

. The response is
strolled by transcriptional regulation of MT synthesis in both cell systems. Serum zinc
reentrations have been shown to respond within a 24 hour period to dietary content of
c“°. Thus as serum zinc concentrations increase, MT synthesis in hepatic cells is
:iated, which in turn allows for more zinc to be moved from the vascular space into
latic cells, to be stored until needed to meet cellular requirementsl ' ‘. In intestinal
helium, if serum zinc concentrations are low, the majority of the zinc that is absorbed

. the mucosal cells is bound to LMW, ZBLs, which appeared to be quickly transferred

1e vascular space. An increase in serum zinc leads to an increase in mucosal cell MT

 

entrations and an increase in the amount of zinc bound to MT, decreasing the

unt of zinc that is absorbed and transferred to the vascular space. The nuclear

lation of zinc absorption would then rely on pathways that sense increases in serum
Foncentrations, both in hepatic and mucosal cells, to initiate the synthesis of MT.

MT would act to sequester newly absorbed zinc when endogenous zinc

:ntrations were elevated, preventing transfer to plasma albumin” ‘. The life of MT in

sal cells is longer than the life of the cell, so the additional zinc content that is being

stered in the cell could be lost into the intestinal lumen by desquamation.

 

 

85

The second theory was that zinc absorption was regulated by endogenous zinc
rcretion and not related to the induction of MT synthesis in the liver or intestinal cells1 13.
he theory was derived fi'om an experiment designed to account for the effects that
otope dilution had on the absorption of orally administered “Zn1 ‘3. Isotope dilution
sts the effects of endogenous concentrations of zinc that is secreted into mucosal cells
om the vascular space and then into the GI tract on the absorption of zinc from the tract.
1e zinc that is secreted back into the GI tract can dilute the “Zn that is present in the
testinal lumen. The higher the endogenous concentrations of zinc, the more dilution

at takes place and the less concentrated “Zn is, causing a decrease in the amount that

n be absorbed. The two previous experiments did not account for the effects that
renteral injections would have on elevating endogenous zinc concentrations and the
:retion of zinc back into the intestinal lumen”°"”.
The experiment used adult and weanling rats that were all given the same basal diet,
varying concentrations of zinc acetate in their water supply. The rats were then either
:cted or given oral boluses of “Zn and the radioactivity in tissues and feces were
:rmined. The intramuscular injections of “Zn were given to assess the secretion of
back into the intestinal lumen. The orally administered “Zn allowed for the

ssment of the effects of endogenously secreted zinc into the intestinal lumen on the
:ntage of “Zn absorbed by the GI tract. The true absorption of dietary zinc, taking
recount endogenous isotope dilution, was calculated by applying the recorded

ty levels, from endogenous sources and percentage of “Zn absorbed to the Weigand

irchgessner formula1 '3. The results revealed that there were no differences in the

 

5 .‘h

‘v
‘A

 

 

otal zinc concentration in the intestine between the rats given deficient, adequate or
xcessive zinc levels. There was a decrease in the amount of “Zn activity detected in
u ucosal cells of weanling rats, given a higher dietary zinc content, after zinc loading by
Intramuscular injection. These results were interpreted to indicate that since the total
mount of zinc in intestinal cells remained constant despite zinc loading and fluctuating
.ietary contents, the decrease in the amount of “Zn absorbed was due to the increase in
re amount of endogenous zinc that was secreted into the intestinal lumen, which diluted

“3. The presence of more zinc being detected in the feces of the rats

re “Zn given orally
ven the higher zinc diets helped to confirm their hypothesis. The rats given zinc
:ficient diets absorbed more of the orally administered “Zn into the mucosal cells than
ts provided adequate or excessive zinc dietsl ‘3.
The calculations of the average daily absorption of zinc, with the isotope dilution
.dies, provided evidence that zinc homeostasis was maintained by excretionl 13. As the
1ke of zinc was increased, the apparent amount of zinc that was absorbed decreased.
wever, the actual amount of zinc that was absorbed on a rig/day basis, indicated that
h the exception of the weanling rats given the lowest intake of zinc, the amounts were
stically equivalent for all the dietary groups in both the adults and weanlings. This

t that fluctuations in dietary contents of zinc did not influence the amount of zinc
rbed from the GI tract only the amount secreted into the feces. The continual

tion of zinc into the intestinal lumen created a stable pool of zinc with which orally

'stered “Zn mixes with and can become diluted if endogenous levels are

team

 

 

87

Although the experimental findings of the isotope dilution studies were interesting, the
investigators left out several factors that would have made their theory more convincing.

The serum zinc concentrations were never investigated in the study. If zinc metabolism

 

' was regulated by excretion and not by MT synthesis and zinc retention in mucosal cells,

then serum zinc concentrations should have remained elevated until zinc was transferred
from the serum back into mucosal cells and the GI tract. The investigators did not
address the effects that administering actinomycin D prior to parenteral injection of “Zn
had on blocking the inhibitory effects of zinc loading which would change the synthesis
of proteins and causes a reduction in zinc absorption“. If zinc absorption was
independent of MT synthesis then the extent of zinc loading of endogenous pools by
parenteral zinc injections should have been comparable in actinomycin D treated and
untreated rats'“.

The third theory utilized the isotope dilution technique to examine the relationship
between MT and zinc absorption. The results indicated that the absorption of zinc from
he intestinal tract was directly proportional to the MT content of mucosal cells. The
:ffects of intraperitoneal injections of zinc on MT induction and the association of zinc
round to MT and “Zn bound to MT were assessed after “Zn was injected into the
uodenal lumen'”. The absorption of “Zn was determined by measuring the liver
ontent after oral administration. Since the induction dose of zinc and even the body

)ntent of zinc greatly exceeded the radioactive tracer dose injected into the duodenum, it

as essential to measure the dilution of the isotope to determine true zinc absorption‘”.

 

 

88

The raw data collected fiom the experiment appeared to support the theory that MT
acted as a mucosal buffer in the presence of elevated endogenous levels of zinc. As the
dosage of zinc used to induce MT synthesis increased to 2 umole, the amount of “Zn that
was absorbed by intestinal cells appeared to decrease 18 hours later, which corresponded
to peak amounts of “Zn bound to MT. Conversely, as the induction dose of zinc was
lowered to dosages that caused deficiency, there was a 200 to 300% increase in “Zn
absorption‘”. However, when the absorption values were applied to the isotope dilution
formula it appeared that the induction of MT increased “Zn absorption by the intestine.
Using the formula it was determined that a 2 umole dose of zinc used to induce MT
synthesis diluted the “Zn bound to MT in the intestine by 500 fold, which meant that the
actual absorption of “Zn was 50—100 times greater than the controls'”. The amount of
“Zn found complexed with mucosal MT was directly proportional to the ratio of zinc
bound to MT/“Zn bound to MT. As the absorption of “Zn increased, the ratio decreased.
Further support of MT facilitating zinc absorption was provided by the effects of
actinomycin D blocking the synthesis of MT and decreasing the absorption of “Zn. The
dilution of the “Zn did not occur in the intestinal lumen as previously determined by the
nvestigators which thought that zinc absorption was independent of MT synthesis, but
ccurred intracellularly in this experimentI ”'1 ‘4.

The combined findings in the study suggested a positive relationship between the
duction of MT synthesis and “Zn absorption from the GI tract. However, the findings
0 not preclude the possibility that an additional zinc binding protein may also be

erational in the mechanisms responsible for the absorption of zinc from the intestinal

 

 

 

89

ren. They also do not disprove the possibility that MT may be acting as a temporary
rage protein to prevent the animal from being exposed to harmful levels of zinc, until
load could be reduced gradually by absorption into the body‘”. The fact that “Zn
sorption was measured indirectly as “Zn uptake by the liver may have biased their
perimental results. Parenteral injections of zinc increase liver uptake of zinc, which
uld have falsely elevated the “Zn that was interpreted to be an increase in the
sorption of zinc due to increasing MT levels. The investigators should have also
eluded serum “Zn and zinc concentrations over time to determine if the increase in “Zn
rund to MT in the intestine coincided with a increase in serum “Zn activity.

The three conflicting theories as to how or if MT functions in the absorption and
stribution of zinc, are understandable when the diverse functions and variety of
.ysiological states with which MT is associated are taken into account. The fourth
|:ory of MT frmction in zinc absorption was derived from a study that was designed to
evaluate the previous theories. None of the investigators in the other studies had
nbined the isotope dilution technique with examining the absorption of “Zn into
cosal cells and its transfer to the vascular space, as well as its effects on MT
centrations. The study made the assumption that since MT was a cytosolic protein, its
: was confined to retention and release of zinc and another mechanism was responsible
.umenal absorption of zinc across the brush border membrane“.

he experimental design utilized simultaneous perfusion of the mesenteric vasculature

lumen of the rat small intestine. The rats were divided into three groups, zinc-

 

:ient, control and fasted group. The experiment consisted of two time periods, the

 

 

 

9O

rrst in which “ZnC12 was perfused for 30 minutes into intestinal lumen and the second
vhich began with the lumenal perfusate being flushed with “Zn-free buffer and
rubsequent collection of the vascular perfusate for 40 minutes at 5 minute intervals‘“.
[he first period allowed the mucosal zinc pool to be labeled with “Zn and the second
period allowed for the rate of “Zn transfer from the mucosal pool to the vascular
perfusate and secretion back into the intestinal lumen to be assessed. The decrease in
“Zn activity from the lumenal perfusate in Period 1, reflected the amount absorbed by the
mucosal cells and the initial “Zn content of mucosal cells at the beginning of Period 2, as
well as “Zn content of lumen and vascular perfusates at the end of Period 2, reflected
what was retained, distributed and secreted by mucosal cells‘“. The eXperiment was
designed to eliminate the affects of isotope dilution in two ways. First, intestinal MT
concentrations were manipulated by short term dietary zinc fluctuations, that did not
uence the endogenous, cytosolic pool of zinc'“. Second, the effects of fluctuating
ietary zinc contents on zinc absorption were assessed by comparing the half-life of
ucosal “Zn pools‘“. The half-life of “Zn was not affected by dilution of the isotope
'th endogenous zinc. The results of the experiment are presented in Table 8. The

ntrol group has the actual measured values obtained by the study, the fasted and zinc-

pleted groups values are exponential approximations of the control values'“.

 

 

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91

Table 8 - The Effects of Rats Fed Three Different Dietary Concentrations of Zinc on
“Zn Uptake, Retention and Transfer to the Vascular Space by Intestinal

 

 

 

 

 

Mucosal Cells
Period 1 Period 2
Dietary “Zn “Zn Mucosal MT “Zn t ,4 of
groups uptake by retained to content mucosal “Zn in
mucosal in vascular of pool mucosal
cells mucosal transfer mucosal available cells
(nmol/ 30min) cells (%) cells for (min)
(nmol/g) (Hg/g) transfer
(%)
Control 5.1 i 0.5 4.4 i 0.6 2.5: 0.6 19.0 i 2.7 3.3 i 0.3 25.7i 2.9
Fasted 1 .5x 1x 1 .5x ' 2x 2x 3x
Zn-depleted 2x 2x 2x 1.5x 1x 0.5x

 

 

 

 

 

 

 

 

 

All three groups of rats transferred less than 3% of the initial “Zn absorbed by the
mucosal cells to the vascular perfusate“. In addition the rate of “Zn transfer from
mucosal cells to the vascular perfusate decreased exponentially Overtime for all three
groups. The fasted and zinc depleted group both absorbed more “Zn into mucosal tissues

than the controls. However, the zinc depleted group retained more of the “Zn in

 

mucosal cells than the control and fasted groups, which were comparable. The fasted

    
  
  
   
 

group excreted more “Zn back into the intestinal lumen‘“. The elevated “Zn uptake by
ucosal tissue in zinc depleted rats supports previous studies that have indicated zinc
epletion stimulates the mucosal cell membrane transport mechanisms responsible for
inc uptake”. It also contradicts investigators that first examined “Zn uptake utilizing

e isotope dilution technique. They stated that previous misinterpretation of

 

 

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perimental results that did not take into account isotope dilution led to the belief that
re depleted rats absorbed and retained more “Zn, but in reality they did not1 ’3.

The data collected in this experiment was somewhat confusing when the MT content
mucosal cells was examined in conjunction with either the “Zn mucosal pool available
rtransfer or the t“2 life of “Zn in mucosal cells. A comparison of MT content and “Zn
rcosal pool available for transfer, to either the intestinal lumen or vascular perfusate,
monstrates that a greater amount of “Zn was associated with the transport pool when
llular MT concentrations were elevated by fasting‘“. This supports the third theory that
T facilitates the absorption of zinc. Fasting while not influencing the amount of “Zn
ained by mucosal tissue, did influence the compartmentalization of mucosal “Zn in

it more was available for mucosal to vascular transfer than the control group'“.

However, a comparison of the MT content of cells and the t1,2 life of “Zn in the

   
  
   
  
  
   
 
  

cosal cells shows a direct relationship between MT content and t1), of “an“. The

sin fasted rats had two times the MT content of controls and three times the t,,2 life of
in mucosal cells. Zinc depleted rats had less MT in their cells and their t1,2 life was
that of controls. Thus these result supports the theory that MT acts as a zinc buffer.

e interpretation of the conflicting results led to an integration of the first and third
ries of MT’s role in zinc absorption and distribution. The inciting cause of MT

ction determined the purpose the protein would serve in relation to intestinal zinc
tion. If MT induction was stimulated by fasting an increased proportion of dietary
en up by the intestine would be associated with the MT pool available for zinc

er, resulting in increased zinc absorption, but at a slower rate‘“. When MT

 

 

93

induction was the result of parenteral zinc loading, elevated endogenous levels of zinc
were found in the mucosal MT pool. In this case, MT would be acting as a zinc sink,
facilitating the transfer of zinc from the vascular space to the mucosal cells and
subsequently reducing the amount of dietary zinc absorbed‘“.

All four of the theories, with regards to the role that MT has in zinc absorption do not
rreclude the possibility of other ZBLs having a functional role in zinc absorption.
\lthough MT is obviously an important component of zinc metabolism, it is a cytosolic
rrotein, which would limit it participation in the movement of zinc from the intestinal
men into epithelial cells. As previously discussed in the “Kinetics of Zinc Absorption
ection”, the absorption of zinc from the lumen is dependent on two mechanisms, one is
nrier—mediated and the other passive. In addition, the same types of mechanisms also
aerate to transfer zinc intracellularly to the vascular space. The ligand requirements for
ese processes are a mystery to researchers. There have been several LMW, ZBLs
entified that appear to functionally assist zinc absorption, but none have been

nitively proven to participate in the process. In 1983, a LMW, ZBL was identified in
intestine of suckling and weanling rats through the use of differential hybridization“.
's differentially expressed message in weanling rats was a cysteine-rich intestinal
tein (CRIP), distinct from MT. Up until this time it had been difficult to distinguish
IP from other metalloenzymes, primarily MT, with low resolution gel-filtration
amatography methods. The difficulty in distinguishing the proteins was attributed to
n having similar molecular weights and metal binding attributes, which caused them

Dmigrate on gels.

 

 

 

94

The identification and characterization of CRIP
An experiment designed to identify proteins whose transcripts increased from birth to

weaning in rodent intestine led to the identification of a novel protein that was rich in
cysteines, and thus became known as the cysteine-rich intestinal protein (CRIP). Isolated
mRNA, fiom mouse intestine was used to make a cDNA library for differential
hybridization with adult mouse cDNA. One clone was identified whose amounts began
to increase during mid-suckling and reached peak amounts during weaning in the mouse.
:7urther characterization of the clone indicated that it contained two internal repeated
:equences of seven conserved cysteine residues and one histidine residue“. The high
ncidence of repeated cysteine residues were the basis for naming the protein. CRIP was
ound to be highly conserved between species such as sea squirts, fish, birds and humans,
ased Southern blot hybridization studies using rodent CRIP probes. The investigation of
re phylogenesis of CRIP in different species suggested that it existed when fish diverged
om other vertebrates 450 million years ago during chordate evolution“. When the

' 0 acid sequence of CRIP was compared to other known proteins it was found to be

'lar to proteins that contained LIM motifs that bind zinc in their stable form.

The increasing levels of CRIP mRN A in the intestine of developing rats, the discovery
t the highest concentrations of the protein was localized to the intestine and the ability
the protein to bind zinc, suggested that it may fimction in the absorption of zinc from
GI tract. The participation of CRIP in zinc absorption fiom the intestinal lumen
uld be consistent with two previous findings. First, the increases in CRIP protein

illeled the development of a saturable pathway for zinc absorption in the intestine“.

 

 

 

95

Second models previously proposed for zinc absorption included the involvement of MT
with other LMW, ZBLs. The possibility that CRIP participated in zinc absorption also
made it a candidate gene to examine as the causative agent in zinc absorption disorders,
such as AE and BHZD, which is the subject of this dissertation.

The coding region of the rat CRIP gene is 404 bp in length. There is a conserved
poly-A signal, aataaa, that begins 18 nucleotides upstream from the poly-A site“. The 5’
proximal initiator codon aug, starts at nucleotide 62 and is contained in the purine
nitiator consensus sequence ccrccaug“. The promoter sequence in the rat has been
rnalyzed 2744 bp upstream from the initiator codon, in a clone isolated from a rat
genomic library. The first 100 bp are 74% GC rich and there are multiple regions that
may serve as binding sites for transcription factors, glucocorticoid response elements
GRE), promoters and negative regulatory elements (NRE)l 16. Table 9 contains the
reations and types of binding sites in the promoter region of rat CRIP gene‘“. The CRIP
romoter region lacks both a tata and caat box for initiation of transcription. However it

es contain a Sp-l site, which has been shown to play a role in the initiation of

cription in other proteins that lack tata boxes1 16.

 

 

 

Table 9 - The Location, Sequence and Types of Binding Sites Identified in the

96

Promoter Sequence of the 5’ Flanking Region of Rat CRIP

 

 

 

 

 

5’ Location Sequence Type of Binding Site
-l750 agcacagactgttct. GRE
-l416 gttcacccctgttct GRE
-1279 caccc caccc box
908 gggcgg Sp-1
-8 67 agagataa gata-2
~816 gcctgggc AP-2
~703 tggca NF-l
-697 aggtcagggtgttca GRE
-677 gcctgggc AP-2
~523 atttgcat Oct
-465 caccc caccc box
-401 ttatc gata-2
-3 72 caccc caccc box
-345 gtgcaaga GRE
-255 gggcggg SP"1
-231 caccc caccc box
-1 69 tatct gata-2
~74 ggggcggggcggggcc gc box
-16 ggcgggcgccaccc gc box

 

Transfection studies of the promoter region with a Chloramphenicol acetyltransferase
(CAT) reporter gene have been conducted to determine the sites responsible for
regulating basal activity of CRIP. Promoter constructs with a CAT recorder were
transfected into intestinal epithelial cells (IEC-6) derived from adult rat intestine. The
ransient expression of CAT in the transfected cells was assayed by liquid scintillation
ounting of CAT products. The results of these studies indicated that the removal of the
lot region, at position ~523 caused a 6.6 fold increase in basal activity of CAT‘“. If the

rccc boxes were deleted in addition to the Oct, the basal activity of CAT increased from

 

 

 

97

6.6 fold to 8 fold'“. Both the Oct region and caccc box have been implicated in
repressing the expression of genes. Oct has been identified as a NRE and caccc box has
been shown to down regulate monoamine oxidase B gene‘“. The identification of
binding sites for NRE in the CRIP promoter sequence may explain low basal expression
of CRIP in specific tissues and during development. A developmental decrease in a
repressor protein may explain the observed increase in CRIP expression with age‘“.
Constructs that contained the GRE sites showed increased activity in the presence of
:lexamethasone. The ability of dexamethasone to increase the expression of CRIP will be
liscussed further in the, “Developmental Regulation of CRIP Expression in the Intestine”
ection .
In the original paper that first identified CRIP, the gene was mapped to chromosome
2 in the mouse and was found to be closely linked to the Igh-C locus, a immunoglobulin
=avy chain constant region complex“. The close linkage of CRIP to the Igh-C locus led
the prediction that CRIP would be present on chromosome 14 in humans. Additional
pping of the chromosome 12 in the mouse has provided information in regards to
er genes present on the chromosome. The subtelomeric murine chromosome 12
tains four known genes; Aat (al-antitrypsin), Ckb (creatine kinase, brain form), CRIP
teine-rich intestinal protein) and Igh-C (immunoglobulin heavy chain constant region
plexm'm.
RIP is a single polypeptide chain that contains 77 amino acids. The protein has a
cular weight of approximately 8.55 kDa when bound to zinc. It contains seven

ines and one histidine residue arranged in a conserved motif. CRIP is a member of

 

 

98

a dispersed multigene family containing structural LIM motifs. The LIM motif

structurally appears as a zinc finger with the conserved cysteines forming the zinc

binding domain. Typically there are nine tandem repeats in thirty amino acid groups, each
containing two pairs of Cys and His residues that are approximately 3 kDa. The zinc atom
forms the basis of tetrahedral coordination of two invariant pairs of Cys and His
residues“. The conserved amino acids between the Cys and His residues may serve as
DNA, RNA or protein binding sites. The coordination of the nuclear binding domain by
zinc is ideal, considering the ion is not easily reduced which can cause hydrolysis of the
DNA or RNA.

There are more than ten classes of zinc-finger binding motifs that are involved in a
Iariety of regulatory functions‘zo'm. The classifications are dependent on gene function
nd the differences in function can be accounted for by structural variations‘2"‘“.
ommon structural variations include; whether the motif contains conserved Cys and
is; whether the amino acids are conserved around the zinc binding sites; whether

drophobic regions are formed; a-helix or B-sheet formation; spatial distances between
c atoms; and overall structural tetrahedral formation. The sixth class of these proteins
ludes genes with LIM motifs. There are three types of LIM genes that are

tinguished from each other structurally by the number and types of LIM motifs the

e contains. LIM motifs contain the cysteine rich sequence,
ZCXUHX2CX2CX2CX17CX2, the seven cysteines and one histidine are spatially aligned
low for them to be metal binding ligands‘“. LIM motifs were first identified in three

es, lin-ll of Caenorhabditis elegans, I SL-l from the rat and mec-3 from

 

 

 

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99

Caenorhabditis elegans. Thus the name arose from the first letter of each gene’ s name.
Table 10 contains examples of six classes of zinc-fingers, their zinc binding capacity,
amino acid sequence, structural features and possible functions"8"2°"2"‘22'”4.

“There are three primary types of LIM proteins, one contains two LIM motifs in
association with one homeodomain, the other has two LIM motifs linked to a protein-
kinase domain and the third only has LIM domainsm. Thus far there have been
numerous proteins containing LIM motifs identified and their proposed functions have
ranged from DNA or RNA binding to nuclear and cytoskeletal localization.

Homeodomains are often involved in DNA or RNA binding. CRIP contains only one

91M motif. The LIM-only genes lack an identifiable DNA or RNA binding domain and

may function in protein-protein interactions.

 

 

 

 

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Ell 3 J

Liz-s.

 

 

 

100

Table 10 - Six Classes of Zinc-finger Proteins, Amino Acid Sequences and
Structural Characteristics

 

 

 

 

 

 

 

 

 

 

 

 

Class and Conserved Sequence Structural Features Function
Representative
protein
Class]
0. fl.
TFIIIA cx,_,cx,,Hx,,,H (‘zufj (:zn’) Nucleic acid
’c’ hvc \bo binding
NH, C H
_Z____g_inc Fin er
Class]!
steroid-thyroid CXZCX13CX2CX,5CX5- )Ié DNA binding;
receptors CX12CX4C oligrnerization
NH:
Iwwd
Class III NH2\ COOH
\ ch/ \zn lc , ,
GAL4 CXZCX6CX6CX2CX6C (:20 ’ \ [m \ l DNA bmdmg
Ucv c
Zinc Clrger
Class 1V
(1 a“.
GATA-l cx,cx,,cx,c (1211’) (:m: DNA binding
c ‘c c
Nit, v cooii
Zi_n<=_Fi_ng§r
Class V NH2 1
‘°<'>° "—3
BRCA l cx,cx,_,,cx,_,H- zn\ Protein-protein
x,,cx,cx,4,cx,c C’Jc Vii"; interaction;
(I /m\ Nucleic acid
0v C binding; 55 nucleic
C09" acid binding
CI V1 Ring Finger
ass
c h c c
(LIM genes) CXZCanHXZCXZC- ( \zn’) ( \zn’ ) Protein-protein
x,,,,cx,,,c ’c’ ‘c vc’ t‘ interactions; DNA
CRIP NH2 COOH binding
Zinc—Fined

 

 

 

 

 

lOl
re structure of the CRIP protein has been determined by high resolution lH-
nuclear and lH—“N heteronuclear-correlated nmr and 3D structural studies of
rbinant rodent CRIP‘“. The protein contains two zinc-fingers, the N-terminal zinc-
is a CCHC module and the C-terminal module is CCCC, both modules are packed

’25. There are four anti-

er in such a way that they form a hydrophobic interface
:1 fl-sheets and an a-helical structure formed as the protein folds’“. Each zinc
binds one zinc atom, which allows CRIP to bind two zinc atoms per molecule of
. CRIP extracts run through continuous elution PAGE columns to strip the
y of bound zinc, then incubated with “Zn and buffer at a pH of 7.5, did not bind
al. However, CRIP did bind “Zn introduced into the intestinal lumen in vivo,
ing that the binding of “Zn is a result of a cellular process rather that metal
:6 at a labile binding site or binding to fill an unoccupied site’“. At pH values

5 and 8 CRIP binds 25% of available “Zn in stable conforrnation‘“. However,
I values are below 4.5 only 8% of “Zn is bound to CRIP and with a pH above
r of “Zn is bound‘“. Although CRIP preferentially binds zinc it also binds
rand copper with a decreasing binding affinity Zn>Cd>Cu, but it does not bind
he ratio of the binding affinity to zinc between MT and CRIP, at a pH of 7.5

respectively, indicating that MT can preferentially compete with CRIP for

sue distribution of CRIP was originally investigated by examining a small
’tissues for relative amounts of CRIP mRNA in rats. CRIP mRNA was

by using dot blot hybridization with a 32P labeled plasmid DNA probe derived

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102

cm a cDNA rat clone“. Additional investigations of rat CRIP tissue distribution used
tRNA preparations from various organs in a Northern blot study that was probed with

’ labeled rat CRIP cDNA. The CRIP mRNA was quantitated by densitometry of
toradiographs produced from the probe hybridizing to CRIP mRNAm. Analysis of
llular proteins in the liver and pancreas were also investigated by injecting “Zn into the
rtal vein and purifying and identifying extracted proteins fiom cellular homogenates to
: if CRIP was present”. All the experiments indicated that the small intestine and

on were the major sites for CRIP mRNA expression‘2'56'127. CRIP was also detected in
iller amounts in the lung, spleen, adrenal, testis, skin, heart, skeletal muscle and

nach, while the highest concentration of CRIP mRN A was detected in the small

stine and colon. The presence of CRIP in several different tissues implies that its
:tion is not unique to the intestine “"27. Almost all the experiments to date have not
cted the presence of CRIP mRNA or protein in the adult liver or pancreas or in 18 to
ay old fetal yolk, liver and placenta of rats”‘“"27.
he distribution of CRIP mRNA along the villus to crypt axis was determined in the
l intestine by sequential rinsing of isolated sections of rat jejunum with a sodium
ide and dithiothreitol solution followed by physiologic buffered saline incubating in
action at 37°C for 5 minutes. The buffer was then collected and the section rinsed

, this allowed for the cells to be harvested from the villus tip descending to the crypt

127

producing seven fractions The tissue concentration of CRIP in the mucosal layer

126

etermined to be 15 to 20 ug/g of tissue The crypt fractions were distinguished

hid-villus and villus fractions by the identification of cryptidin in the cytosol

 

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103

genates. Cryptidin is exclusively expressed in crypt cells of the small intestine‘“.
tains a consensus sequence found in corticostatins and defensins, and is
opmentally regulated in crypt epithelial cells below the level of the stem cells'“.
’ mRNA levels were normalized using B-actin mRNA levels. The results of the
riment indicated that Fractions 6 and 7 represented crypt cells and had the highest

127. The mid-villus regions represented by Fractions 4 and 5

:ntration of B-actin
ined the highest CRIP mRNA content. The findings that CRIP mRNA was
minantly found in the mid-villus region was consistent with the synthesis of CRIP
erocytes migrate along the villus axis and the degradation of cellular RNA as

sal cells reached the villus tipm.
opmental regulation of CRIP expression in the intestine

3 expression of CRIP in developing rat intestine increased from when it was first
ed in the 21 day old fetus to when the rats reached maturity“'m. There was more
nal CRIP mRNA detected in adult rats than 9 day old pups“. A small rise in CRIP
t levels in the intestine occurred at 2 days of age followed by a two fold increase by
and an eight fold increase above the levels at birth by day 24“. Endogenous

)f corticosteriods peak between 14 and 24 days of age in rats and one can speculate
he correlation between the increasing CRIP levels at this time“. Another

nent’s results correlated well with the previous one above, indicating a general

3 in CRIP mRNA levels up to 21 days of age in the rat intestinem. These findings

56,126,127

1e hypothesis that CRIP is a developmentally regulated protein

 

 

 

 

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rere are some physiologic factors that need to be taken into account when evaluating
aparent increases in CRIP mRNA levels that have been reported in developing rat
ine. The intestinal mucosal layer is continuously regenerating from progenitor crypt
that are migrating towards the villus tip. Between birth and weaning the depth of
cells increases causing a relative decrease in the population of villus cells. This

1565 the ratio of villus height to crypt depth from 6.5 to 2. There is also an increase
turnover rate of mucosal cells, a 21 day old rat turns over cells four times faster

5 day old pup“. Thus the increase in CRIP mRNA levels detected in the two
ments above, could be due to several mechanisms: First, there could be an increase
olute CRIP mRNA in each cell; second, there could be an increase in the number of
ontaining CRIP, without a change in message levels within the cell; third, a change

relative number of crypt versus villus cells; and forth, a combination of the first

cocorticoids are regulators of intestinal development in many species, therefore it
ible that they also may regulate CRIP“. Corticosterone is the major circulating
articoid in neonatal rats, its concentration gradually increases during suckling up to

3 of age127

. The presence of four potential GRE sites in the promoter region of
long with the coincidental rise in CRIP mRNA paralleling increased

terone levels, provides suggestive evidence that the protein is in part regulated by
rticoidsZ4'1“. There have been several experiments conducted to examine the

hip between glucocorticoid induction of CRIP expression and intestinal

'on in neonatal rats. The results of the experiments all support the hypothesis that

 

105
’ in neonatal rat intestine is developmentally regulated by endogenous levels of
rcorticoidsl'6"27"29.
r determine the effects that glucocorticoids have on CRIP expression in neonatal and
rats, experiments have been conducted using dexamethasone. Dexamethasone is
red over other corticosteriods due to the fact that circulating concentrations are not
need by corticosteriod-binding globulin; therefore, the plasma levels can be
rted accurately and consistently in experiments.
rec separate experiments have demonstrated that the administration of
rethasone induces the synthesis of CRIP24’116'127. One experiment utilized IEC-6
hat were treated with 5 uM of dexamethasone, the other two experiments used
in] rats injected with dexamethasone or hydrocortisone. In all three experiments
rous administration of corticosteriods induced CRIP expression 12 hours after
istration, causing a two fold increase in CRIP mRNAlevelSZ4’ll6'127. The CRIP
L levels continued to increase up to 72 hours post-injection, reaching 70% of the
1 message levels in a 24 day old rat“.
iefine the importance of endogenous corticosteriods in CRIP expression,
iectomies were performed on 9 day old rat pups and the jejunal expression of
[tRNA was measured. Four litters of pups were used, half the litters were
ectomized, the other half sham operated. The removal of the adrenal gland
ed the developmental surge of endogenous corticosteriods that occurs between 14
days of age“. The pups’ jejunal mucosal cells were collected on days 17, 20, 23

post-gestation, for RNA isolation and northern blot analysis. The blots were

 

 

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106

:d with 32P labeled cDNA from rat CRIP clone“. The sham operated pups displayed
al increases in corticosterone plasma levels and subsequent increases in CRIP

A levels between 14 and 24 days of age. Adrenalectomized pups displayed

icant reductions in CRIP mRNA levels compared to the controls". However,

:en 23 and 26 days post-gestation in adrenalectomized pups the CRIP mRNA levels
rsed to comparable levels found in controls. The reason why the CRIP mRNA
elevated in the adrenalectomized rats after 23 days of age were unclear, but could

a to the developmental profile of CRIP not being solely reliant on glucocorticoids,
:itive or negative modulators of transcription or post-translational stabilization“.

1 experiment examining the effects of exogenous dexamethasone injections on

tal and adult rat intestinal expression of CRIP was also conducted. The neonatal rats
njected subcutaneously with 1 mg/kg of dexamethasone at 1 and 2 days of age.
ntestinal tissues were then collected on day 3 to assess protein expression and on
,3,7,14 and 21 for CRIP mRNA analysism. The intestinal cytosol fractions were
ed by HPLC and the isolated CRIP containing fraction was incubated in “Zn. The
indicated that there was a 2-fold increase in CRIP mRNA and CRIP protein levels
[1 binding to CRIP in pups injected with dexamethasone when compared to

i. Adult rats that were given 1 and 2 mg/kg doses of dexamethasone by

itoneal injection, had no significant changes in intestinal CRIP mRNA expression
1 up to 12 hours post-injection in their intestinal cytosolm.

eared that CRIP mRNA expression in response to glucocorticoids was an age

nt process. To confirm this hypothesis 3 series of three dexamethasone injections

 

 

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107
given to three litters of rat pups beginning on days 10, 16 and 18 post-gestation“.
reared pups and controls were killed 4 days after the injections. Total RNA from the
was isolated for Northern blot hybridization by radiolabeled rat CRIP clones.
ification of CRIP mRNA expression was performed by counting labeled dots with
scintillation. The results indicate that significant elevations of CRIP mRNA
sion occurred in pups given injections of dexamethasone on day 10 but the amount
sage expressed declined in the same animals after that until they were
onsive to the injections by 26 days of age. Pups had a 3.8 fold increase in CRIP
expression above controls at 10 days of age, and 1.9 and 1.5 fold increase in
sion at 16 and 18 days of age“. Thus it appeared that CRIP mRNA expression was
iced by glucocorticoids up to 18 days of age, but the mechanisms that regulated the
sion thereafter remain unknown.
Jramphenicol acetlytransferase reporter constructs were used to help define the
ane of the four GRE sites in the 5’ promoter region of CRIP‘“. The constructs
cubated with nuclear extracts from the small intestine of adult rats that had been
sly treated with dexamethasone for 30 minutes. Afterwards, gel electrophoresis
'-shift assays were conducted on the treated and control groups to detect the
t of nuclear proteins bound to the construct that were responsive to glucocorticoid
t‘“. The treated group had nuclear extracts bound to the promoter constructs that
their migration in the gels. The results indicated that a glucocorticoid induced
y be involved in the up-regulation of CRIP expression during suckling“. One

2d experimental result was that the CAT reporter constructs with various

 

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iii

108
:tions in the 5’ region, which eliminated the four GRE sites did not have a significant
rction the dexamethasone induced activity of CAT‘“.
he experimental results from the age responsiveness study and the CAT reporter
:tructs implied that the GRE sites alone were not responsible for gene expression.
'e are a number of possibilities as to why CRIP expression appears to have multiple
lating factors. The repressor binding sites that are actively controlling developmental
lation of CRIP may also impose tissue specificity of gene expression’“. There also
i be high levels of repressor binding factors that occur in tissues with low levels of
’ expression. Adult rats may no longer have the binding factors that neonates do to
to the promoter region of the CAT constructs, thus the promoter region may only be
3 during developmental stages. The cells in the experiment were also obtained from
mucosa that may not produce glucocorticoid nuclear factors to bind to the GRE
6
CRIP is regulated by a variety of mechanisms, then examining the elevations of
rs hormonal levels in neonates may reveal additional regulators of expression.
21 levels of another hormone, L-thyroxine (T4), elevate prior to corticosterone and it
wn to induce the production of corticosterone-binding globulin. There is a
ility that glucocorticoids may regulate the expression of CRIP mRNA during the
of intestinal maturation and that T4 acts synergistically with the glucocorticoids“.
estigate this possibility a combination of dexamethasone and T4 treatments were
0 four litters of rat pups, and CRIP mRNA was measured in the intestine. Each

as given a separate treatment protocol: 1. Vehicle injections on days 5 through 12

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“1
IN
.

it

 

109

est-gestation; 2. T4 injections on days 5 through 12; 3. Dexamethasone injections given
ays 8 through 12; 4. T, injections on days 5 through 12 and dexamethasone injections
'ven on days 8 through 12. The timing of the injections was designed to simulate in vivo
ievations of plasma T4 levels that normally proceed plasma corticosterone increases,
rrcept at a younger age“. Total cellular RNA was collected from the mucosa of the
junum and CRIP mRNA was quantified on day 13. L-thyroxine administration alone
rused a small but significant increase in CRIP mRNA expression above the control
rimals. Dexamethasone caused an increase in message expression as previously
edicted, the T1 and dexamethasone treatment combined had a response greater than
her of the two alone, suggesting a synergistic response“. Therefore the expression of
UP may be regulated by both hormonal factors during intestinal maturation.

The regulation of CRIP expression during development by glucocorticoids could

cur by the hormone excellerating the rate of mature intestinal epithelial cells replacing
mature cells indirectly increasing CRIP intestinal levels. Glucocorticoids could also
iuce nuclear factors that bind to the promoter region of CRIP, decreasing the mRNA
nover of CRIP. Further research examining the protein levels of CRIP in parallel to its
LNA levels will aid in determining the overall effects of hormonal regulation.

f CRIP is expected to participate in the absorption of zinc from the GI tract, then it
11d be expected that its expression would be influenced by dietary levels of zinc, this
vever, does not appear to be the case. Experiments were designed to measure the

cts of dietary, intraperitoneal injections, incubation of IEC-6 cells and treatment of

129

recorder constructs with varying concentrations of zinc In the first experiment,

 

 

:51

fl)" '

 

110

rec groups of adult rats were fed a deficient, adequate and excessive zinc diets for up to
i days prior to harvesting cells of the jejunum and ileum for totRNA extraction. The
cond experiment examined the affects of incubating IEC-6 cells in solutions containing
fferent zinc concentrations, then measuring the CRIP and MT mRNA levels. The third
ed CAT reporter constructs transfected into IEC-6 cells and incubated in various zinc
ncentrations to assess transient expression of CAT activity. The results of all three
Jeriments indicated that there was no change in CRIP mRNA expression, but the

)ected increases and decreases in MT mRNA expression occurred‘”. The fact that

 

IP was not regulated by zinc, but did require zinc for structural integrity and probable
iular function does not preclude the hypothesis that it may be involved in trafficking
: for intestinal absorption, but does suggest that it may have more diverse functions.
IP’s function in the regulation of zinc absorption from the GI tract

CRIP’s potential role in zinc absorption from the intestinal lumen was first
othesized by Birkenmeier and Gordon the individuals who discovered the protein“.
identification of CRIP in increasing amounts in the villus cells of rodent small

stine from birth to weaning, along with the ability of the protein to bind two zinc

rs, suggested that it may play an important function in the nutritional absorption or
:tinal metabolism of zinc“. The first experiment that established an association

'een CRIP and the absorption of zinc from the GI tract, used in viva ligated loops of
.enum injected with “Zn. The experiment was designed to identify LMW proteins
)ound “Zn in mucosal cells shortly after it was introduced into the duodenum”.

i
'collecting the intestinal cell mucosal layer, it was homogenized and passed through

111
a gel-filtration HPLC colunm. A large portion of the “Zn injected into the intestine was
associated with one peak detected between 6 and 9 kDa, that was unique from MT”.
After further isolation, sequence analysis of the protein found it to be identical to the
RIP. The fact that such a large portion of “Zn introduced into the intestinal lumen was
ssociated with CRIP 15 minutes after injection, suggested that it was involved in zinc
rinding during transmucosal transport.

As previously discussed in the “Kinetics of Zinc Absorption” section, zinc transport
cm the lumen of the intestine into mucosal cells is facilitated by a carrier-mediated
athway and a non-saturable pathway. Two similar mechanisms also operate in the
ansmucosal transport of zinc through mucosal cells to the vascular space. Evaluating
e intracellular distribution of total zinc and “Zn by varying the intestinal lumen
rncentrations of zinc injected in the duodenal sacs, allowed for the binding kinetics of
UP to be assessed. When lumenal zinc concentrations were below 5 uM, which was
:0 the saturation concentration for the carrier-mediated pathway for zinc transport from
: GI lumen into mucosal cells, CRIP bound almost 50% of the “Zn entering the
rcosal cells‘m. This represented l 1% of the total zinc content in the cell cytosol. This
Licated that the majority of “Zn was associated with CRIP compared to other LMW,
Ls in intestinal cells. This finding would be expected of a protein that was involved in
carrier-mediated process of zinc transport. When lumenal zinc concentrations were
1 11M, CRIP bound 25% of the “Zn in cell cytosol, but the total cellular zinc content
the proportion of total zinc bound to CRIP remained at 11%”. The amount of “Zn

nd to CRIP decreased as the intestinal lumen concentrations of zinc increased,

 

112
suggesting that CRIP had a limited binding capacity and became saturated in the presence
of excess zinc”.

If CRIP was involved in zinc absorption, then its mechanical function and regulation
of absorption still remained to be determined. The focus of the following investigation
was to further evaluate the role of CRIP in zinc absorption and the relationship it may
have with MT or other ZBLs in regulating zinc transport“.

Different groups of adult rats were fed diets with low and high zinc concentrations for
20 days. At the conclusion of the feeding trial, ligated loops of their proximal small
,ntestine were injected with “Zn for 15 minutes, then flushed. The mucosal cells were
:ollected and homogenized to identify the interactions between CRIP, MT and
ronspecific ZBLS“. Some of the rats fed low zinc concentrations were also given
ntraperitoneal injections of 25 pmol of zinc 20 hours prior to intestinal infusion. The
iZn absorption in the homogenized mucosal cells’ cytosol was determined by the
ifference between the disappearance of “Zn from the lumen and “Zn injected into the
rtestinal loop“. Table 11 contains the results of “Zn association with MT and CRIP in

re three groups of rats.

 

H—n-v-

 

lib]

 

I}

 

113

Table 11 - The Percentage of “Zn in Intestinal Mucosal Cells Cytosol Associated
with either Metallothionein or CRIP in Rats Fed Different Zinc Diets

Dietary Group % of “Zn bound to MT % of “Zn bound to
CRIP

 

The absorption of “Zn in rats fed low dietary zinc concentrations was greater than that
f rats fed high zinc concentrations or parentally treated with zinc“. There was an
icrease in the association of cytosolic “Zn with MT and a decrease in the amount of “Zn
ssociated with CRIP as dietary zinc contents increased“. The amount of CRIP in
ucosal cells was not affected by the dietary zinc changes, but MT concentrations were
arkedly increased along with the amount of bound “Zn. An increase in the amount of
T available to bind zinc may have caused a redistribution of “Zn in the cytosol,
‘ulting in markedly less “Zn being associated with CRIP“. The data also suggests that
ompetitive relationship existed between CRIP and MT. If CRIP was responsible for
carrier-mediated pathway of zinc absorption then decreasing the proportion bound to
protein would decrease the amount of “Zn absorbed into the vascular space.
The concept of CRIP and MT competitively regulating zinc absorption was also
estigated in an experiment that used adult rats with induced insulin dependent diabetes
litus'“. Diabetes is often characterized by hyperzincuria and a decrease in the amount

' c absorbed by the GI tract. The study focused on how rats regulated their zinc

ll4

htake to accommodate their increase in urinary excretion. Two groups of rats were fed
liets containing ZnCO3 for 5 days. One group was injected with streptozotocin to induce
liabetes'“. The zinc contents of the urine, feces and plasma were measured daily by
tAS. On day 20 of the experiment, in vivo ligated sacs of duodenum were injected with
25 uM solution of “Zn for 30 minutes, to assess the rate of absorption and cellular
istribution of zinc in the mucosa'“.
The results indicated that the diabetic rats had twice the food intake of controls. The
at absorption of “Zn from the lumen was the same for both groups, but the mucosal
intents was greater in the diabetic rats that the controls. In addition, almost twice as
uch zinc was transferred to the vascular space in the control animals when compared to
e diabetic rats. Thus, it appeared that the increased amount of zinc absorbed by diabetic
is was being retained in their mucosal cells. The cellular distribution of “Zn
monstrated that the majority of the “Zn was bound to MT as compared to CRIP or
1er HMW, ZBLs in the cytosol of the diabetic rats. There was also more MT present in
= cytosol of diabetic rats than controls. The investigators concluded that higher
stinal MT levels resulted in less lumenal zinc being bound to CRIP which inhibited
transport’“.
he effects of increased lumenal zinc concentrations on the distribution of “Zn to
s in the cytosol was also examined by gel-filtration HPLC, electrophoresis and
tern blots. The results indicated that as the lumenal zinc concentrations increased
1 to 5 pM, “Zn was primarily bound to CRIP. As lumenal concentrations

ased from S to 200 pM, the amount of cytosolic zinc associated with CRIP

 

 

115

lecreased by 14%“. However, the total amount of zinc bound to CRIP did not change.
he total amount of cytosolic zinc and the proportion of “Zn bound to MT did not change
ither, but the distribution of “Zn to other ZBLs increased“. The amount of “Zn bound
) HMW, ZBLs increased, but it did not appear to be associated with any specific
actions. Distribution of “Zn among the HMW, ZBLs shifts over time, suggesting a
rriety of intracellular constituents are involved in zinc absorption“.

Although CRIP appears to be well suited for facilitating the absorption of zinc from
c GI tract, it has not been definitively proven to act in such a manner to date. A study
at disputes the relationship between CRIP facilitating zinc absorption and MT
mpetitively inhibiting zinc transport intracellularly was conducted in human

enocarcinoma intestinal cells (Caco-2)m. The effects of vitamin D on zinc transport

 

i CRIP and MT mRNA induction were assessed. Vitamin D has been shown to

rease “Zn uptake and transfer from mucosal to serosal surface in in vitro ligated

idenal sac studies‘“. The Caco-2 cells were grown in a monolayer supported by a
meable filter. The cells were incubated in a solution containing vitamin D for 72

rs prior to the cells on the filter support being transferred to 6 well plates‘“. The

is contained a serosal buffer that the monolayer of cells and filter were place over.

e the filter and cells were in place a mucosal buffer was added on top of the

olayer that contained “Zn. The transfer of “Zn from the mucosal buffer into the cells
transcellular movement of the “Zn into the serosal buffer were then measured.

he vitamin D treated cells had an increase in the amount of “Zn transported through

ellular layer after incubation for 72 hours, but did not show a significant increase in

 

I'——'

 

 

Ii:

 

116

the amount of “Zn transported if the cells were incubated less then 24 hours’“.
Metallothionein mRNA levels were four times higher than controls in cells incubated 24
hours and ten times higher after 72 hours of incubation‘“. However, CRIP mRNA levels
decreased to 78% of the control values after 72 hours of incubation. It would be expected
that the increased transport rate of “Zn would have been accompanied by an increase in
CRIP mRNA expression, if the protein was solely responsible for the up—regulation of
zinc transport. The findings do not exclude the possibility of CRIP participating in zinc
absorption, but do indicate that an additional mediator of absorption is also frmctioning.
The lag in response time for “Zn transport to increase after incubation in the vitamin D
iolution suggests that the vitamin induces a genomic event that results in up—regulation‘“.

The experimental results that have supported the interaction of CRIP and MT in the
egulation of zinc absorption from the GI tract have led to the formation of a model that
epicts the function of CRIP and its interactions with MT and other nonspecific proteins
uring zinc transport in mucosal cells, shown in Figure 2. In the model, CRIP acts as an
rtracellular zinc carrier. The ability of CRIP to mediate both lumenal zinc absorption
rd intracellular transport fits the criteria proposed for carrier-mediated lumenal zinc
rsorption and the rapidly transferred pool of intracellular zinc in kinetic studies
1
scussed earlier”. CRIP becomes saturated with zinc at the same lumenal concentration
it the carrier-mediated mechanism for zinc absorption does in the zinc flux from the
estinal lumen into mucosal cells. Theoretically, the regulation of zinc absorption from
GI tract would involve CRIP binding zinc at the apical border of enterocytes, possibly

:ompetition with nonspecific ZBLs of high or low molecular weight. CRIP may also

 

 

117
exchange zinc at the basolateral membrane with another ligand that is responsible for
transfer to the vascular space“. The possibility of the additional transfer protein was
depicted as (E) in Figure 2. As the concentration of lumenal zinc increases and CRIP

becomes saturated, more zinc would be available to bind to other ZBLs that may induce

 

MT synthesis or facilitate zinc transmural movement independently. These ZBLs are
represented as nonspecific binding ligands (N SB) in Figure 2. Zinc would bind MT
preferentially in enterocytes which would competitively decrease the amount bound to
CRIP“. The fate of zinc bound to MT is not completely known. It is believed that MT
acts as a storage pool that allows for the labile exchange of zinc with other proteins for
netabolic functions intracellularly". However, the vast majority of zinc bound to MT is
bought to be lost into the GI tract during desquamation of the enterocytess. It has not
reen determined if zinc bound to MT participates in the pool of zinc that is slowly
:ansferred to the vascular space. Currently, the investigators of the experiment described

bove indicate that zinc is bound intracellularly to nonspecific HMW, ZBLs, which act as

re pool of zinc that is slowly transferred to the vascular space“.

lur

Figurel
Nonspec

The p
Prisentet
palhli‘ay
adequite

The CR

 

 

   
   

 

 

. Paracellular -——-

2n sf?

Abumin—Zn

Tronscellulor

Figure 2 - Model of Zinc Absorption by an Enterocytes Involving CRIP, MT and
Nonspecific Zinc Binding Ligands, and a Paracellular Pathway © J Nutr (122:89-95)

The paracellular pathway for zinc absorption from the lumen to the vascular space as
resented in Figure 2 relies on the passive diffusion of zinc between enterocytes. This
athway is believed to be what allows for the dietary supplementation of zinc to maintain
iequate endogenous concentrations in individuals affected with BHZD and AE.
he CRIP locus as a candidate gene for bovine hereditary zinc deficiency
i Since CRIP appears to be involved in the absorption and transcellular transport of zinc
the GI tract of rats, it is an excellent candidate gene for a possible mutation in cattle
ected with BHZD. By employing molecular genetic techniques, the research presented

'5 dissertation investigates the genetic sequence of CRIP and its tissue distribution.
comparing the genetic sequence of the coding region of CRIP between heterozygous
i homozygous BHZD cattle, as well as unaffected, unrelated cattle a mutation can be
ntified. If a mutation is not identified in the coding region of CRIP, then the

estigation into the relative tissue distribution of CRIP will aid in determining if there

 

 

isamurarion occurring in t
suppressing CRIP expressir
deficient and zinc adequate

irrher insight into additior

 

 

119
is a mutation occurring in the 5’ promoter region of the gene or if a trans acting factor is
suppressing CRIP expression. The tissue distribution of bovine CRIP in affected zinc
deficient and zinc adequate as well as unaffected, unrelated bulls and heifers, will give

further insight into additional functions the protein may have in physiology.

 

 

 

lhe research conducter
approach; therefore the In
sprite sections. The firs
used to produce and evalr
include the methods used
edllerinreutil protocols ar

manufactures and supplie

Materials and Meth

Hereditary Zinc Del

Producing a BHZD W1

The pedigree at Michi
aBlack Pied Danish, her
run Z-24 and Z-33 don

designed to obtain seven

Chapter 2

MATERIALS AND METHODS

The research conducted to study BHZD included both a clinical and laboratory
approach; therefore the materials and methods descriptions will be divided into two
separate sections. The first section will focus on the clinical applications and diagnostics
used to produce and evaluate the pedigree of affected cattle. The second section will
include the methods used in the molecular analysis of CRIP. Detailed information on
experimental protocols and clinical procedures will be provided in Appendix B. All

manufactures and suppliers of materials used are referenced in Appendix C.

Materials and Methods for the clinical investigation of Bovine
Hereditary Zinc Deficiency:

Droducing a BHZD pedigree

The pedigree at Michigan State University was established with semen from Denmark,
Black Pied Danish, homozygous affected bull and two related obligate heterozygote
ows Z-24 and Z-33 donated by Dr. Lance Perryman". An embryo transfer program was
esigned to obtain seven offspring for the study of hereditary zinc deficiency, utilizing

elated recipient cows. The embryo transfer protocol involved the synchronization of

 

nor cows with progesterone ear implants and inducing superovulation through a series

intramuscular injections of lutalyse and follicle stimulating hormone (F SH)‘“"“ 1“.
120

 

 

The donor cows were thCD
affected bull upon demons
donor cows 7 days after A]
one implanted into a recip
recipient cows were previc
implanted into the recipier

mding was based on the 1
Variation; presence of vesi

oldie zona pellucidaj pres

page of development133 . '
Cue and management c
The pregnant recipient

day prior to calving. Thn

isolation facility for close

”ditions of 2 ml of rota

“his Prior to parturition
ud selenium intramuscu
Afterlilrturirion, the t

ldllllldUa] Stalls. The ca.

die of rota ‘

and cor

allowed to suckle 12% 0

r.

“d b)’ a °°1°$Uumere
h
“Hillscular inJ'ectiorr (

 

121
The donor cows were then artificially inseminated (A1) with semen from a homozygous

affected bull upon demonstrating standing heat. The embryos were flushed from the
donor cows 7 days after A1. The embryos were graded for viability and grade 1 through 3
were implanted into a recipient cow’s uterine horn that had a corpus luteum. The
recipient cows were previously synchronized with lutalyse. The embryos that were
implanted into the recipients were primarily in the morula or early blastocyst stage. The
grading was based on the compactness of the cells; regularity of shape; cell size and
variation; presence of vesicles in the cell cytoplasm; presence of extruded cells, regularity
of the zona pellucida; presence or absence of cellular debris and evidence of the proper
stage of development133 . This procedure is outlined in Appendix B.
Care and management of the calves

The pregnant recipient cows were maintained on pasture and provided supplemental

hay prior to calving. Three weeks before their calving date, they were brought into an

  
  
  
   
 
  
  
  
  
  

isolation facility for close observation. The pregnant cows were immunized with two
injections of 2 ml of rotavirus, coronavirus and Escherichia coli vaccine”, at 8 and 4
weeks prior to parturition. They concurrently received two 10 ml injections of vitamin E
and selenium intramuscularly.

After parturition, the calves were immediately removed from the dams and placed in
individual stalls. The calves navels were dipped in 2% iodine and they received one oral
dose of rotavirus and coronavirusa vaccine, prior to being fed colostrum. They were
allowed to suckle 12% of their body weight in banked, superior quality colostrum, as
tested by a colostrometer, within the first 12 hours of life. In addition they received a lml

intramuscular injection of vitamin E and selenium and Vitamin A and D. The calves

 

 

WWW,“ y..- ..w...*__~m

m adminiStflCd 1 g of 51
as aprophylactic precallti0
calves were born, serum IE
transfer of immunogloblfli

lhe calves were fed mi
feedings from a nipple bot
beginning at 1 week of ag'
physiologic status, and the
were 6 to 8 weeks old. A
facility where they were p

The calves were deho
routinely vith 1% Ivomet
vitamin E and selenium ll
lhil‘ reached 4 months of
and selenium enriched trz

Trace element analvsis

T e
race mineral concen'

[h ,
ecalv es were 12 weelo

lle blood was draWn int

1‘3 t
note the plasma for at

headlined methods on '

Spectrometer‘, by the To

t
Woo. College ofl

 

122
were administered 1 g of sterile sodium ampicillin intramuscularly twice a day for 7 days

as a prophylactic precaution against bacterial infection. Twenty four hours after the
calves were born, serum IgG concentrations were assessed to ensure adequate passive
transfer of immunoglobulins had taken place.

The calves were fed milk replacerc, at 12% of their body weight divided into two
feedings from a nipple bottle They were also allowed free access to hay and grain0
beginning at 1 week of age. Daily physical exams were conducted to monitor their
physiologic status, and their body weights were measured every two days until the calves
were 6 to 8 weeks old. As the calves became older, they were moved to an outdoor
facility where they were placed on a grainc diet and provided free access to bay.

The calves were dehorned at 3 to 4 weeks of age. They were also dewormed
routinely with 1% Ivomectind by subcutaneous injection until they matured. In addition,
vitamin E and selenium injections were given intramuscularly at 30 day intervals until
they reached 4 months of age. At that time, they were provided free access to vitamin E
and selenium enriched trace mineral salt blocks.

Trace element analysis

Trace mineral concentrations were monitored using biweekly plasma samples until

e calves were 12 weeks of age, then once a week thereafter or as clinically indicated.

e blood was drawn into trace element free heparinized tubese and centrifuged to

emove the plasma for analysis. Trace element analysis was carried out using
stablished methods on a Poly Scan 61E Inductively Coupled Plasma-Argon Emission
pectrometer‘, by the Toxicology Section of the Animal Health and Diagnostic

I36

aboratory, College of Veterinary Medicine, Michigan State University

 

 

,_ .s-_.,_.. __
w— ——~— __ F - *» --""';_.. _--. a..- ..
"- -~ “ ~r7:,-s_._ - 2 .a .. .
,-. ....-. a ....t ~~ .. ..-.-,._

Clinical chem“? mdi
Complete blood count
intervals through the calv
of the samples was done I
Laboratories, College Of‘
Histopathologic cumin
Prior to and do
biopsies were obtained f0
anesthetized with a subcu
used to obtain the skin sa
Embedded, sectioned at 6
microscopic evaluation. l
Health and Diagnostics L
l'niversity.
Trtatlnent of affected ca
Calves were identified
lllttttttations decreased
“m Weeks, Upon initiati
lhe f0th of a limo acetate
his dOSage was then adji

“d ‘5 tom. Once the af

litt2273 IU/ kg of pros

 

123
Ilinical chemistry studies

Complete blood counts and clinical chemistry profiles were performed at weekly
ntervals through the calves first 3 months of life or more often when indicated. Analysis
)f the samples was done using routine methods by the Clinical Pathology Service
Laboratories, College of Veterinary Medicine, Michigan State University.
Histopathologic examination of the skin

Prior to and during the development of skin lesions on the affected calves, skin
biopsies were obtained for histologic evaluation. The areas biopsied were locally
anesthetized with a subcutaneous injection of 2% lidocaine and a 6 mm punch biopsy was
used to obtain the skin sample. The samples were fixed in 10% formalin, paraffin
embedded, sectioned at 6 microns and stained with hematoxyln-eosin for light
microscopic evaluation. Tissue preparation and analysis was performed by the Animal

ealth and Diagnostics Laboratory, College of Veterinary Medicine, Michigan state

niversity.

reatment of affected calves

Calves were identified as being homozygous affected, when their plasma zinc

ncentrations decreased below 0.5 ppm and remained below this value for more than

0 weeks. Upon initiating zinc therapy, the calves were given 1 g of elemental zinc in
re form of a zinc acetate solution mixed in their milk replacer twice a day for three days.
his dosage was then adjusted to maintain plasma zinc concentrations between 0.8 ppm
d 1.5 ppm. Once the affected animals’ plasma zinc concentrations decreased below 0.5

m, 2273 IU/ kg of procaine penicillin was administered intramuscularly once a day, as

 

 

 

 

 

aprophllaetic pmmn
concentrations returned to

hpehence in handling
management procedllleS-
ofphmmaceutical grade 2
rhinistration of the bolu
double distilled water (DI
his solution was mixed ‘
bottle. The mixture was ‘
dlthe affected offspring;
attttioistered in this main
to sickle. As a conseque:
acetate placed in 1 02 gel
diluted to once a week a
Clittital presentation of th
Discontinuing zinc treat

Four of the affected ar

the affected animals \

long sacrificed, their p13

h‘eg' .
ll) for approxirnatelt

h
thanely euthanized

 

124
a prophylactic precaution against bacterial infection, until their plasma zinc

concentrations returned to the normal range.

Experience in handling VYG—l , aided in establishing the majority of the calves'
management procedures. Zinc treatment of VYG-l was initiated with 50 mg oral bolus
of pharmaceutical grade zinc once a day. After 5 weeks he refused to allow
administration of the boluses. A reagent grade zinc acetateg solution was prepared using
double distilled water (DDW) at a concentration of 1g of elemental zinc/10 ml of DDW.
This solution was mixed with a small amount of milk replacer and offered in a nipple
bottle. The mixture was well tolerated, consequently the combination was used to treat
all the affected offspring; a typical maintenance dose of 1 g of elemental zinc/day was
administered in this manner. At approximately 1 year of age VYG-l completely refused
to suckle. As a consequence he was medicated with oral boluses of 14 g of dihydrate zinc
acetate placed in 1 oz gel capsules”, every other day. The frequency of dosing was then
adjusted to once a week and the amount of zinc given was adjusted according to the
clinical presentation of the animals.

Discontinuing zinc treatment in adult animals

Four of the affected animals were sacrificed as adults, VYG-l through VYG-4. Two

of the affected animals, VYG-3 and VYG—4, had their zinc therapy discontinued prior to
being sacrificed, their plasma zinc levels and their physical condition were monitored
weekly for approximately 8 weeks. When their physical status deteriorated, they were

lumanely euthanized.

 

 

 

Molecular inmfigat

Primer Selection

Initially, primers used I
sequencing, of the bovine
ofCRlP‘l‘”. lhe sequenc
were designed from the bt
determined by visually in

l. insuring that the primt
anneal to their intende
needed to be no less ti

3. Selecting primer pairs

‘ did not have a gc com

t The percentage of nor
20 nucleotide primer 2

- Primer pairs were sele

_ ettilts gc content an

1 er locations on th
nucleou'de codons per

Ptimers were synthesi;

tatt'ersity, using an APP]

mi piiIIIEl‘S Were ammoni

olution concentrations W

h ‘ .
em“ Purification p:

stipends B.

125
Molecular Investigation of the CRIP Locus:

Primer Selection
Initially, primers used for polymerase chain reaction (PCR) amplification and DNA

sequencing, of the bovine CRIP, were designed fiom the cDNA rat and mouse sequence

of CRIP‘Z'”. The sequence contained the coding region of the gene. Subsequent primers

were designed from the bovine CRIP gene as it was sequenced. Primer selection was

determined by visually inspecting the gene sequence and applying the following criteria:

l. Insuring that the primers selected were unique enough and of sufficient length to
anneal to their intended target; which meant that the average length of the primers
needed to be no less than 16 nucleotides.

2. Selecting primer pairs that would not form a homodimer, by insuring that the 3’ end
did not have a gc combination or the possibility of forming a complimentary hair pin.

5. The percentage of non-complimentary base pair mismatches was less than 20% for a
20 nucleotide primer and no mismatches were tolerated at the 3’ end.

1. Primer pairs were selected to have similar annealing temperatures, by having similar
lengths, gc content and the same number of hydrogen bonds.

5. Primer locations on the template were selected to have the fewest number of

nucleotide codons per amino acid.

Primers were synthesized at the Macromolecular Structure Facility of Michigan State

niversity, using an Applied Biosystems 394 DNA/RNA synthesizer“. Prior to their use

te primers were ammonium acetate and ethanol precipitated to purify them. The stock

elution concentrations were determined and a 20 uM working solution was prepared.

e primer purification protocol and the formula for determining the 20 uM solution is in

 

pendix B.

 

 

Table 12 - Prim“ Used
sequence ““1

 

Printer 1
14
75
l

 

 

 

 

1

0°

 

s
5
$66
peanSEXMD)
ngESEXISU)
tam

lain

,_
\O

 

 

 

 

 

 

 

 

Genomic DNA emfli”
Whole blood was 0011'
separate red blood cells. l
the tubes and placed in a
using a Genomix DNA e
titration of proteins Wi‘
aqueous phase by ethane
B. Conformation of the
gel electrophoresis descr
Concentration was deterr
Ultraviolet (UV) light Sp
flit the ratio of Amy/A2;

it the reading at A

zool

126

Table 12 - Primers Used for PCR Reactions, Identification Numbers, Nucleotide
Sequence and Origin

 

Genomic DNA extraction from blood
Whole blood was collected in acid citrate dextrose tubes (ACD)e and centrifuged to
separate red blood cells, buffy coat and plasma. The buffy coat was then aspirated from

the tubes and placed in a 12 ml polypropylene tubes‘. The DNA extraction was done by

  
  
  
  
  
  
  
  
  

using a Genomix DNA extraction kit“, which involved the lysis of white blood cells and
extraction of proteins with chloroform followed by precipitation of DNA from the
aqueous phase by ethanol‘”. The protocol and solutions utilized are detailed in Appendix
B. Conformation of the presence of high molecular weight DNA was done by agarose
gel electrophoresis described in Appendix B. Once the DNA was extracted, the
concentration was determined reading the absorption at 260 and 280 angstroms with a

ultraviolet (UV) light spectrophotometer”. To determine if the DNA preparations were

 

Ute the ratio of Am/A280 had to range between 1.8-2.0. If the reading at A280 was higher

an the reading at A260 producing a ratio less than 1.8, then the samples contained

 

 

 

mains in them and “e“
Plotocol detailed in App e
needed. The folloWiflg f0

on from blood emcfi

 

AmOunt 0f DN‘Tmll

(50 rig/ml)

Amount Of DNA m l

factor)

unison factor = AII
W

 

Harvesting tissues

Tissues were harveSte
tone was removed from
double packaged in 31313
liquid nitrogen. Once tht
her were needed. lntest
titty rinsed with physit
hind; 0.24 % KHth
Total RNA extraction f

Total RNA was extra
Mid guanidium thioc:
exilittion protocol were

(DEPC) treated DDW ar

 

127
proteins in them and needed to be further purified with the phenol/chloroform extraction

protocol detailed in Appendix B. The extracted genomic DNA was stored at 4°C until
needed. The following formulas were used to calculate the concentration of genomic

DNA from blood extraction.

 

Amount of DNA in pig/ml indilution = (Optical Density of the sample at A260)
(50 rig/ml)

Amount of DNA in pig/ml in the original solution = (pig/ml in dilution) (dilution
factor)

Dilution factor = Amount of TE that sample was diluted in = 1000 pol/5 ul

To determine the amount of DNA in ug/ul = (pig/ml in solution) (103)

 

 

 

Harvesting tissues
Tissues were harvested from animals immediately after they were sacrificed. After a

tissue was removed from the animal it was cut into approximately three 1 g pieces and

  
  
  
  
  
   
 
  
  

double packaged in a plastic bag. The packages were then immediately submerged in
liquid nitrogen. Once the tissues were frozen, they were stored in a -70°C freezer until
they were needed. Intestinal tissues were everted, exposing the mucosal surface, and
gently rinsed with physiologic buffered saline (PBS) (0.8 % NaCl; 0.2 % KCl; 1.44 %
NazHPOg 0.24 % KH2P04) to remove any digesta prior to being packaged.
Total RNA extraction from tissues

Total RNA was extracted from tissues using a single step method of RNA isolation
y acid guanidium thiocyanate-phenol-chloroforml4°. All the supplies used in the RNA
xtraction protocol were commercial prepackaged, or rinsed with diethylprocarbonate

EPC) treated DDW and RN Ase Zapl then autoclaved. The tissues were kept frozen at -

 

 

rot until they were cut a
mi of tissuC was homogef
solution ofpheno1 and gut
they were centrifuged T]
piqceutriiugafion' Thet
gopmpanol and pelleted i
rented now. the TRIZO
lhe quality and quantity 0
and W spectrophotomete
examining the integrity 0f
electrophoreses on an 3831
and quality they were the:

To ensure that there
tiquots of totRNA were i
litSamples also had lul
itgadation. After the sat
thiol/chloroform extract

DiAse proteins.

its
the transcription of

 

128
70°C until they were cut and placed in 50 ml polypropylene tubes‘. Approximately 300

mg of tissue was homogenized in 4 ml of TRIzol reagentm , which is a monophasic
solution of phenol and guanidine isothiocyanate, chloroform was added to the tubes and
they were centrifuged. The totRNA was extracted into the aqueous phase of the tubes
after centrifugation. The totRNA was precipitated from the aqueous phase with
isopropanol and pelleted by centrifugation and then resuspended in 500 pl of DEPC
treated DDW. The TRIzol reagent RNA extraction protocol is outlined in Appendix B.
The quality and quantity of RNA present was determined by agarose gel electrophoresis
and UV spectrophotometer. The quality of the totRNA preparation was assessed by
examining the integrity of the 28S and 18S ribosomal bands of the sample after they were
electrophoreses on an agarose gel. If the samples appeared to be of sufficient quantity
and quality they were then stored at -20°C.

To ensure that there was no genomic DNA present in the RNA samples, 20 pl
aliquots of totRNA were DNAse treated with 4 gal of RQI RNAse-Free DNAsell ”2’ m.
The samples also had lul of RNAse Inhibitor° added to them to prevent RNA
degradation. After the samples were incubated at 37°C overnight they were
thenol/chloroform extracted as per the protocol detailed in Appendix B, to remove the
)NAse proteins.
leverse transcription of total RNA preparations to produce cDNA

Four microliters of totRNA preparations were incubated with Moloney Murine

eukemia Virus (M—MLV) Reverse transcriptase” (RT), random priming hexarners and

r—-—?*

 

heat denatured RNA, to pr
cDNA was subsequently t
Polymerase chain reacti

A standard PCR proto
were either 25 pl or 50 pl

thennocyclersp'“. The rea

Table 13 - Primer Pairs

 

 

Primers lnitial/
Timc(min)

e:
94°Q4m

\

1581159 W

and

tan ~

.\ I

the Tee/7.:

 

 

 

129
heat denatured RNA, to produce a complementary single strand of cDNA'“"‘“. The

cDNA was subsequently used in PCR reactions.
Polymerase chain reactions

A standard PCR protocol was used in all experiments“. The total reaction volumes
were either 25 pl or 50 [41. The PCR reactions were performed in one of several

thermocyclersp'q. The reaction reagents and concentrations are listed in Appendix B.

Table 13 - Primer Pairs and their Cycle Sequencing Times and Temperatures

 

Once the PCR product had been generated, an aliquot was agarose gel
electrophoresed, stained with ethidium bromide and examined on a UV box light source
to assess whether the appropriate band size was present.

Reverse transcriptase-PCR was done with the cDNA and mRN A hybrids produced
from the totRNA preparations, as previously described. This study included the use of

both non-radioactive and radioactive RT-PCR. The PCR reactions were performed With

 

the standafd protocol alre
template. The radioactivt
expressed in and the degr
PTOtocol for this study is '
reactions were supplied a
allowed the ittcol'l’oratioI
generated.
Extracting amplificatio‘
When it was determin
entire PCR sample was r
M) gel and the appIOj
ng was then placed in a
from the agarose plug, b‘
beads, then rinsing the b
then pelleted, air dried a
protocol for the procedu
DNA sequencing proto

The different types

let '
enlune the nucleotide

ll Pnlrnucleotide kinas
in . ‘.
drudunl protocols for

Si(illeliase Version 2 0 l

 

1 30
the standard protocol already referenced with the exception of cDNA being used as

template. The radioactive RT-PCR was conducted to determine which tissues CRIP was
expressed in and the degree of expression in those tissues in relation to one another. The
protocol for this study is outlined in Appendix B. Half of the dCTP dinucleotides in the
reactions were supplied as or -32P dCTPr and half were non-radiolabeled dCTPs. This
allowed the incorporation of radiolabeled dinucleotides into the PCR product being
generated.
Extracting amplification products from agarose gels
When it was determined that the expected PCR product had been generated, then the
entire PCR sample was run on a Tris-acetate disodium ethyl...” " ‘ ‘“ t ‘
(TAB) gel and the appropriate sized band was cut out of the gel. The agarose plug of 300
mg was then placed in a 1.5 ml eppendorf tube. A Qiaex kits was used to isolate the DNA
‘ from the agarose plug, by dissolving the plug in a solution containing DNA binding
beads, then rinsing the beads several times eliminating all the agarose. The beads are
then pelleted, air dried and the DNA eluted by incubating the beads in DDW. The
protocol for the procedure is explained in Appendix B.
DNA sequencing protocols
Three different types of commercially produced sequencing kits were used to
determine the nucleotide sequence of CRIP. Primers were labeled with y-P33 dATPr using
T4 Polynucleotide kinase enzymem and the labeling listed in Appendix B, along with the

' dividual protocols for the sequencing kits. The first type of sequencing kit used was the

 

equenase Version 2.0 DNA sequencing kitt which used radiolabeled primers. The

F“...—

 

second methOd used was
primers. The cycle seq“
nith less template and p1
Thermo Sequenase kit
termination mixes coma
Agarose gels

Primarily two Wes ‘
Study. The gels that we!
tentacetate (TBE) were
lxlAE solution were ‘
PCR products. The gel
appropriate loading dye
asize reference. The ge
appropriate running bu‘
electrophoresed, they or
ladder lane had incorpc
Placed on a UV light be
photographing the gel 1
Acrylamide gels

Aerl’l'rllnide gels we
tendons and PCR pro

Co
" ck sequeueing reac'

131
second method used was the ATaq Cycle Sequencing kit‘, which also used radiolabeled

primers. The cycle sequencing kit allowed for longer pieces of DNA to be sequenced,
with less template and primer than the Sequenase protocol. The third method used a
Thermo Sequenase kit’. This method did not use radiolabeled primers, instead each of the
termination mixes contained radiolabeled terminal dideoxynucleotides (ddNTP).
Agarose gels

Primarily two types of agarose gels of varying concentrations were utilized in this
study. The gels that were composed of 1 x Tris-boric acid disodium ethylene-diamine-
tetraacetate (T BB) were used to detect both DNA and RNA products. Gels composed of
l x TAE solution were used in the process of isolating DNA bands of appropriate size in
PCR products. The gel wells were loaded with an aliquot of the sample mixed with the
appropriate loading dyes. In addition a 100 base pair ladder was also loaded in a well as
a size reference. The gels were then submerged in a one time concentration of the
appropriate running buffer and subjected to electrophoresis. After the gels were
electrophoresed, they were stained in an ethidium bromide solution. When the DNA
ladder lane had incorporated enough of the dye to be visually detectable, the gels were
placed on a UV light box for imaging. Gel hand information was recorded by
photographing the gel using a Photo-Documentation camera“ and Polariod film“.
Acrylamide gels

Acrylamide gels were used for the purpose of electrophoresing DNA sequencing
reactions and PCR products. Both the Sequenase sequencing reactions and the ATaq

Cycle sequencing reactions were run on 6% acrylamide, 8 M urea sequencing gels. The

 

 

 

electrophoresis buffers m
not buffer at the cathod'
glycerol tolerant W131“
reactions were run 011 th‘
high concentration of g1)
through the gels. All tht
electrophoresis boxes“.
Sodium dodecyl sulfl
Stacking gels were used
lhe gels were run in 31:
buffer. A4 til RT-PCR
wells using a Hamilton
watts and 35 ma. Afier
gels were fixed to the a
aetetie acid described ir
gels were silver stained

All the acrylarnide g

132
electrophoresis buffers used were a 0.6 x TBE buffer at the anode electrode and a l x

TBE buffer at the cathode electrode. The Thermo Sequenase reactions were run on 6%
glycerol tolerant acrylamide, 8 M urea gels. The products of the Thermo Sequenase
reactions were run on the glycerol tolerant gels due to the reaction enzyme containing a
high concentration of glycerol which could cause distortion in the bands as they migrated
through the gels. All the sequencing reaction gels were run on vertical sequencing gel
electrophoresis boxes“.

Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) with
stacking gels were used to run RT-PCR products in the CRIP tissue expression study.
The gels were run in a Protean Il xi Vertical Electrophoresis Cell", with 1 x SDS running
buffer. A 4 ul RT-PCR aliquot with 2 pl of 2 x SDS loading buffer was injected into the
wells using a Hamilton syringey. The gels were then run for 8 to 10 hours at 200 volts, 40

watts and 35 ma. After electrophoresis of the gels was completed, the DNA bands in the

  
  
   
 
   
 
  

gels were fixed to the acrylamide with a fixative solution", containing methanol and
actetic acid described in Appendix B. Radioactive gels were then dried, nonradioactive
gels were silver stainedx prior to being dried.

All the acrylamide gels were vacuum dried on gel driers‘“. Sodium dodecyl sulfate
polyacrylamide gels were supported between two sheets of wet mylar and vacuum dried
for 1 hour at 75°C. The sequencing gels were mounted on 3 M chromatography paperbb
(1 also vacuumed dried at 90°C for 3 hours. Both the sequencing gels and the

adioactive SDS-PAGE gels were exposed radiographic filmcc and developed.

 

Determining the “lam

Quantitation of each l
pallet and inserting it int
for counting were set at
resolution plate of 0.80 I
recalled by the compute]
allowed for the creation
the scanned image, With
the computer. To create
the band that had the mo
size of the largest bands
detection field that was
on the image. In placin
made to center the field
npossible. In additio
l0 Produce an average 1
lnpner quantitated to

background CPM was

background detection 1

Ed background CPMr
The linear range of

frontingS ul aliquots

 

133
Determining the relative expression of CRIP in tissues

Quantitation of each band was determined by placing the SDS-PAGE gel was on a
pallet and inserting it into a AMBIS Radioanalytic Imaging Systemdd ”5. The parameters
for counting were set at 10 minutes of detection time with 32 X 9 movements on a
resolution plate of 0.80 x 3.20. When the gel scan had been completed, the image was
recalled by the computer system and reproduced on a monitor. The AMBIS software
allowed for the creation of a rectangular detection field to be drawn around the bands on
the scanned image, within which the counts per minute (CPM) could be determined by
the computer. To create a standard size rectangular field for all the bands in the same gel,
the band that had the most distinctive edges and appeared to be most representative in
size of the largest bands on the monitor, was selected by the investigator. The same
detection field that was created for the standard band was then placed around each band
on the image. In placing the detection field around the sample bands, an attempt was
made to center the field around the darkest visible area and incorporate as much activity
as possible. In addition, random detection fields were placed throughout the gels image,
to produce an average background CPM. Once all the detection fields were in place, the
computer quantitated the counts in each field and printed the results. An average
background CPM was determined by eliminating the highest and lowest CPM in the

ackground detection fields and averaging the remaining values together. The average

 

el background CPM was then subtracted from each of the sample bands CPM.
The linear range of the PCR reactions for CRIP and B-actin were determined by

emoving 5 pl aliquots of the reaction mix at 5 cycle intervals, from the therrnocycler.

 

The aliquots were then
determined on the AM]
had the background avr
of cycles at which they
which cycle produced l
subsequent sample PCl
Radiolabeled RT-Pt
both CRIP 158/159 am
and drying, autoradiog
above. Once the CPM
subtracted from it, the
than the average backg

measurements had the

134
The aliquots were then run on an SDS—PAGE gel and the bands CPM values were

determined on the AMBIS machine, as described above. Once the sample band CPMs
had the background averages subtracted from them they were plotted against the number
of cycles at which they were taken. The analysis of this plot led to the determination of
which cycle produced PCR products that were within the linear range of the reaction. All
subsequent sample PCR reactions in the study were then terminated at this cycle number.

Radiolabeled RT-PCR products were generated from 30 tissues using primer sets for
both CRIP 158/159 and B-actin. They were then loaded and run on the SDS-PAGE gels
and drying, autoradiography and quantitation of the products was completed as described
above. Once the CPMs for each band was determined and the average background CPM
subtracted from it, the tissues that had activity levels that were not three times greater
than the average background count were eliminated from the study. The remaining

measurements had the ratio of 158/159:B-actin products was determined and recorded.

 

 

Results of Clinical

The results of the Cl
sections. The first secti
lhe second section Will
CRIP locus as a candid
Embryo transfer stud

Atotal of six embry
averaged 0.83 fertilizer
unfertilized ova was 5/
33 average yield of fen
125/flush and 1.62 deg
ndZ-33 were 0.6 and

l0n both donors were

nsulted.

 

Chapter 3
RESULTS

Results of Clinical Investigation of Bovine Hereditary Zinc Deficiency:

The results of the clinical and molecular investigations will be presented in two
sections. The first section will present the data obtained from the clinical investigation.
The second section will present the data obtained from the molecular investigation of the
CRIP locus as a candidate gene for a mutation responsible for BHZD.

Embryo transfer study

A total of six embryo flushes were performed in both cows. Donor cow Z-24,
averaged 0.83 fertilized embryos per flush, the average yield of non-degenerate,
unfertilized ova was 5/flush and degenerate, fertilized embryos was 0.5/flush. Donor Z-
33 average yield of fertilized embryos was l/flush, non-degenerate, unfertilized ova was
125/flush and 1.62 degenerate fertilized embryos/flush. The conception rates for Z-24
and Z-33 were 0.6 and 0.5 respectively. In two years a total of thirteen fertilized embryos
from both donors were implanted into recipients, of which seven full term pregnancies
resulted.

The seven calves of the BHZD pedigree were identified as VYG-O through VYG-6.

There were two affected bulls, VYG-l and VYG-3, three affected heifers, VYG-2,4,6,
and one obligate heterozygote heifer, VYG-S. A bull calf, VYG-O, died two days after

135

 

birth from pneumonia be
llYG-C, was unafl‘ected
2.5 months of age. The '.

Figure 3.

 

136
birth from pneumonia before his phenotype could be determined. A control bull calf,

VYG-C, was unaffected and unrelated to the pedigree described above, was raised until
2.5 months of age. The BHZD pedigree that was produced for the study is depicted in

Figure 3.

 

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Chiral manifestations

Table 14 - The Chronole
Zinc Deficienc

Tattoo identification WC

 

Genotype [2
Dan 2.

on Weight (kg) 3

zndrOP < 0.5 ppm

Alli Phos drop

Diallhea

Excessive lacn'mation

Nasal Discharge
Plylsm

PW Suckle Reflex
”inclemenrahon

Sites of Skin Lesions

Lama] Co

m .
Ollh mISUl‘es

eoral cavity
Behind the Pine
Flank

Pedanal

3
led Oi Zn Treatment

“leaned

1 3 8
Ilinical manifestations

I‘ able l4 - The Chronological Order of Development of Clinical Manifestations of
Zinc Deficiency in Affected BHZD Calves

Tattoo Identification WG-0 WG-1 WG-2 WG-3 WG-4 WG-6 WG-5 WG-0

Genotype I . I . . O D

 

Dam Z-33 Z-33 Z-24 Z-33 Z-33 Z-24 Z-24 NA
Birth Weight (kg) 32.2 40 40.4 31.8 36.3 35 33.6 52.7
Days of Age Postpartum
Zn dror: <o.5 ppm 16 18 19 25 31
Alk Phos drop - - - 27 29 51 NA NA - - - - - -
Diarrhea - - - 20 23 19 49 34 - - - - - -
Excessive lacrimation - - - 39 - — - - - - 44 40 - - - - - -
Nasal Discharge - - - 39 - - - - - - 44 40 - . - - - -
Ptylism - - - 4o - - - 64 46 43 - - - - - -
p Poor Suckle Reflex — - - 40 40 64 51 43 - - - - - —
l HYPOpigmentatlon - - - 4o - - ~ 64 4o 46 - - - - - -

l Sites of Skin Lesions

Lateral Commisures - - - 4O 40 63 42 41 - - - — — —

of the oral cavity

Behind the Pina --- 41 --- 64 --- 56 --- ---

Flank --- 39 --- --- --- 63 --- ---

ExternalNares --- --- --- 62 "' "' "-
l Perianal --- 39 --- —-— 46 63 --- ---
Start of Zn Treatment - - - 47 4O 75 67 58 — - _ - - -

Weaned - - - 71 53 89 96 72 64 73

 

 

Dianhea was the earl:
three days aftCI plasma 2
typically dark green and
became lethargic and de]
sn'nuli. Four out of the
lacnmation and a clear n

lhe skin lesions were

hipokeratotic lesions ar

bilaterally Symmetrical, .

tithe oral cavity, with s
began as patches of dry 5
in considerable variabi
he lesions became more
loll. and the external pi:
medial flank, the periana
illOiled to remain deficil
i“ flail skin along botl
his? areas never produc

Developing CODCUlTel
fill'inished suckling abil

linjpple 0fthe bottle

nththeir tongue flat agv

139

Diarrhea was the earliest clinical sign noted in all affected calves, typically developing
three days after plasma zinc concentrations decreased below 0.5 ppm. The diarrhea was
typically dark green and watery with a sweet metallic odor. Concurrently , the calves
became lethargic and depressed in both their motor skills and response to external
stimuli. Four out of the five affected calves subsequently developed excessive
lacrimation and a clear nasal discharge.

The skin lesions were first observed on the muzzle in affected calves. Typical gross
hyperkeratotic lesions are depicted in Figure 4a,b,c. The distribution of the lesions was
bilaterally symmetrical, affecting the skin peripheral to the nares and lateral commisures
of the oral cavity, with subsequent involvement of the intramandibular space. Lesions
began as patches of dry scaly skin that fissured within 24 hours and exuded serum. There
was considerable variability in the distribution of skin lesion away from the muzzle. As
the lesions became more extensive, they developed around the eyes, caudal portion of the
poll, and the external pinna. Four of the five affected calves developed lesions on the
medial flank, the perianal area and the ventral abdomen. Two of the calves that were
allowed to remain deficient for a prolonged period of time, VYG-l and VYG-4, had very
dry flaky skin along both jugular furrows, the thoracic inlet and whithers. However,
these areas never produced a serous exudate.

Developing concurrently with, but not attributed to the perioral lesions, was a

diminished suckling ability. Affected calves had difficulty curving their tongue around
e nipple of the bottle. Instead of suckling, they combined a chewing and pulling motion

'th their tongue flat against the nipple to ingest their milk. As a result of the affected

 

calves’ inability to sue
replacer increased con
reveal lesions. Exces-
difiiculties. Once trea
drooling resolved witl
Commisures of the cal
difficulties, because ti
resolved.

The hair coat of all
Shaft Pigmentation in
Wilmi- The poliosis

“5151158

. The rest oft]

exintimation of the ha

140
alves’ inability to suckle, the amount of time it took for them to ingest their milk

eplacer increased considerably. A thorough examination of their oral cavities did not
'eveal lesions. Excessive drooling was also noted simultaneously with their suckling
lifficulties. Once treatment with zinc began, their suckling difficulties and excessive
drooling resolved within 24 hours. Although there were lesions present in the lateral
commisures of the calves’ oral cavity, they were not considered the cause of the suckling
difficulties, because the lesions were still present after the suckling complications
resolved.

The hair coat of affected calves was dry and there was a noticeable decrease in hair
shaft pigmentation in dark coated areas. This was particularly apparent around the orbital
regions. The poliosis was analogous to the "spectacle disease" of copper deficiency
”8'152'158. The rest of the hair coat was characterized by a rough texture with close

examination of the hairs revealing crimped ends.

 

 

 

 

Filire 4a - Cntaneo
Calves: l
Pinna am

 

 

141

 

Figure 4a - Cutaneous Lesions that Developed on the Cranium of Affected BHZD
Calves: Note patches of dry scaly skin in the intramandibular space, caudal
pinna and orbital region.

‘igure 4b - Advanced Cutaneous Lesions Along the Stifle of BHZD Calves: These
occurred later in the onset of disease and were characterized by dried serum
and clumped hair.

 

 

 

 

 

 

142

 

Figure 4c - Advanced Cutaneous Lesions on the Rear Fetlocks of the Affected
BHZD Calves

 

 

Histopathology 1

Biopsies from
ligurei . In full
perirascular and
This was associa
erocytosis and e
Siperficially, m}
idiot the stratr
linens and gra
ChanEes in the C

comPact and or

 

143
Histopathology findings

Biopsies from fully developed and resolving skin lesions were examined as shown in
Figure 5 . In fully developed lesions there was an intense superficial to midlevel
perivascular and interstitial mixed inflammatory cell dermatitis with mononuclear cells.
This was associated with moderate irregular epidermal hyperplasia, marked neutrophilic
exocytosis and extensive confluent parakeratosis with entrapped neutrophils and
superficially, myriads of gram positive cocci. There was mild basophilia of the lower
half of the stratum malphigii with pallor and focally prominent vaculation of the upper
spinous and granular cell zones depicted in Figure 5a. In resolving lesions, the tinctorial
changes in the different regions of the epidermis were lost and the stratum corneum was

compact and orthokeratotic as shown in Figure 5b.

 

 

tire Sa - Photomi
Zinc De:
“nth ext:
zones (8
layers (I
ll & E; l

iii 5b - Photon
Zinc Dr
treatme:
epideru
Compac
is lost, :
H & E;

 

144

5a - Photomicrograph of a Characteristic Skin Lesion in Bovine Hereditary
Zinc Deficiency: There is a perivascular inflammatory reaction associated
with extensive parakeratosis (P), palor of the granular and upper spinous
zones (S), and an increased basophilia of the lower spinous and basal cell
layers (B).
H&E; Bar=300 p.

5b - Photomicrograph of the Resolving Skin Lesion in Bovine Hereditary
Zinc Deficiency: Biopsied at the same site as in-Figure 5a after zinc
treatment. Compare with Figure 5a. Although the inflammation and
epidermal hyperplasia persists, the parakeratosis has been replaced by
compact stratum corneum, the palor of the granular and upper spinous zones
is lost, and the lower spinous and basal cell layers are not as basophilic.
H&E;Bar=300 u.

 

 

 

Figill'e 5a

 

 

 

145

 

 

Figure 5a

 

Figure 5b

 

Biochemical findint
The plasma zinc 5
affected and unaffeci
concentrations gradu
4to 8 weeks. This P
(V YG-l through 4 E
activity. on average a
activity consistently
VYG-C, and VYG-.
alkaline phosphatase
plasma copper conce
range; however, the

concentrations of the

146
Biochemical findings

The plasma zinc and copper concentrations and the alkaline phosphatase activity in
affected and unaffected calves are presented in Figure 6. As indicated, plasma zinc
concentrations gradually decreased from normal levels at birth to below 0.5 ppm within
4 to 8 weeks. This preceded the development of any clinical signs in all affected animals
(V YG-l through 4 and 6). A consistent finding was the drop in alkaline phosphatase
activity_on average about ten days after the decline in plasma zinc. Alkaline phosphatase
activity consistently paralleled changes in plasma zinc concentrations. Both the control,
VYG—C, and VYG-S, an obligate heterozygote, maintained zinc concentrations and
alkaline phosphatase levels within normal ranges throughout their development. The
plasma copper concentrations in the calves in our pedigree were slightly below normal
range; however, the affected calves copper concentrations were similar to the plasma

concentrations of the unaffected calves for the same time period.

 

 

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Hen-0 P

 

 

 

 

 

 

 

52:3 6 2:3,.—
Estnn «we: 9.035 Eacna .mon 9325
R N S 3 m m m o R S cm a t 2 9 a m n o
9mg 0
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8 E33... .35 9.035
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1 osmod 0 9m?
Nomad md memoa
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o o m o 3 2. 5 an Nn R N t n. o o n o
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lndividual animiiis
The most commOIi
pneumonia. Both VY
contracted pneumonii
concentrations were i
below 0.5 ppm when
supportive care, antii
Affected calves al
calves were dehornec
three weeks time. ll
over the course of si:
immediately after thr
longer period of timr
There we a nota'
lliial’fectcd calves. F:
control calf. Figure
Unaffected heterozy;
by CO“liming the hr
homozygous heifer
“litre 3. Wm
34kg and 35 kg. A
281 kg and WG-6

dlscleliancieg, at 3‘

149

Individual animals

The most common secondary complication that occurred in the affected calves was
pneumonia. Both VYG—l and VYG-2 developed respiratory infections. VYG-l
contracted pneumonia twice, both episodes occurring when his zinc plasma
concentrations were below 0.5 ppm. Plasma zinc concentrations of VYG-2 were also
below 0.5 ppm when she developed pneumonia. Both the calves recovered with
supportive care, antibiotic therapy, and increases in their daily oral zinc doses.

Affected calves also experienced delayed wound healing. When affected and normal
calves were dehomed, the dehom sites on the unaffected calf healed completely within
three weeks time. The two affected calves that were dehomed at the same time healed
over the course of six weeks. During this time, they were zinc deficient for two weeks
immediately after they were dehorned. The hair shaved around the dehom sites also took
longer period of time to regrow.

There was a notable difference in over all growth rates between affected and
unaffected calves. Figure 7 is a chart of the weight gains of the six BHZD calves and the
control calf. Figure 8 is a photograph of two affected BHZD heifers and the one
unaffected heterozygote heifer. The distinction in growth rates is illustrated dramatically
by comparing the body weights of VYG-S, a heterozygote heifer calf and, VYG-6, a
homozygous heifer calf. Both calves were offspring of the same dam and sire, as shown
in Figure 3. VYG-5 was born 9 days prior to VYG-6, their respective birth weights were
34 kg and 35 kg. At approximately 12 and 36 weeks of age, VYG-S weighed 90 kg and
281 kg and VYG-6 weighed 71 kg and 190 kg. In addition to their body weight

iiscrepancies, at 36 weeks of age, the whither height of VYG-5 was 116.8 cm and VYG-

 

 

6 was 101.6 cm. The v
photograph of VYG-4,5
days older than VYG-S,
heifer. As stated previc
they are almost the sam
lhe two bulls, VYG
continued to grow and
zinc acetate per week 21
Currently, there is one

is being dosed at 1 12 g

 

_ 150
6 was 101.6 cm. The variation in growth rates is also apparent when examining the

photograph of VYG-4,5,6 in Figure 8. VYG-4 is a homozygous affected heifer, and is 80
days older than VYG—S, yet she is substantially smaller than the heterozygous unaffected
heifer. As stated previously VYG-6 appears much smaller than VYG-5, even though
they are almost the same age.

The two bulls, VYG—l and VYG-3, required more zinc than the heifers as they
continued to grow and mature. The bulls at 2 years of age averaged 84 g of dihydrate
zinc acetate per week and the heifers at the same age averaged 56 to 70 g per week.
Currently, there is one affected heifer remaining from pedigree, she is 3 years of age and

is being dosed at 112 g once a week.

 

 

 

151

 

x.

ow< .«o $303 2 3 moi—«U 2: he mEnU Emma?» >333 - \- Paw—m

om< be 9.035
m

m v m N

0—

ON

on

ov

o 0 o
is to '0
(Bx) suBIOM

O
(D

 

 

iiturea.

Photograp
Hetemzyg
and is 80 d
unaffected.
8 days you

 

 

‘igure 8 - Photograph of Two Affected BHZD Heifers and One Unaffected
Heterozygote Heifer: VYG-4 is a homozygous affected, she is on the left
and is 80 days older than VYG-5, who is in the middle and is a heterozygote
unaffected. VYG-6 is on the right, she is a homozygous affected heifer and is
8 days younger than VYG—S.

 

Necropsy findings
When two of the l
treatments discontinu
manifestations as not
chnical symptoms to
diSplayed about two r
with what has been pr
fat and were dehydral
ulcerative, hemorrhag
The majority of their
the viscosity of the sy
haddition, VYG.4 d
about5 weeks after 21'

3limit with a packed

 

 

Necropsy findings

When two of the homozygous BHZD animals, a bull and a heifer, had their zinc
treatments discontinued prior to being sacrificed, they developed typical clinical
manifestations as noted before. What was interesting was that it took 5 to 7 weeks for the
clinical symptoms to become evident, with the exception of lethargy which both
displayed about two week after zinc withdraw. Gross necropsy findings were consistent
with what has been previously reported33’42'43'44’45'46. Both the animals had very little body
fat and were dehydrated at presentation. They had parakeratosis of the esophagus and
ulcerative, hemorrhagic lesions diffusely throughout the mucosal lining of their GI tract.
The majority of their peripheral lymph nodes were enlarged. VYG—4 had a decrease in
the viscosity of the synovial fluid in her carpus and hock joints, indicative of synovitis.
In addition, VYG—4 developed profuse, watery, bloody diarrhea and became dehydrated
about 5 weeks after zinc withdrawal. The bloody diarrhea resulted in VYG-4 being

anemic with a packed cell volume of 20 prior to being euthanized.

 

Results of the Mr

identifying the codir

Primers were desi
with bovine DNA ter
and unaffected, unrel

A total of six prirr
these primers were dr
literature and one wa
sequence of CRIP‘EJ‘
the bovine coding reg
secliltnce of the codi;
3, flanking Primers a
cDNA and would nor.
intheirICgion. This
Table 16 liSts the prir

CDNA, alOng with W:

 

 

Results of the Molecular Investigation of the CRIP Locus:

Identifying the coding region of the bovine homologue of CRIP

Primers were designed from the coding region of rat CRIP, to use in PCR reactions
with bovine DNA template from homozygote and heterozygote BHZD bulls and heifers
and unaffected, unrelated cattle.

A total of six primers were used in determining the sequence of the CRIP gene, five of
these primers were designed from the rodent sequence previously described in the
literature and one was subsequently designed from a genomic PCR product of the bovine
sequence of CRIP‘Z'“. Table 15 includes the primer identification numbers, locations in
the bovine coding region, origins and priming direction. Figure 9 is the nucleotide
sequence of the coding region of CRIP from a phenotypically affected heifer, the 5’ and
3’ flanking primers are from rodent origin. Several of the primer pairs only amplified
CDNA and would not amplify in genomic DNA, implying that there was an intron located
in their region. This will be discussed further in the gene structure of CRIP section.

Table 16 lists the primer pairs used to amplify PCR products from both genomic and

CDNA, along with whether they produced a product that was sequenced.

 

Table 15 - Primers l

 

Primer
Identification
Number

74
75
76
158
159
566

 

155

Table 15 - Primers Used to Sequence the Coding Region of CRIP

 

 

 

 

 

template

 

Primer Primer Origin Priming Direction
Identification location
Number
74 300 rat/mouse cDNA Sense
75 157 rat/mouse cDNA Sense
76 394 rat/mouse cDNA Anti-sense
158 62 rat/mouse cDNA Sense
1 59 l 78 rat/mouse cDNA Anti-sense
566 272 PCR product of Anti-sense
primer set 75/76 using
a genomic DNA

 

 

 

 

 

158(R
am
so

GCTGAGCGGG TGA
110

75 (Rat Origin
orcrcacm ms
160

noccarccc crfi
zro

<0
MAGGCITIA GGC
260

GGTTAAAGAA ACT.
310

AGAGACAGAC AG(
360

Figure 9 . Nileleotid
iHdicated j
underlinec
codon and
their ofigi
direction 2
13081110113 1

 

 

156

158(Rat Origin)>
ATGCCGAAG TGCCCCAAGT GCAGCAAAGA GGTGTACTTT
60 7o 80 90 100

GCTGAGCGGG TGACTTCCCT GGGGAAGGAC TGGCATCGGC CTTGTCTCAA
140

110 120 130 150
75 (Rat Origin)> <(Rat Origin)159
GTGTGAGAAA TGTGGAAAGA CACTGACCTT GGGGGGTCAT GCAGAACATG
160 170 180 190 zoo

AAGGCAAGCC crficcmc CACCCCTGCT ACACAACCAT GTTTGGACCC
220

210 230 240 250
<(Bovine Origin)566 <(Rat Origin)74
AAAGGCTTTA GGCACEGTGG AGCTAAGAGC CACACTTTCA AGTAGACCGA
260 270 280 290 300

GGTTAAAGAA ACTCTCCCTA CCAACACAGG GACAGTGCCA AGCCTTACCC

 

310 320 330 340 350
<(Rat0rigin)76
AGAGACAGAC AGGCTCTCAA TAGACCTCCA TGCCT'ITAAT AAAC
360 370 380 390

Figure 9 - Nucleotide Sequence of the Coding Region of the Bovine CRIP Gene: Is
indicated in bold, the 5’ and 3’ flanking primers that are not in bold and
underlined are designed from the rat/mouse cDNA sequence. The initiation
codon and terminal codon are italicized. Primer identification numbers and
their origins are above the nucleotide sequence, their sense (>) or antrsense (<)
direction are indicated by the symbols. The boxes denote the polymorphic
positions that were identified between two unaffected, unrelated cows.

 

 

Table ro-Primer P
CRIP GI

 

Prim Set
1581159

 

 

158/566

 

74175

 

75/76

 

 

\‘

There were no dif.
CRIP between the ho
unaffected. unrelated
between two unaffect
when translated. The

nucleotide variations

inflected, unrelated

13113110118 are denoted
In the original inv
and mouse clone and

Gill) nucleotide and a

 

157

Table 16 - Primer Pairs and Templates Used to Amplify and Sequence the Bovine
CRIP Gene

 

There were no differences detected in the nucleotide sequence of the coding region of
CRIP between the homozygous affected or heterozygous unaffected BHZD cattle or
unaffected, unrelated cattle. There were four nucleotide polymorphisms detected
between two unaffected, unrelated cows, which resulted in three amino acid substitutions
when translated. The nucleotide sequence of cattle in the BHZD pedigree, had the
nucleotide variations that resulted in the polymorphic sites, but since these were in the
unaffected, unrelated cattle too, they were not considered mutations. The nucleotide
variations are denoted by the box in Figure 10.

In the original investigation of CRIP, cDNA sequences were identified in both a rat
1nd mouse clone and showed 95% identity between them“. Since that time, the human
IRIP nucleotide and amino acid sequence has also been determinedl‘6'm. A comparison
if the 240 base coding region of CRIP between the rat, mouse, human and bovine gene is

resented in Figure 10. The gene is highly conserved between species. Table 17

 

 

contains the percent

sequences.

 

 

158
contains the percent homologies between species for the nucleotide and amino acid

sequences.

 

 

 

 

vawmIP

m1?

MRE

MRE

MRE

MRE

KN?
MRE

ATGCCGAAl
ATGCCGAAl

GCCGAA
ATGCCCAAl

GACGTCAC
GACGTCAC
GACL'TCCC
oAcgrcrc
c

croomc
croomc
GTGGAAAt
GTGGQAAt

crc

TACTGCA}
rncrocm
lACl‘GCAi
. m , , . .
TACTGCA:

GCGAGGTl
GCGAGGTI
GCACGGTc

. .... A"
GCGGGGC

Figure 10 . A com

Humar
cDNA]
the bCI
inunan‘
from th
sequen
binding
abOve t

 

 

rCRIP
mCRW
bCRIP
hCRW

rCRIP

mCRW
bCRIP
hCRIP

rCRIP

mCRIP
bCRIP
bCRIP

rCRIP
mCRP
bCRIP
bCRIP
rCRIP
mCRIP
bCRIP

bCRIP

A T GCCGAAGT
A TGCCGAAGT

GCCGAAGT
A TGCCCAAGT

GACGTCACTA
GACGTCACTA
GACITCCCTQ
GAcgrcrcrg
c
GTGGAAAGAC
GTGGAAAGAC
GTGGAAAGAC
GTGGQAAGAC
ca:
TACTGCAACC
TACTGCAAIC
TACTGCAACC

- TTG .......

TACTGCAACC
GCGAGGTGGA
GCGAGGTGGA
GCACGGTGGA

..... A - - - - -
GCGGGGCGGA

GCCCCAAGTG
GCCCCAAGTG
GCCCCAAGTG
GICCCAAGTG

GGCAAGGACT
GGCAAGGACT
GGQAAGGACT
GGCAAGGACT

ACTGACCTCT
ACTGACCTCT
ACTGACCTIQ
QCTGACCTCT
c
ATCCCTGCTA
ATCCCTGCTA
AQCCCTGCTA

AQCCCTGCTA
GCTGAAAGCC
GCTGAQAGCC
GCTAAQAGCC

GchAgAGcc

159

C

CGACAAGGAG
CGACAAGGAG
CAQCAAAGAG
CAACAAGGAG
H
GGCATCGTCC
GGCATCGTCC
GGCATCGQCC
GGCATCGQCC

GGGGGTCATG
GGGGGTCATG
GGGGGTCATG
GGGGGQCAQG

CTCCGCCATG
CTCCGCCATG

CACAACCATG
CQCAQCCATG
ACACTTTCAA

ACACTTTCAA
ACACTTTCAA

ACACTTTCAA

GTGTATTTCG

GTGTATTTCG

GTGTAQTTIG

GTGrAgrrIG
c

CTGCCTGAAG
CTGCCTGAAG
ITGICTQAAG
CTGCCTGAAG

CTGAGCATGA
CTGAGCATGA
CAGAACATGA
CTGAGCAQGA

TTTGGGCCCA
TTTGGGCCCA
TTTGGACCCA
TTTGGGCCIA
GTMGACTGAG
GTAGACTGAG
GTAGACTGAG

G TA GACTGAG

CTGAGCGAGT
CTGAGCGAGT
CTGAGCGQGT
CTGAGAGQGT
c
TGCGAGAAAT
TGIGAGAAAT
TGIGAGAAAT
TGCGAGAAAT

AGGCAAGCCC
AGGCAAGCCC
AGGCAAGCCC
AGGCAAACCC

AAGGCTTTGG
AAGGCUTGG
AAGGCTTTAG

AAGGCTITGG

Figure 10 - A Comparison of the Nucleotide Sequences of CRIP in the Rat, Mouse,
Human and Bovine: The rCRIP nucleic acid sequence is the rat clone pRI3
cDNA published sequence; then mCRIP sequence is the mouse clone pMI3;
the bCRIP sequence is the bovine sequence; and the hCRIP sequence is the
human'“. The underlined letters in the sequence are nucleic acids that vary
from the rat nucleotide sequence. The nucleotide variations in the bovine
sequence are written underneath their counterparts and underlined. The zinc
binding sites are bolded with the corresponding amino acid designation
abovethecodon.

 

 

 

Table 17 - The Pen
Nucleoti

percenta

Mouse
Bovine

Human I

 

 

A linear model of
are three domains in
consistent uith Other
C-terminus. Underlir
The three domains 0-

The 77 antino aci
12' The anion acid
some nucleotide vari
Specifically llIlique it
have substitutions wi
Zinc binding Sites in
compared to the rat 5
binding finger 3101111
run codons that are a
phenylalanine at Dos

unrelatedcows Intl

 

 

160

Table 17 - The Percentage of Homology that is Shared Between Species for the
Nucleotide Coding Region of CRIP and the Amino Acid Sequence: The
percentage of amino acid homologies between species are in the shaded area.

 

A linear model of the bovine CRIP protein structure is depicted in Figure 11. There
are three domains in the protein, the first two are the zinc binding fingers, that are
consistent with other LIM zinc binding motifs, and the third domain, is the glycine-rich
C-terminus. Underlined amino acids are those that are unique in bovine CRIP protein.

The three domains of the protein are noted underneath.

  
  
  
  
  
  
  
  
  
  
  

The 77 amino acid sequences of the rodent, bovine and human are compared in Figure
12. The amino acid sequence of the mouse and the rat are identical, even though there are
some nucleotide variations between the two. There are six amino acids that are
specifically unique to the bovine protein. There are also three amino acid positions that
have substitutions which are different from the rat sequence. The boxed residues are the
zinc binding sites in the protein. Amino acids in bold type are unique to that species as
compared to the rat sequence. The first three polymorphic sites occur in the second zinc

inding finger around and including the third zinc chelating residue. Translation of the

 

o codons that are affected by the polymorphisms indicate substitutions of a tyrosine by
henylalanine at position 51 and a cysteine by glycine at position 52, in two, unaffected,

elated cows. In the BHZD pedigree, the codons translated as a phenylalanine and a

 

 

undated cows. In tl
glycine. The fourth 1
guanine is replaced 1:

pedigree. This result

 

 

 

161
unrelated cows. In the BHZD pedigree, the codons translated as a phenylalanine and a

glycine. The fourth polymorphic site occurred at the carboxyl terminus, in which a
guanine is replaced by a adenine in two unaffected, unrelated cows and the BHZD

pedigree. This resulted in the substitution of serine a glycine at position 69.

 

 

NH;—

s

Do:

Figure 11 -A Linea
Fingers

 

by X/x; h

 

 

162
C c G/C
(‘zn’H) (\Zn< ) g SIG _G_ K
anh— C’ ‘C _.___.. C’ C coon
P K F n - A
‘ Domain 1 Domain Zfi V Domain3 7

'igure ll - A Linear Model of the Bovine CRIP Protein with Two Zinc-binding

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fingers
l 10 20
IRIPMPKCPKCDKEVYFAERVTSLGKDWHRP
ZRIPMPKCPKCSKEVYFAERVTSLGKDWHRP
ZRIPMPKCPKCNKEVYFAERVTSLGKDWHRP
Domain] ‘7
30 40 50
RIPLKCEKCGKTLTSGGHAEHEGKPY NHP
IRIPLKCEKCGKTLTLGGHAEHEGKPY’f NHP
5RIPLKCEKCGKTLTSGGHAEHEGKPY NHP
V Domain2 fi
60 70
UPYSAMFGPKGFGRGGAESHTFK
RIPYTTMFGPKGFRHS/SGAKSHTFK
RIPYAAMFGPKGFGRGGAESHTFK
‘ Domain3

 

ure 12 - Amino Acid Sequences of CRIP in the Rat/Mouse, Bovine and Human.

 

rCRIP represents the both the rat and mouse amino acid sequences; bCRIP is
the bovine amino acid sequence, with the amino acid substitutions indicated

by x/x; hCRIP is the published amino acid sequence for humans'47

 

 

 

 

Gene structure 0f

lhe PCR produe'
amplify the appropri
intron between the p
two experiments we.
templates with both
158/159, which proc
product were utilize:
The first control was
220 hp product. The

protooncogene. The

,_,-n.-...¢._¢--v. _.- ._ -4 v'

 

163

Gene structure of CRIP

The PCR products from both the primer sets of 158/159 and 15 8/566 would only
mplify the appropriate product with a cDNA template, therefore the presence of an
ntron between the primer locations became evident. In order to confirm this hypothesis,
wo experiments were conducted. The first involved the use of several different
emplates with both radioactive and non-radioactive PCR reactions. The primer pairs
58/159, which produced a 117 bp product and 158/566, which produced a 211 bp
troduct were utilized. Two additional primer sets FES and B-actin were used as controls.
‘he first control was B-actin, which amplifies from cDNA template only, producing a
20 bp product. The second control was a primer pair designed from c-FES
rotooncogene. The FES primers were designed to amplify a gDNA specific target that
)anned an intron, their product was approximately 500 bp in length. Table 18 lists the

mplates and primer sets along with their PCR products.

able l8 - PCR Products of Primers and Templates Used to Characterize CRIP’s
Gene Structure

 

 

 

 

 

 

 

:MPLATES Primers 158/159 Primers 158/566 Primer B-Actin Primer FES
BEA Weak Product Weak Product No Product Strong Product
fie tot RNA Weak Product Weak Product No Product No Product
%_ Strong Product Strong Product Strong Product No Product
9“; Weak Product Weak Product No Product Strong Product
lied gDNA No Product No Product No Product Strong Product

 

 

 

 

 

 

 

 

In addition, Figu
To confirm that the

and sequenced.

158/159
L12 3 4 5

 

Figure 13 . PhOtog

the Ge
RNA; 1

templat

 

 

164
In addition, Figure 13 is a photograph of the PCR products from the above reactions.

To confirm that the PCR products were the expected sequence, the products were isolated
and sequenced.
Primer Pairs

158/566 B-actin c-FES
1 2 3 4 5 l 2 3 4 5

158/159
L12345 L12345

lit-s

    

illllillilfl t"
I

 
 

l l llllllll

Y
O

7
l
f
t

Figure 13 - Photograph of PCR Products of the Primer Sets Used to Characterize
the Gene Structure of CRIP: L, 100 bp ladder; template for lane 1, tot
RNA; template for lane 2, DNAsed tot RNA; template for lane 3, cDNA;
template for lane 4, gDNA; template for lane 5, RNAsed gDNA.

 

 

 

CRIP tissue expm

lhirt)’ tissues We]
homozygous afi‘ecte
unrelated 00‘“ th
zinc Status at the tin
WG-2 and VYG'4

As discussed abc
thus they were used
product acted as an
differences in indi"
benveen the difleret
of both the PCR rea
the exponential pha
for 40 cycles, every
vote subsequently I
was quantitated on :
temperatures every
annealing temperate

forthe158/159 and

165
iRIP tissue expression study

Thirty tissues were tested for the presence of the CRIP message, in four BHZD

.omozygous affected animals [VYG-l , VYG-2, VYG-3 and VYG-4] and one unaffected,

nrelated cow. Two of the affected animals, VYG-l and VYG-2, had adequate dietary
.inc status at the time of sacrifice and two were zinc depleted, VYG—3 and VYG—4.
fYG-Z and VYG-4 were heifers and VYG-l and VYG-3 were bulls.

As discussed above, the PCR product of 158/159 and B-actin only amplified cDNA,
hus they were used to detect CRIP cDNA expression in the tissues. The B-actin primer
troduct acted as an endogenous standard that permitted the detection of relative
lifferences in individual RNA samples resulting from variations in cellular density

Ietween the different tissue types and RNA degradation of samples145

. The linear range
f both the PCR reactions was determined to assure that the samples were terminated in
re exponential phase of amplification. A 50 ul radioactive reaction was set up and run
)r 40 cycles, every 5 cycles, a 5 ul aliquot was removed from the sample. The aliquots
'ere subsequently run on a SDS-PAGE gel and the radioactivity in each PCR product
as quantitated on an AMBIS machine. The first 15 cycles had decreasing annealing
mperatures every 5 cycles, until the reactions reached cycle 20, at that time the

nealing temperature remained at 43°C. Figure 14 shows the charts of the linear ranges

r the 158/159 and B-actin PCR products.

 

 

166

Figure 14 - Linear Range of Amplification of PCR Products for Primer Pairs
158/159 and B-actin: Graph a represents the linear range for B-actm; Graph
b represents the linear range for 158/159

I5

5000--

4000--

2000..-.

3000‘“

2500“.

20005--

CPM

1500‘“

1000c.‘

SOO‘N

 

167

 

 

Figure 143: B-actin

16000

14000

12000 4

10000 -

CPM

6000 q
4000 l

2000 ~

8000 —

O
A

 

 

5 10 15 20 25 30 35 40 45
Cycle Number

 

CPM

Figure 14b: 158/159

3500

3000

2500

2000

1 500

1000

0v V v V f

  
 
   

 

 
 

5 10 15 20 25
Cycle Number

 

 

 

Both of the grapl
cycles 20 and 25. T
actin PCR product a
to assure that the PC
linear limits of the I
both primer sets.

Almost all the ti:
appropriate PCR. 1
results were within
times the average b
only CPM values tl
35 indicated by Fig
the 158/159 PCRp
products Was 381 a
resulted in twenty 1

within the experim
Products Were then

Table 19 cohtains l

 

168
Both of the graphs indicate that the linear range of the PCR reactions begins between

ycles 20 and 25. The primer pairs 158/159 appears to plateau around cycle 40 and the [5-
:tin PCR product appears to remain linear throughout the full range of cycles. In order

) assure that the PCR products produced from the cDNA of various tissues are within the
near limits of the reactions, all subsequent reactions were terminated at 30 cycles for

0th primer sets.

Almost all the tissues tested, for the presence of CRIP and B-actin amplified the
ppropriate PCR. However, in order to ensure that the sensitivity of the experimental
:sults were within detectable limits, only bands that had CPM values greater than three
mes the average background count for the individual gels were recorded. In addition,
11y CPM values that were within the linear ranges of the 158/159 and B-actin reactions,
: indicated by Figure 14 were included in the study. The approximate linear range for
e 158/159 PCR products was from 295 to 2500 CPM, and the range for the-B-actin PCR
oducts was 381 and up. The sensitivity and linear range limits on the data collected
sulted in twenty two of the original thirty tissue samples having CPM values that fell
thin the experimental criteria. A ratio of the CPM for 158/159 and B-actin PCR
)ducts were then used to normalize the data for each of the individual RNA samples.

ble 19 contains the ratios of each of the tissues examined in five different animals.

 

 

 

Table 19 - The Rati
Tissues I
status: V
lelC-dCfit

a COW W1

 

 

 

 

 

Ileum

Duodenum
Mammaw

Skin

Skeletal Muscpe
‘ Hmasum

' renal

Bladder
Brainslem

but Node
Cecum
' ongue

hold
eshcle
Range
Verane ratio

 

169

Table 19 - The Ratios of the CPM of PCR Products from 158/159 and B-actin for 23
Tissues Examined in Five Animals: VYG—l is a bull with zinc-adequate
status: VYG-Z is a heifer with zinc—adequate statue: VYG—3 is a bull with
zinc-deficient status: VYG-4 is a heifer with zinc-deficient status: Control is
a cow with adequate zinc status.

 

 

 

 

lhe CRIP to [S-act
uith an overall avera;
produced a visible P(
nithin the sensitivity
the expression of CR
the animal. A comp:
hmph node tissue w;
srpression of CRIP i

than amount in an

170
The CRIP to B-actin ratios ranged from 0.11 to 2.38 for all the animals and tissues,

rith an overall average ratio of 0.56. With the exception of fat, all the tissues tested
roduced a visible PCR product for CRIP, even if the detection of the band was not

(ithin the sensitivity limits of the experiment. There were no consistencies detected in

re expression of CRIP in the tissues when sorted by zinc status of the animal or sex of
re animal. A comparison between sample sets of the individual tissues indicated that the
vmph node tissue was the only one that consistently appeared to have a higher level of
xpression of CRIP in its cells. Otherwise, CRIP did not appear to be expressed in

reater amount in any one tissue as compared to others.

 

 

 

 

Clinical Investii

lt is clear that th'
to the basic defect i
absorption of zinc i
undies”. One of th
effective at low cor
probably involves t
gastrointestinal trac
cheered cattle resul
lumen the non-San
it is most likely tha
The first part of thi
biochemical manifi
accomplish this a F
produced. The het
than Per flush o

Injections of follicl

 

Chapter 4
DISCUSSION

Ilinical Investigation of Bovine Hereditary Zinc Deficiency:

It is clear that the failure to absorb zinc efficiently from normal food sources is central
t the basic defect in BHZD. The existence of at least two biochemical pathways for the
)sorption of zinc in the intestinal lumen have been defined, mainly through rodent
udies”. One of these pathways is a transporter-dependent, saturable system which is
fective at low concentrations of lumenal zinc; the other is a non-saturable system that
“obably involves the passive diffusion of zinc between the enterocytes of the
strointestinal tract. Since the administration of large amounts of elemental zinc to
fected cattle results in sufficient amounts of zinc being absorbed from the intestinal
nen, the non-saturable component is probably functioning in cattle with BHZD”. Thus
.8 most likely that the transporter-dependent component is impaired in this disease.
‘e first part of this study was to chronologically document the variety of clinical and
ichemical manifestations of zinc deficiency as observed in five calves with BHZD. To
complish this a pedigree of BHZD animals with five affected and one heterozygote was
duced. The heterozygote donor cows for the study only produced one fertilized viable
bryo per flush on average. This was after the cows were induced to superovulate with

ctions of follicle stimulating hormone. The reasons for such low yields of embryos

171

 

 

 

could have been two i
uhich has been shown
been that the cows we
affected their fertility
BHZD on obligate he
fertility in both cows
study.

The information 1
increased diagnostic
homologue AE. Th
Eithth retardation,
greater detail the ag
Wee“ Plasma zit
have consistently b
repolled; a decree

Diarrhea Was 11:
l°l°thtrations to l
descriptions of the
allllhuted to line ‘
harder to replace 1
llanhealus . Z
high metaboliC ra

 

172
ould have been two fold. The first was that the donor cows were grossly overweight,

”4. The second reason could have

hich has been shown to decrease fertility in bovines
een that the cows were both heterozygote carriers of BHZD, which could have also
ected their fertility. This has never been documented in the literature, but the effects of

pHZD on obligate heterozygotes has not been studied. The end result of the decreased
l
l

srtrlity in both cows was that it took 2 V2 years to establish the pedigree necessary for the
tudy.

The information provided in this study should allow for a better understanding and
lcreased diagnostic ability for the early detection of BHZD as well as its human
omologue AB. The study has confirmed the classical findings of diarrhea, parakeratosis,
rowth retardation, and delayed wound healing, as well as been able to document in
reater detail the age at which plasma zinc concentrations decline and the parallel
:tween plasma zinc and serum alkaline phosphatase37’41'43’52'”. Two additional findings
we consistently been observed in all the affected calves that have not previously been
ported; a decrease in the ability to suckle and poliosis around the orbits.

Diarrhea was the first clinical sign noted; and followed a drop in plasma zinc
incentrations to below 0.5 ppm. This observation was consistent with classical
SCriptions of the development of BHZD in calves40’4"43'”8 . The diarrhea can be

'buted to zinc deficiency impairing the regeneration of epithelial cells and their brush
rder to replace those that have sloughed into the intestine, resulting in a malabsorptive

heal48

. Zinc plays a crucial role in cellular metabolism, thus any tissues that have a
h metabolic rate would require more zinc dependent processes to regenerate. Zinc

ciency also results in the decreased activity of zinc dependent digestive enzymes from

 

 

 

lheliver, pancreas and
maldigestive diarrhea1
The development t
Zinc is critical to the
cell proliferation and
epithelial cells in the
pattern of confluent j
heaphilia of the 10v
Pathognomonic for :
lesions can occur in
cinhosis and glucag
hhpaired in these Ct
There have beer
The affected Calves
ulhtiected calves,
when ”Waring t
heifer Whose mean
homozygous heifi

respeCIIVely' lel

eflTCTeflcy 0f ullll

grow and main

Wilght gains Ofl

 

173
he liver, pancreas and epithelial cells in the gastrointestinal tract, which results in a

naldigestive diarrhea”).

The development of parakeratosis in a zinc deficient state has been well documented.
Zinc is critical to the activities of enzymes and transcription factors involved in epithelial
ell proliferation and development“. In a zinc deficient state, the rapid turnover of
pithelial cells in the dermas is dramatically altered, producing skin lesions. However the
tattem of confluent parakeratosis, pallor/vacuolization of the upper spinous zones and
tasophilia of the lower half of the nucleated epidermis should not be considered
tathognomonic for zinc deficiency. In non-ruminant mammals, morphologically similar
:sions can occur in a number of diseases including niacin and riboflavin deficiency,
irrhosis and glucagon secreting tumors, perhaps indicating that similar pathways are
npaired in these conditions34'150'15‘.

There have been a number of reports that support the observations in this study that
re affected calves have a decrease in growth rate and weight gain as compared to the
naffected calves, as shown in Figure 7 and 84°54’41”. These observations are apparent
'hen comparing the weight and whither height of VYG-S, the unaffected, heterozygous
‘ ifer whose measurements were substantially greater than VYG- 4 and 6 the affected,

mozygous heifers, that were 80 days older and several days younger than VYG-S
spectively. Zinc deficiency is known to cause a decrease in feed consumption and
lciency of utilization in addition to retardation of metabolic fimctions responsible for

owth and ma‘ruration“"”53"52 . This is also dramatically illustrated when the average daily

ight gains of VYG-C, VYG-l and VYG-3 are compared. All three of these animals

 

 

 

were bu” calves that
weight gain of5 kg 1

Healing of the de
corroborates with de
deficient states and i
fimaionsfl'm‘

The age at WhiCh
was variable. The F
however a gradual (
1013de 0.5 PPm' I
zinc stores acqufied
all of the graphs 0f
alkaline phosphate:
phosphatase is a 21
observed in a PM
chemiStries are a II
analysis, and decre
BHZD in cattle 2111*

are often containin

the values may gt
lhe decline in 1
development of pr.-

Dfltllmonia Was dt

 

174
vere bull calves that were fed the same diets, yet VYG-C, the control calf, averaged a

veight gain of 5 kg per week and VYG-l and VYG—3 averaged 3.5 kg per week.

Healing of the dehom sites was greatly delayed in the affected calves. This
orroborates with delayed wound healing responses that are documented under other zinc
Leficient states and is consistent with the importance of zinc in diverse cellular
unctionsm”.

The age at which affected calves began to display clinical signs of zinc deficiency
was variable. The plasma zinc concentrations of all the calves were normal at birth;
owever a gradual decrease was noted in affected calves over the course of 3 to 5 weeks
) below 0.5 ppm. This variation in onset is probably attributable to differences in their
.nc stores acquired in utero, overall growth rate and physical health. When examining
l of the graphs of the affected and unaffected calves in Figure 6, it can be noted that the
kaline phosphatase concentrations parallel the plasma zinc concentrations. Alkaline
tosphatase is a zinc dependent enzyme and its parallel with plasma zinc was also
served in a previous study which involved calves fed zinc deficient diets'”. Blood
emistn‘es are a more frequently performed diagnostic procedure than trace mineral

ysis, and decreases in this enzyme can be useful in alerting to the possibility of

D in cattle and AB in humans. Furthermore, plasma samples taken for zinc analysis

often contaminated by the use of inappropriate blood collection procedures and low

c values may go undetectedls“.
The decline in plasma zinc concentrations to below 0.5 ppm also correlated with the
elopment of pneumonia in two of the homozygous affected calves. The occurrence of

umonia was due to the impairment of the calves’ immune functions that results from

 

 

 

 

 

zinc deficiency. Th
discussed in the “Rt
It is interesting t
were slightly belovt
selenium, which ca
effect and a lower 6
also may be attribu
naffected heifer, \
the affected calves.
01" managemer
his behavior. As 5]
concentrations and
Containing milk re}
this time, he was tr
atetate, in gelatin c
received in his mil
range. His refusal
‘0th decrease in 2
even in adult rumi]
solution to bl'pass
there it can be ab:
utiliZed by baCteri;

 

175
zinc deficiency. The pathophysiology of the immune impairment was previously

discussed in the “Review of the Literature Section”.

It is interesting to note that plasma copper concentrations of the calves in this study
were slightly below or in the low normal range. All the calves were supplemented with
selenium, which can reduce serum copper concentrations, resulting in a copper sparring
effect and a lower expected range for adequate plasma copper concentrations” . This
tlSO may be attributed to their dietary intake as the control calf, VYG-C and the
maffected heifer, VYG-S had plasma copper concentrations that were similar to those of
he affected calves.

Our management protocols were continually modified as VYG-l matured and altered
liS behavior. As shown in Figure 6, he had noticeable decreases in his plasma zinc
oncentrations and alkaline phosphatase when he refused to suckle from the nipple bottle
ontaining milk replacer and 1 g of elemental zinc, at approximately 50 weeks of age. At
Fis time, he was treated with oral boluses of 1 g of elemental zinc, in the form of zinc

:cetate, in gelatin capsules. Although the dosage was equivalent to the total dose he
ceived in his milk replacer, his plasma zinc concentrations remained below normal
ge. His refusal to suckle the formula as opposed to being bolused contributed further
the decrease in zinc absorption by the gastrointestinal tract. The process of suckling,
en in adult ruminants, stimulates the esophageal groove to close allowing the zinc
lution to bypass the rumen and go into the abomasum, omasum and small intestine

ere it can be absorbed‘”. In the rumen, zinc can be bound by phytates in fiber and

tlized by bacteria which decreases the availability for absorption from the

 

l . . . . . .
ftromtestinal tract‘”. By comparing affected animals of the same age that are receivmg

‘

zinc in solution
related to matumti
This is obvious
have maintained
suckle their zinc so
Administration of a
day was needed to
bull calves required
semen losses.

At the conclusio
heifer and bull had
onavcrage 5 to 7

deficiency as com

 

deficiency in adults
We in the body

blrbothofwhicht

thtweprovit

BHZDaddtanima

“lid Once the

 

 

176
zinc in solution versus the oral boluses, the possibility of a physiologic phenomena

related to maturation of the animal being the cause of decreased absorption was ruled out.
This is obvious when examining the graphs of VYG-2 and VYG—3 in Figure 6, who both
have maintained their plasma zinc concentrations within normal range. They continue to
suckle their zinc solutions, and are older than the age at which VYG-l stopped.
Administration of a much higher dose, 14 g of elemental zinc in oral boluses every other
lay was needed to sustain normal plasma zinc levels in the absence of suckling. The
tull calves required more zinc than the heifers due to a higher growth rate and additional
emen losses.

At the conclusion of the clinical portion of this study, two of the affected cattle, a

 

eifer and bull had their zinc supplementation suspended prior to being sacrificed. It took
n average 5 to 7 additional weeks for them to show severe signs of clinical zinc
:ficiency as compared to when they were calves. The longer time of onset of zinc
:ficiency in adults as compared to the calves was due to the presence of more zinc
serves in the body. In addition, the adult animals were allowed to forage and provided
.y, both of which would have contained zinc, where as the calves only source of zinc
is what we provided them in their milk replacer. However, the amounts of zinc that the
D adult animals would have been able to absorb from their diet would have been
' a1. Once the adults did develop clinical manifestations, they were typical of what
been previously described.
A unique observation to this study of calves with BHZD was a decrease in suckling
ity when their plasma zinc concentrations fell below 0.5 ppm. Close physical

' ation of the calves' oral cavities did not reveal lesions that could account for the

 

 

 

 

BHZD. It may als
iththedifferentia
Both alopecia t

dogsareindist

limitation that

llhlosislhet

blood

 

177
inability to suckle milk replacer from a nipple bottle. In addition, the prompt

improvement in suckling ability after the administration of zinc therapy suggests that
zinc deficiency impairs glossal motor function. Although the calves did have lesion
along the lateral commisures of their oral cavity, this did not appear to be the cause of
their suckling difficulties, since the lesions were still present after the resolution of the
suckling difficulties. Vitamin E and selenium injections were adtninistered
intramuscularly every 30 days until the calves were 4 months of age, ruling out the
possibility of white muscle disease as the cause of their suckling difficulties’57'm.
Furthermore, serum vitamin E and selenium concentrations were investigated in one calf
with poor suckling ability and were within normal limits. Poor suckling ability has not
been reported in earlier studies regarding zinc deficiency in cattle. Bull Terriers with
lethal acroderrnatitis however were also reported to have difficulty suckling and

chewing“. The present study indicates that it is an important early clinical indicator of

  
  
  
  
  
   
 
  
  

BHZD. It may also be useful in alerting clinicians to the possibility of AE and including
it in the differential diagnosis of diseases in infants.

Both alopecia and poliosis were noted in non-treated affected calves. These hair
changes are indistinguishable in both pattern and appearance from the loss of hair shaft
pigmentation that occurs in copper deficient cattle. During the development of alopecia

d poliosis, the plasma zinc concentrations were considerably below the normal range.

 

en the coat changes resolved, the zinc concentrations were above or within normal
ange. Plasma copper concentrations, although in the low normal range in all the calves
this study, remained stable during and afier the resolution of poliosis. Therefore it is

ikely that copper was a factor in the hair changes. These findings challenge the

 

without the veil of

atelollowed. The:

affects of the deli

 

 

178
concept that a "spectacle" appearance to the pelage in the area surrounding the orbits is

diagnostic for copper deficiency'“"52"”.
The clinical investigation has focused on the manifestations of zinc deficiency as
observed in calves and the diagnostic criteria, as well as effective treatment and
management protocols for BHZD. A host of secondary complications often occurs with
affected calves and includes thymic hypoplasia, a decrease in their immune response

leading to septicemia and diarrhea that subsequently can cause dehydration, acidosis and

death“°'43 . This study was designed to observe as pure a zinc deficient state as possible,

 

preventing the deveIOpment of many secondary complications through measures such as
the use of prophylactic antibiotics as well as ensuring proper nutrition and a clean
environment. In the field, these animals would be susceptible to developing systemic
infections which could quickly become life threatening. Cattle with this hereditary
disorder provide a very useful model to delineate the manifeStations of zinc deficiency
without the veil of secondary complications provided adequate management procedures

are followed. These animals can also be a valuable research source for investigating the

affects of zinc deficiency on the immune system.

 

 

 

 

 

 

there is abnormal l

boxes to down reg

lasers for this. C

Furthermore dive

3WD atticthot

inedible

Mil 4

 

179
Molecular Investigation of the CRIP Locus:

The hypothesis of this dissertation, “that a mutation at the CRIP locus is responsible
for the defect causing BHZD in cattle” was not proven. There was one 20 bp primer at
the 5’ end of the coding region of CRIP that was not sequenced in the bovine. The
primer was number 158 and was from rat origin. It is very unlikely that there is a
mutation in this region that would translate to an amino acid variation causing a mutation
at the locus. If there were more than 1 or 2 base pair mismatches in the primer, it would
not have annealed to the cDNA template and produced a PCR product during
amplification, which it did consistently. The results of the molecular investigation
determined that there were no differences in the nucleotide sequence in the coding region
of CRIP between the heterozygous unaffected, homozygous affected and unaffected,
unrelated cattle. Although the 5’ promoter region of CRIP in the BHZD cattle has not
been determined yet, it is unlikely that the region contains a defect. It is also unlikely that

there is abnormal expression of a repressor protein that binds to the Oct region or cacc
118

boxes to down regulate CRIP expression in the affected animals The are several

reasons for this. CRIP expression was detected in both affected and unaffected animals.
Furthermore diverse tissue distribution of CRIP mRNA expression was observed in
HZD cattle that were zinc deficient and zinc adequate at the time of tissue collection, as
ell as control animals. The results of the experiments in which CRIP cDNA was
roduced to examine relative CRIP expression, showed no evidence of a decrease in
RIP expression in the affected zinc deficient BHZD cattle as compared to zinc adequate

d controls. It is therefore unlikely that an alteration in the expression of CRIP in zinc

 

 

 

dwmparison of the

Ind bovine reveals :

wwbetwcen thet

lenient are idem

ithins occurwi

litter bowed

 

1 80
deficient animals resulted from a trans acting factor suppressing protein expression or a

mutation in the promoter region. This also tends to discredit the possibility that CRIP is
the primary transport protein responsible for zinc absorption in the GI tract, as
hypomesized56’57.
The possibility that CRIP may be one of the factors which may indirectly influence the
absorption of zinc from the GI tract during maturation still exists. The developmental
egulation of CRIP by glucocorticoids and T4 could be interpreted to indicate that CRIP
imctions in a zinc dependent developmentally regulated process during intestinal
naturation in conjunction with the two hormones“. Direct evidence of CRIP mediation

n intestinal maturation would require demonstrating that stimulation of maturation by

:lucocorticoids is inhibited if CRIP expression is blocked in some way“.

CRIP is highly conserved between the rodent and the bovine with an 88% homology
etween the amino acid sequences and the same percent identity at the nucleotide level.

. comparison of the nucleotide and amino acid sequences of CRIP in the rodent, human
d bovine reveals several interesting similarities. Half of the nucleotide variations that
cur between the three species are at the same positions and 75% of these compared to

rodent are identical in the human and bovine. The remaining 50% of the nucleotide
'ations occur within 5 to 10 bp of each other. The variations in the nucleotide
uences between the rodent and bovid only resulted in nine amino acid variations and
66 amino acid substitutions at positions X51, ,2 and 69 in cattle. Five of the amino acid
'ations occurred in the third domain following the two zinc-binding fingers, this is to
expected since the zinc finger motifs are highly conserved in evolution. It is also

resting to note that almost all of the amino acid residue variations are conservative.

 

 

The conservation of
preserve the charact
three species to be 3
activity. The one ct
domain. in the hurt
the bovine, the resic
and glycine is a 3m:
since there were no
can be assumed tha
The significance
second zinc-bindin;
cl'SIeine or glycine
[W0 Wlim0rphism
”llmowhism at p
“morphisms We
the “duals in the 1

 

181
The conservation of the chemical characteristics of the amino acid residues would

preserve the characteristics of the domain in which they occur allowing the protein in all
three species to be able to functionally fold in the proper configuration for metabolic
activity. The one exception is at position X67, which occurs in the glycine rich third
domain. In the human and the rodent the residue at this position is a glycine; however, in
the bovine, the residue is an arginine. Arginine is a positively charged polar amino acid
and glycine is a small, hydrophobic amino acid. The significance of this is unknown, but
since there were no apparent aberrant effects in the clinical presentation of the cattle, it
:an be assumed that it is a normal occurrence in the bovine CRIP protein.

The significance of the first two amino acid substitutions detected at X51 and X52 in the
econd zinc-binding finger of CRIP, which are a tyrosine or phenylalanine at X51 and a
ysteine or glycine at X52, is unknown. The amino acid substitution at X51 resulted from
wo polymorphisms, in which an adenine and a cytosine were also two thymines and the
olymorphism at position X52 there was guanine instead of a thymine. All three of these

lymorphisms were found to occur between two unaffected, unrelated cows and all of
e animals in the BHZD pedigree. The fact that both homozygous and heterozygous

'mals with BHZD were homozygous for the variant, is significant because it can be
smissed as a mutational defect in the protein.

The first amino acid substitution in which a tyrosine is substituted for by a
enylalanine probably does not alter protein function. Both of the residues are aromatic
d hydrophobic in nature; however, tyrosine is polar and phenylalanine is not. The
nificance of the substitution lies in it being adjacent to the third zinc chelating residue

e second zinc-binding finger. Although individually both the X5l and X52

 

-r awg{

 

 

 

substitutions maybe ‘
causing structural in:
to be more susceptih
netabolic function.
preferentially bind 2
adjacent snbstitutio:
udigree, thus they
There is no indi.
has been detected 2
sequence has been
afiiuities for zinc (
llulnoun‘“. The 1
conf(urination char
Preferentially, Er
different line che
Proteins that Char

in the UM far
chewing the 2th
Wm Protein j
Sllbfa111in Allin
second Zinc-him

metal mm are 1'1

182
substitutions maybe inert, combined they may alter the zinc binding ability of the protein

causing structural instability. The structural instability in turn could allow for the protein
to be more susceptible to degradation, or unable to attain the proper conformation for its
metabolic function. The combined amino acids may also allow the protein to
preferentially bind a DNA, RNA or protein target. Whatever the result of the two
adjacent substitutions, they occurred in two unaffected, unrelated cows and the BHZD
pedigree, thus they can not be considered mutations.

There is no indication in the literature that the substitution of glycine for the cysteine
has been detected as a polymorphism in any other species in which the CRIP amino acid
sequence has been determined. Although different amino acids have higher binding
affinities for zinc (histidine>>glutamate>aspartate_=_cysteine), glycine’s affinity is
unknown'éo. The presence of glycine in the second zinc finger of CRIP may allow for a
conformation change that could facilitate DNA or RNA binding of the protein
preferentially. Experiments utilizing site directed mutagenesis have determined that
‘different zinc chelating amino acid residues confer specific conformational changes in
proteins that change their binding characteristics'“.

In the LIM family of proteins there are three proteins that have a glycine residue
chelating the zinc ion in one of their zinc fingers. The first protein SF 3, is a pollen
specific protein in sunflowers which belongs to the same family that CRIP does,
Subfamily Ami“. In the SF 3 protein, glycine is the third zinc chelating residue in the

econd zinc-binding finger. The two other proteins that have a glycine residues chelating

eta] ions are rubredoxin and azurin. Both of these proteins have the metal binding

 

 

 

sequences of (CXZC
binding protein in t
the protein to 2135111
are believed to bin
The third poiyr
position X69 when
adenine or a goat
unrelated cows. .
The glycine is cc
CRIP. Serine ha
hahfle.
The gene stn
hat there is an 5
inveShgations i
primarily CDN,
character‘ue th
gehwated from
mlmeroug me
Notions inve
genomic Lair
the Presence
films which 5

CYSieineS an.

 

l 83
sequences of (CX2CG) and (CX3GH) respectively'“. Rubredoxin is an iron-sulfur

binding protein in Clostridium pasteurianum, the glycine residue is thought to allow for
the protein to assume the proper configuration to bind iron'“. All three of these proteins
are believed to bind DNA or RNA.

The third polymorphic site occurred in the glycine rich C-terminus of the protein, at
position X69 where a glycine or a serine was observed, resulting from the presence of an
adenine or a guanine, respectively. The polymorphism occurred between two unaffected,
unrelated cows. All of the animals in the BHZD pedigree had a serine at this position.
The glycine is conserved in both the reported human and rodent amino acid sequences of
CRIP. Serine has very similar chemical characteristics to glycine, however, it is polar in
nature.

The gene structure of CRIP has not been previously reported. This study indicates

‘ that there is an intron between the first and second zinc-binding fingers. Previous
investigations into the nucleotide sequence of rodent and human CRIP have utilized
primarily cDNA preparations for PCR and sequencing. Attempts were made to further
characterize the intron by screening a bovine genomic library with a 220 bp probe
generated from a genomic bovine template and sequencing through the intron; however,
numerous attempts to isolate the CRIP gene in the library failed. There has only been one
previous investigation into rCRIP that has isolated genomic CRIP sequence from a rat
genomic Lambda Dash 11 vector“. The homology between adjacent finger modules and
the presence of an intron suggests that the second finger is an internal duplication of the
first, which subsequently diverged in structure and class to one that contained the three

165

cysteines and one histidine and another that contained four cysteines . Other genes in

 

 

 

the LIM subfamily ‘
motifs consistently
themselves‘w. The
has the (CCHC) m:
the LIM family of
together and form

clusters. These ex
recognition”.

In the original
determined by do
tissues, with a ”I
amhunts of CR1}
levels detected i1
adult rat brain, It
blot analysis has
Recently, CRIP
mononuclear cg
has been able tr
heteroznguS a

21Incdhficiem a
tissues of We:
c0111(there

observable bar

 

184
the LIM subfamily that CRIP belongs to have had introns detected between the LIM

motifs consistently and there are some genes that have introns in the zinc-binding fingers
themselves“. The LIM motif that CRIP contains, in which the first zinc-binding finger
has the (CCHC) module and the second (CCC/GC) module is highly conserved within
the LIM family of proteins. The (CCHC) and (CCCC) modules are tightly packed
together and form a hydrophobic core that is surrounded by positive and negative charged
clusters. These exposed surfaces provide potential candidate sites for molecular
recognition”.

In the original paper that identified CRIP, tissue specificity of the protein was
determined by dot blot hybridization of mRNA from a variety of adult and fetal rat
tissues, with a 32P labeled rat clone probe. The study found that the highest relative
amounts of CRIP were in the duodenum, jejunum, ileum, cecum and colon, with lower
levels detected in the lungs, spleen, adrenal and testis“. CRIP was not detected in the
adult rat brain, kidney or liver in this investigation. Additional studies utilizing Northern
blot analysis have detected CRIP in the skin, heart, skeletal muscle and stomach2"'26.
Recently, CRIP has been detected in peritoneal macrophages, peripheral blood
mononuclear cells and in small concentrations in liver and plasma‘66"67. This investigation
has been able to definitively detect CRIP in 22 of 30 tissues tested in adult obligate
heterozygous and homozygous affected BHZD cattle that were both zinc adequate and
zinc deficient at the time of tissue collection. CRIP was also found in the corresponding
tissues of unrelated, unaffected cows and a calf. In several of the tissues in which CRIP
could not be reliably quantified due to low signal to background ratio, there were visually

observable bands on the autoradiographs that corresponded to CRIP. These tissues were

 

 

 

 

 

the uterus, pancrea
autoradiography or
or adipose tissue. ‘
It is unlikely th
been reported not
differential tissue
eXperimental tech
isolation and Nor
tissue53‘56"16"26, j
mRNA than Nort
present in order t
by the RT-PCRi
amplification of
rehoned not to c
Although N0
Sim11hr results, t
compromised, ]
as he handling
RNAses. None
inhibitors to de
handling. Spec

undigTaded am

 

185
the uterus, pancreas, eyes and ovaries. A CRIP band was not however, detected by

autoradiography or by B counting of RT—PCR products of CRIP from the aorta, pituitary
or adipose tissue. '

It is unlikely that the variation in ability to detect CRIP in tissues that have previously
been reported not to contain the protein, such as kidney and brain in the rat, is due to
differential tissue distribution within bovines, but more likely due to the variation in
experimental technique. Thus far, all the studies have utilized a variation of totRNA
isolation and Northern or dot blot analysis to detect the presence of CRIP mRNA in
tissues”‘“"‘6'm. Reverse transcription-PCR is thousand fold more sensitive in detecting
mRNA than Northern blot analysis and requires very small amounts of a message to be
present in order to amplify a product’“. The increased sensitivity of detection afforded
by the RT-PCR technique and the use of or -32P dCTP during the subsequent
amplification of the cDNA template allowed detection of the protein in tissues previously
reported not to contain CRIP.

Although Northern blot analysis is a common technique that has consistently produced
similar results, there are many steps in which the sensitivity of the technique can be
compromised. The extraction and purification of totRNA from the tissue samples as well
as the handling of the samples during blot analysis, is susceptible to RNA degradation by
RNAses. None of the protocols of the previous studies mentioned the use of RNAse
inhibitors to decrease the amount of RNA degradation that occurs in the samples during
handling. Speetrometric analysis of the totRNA samples can not distinguish between

undegraded and degraded RNA, so even if the samples were loaded in equivalent

 

 

 

 

concentrations intr
If the concentratio
comparisons betw
amuse-keeping g
cell cycle”. This
h-actin. By stand
keeping gene, the
into account. Th.
that occur in the .
degradation, Taq
amolification prr
This particula
comhhrison of ti
able to assess re
Process had to b
plateau effect fr.
would become 1
linear range of z
Shlined With eth
concentratiOn 0
radioactive nuc

”ports that rad

186
concentrations into denaturing gels, a large portion of the RNA may have been degraded.

If the concentration of undegraded RNA loaded onto the gel is not the same, then
comparisons between samples is inaccurate. It is therefore important to include the use of
a house-keeping gene which is constitutively expressed in all tissues without variation in
cell cycle‘“. This was accomplished in this study by the use of the house-keeping gene
B-actin. By standardizing the sample concentration to the concentration of the house—
keeping gene, the integrity, purity and variation in tissue RNA concentration can be taken
into account. The house-keeping gene also allows for the standardization of differences
that occur in the amplification efficiency of PCR reactions, caused by nucleotide
degradation, Taq inactivation, excess template and competition from nonspecific
amplification products‘“"“.
This particular study attempted to evaluate CRIP tissue distribution and a general
comparison of tissue content within and between the animals in the study. In order to be
able to assess relative amounts of mRN A in the original tissue, the PCR amplification
process had to be terminated within the linear range of the reaction. This would prevent a
plateau effect from occurring, in which one or more of the reagents in the reaction mix
would become limited hindering amplification. Often however, PCR products within the
linear range of a reaction are not concentrated enough to produce a visible band when
stained with ethidium bromide. This would prevent a subjective assessment of the
concentration of the product by ethidium bromide stained standards. Therefore a
radioactive nucleotide, (it-”P dCTP, was added to the PCR reaction mix. There have been

eports that radioactive nucleotides added to reaction mixtures that are not incorporated

 

 

 

 

into the PCR prod
the CPM values 0
others, there may
possibility, backg
bands being meat
subtracted from t
This study are
expression. Corr
first was looking
determine which
he comparing t
ahihlrtl with the
Some of the com
Proteins levels i
CDNA PTOductit
rarrdom Primitig
targets and the i
cDNA Pr0ducti
IherlmlcYCIer a1
Mum belWe.
qua”titative Va]
With the ab

bemwn from

 

187
into the PCR product produce a trail in the lanes of gels, which will artificially increase

the CPM values of the bands being measured‘“. If some reactions were more robust than
others, there may or may not be more smearing in the sample lanes. To account for this
possibility, background CPM values were taken throughout the gels, above and below the
bands being measured. The background readings were then averaged and the average
subtracted from the CPM count of the sample measured.

This study attempted only to determine relative, not absolute, tissue levels of CRIP
expression. Comparisons of the relative CRIP tissue levels were made in two ways. The
first was looking at the expression levels of CRIP within the tissues of an animal to
determine which organs had a higher concentration of CRIP for that animal. The second,
was comparing the relative levels of CRIP expression within a particular tissue in one
animal with the same tissue in another animal. By making these relative comparisons,
some of the complications that occur, which prevent quantitative determinations for
proteins levels in tissues were avoided. Several of the complications are, the variation in
cDNA production between sample and standard, due to a decrease in efficiency of
random priming of the specific target as it becomes diluted with nonspecific cDNA
targets and the interference of secondary structure in the mRNA used as a template for
cDNA production”. A variation in temperature cycles between wells in the
therrnocycler and sample tube thickness, can result in non-equivalent amplification of
products between the standard and the sample. All of these factors would alter the
quantitative values used for the comparison of tissue concentrations of CRIP.

With the above considerations in mind there are some important conclusions that can

3e drawn from evaluating the tissue distribution data. As stated, CRIP mRNA was

 

 

 

 

 

 

identified in sever
presence of CRIP
affected and heter
depleted status in
The lack of varia
cows during tissr
induction is inde
reports of other :
deficiency in str
increasing evide
an unlikely cant
mature humans
Other studies ar
m8 and bovine
aZinc haltspor
that lime to no

The Present

 

l 88
identified in several tissues that it previously had been reported to be absent. The

presence of CRIP in the variety and range of concentrations found in tissues of the
affected and heterozygote BHZD pedigree and controls with both zinc adequate and zinc
depleted status indicates it is unlikely that there is a mutation at the CRIP locus in BHZD.
The lack of variation in CRIP mRNA between zinc adequate and zinc depleted BHZD
cows during tissue collection also reaffirms previous experimental findings that CRIP

127' These results are also consistent with

induction is independent of dietary zinc status
reports of other zinc-fmger proteins such as TF IIIA, which are not being affected by zinc
deficiency in structure, in vivo‘. The wide tissue distribution of CRIP along with
increasing evidence of the role of LIM proteins in cellular metabolism makes the protein
an unlikely candidate as being solely responsible for carrier-mediated zinc absorption in
mature humans and animals. CRIP’s presence in fairly high concentrations reported in
other studies and verified in this study in the stomach, abomasum, cecum and colon of
rats and bovines, which at best are sites of low zinc absorption, also contradicts its role as

a zinc transport proteinll'smé. Experiments in both rats, calves and sheep have reported
that little to no zinc absorption occurs in these organs69'71'75'76.
The presence of CRIP in relatively high concentrations in tissues that have high
cellular turnover rates such as the intestine, suggests that its fimction may be related to
cellular proliferation or differentiation. Two recent investigations have examined CRIP’s
role in tissues that are highly regenerative and in tissues that require repair afier an insult.
The first investigation looked at the expression levels of CRIP in immune cells of rats,

er they were injected with lipopolysaccharides, to elicit an acute immune response‘“.

e results of the experiment showed a dramatic increase in the CRIP mRNA levels in

 

 

 

peritoneal macrc
may be responsi
activated who
consistently higi
tissue distributie
inthe body, it rr
concentrations i
In the secont
liver by injectio
undetectable lei
ce1h; intestinal
CRIP CXpressio
“hitching that
tissue distributi
thricient prior 1
diarrhea and pn
health Status w:
ahrrost “Vice as
cellular We Over
halve high 0011c
Both 0f the

OfCRIP in the

 

189
peritoneal macrophages as well lymphoid tissues in general. They concluded that CRIP

may be responsible for the differentiation of blood neutrophils and monocytes into
activated neutrophils and peripheral monocytes'“. In this dissertation, there were
consistently high levels of CRIP detected in the lymph nodes of all the animals in the
tissue distribution study. Since the intestine contains up to 80% of the lymph node tissue
in the body, it may explain why CRIP has been found historically in such high
concentrations in the intestine compared to other organs.

In the second investigation, the expression of CRIP was stimulated in the intestine and
liver by injections of carbon tretrachloride (CC14) '67. The liver initially contained almost
undetectable levels of CRIP, but in response to CCl4 CRIP expression doubled in these
cells; intestinal levels of CRIP also increased. The results of this experiment showed that
CRIP expression increased in tissues that were most affected by a stress challenge,

y suggesting that CRIP may contribute to the process of cell recovery. In the results of the
tissue distribution study, VYG-4, an affected heifer, was allowed to become zinc
deficient prior to being sacrificed. She was very debilitated and had profuse bloody
diarrhea and pneumonia at the time of sacrifice, as compared to the other animals whose
health status was not as compromised. The average ratio of CRIPzfi-actin in VYG-4, was
most twice as high as VYG-l, VYG-2 and VYG-3. Thus if CRIP is responsible for
ellular recovery and proliferation, it would be reasonable to expect debilitated animals to
ave high concentration of the protein present in proliferating or damaged tissues.
Both of the studies eliciting a proliferative response, have also confirmed the presence

f CRIP in the plasma of rats. CRIP has been suggested to have a unique signal

 

consensus seqr
secreted into tl
cellular repair
concentrations
observed to dc
To date, It
transcellularr
transports it it
CRIP may be
production 01
intracellular t
transcellular
protein inter;
diStribution 2
indicate that
Several to
absorption h
then is a p03
tells. As Su
transPort of
hyhndimfio
these metho

“Sing 36pm

 

190
consensus sequence common to exported proteins at its N-tenninus'“. If CRIP is actively

secreted into the plasma to act on target tissues elsewhere, it could also be responsible for
cellular repair in tissues that do not normally contain the protein or express it in very low
concentrations. Current evidence supports this theory because CRIP plasma levels were
observed to decrease after immunostimulation'“.

To date, no experimental data has been published that demonstrates CRIP is a
transcellular membrane protein that actually binds zinc in the intestinal lumen and
transports it to the vascular space. The evidence does not preclude the possibility that
CRIP may be involved indirectly in the regulation of zinc absorption by influencing the
production of other transport proteins, or being one of many LMW, ZBLs that facilitate
intracellular trafficking of zinc in mucosal cells. The proposed function of CRIP as a
transcellular transporter of zinc in mucosal cells could be facilitated through protein-
protein interactions that allow for the exchange of zinc ions. However, CRIP’s tissue
distribution and structural similarities to other proteins with known functions tends to
indicate that its primary function is not related to zinc absorption.

Several additional experiments could be conducted to further characterize zinc
absorption in the GI tract. It is apparent from the results of previous investigations that
there is a population of LMW, ZBLs present in the intestinal lumen and within mucosal
cells. As such, one or all of these ligands could be responsible in part or solely for the
transport of zinc in the GI tract. CRIP’s discovery was the result of advanced cDNA
hybridization and protein separation techniques by gel electrophoresis. Prior to the use of
these methods, CRIP could not be differentiated from MT. Therefore additional studies

using separation media that has a higher power of resolution for LMW, ZBLs should be

 

 

 

used to contint
extracts from 1:
within 30 mint
media, to colic
been isolated tl
sequences can

Utilizing th
unaffected and
in the intestine
cDNA intestin
pith cDNA pn
With probes in
Candidate gene

Informative
heterozltgote a
genes that are
Sartre farnilyns
intervals, to as
idetttified. 0n
antlined for a
name genor

depending 011‘

 

191
used to continue to screen the protein populations of mucosal cells. Homogenized cell

extracts from both neonatal and adult rats that have been treated with 65Zn then harvested
within 30 minutes of administration can be run through the higher resolution separating
media, to collect LMW proteins involved in the zinc transport. Once the proteins have
been isolated through further separation and purification systems, their amino acid
sequences can be determined and their potential zinc binding capacity assessed.

Utilizing the pedigree of BHZD cattle, differential hybridization between unrelated,
unaffected and BHZD cattle can be conducted, to identify cDNA clones that are present
in the intestinal cells of unaffected, unrelated cows as compared to affected cows. A
cDNA intestinal library from the unaffected, unrelated cow can be produced and screened
with cDNA probes from both normal and affected animals, those clones that hybridize
with probes from the normal cows, but not the affected cows would be potential
candidate genes that may be absent in the BHZD pedigree.

Informative polymorphic markers can be utilized in linkage analysis of the
heterozygote animals in the BHZD pedigree. Genetic linkage reflects the fact that two
genes that are near one another on the same chromosome are inherited together in the
same familym. Ideally markers should be spread throughout the genome at .1 morgan
intervals, to assure that a polymorphic marker segregating with a gene of interest can be
.dentified. Once linkage is established to a polymorphic site, then the area can be
analyzed for a defective transcript. There have been a number of methods employed to

xamine genomic loci for mutations, each has its advantages and disadvantages

 

epending on the type of mutation that is being identified.

 

 

Summary:
hhmhms
histopathalogical '.
treatment and mat
maturity in the B‘;
of zinc treatment
hhwflmm
earlier detection
The moleculr
hmmmhhfi
however, the pc

.4.

lhe researcl
Stquence CR1}
fingers of the 1
Can he used in
four 1,01),qu
hhnhuh0n&
Subtle Change
changes that
examined cr

Present in In

 

 

192
Summary:

This study has determined the chronological development of clinical manifestations,
histopathalogical lesions and biochemical alterations of BHZD. In addition, detailed
treatment and management protocols have been developed to sustain affected animals to
maturity in the BHZD pedigree. The resolution of clinical manifestations after initiation
of zinc treatment and subsequent redevelopment of these symptoms in adult cattle with
the discontinuation of treatment has also been documented. Through this information
earlier detection of hereditary zinc deficiency in both humans and cattle can be facilitated.

The molecular investigation has ruled out CRIP as a candidate gene for a mutation that
is responsible for BHZD. CRIP’s physiologic function remains to be determined;
however, the possible roles it may have in cellular metabolism are discussed in Appendix
A.

The research in this dissertation is unique in that it is the first time that the bovine
sequence CRIP has been reported. The discovery of an intron between the two zinc-
fmgers of the protein has never been reported in previous CRIP nucleotide sequences, and
can be used in further gene mapping studies. The investigation was also able to identify
four polymorphisms in the bovine nucleotide sequence of CRIP and three amino acid
substitutions. The polymorphisms and their resulting amino acid substitutions may cause
subtle changes in the affinity of the protein to bind zinc, as well as conformational
Changes that can effect the interaction of CRIP with other proteins. To date this study has

examined CRIP expression in more tissues than any previous studies, and found it to be

present in most.

 

an:
L fir. .,_. 9"

 

The cause of ti
isclear is that it i
lumen at the brus
one or several pr:
primary zinc tra

Future research e

 

193
The cause of the hereditary defect involved in BHZD remains to be determined. What

is clear is that it is a mutation in the ability of zinc to be absorbed from the intestinal
lumen at the brush border membrane. Whether the absorption process in facilitated by
one or several proteins is not known, as well as if the defect is a result of a mutation in a
primary zinc transporter protein or a regulatory protein within the intestinal enterocytes.

Future research endeavors will undoubtedly be directed at trying to unravel this mystery.

 

 

 

 

APPENDICES

 

 

 

Possible Fun
Examining 5
characteristics a
Only has one LI
one LIM motif,
first two zinc-l
high to be a
now this famfl.
binding motifs
RNA and DNr
binding protei
that binds sh,
binds Single st
nodulestts. Si

RNA retrovh

APPENDIX A

Possible Functions of CRIP:

Examining structural similarities of other LIM containing proteins and their functional
characteristics allows for further characterization of CRIP’s function. Although CRIP
only has one LIM motif with two zinc-binding fingers, proteins that contain more than
one LIM motif, that are in the same subfamily as CRIP, have an exact duplication of the
first two zinc-binding fingers for their second or third motif. Originally LIM motifs were
thought to be associated with primarily regulatory proteins related closely to TF IIIA, but
now this family has been broadened in scope to include nuclear receptors and nucleic acid
binding motifs168 . One of the salient features of these proteins is their ability to bind
RNA and DNA with the (CCHC) zinc-binding finger. Mammalian cellular nucleic
binding proteins contain seven (CCHC) zinc-binding fingers and a glycine rich region
that binds single stranded DNA and RNA‘”. Human polymerase enzyme (ADP-ribose)
binds single stranded DNA with the N-terminal LIM motif that contains two (CCHC)
modules”. Single stranded DNA binding proteins are important in DNA replication and
recombination'”. Retroviral nucleic acid binding proteins interact directly with genomic

RNA retroviral particles”. The similarities between CRIP’s LIM motif and the

 

 

 

aforementioned [
through direct in
Figure 15, is .
LlM containing
CRlP‘m-m'm?
their LIM motif
containing doul
motifs. in the I
first LIM motif
ttrminns of CF
sltttilar to the a
htttnan fetal he
also discovere
has been hypo
KKYGPK, w]
Zinc-binding 1

identified as i

 

19S
aforementioned proteins may give insight to CRIP functioning in a cellular regulation

through direct interaction with nucleic acids.

Figure 15, is a schematic comparison of the amino acid sequences and structures of
LIM containing proteins that are in the same family and subfamily A as
CRIP16°"°5"7"”2"73. These proteins have a conserved amino acid sequence that follows
their LIM motifs. Single LIM containing proteins and the second LIM motif in proteins
containing double LIM motifs have a (FGPKG) amino acid sequence that follows their
motifs. In the proteins that have two LIM motifs, the sequence (YGPKG) follows the
first LIM motif, where a tyrosine replaces the phenylalanine. The glycine rich C-
terrninus of CRIP contains seven hydrophobic and two basic residues. The sequence is
similar to the amino acid sequence of hCRHP, a cysteine-rich protein discovered in
human fetal heart tissue, and hCRP a cysteine rich protein that is distinct from hCRI-[P
also discovered in human tissue'65'172. The proposed function of the glycine rich domain
has been hypothesized to be involved in nuclear localization. The amino acid motif
KKYGPK, which is similar to the motif of the cysteine rich proteins and occurs after the

zinc-binding finger domains in Saccharomyces cerevisiae MAToe2 protein, has been

identified as a nuclear localization signal’“.

 

 

 

 

 

 

bCRIP I
n—(c

rCRIP
l
c
, l
n —c

hCRHP

l
c
rr~ (

hCRP
(l
N“ ‘ .

cCRP
N'fi-i

rESPl
NJ
hearers. A
St

 

 

196

bCRIP m
n C ’C/G
\ )

 

 

 

 

 

 

 

N,___(C>Z“ ‘c’ (0)“ c FGPKG -—- coon
rCRIP fl

..__<Z>zn<2) (inf; ....__ a...
hCRHP n

W]

N’ (guild) (::Zn<c romeo—coon

hCRP m m
fl ”1 ~
——(C>Zn<cHl—-(c:zn<:)—YGPKG 5C\Zn<c) C7" ::)-— romeo—coon

cCRP

c H c c, H c c
\Zn/ ) ( \ Zn’ ( ’Zn"<.) i :31: )
N .._... C’ ‘c— c’ ‘ C-YGPKG—C —-—— C -— FGPKG— coon

rESPl A m A m

C H C C C C C
~ C:Zn<CZ—_(C: Zn: ( :anfl) C:Zn< )

N’ c-vopxoé- c c —-

Figure 15 - A Comparison of Amino Acid Sequences of LIM Containing Proteins in
Subfamily A ‘

 

 

 

 

 

 

 

 

Human cyste
sequence novel I
amino acids in 1'
88.7% nucleotid
Shuclurcm. It is
similarity, that t
same consensus
studies utilizing
radiolabeled hC
Primarily small
Spleen, indicatii
fear and adult 1
“Imitation”?
existence of a I
reghltttion of g5

l'ltrmah cyste
hOmOlogue des;
several strum"
duplication 0ft
revealed that it
motif fOHOWinE

duplicatim Ho

197
Human cysteine-rich heart protein (hCRHP) was identified in a study designed to

sequence novel human heart cDNA genes during fetal development. The protein is 77
amino acids in length and contains a single LIM motif. The rCRIP and hCRHP share an
88.7% nucleotide identity in the open reading frame and a 97.4% homologous amino acid
structurem. It is believed that because rCRIP and hCRHP have such a high degree of
similarity, that they are both structurally and ftmctionally related. The two genes have the
same consensus initiation sequence and a glycine rich third domain. Northern blot
studies utilizing RNA extracts from both rodent and human tissues probed with
radiolabeled hCRHP, resulted in two discoveries. First, the hCRHP probe bound to
primarily small intestinal extracts from rodent tissue and to a lesser extent the heart and
spleen, indicating a lack of specificity to the heart. Second, RNA extracted from human
fetal and adult heart tissue showed a decrease in expression of the protein during
maturationm. The change in expression during development, excess basic residues and
existence of a LIM motif suggests that hCRHP may be involved in transcriptional
regulation of gene expression during development of the heart in the human fetusm.
Human cysteine-rich protein (hCRP) is distinct from hCRHP and has a rodent
homologue designated rCRP2. Although it is considered unique from rCRIP, it as
several structural similarities that indicate the protein may have involved from a
duplication of the single LIM motif that CRIP contains. Genomic investigation of hCRP
revealed that it contains six exons and the largest intron occurs after the glycine rich
motif following the first zinc-binding finger, which could have resulted from internal
duplication. Human cysteine-rich protein is 193 amino acid residues in length, it contains

Wo identical LIM motifs both followed by glycine rich domains that are very similar to

 

 

 

both CRIP and
binding histidir
promoter regio
or caat box for
rich”). The sit
the proper fun:
Human cys
number of box
Specificityhh,
wide distribut
in tissue organ
DNA from fla
Preterm highly
Phi’SiOIOgio h
The 1hrrcti
h Shana r
hhhhhtlatedi
important for
“short lived
1hhlttt‘rsenoe
httract With
residueS in ti

specihhhy oi

 

 

1 98
both CRIP and hCRHP165 . It also has a conserved glycine four residues prior to the zinc—

binding histidine in the first zinc finger that is present in both CRIP and hCRHP. The 5’
promoter region of the gene is also similar to CRIP in that the region does not have a tata
or caat box for initiation, and the area immediately 5’ to the open reading frame is gc
rich‘°°. The significance of these structural similarities is that they may be necessary for
the proper function of the proteins in this subfamily in cellular metabolism.

Human cysteine-rich protein has been identified through Northern blot analysis in a
number of human nucleated cells and appears to lack any tissue or developmental
specificity’éo. hCRP probes also hybridize to RNA extracted from rodent issues. The
wide distribution of CRIP and hCRP in tissues suggests that they may serve related roles
in tissue organization and function'ho. Southern blot screening, with hCRP probes of
DNA fiom flatworms, arthropods, chordates and mammals indicates that not only is the
protein highly conserved between species, but it is probably responsible for a basic
physiologic function of cellular metabolism.

The function of the protein has been hypothesized to be a number of activities due to
its structural characteristics. The presence of a two adjacent auuua sequences in the 3’
untranslated region and three potential E2F binding sites in the 5’ region, which are
important for gene transcription during the GO to S cell cycle phase, suggests coregulation
of short lived mRNAs critical in primary responses of the cell to environmental stimuli“).
The presence of two LIM motifs could allow hCRP to bind either RNA, DNA or possibly
interact with other LIM containing proteins. It appears that the last zinc chelating

residues in the second zinc-binding finger is the most divergent and may impart binding

specificity of the protein.

 

 

One experir
pn'mary mp0s
serum inductio
G0 to G1 or int:
then stimulater
of cycloheximi
samples were 1
establish if the
ftprobed with
Strum repletim
Presence of cyt
hCRP is regulg
Shithesis, requ
the Other Subf;
Possibility 0ft
ml'esfigation.

The ability
recently demo]
3“ aClhesion p11.

induce Change:

 

199
One experiment that was conducted to examine the possibility that hCRP was a

primary response gene utilized a human skin fibroblasts cell culture growth arrest and
serum induction system, to determine if the gene was induced in cells as they went from
G0 to G] or into S phase of the cell cycle. The cells were starved of serum for 96 hours
then stimulated with 20% fetal bovine serum for 30 minutes in the presence and absence
of cycloheximide, which inhibits protein synthesis. Northern blots of RNA fiom the
samples were then probed with two known early response indicators, c-myc and c-fos, to
establish if the cell lines were in the transition phase fiom Go to S. The blots were then
reprobed with hCRP, the results indicated that the hCRP mRNA was present prior to
serum repletion and gradually increased 3.8 fold after 3 hours of serum stimulation in the
presence of cycloheximide and 6.1 fold in its absencem. It was therefore concluded that
hCRP is regulated from G0 to S phase in the cell cycle and is independent of protein
synthesis, requiring only the modification of pre-existing transactivators. To date none of
the other Subfamily A proteins have been identified as early response genes, but the
possibility of them also having this function is very intriguing and warrants further
investigation.

The ability of LIM motif containing proteins to interact with each other has also been
recently demonstrated between zyxin and chicken cysteine-rich protein (cCRP). Zyxin is
an adhesion plaque protein that is believed to function as a signal transducer and can
induce changes in cellular behavior through coordinate activation of other proteins‘".
Zyxin has a proline rich N-terminus and three LIM motifs at its C-terminus. The first
zinc-binding finger of each of the LIM motifs is identical to that of all the proteins in

igure 15, containing an (CCHC) module, but the forth zinc chelating residue is different

 

 

in the second 21'
cysteine, histid
homologue of 1
Through the us
with two prote
kD protein ide
zyxin was con
ferredoxin, wl
their LIM mot
The eXperime
subcellular lo
that both cc};
Mistral fee
The last I)
ESPl was id:
C6 glioma Ce
length and Ct
Although the
a“ Only 75.:

Bite to the n

not Originat‘

 

200
in the second zinc-binding finger of each of the LIM motifs, being respectively a

cysteine, histidine and phenylalanine. Chicken cysteine-rich protein is the chicken
homologue of hCRP and there is less than a 95% homology between the proteins.
Through the use of an in vitro blot overlay assay it was discovered that zyxin co-localized
with two proteins, one was 100-kD protein identified as a-actin, and the other was a 23-
kD protein identified as cCRP‘”. The specificity of the interaction between cCRP and
zyxin was confirmed by the failure of zyxin to bind histone H1, cytochrome c and

ferredoxin, which are similar to cCRPm

. The interaction between these two proteins via
their LIM motifs would allow for induction of gene expression or cellular differentiation.
The experiments also postulated that the LIM motifs may have been responsible for
subcellular localization of both proteins. This speculation was based on the observation
that both cCRP and zyxin were associated with actin cytoskeleton and their common
structural feature was the LIM motifsm.

The last protein shown in Figure 15 is an estradiol-stimulated rat brain protein (ESPl).
ESPl was identified through the use of differential hybridization of a rat cDNA library of
C6 glioma cells, that were exposed to l7B-estradiolm. The protein is 164 amino acids in
length and contains two LIM motifs that have the same zinc chelating residues as CRIP.
Although the two proteins have similar zinc chelating residues in their LIM motifs, they
are only 75.5% homologous and have a 69% identity between their nucleotide sequences.
Due to the number of nucleotide variations between the two genes, ESPI probably did

not originate from CRIP, however both probably evolved from a common ancestor‘”.

 

 

There is a bi
motif within
The genc
promoter re,
ribosomal a
tata OI caat
for ESP 1 c
family are l
Estradic

in greater 2
are higher
muscle ant
Characteri:
protein w;
0f protein
Proteins 3
early res;
fefledoxi
inmlved
Stimulati.
The h

 

201
There is a high percentage of homology between the first LIM motif with the second LIM

motif within ESPl implying that the gene evolved from an internal duplication event.

The genomic structures of the two genes share some common attributes in that the 5’
promoter region of the gene is go rich, they both have a purine consensus sequences for
ribosomal attachment and an AUG initiation codon. There also does not appear to be a
tata or cat box for initiation in the 5’ promoter region, that has been published thus far
for ESP 1 once again implying that the induction characteristics of all the genes in this
family are unique and may be essential to their function.

Estradiol-stimulated rat brain protein has a unique tissue distribution. It is expressed
in greater amounts in the heart and liver of females than in males173 . In the rat brain, there
are higher levels detectable in the cortex, with very little being expressed in the kidney,
muscle and spleen. The function of the ESP 1 is unknown, but it does have some
characteristics that are common to other genes in this family. The expression of the
protein was not suppressed by cycloheximide, meaning that its expression is independent
of protein synthesis and may require the modification or activation of pre-existing
proteins similar to hCRP. This brings up the possibility that the protein may also be an
early response gene in cell cycle growth. The similarities that the protein has to
ferredoxin, an iron containing protein, could allude to the possibility that ESPl could be
involved in electron transport or energy expenditure that is a result of 17B-estradiol
stimulation173 .

The high number of correlations between CRIP and other proteins in its subfamily

between amino acid and nucleotide sequence, structure , tissue distribution, evolutionary

 

 

 

 

origins and
metabolisn
facilitate 2'
primary ro
experimen
metabolisr

First ph
that the ev
also reveal
in almost a
in Which g
to the basi.

The cor
family she

means that

 

 

T 202
origins and promoter region, provide strong evidence that CRIP’s functional role'in cell
metabolism is probably unrelated to being a primary zinc transport protein. If it does
facilitate zinc transport in any way, it would probably be a secondary consequence to its
primary role. So the question now is what is CRIP’s function? By summarizing the
experimental data from this study and data from related proteins, CRIP’s role in
metabolism can begin to be deciphered.

First phylogenetic analysis of LIM motif containing proteins similar to CRIP suggests
that the evolutionary origins of the proteins date back some 450 million years“. They
also reveal that the LIM motif containing the (CCHC) and (CCCC) modules are present
in almost all phyla. The detection of the LIM only proteins in such a variety of species,
in which genetic divergence occurred far back in evolution reveals that they are important
to the basic functions of cellular metabolism in all species.

The common amino acid sequences and protein structural characteristics that the
family shares implies that they originated from a common evolutionary precursor. It also
means that the cellular functions of these proteins are probably related. Thus if we
examine the fimctions of known proteins and look at the common features they share with
those that are not known, it will give insight into there metabolic roles. Whether the
functions are related in that they have the ability to bind and transfer metal ions for
metabolic utilization, bind zinc to assume the proper conformation for activation of a
protein or induction of nuclear replication or signal transduction to cellular systems,
remains to be determined.

The fact that CRIP, along with many other members of its protein family, is widely

distributed in tissues, and that with the exception of hCRP, all members of this family

 

appear to b
organizatio
interesting
An experirr
gene. Hum
withholdint
media with
by Northen
cyclohexim

The abil
“'33 an into:
functions an
endless, am
this cahabit
then there i:
facilitating ;
is Either iso;
can be incul
Preteins. To
can be Tune
can be furth

Addition

to exaltline i

 

appear to be developmentally regulated, suggests that it is involved in cellular
organization of tissues in response to maturation and/or external stimuli. It is also
interesting that hCRP was an early response gene and that ESP 1 may also be one too.
An experiment can be designed to examine the potential of CRIP to be an early response
gene. _ Human intestinal epithelial cells (IEC-6) can be arrested in G0 phase by
withholding nutrients for up to 96 hours, then inducing growth by incubating the cells in
media with and without cycloheximide and measuring the CRIP mRNA levels in the cells
by Northern blot analysis. If CRIP expression in the cells increases regardless of
cycloheximide, then it would be indicative of an early response gene.

The ability of LIM containing proteins to interact with each other at their LIM motifs
was an interesting discovery as demonstrated between cCRP and zyxin. The cellular
functions and signaling mechanisms that could be initiated through these interactions are
endless, and it warrants further investigation to see if other proteins in the farmly have
this capability. If CRIP interacts with other LIM containing proteins in the same fashion,
then there is the possibility that it could exchange zinc with these proteins possibly
facilitating absorption or have involvement in intracellular trafficking of zinc. CRIP that
is either isolated by gel filtration columns or produced by in vitro recombinant methods,
can be incubated with homogenates from mucosal cells to see if the protein binds other
proteins. To determine if CRIP binds to other proteins in mucosal cytosol the incubate
can be run on gel shift mobility or blot overlay assays. If a protein is identified, then it
can be further characterized to determine its amino acid sequence and structure.

Additional studies that would aid in characterizing CRIP’S cellular function would be

to examine its subcellular localization. Although CRIP is suppose to be primarily located

 

 

in the cytoplz
have been (13
of mediating
An in vitro e
response ger.
cycloheximi-
with 65Zn on
the CRIP 652
washed awa
elute out CF
that extracel
have the abi
Stimuli.
Since thr
number and
that there 3.1
may not be
1hatttlnetit
CRIP’S role

fimCilOI] is

204
in the cytoplasm of cells, it has been detected in the nucleus of cells in which the tissues

have been damaged‘“. The possibility that CRIP may also be a secreted protein capable
of mediating molecular mechanisms of cell recovery also needs to be further examined.
An in vitro experiment that could be conducted, if CRIP proves not to be an early
response gene, would be to acquire a liver cell line and incubate it in media containing
cycloheximide, to prevent protein synthesis. Recombinantly synthesized CRIP labeled
with 65Zn could then be added to that media. The liver cells would then be incubated in
the CRIP 652n labeled media. After some incubation period, the media would be gently
washed away and the liver cells homogenized and run through gel filtration columns to
elute out CRIP. If the CRIP that was purified was bound to 65Zn then it would indicate
that extracellular CRIP was capable of being transported intracellularly, and thus could

have the ability to migrate into tissues that have low levels of CRIP in response to

 

stimuli.

Since the discovery of the initial three LIM containing proteins that bind zinc, the
number and classes of these proteins have increased to the hundreds and there is no doubt
that there are more to be found. Although they occur in a variety of tissues and may or
may not be developmentally regulated, they appear to all have one primary function.
That function is to facilitate in some way the differentiation and proliferation of cells.
CRIP’s role in this function is yet to be determined, but it is unlikely that its primary

fitnction is as an intestinal zinc transport protein.

 

APPENDIX B

Protocols

This app

both the me

 

gently

 

APPENDIX B

Protocols for the Molecular Investigation of CRIP:

This appendix contains the protocols, solution concentrations and reagents used in
both the molecular investigation of CRIP and the clinical study.

Purifying and preparing oligos for PCR
The PCR primers were produced in 0.2 pmol quantities from the synthesizing facility.

Prior to their use in experiments, they were purified by the following protocol.

1. 200 pl of TE, pH 7.5, was added to each primer tube and heated in a water bath to
55°C for 20 minutes. Occasionally the tubes were vortexed to aid in dissolving the
pellet.

2. After the pellet had dissolved, 50 ul of 10 M ammonium acetate (NH4OAC) and 1 ml
of chilled 95% Ethanol (ETOH) was added to the tube. The contents of the tube were
then mixed and it was placed at 20° C for 20 minutes.

3. The tube was then centrifuged at 13,000 rpm for 15 minutes.

4. The supernatant was decanted off the pellet and it was allow to air dry.

5. Once the pellet air dried, 500 pl of TE pH 7.5 was add to the pellet and the tube was

gently vortexed until the pellet was completely dissolved.

205

Calculatint
1. As prev
primers
concent
2. Thefoh
needed
a hf“

b. (321

 

to x

DNA extra
1- After w
minutes

2' 4-8 IIilt
caPped

for 5 In:

 

 

_ HT'E’WL'T‘FMT time‘s—’i‘éfi "—- - . -

206

Calculating a 20 uM concentration of primers for PCR
1. As previously described in the Materials and Methods section, a 5 ul sample of
primer was diluted into 1 ml of TE and read on the UV spectrophotometer. The
concentration in the solution in rig/pl was calculated.
2. The following formula and information was used to determine the volume of primer
needed to produce a 20 uM concentration of primer.
3. MW of 1 nucleotide z 325 g
b. (325 g) (# nucleotide in the primer)=MW of the primer
c. lM/x mol of primer = MW of primer g/l g
d. x mol=1fMW of primer= moles/liter = M
e. (M)'3=mM and (mM)'3=uM
f. Using your uM volume, insert it into a V1C1=V2C2 formula
g. (x volume of primer)(y.tM concentration calculated above)=(1 ml)(20 uM)
h. uM concentration calculated above = 20 uM/uM concentration calculated above

i. 1 ml - uM concentration calculated above = volume of TE that needs to be added

to x in order to make a total volume of 1 ml.

DNA extraction from blood using a Genomix kit“

1. After whole blood was collected in an ACD tube it was centrifuged at 2000 x g for 25

minutes, the buffy coat was removed and placed in a 15 ml polyprOpylene tube.

2. 4.8 ml of Solution 1 (Lysing solution) was added to the buffy coat and the tube was

Capped and inverted several times to mix the contents. The tube was then mcubated

for 5 minutes in a water bath set 68° C.

 

3. Aftert

 

and it

at 260

4. Centri

 

DNA,
aqueo
Soluti
thin ii
placed
5. The tn
remov
6- The pt
overnj
7- Oncet

ETOH

 

8- The st

rCSuSp

 

207

3. After the incubation in the lysate solution, 7.2 ml of chloroform was added to the tube
and it was inverted several times to mix the contents. The tube was then centrifuged
at 2600 x g for 15 minutes.

4. Centrifuging the tube created three phases, an upper aqueous phase containing the
DNA, a thicker pink liquid and a proteinaceous plug on the bottom. The upper
aqueous phase was transferred by a pipette to a clean 15 ml tube and had 7 ml of
Solution 2 (Precipitating Solution) added to it. The tube was inverted gently until a
thin filamentous precipitate of DNA became visible. Sometimes the tube had to be
placed at 20° C for several hours in order for the DNA to precipitate completely.

5. The tube was then centrifuged for 5 minutes at 1300 x g. The supernatant was
removed and the pellet allowed to air dry for 2 minutes.

6. The pellet was resuspended in 4 ml of Solution 3 (Ionic Exchange Solution)
overnight.

7. Once the DNA pellet had been resuspended, it was precipitated in 8 ml of chilled 95%
ETOH and centrifuged at 1000 x g for 5 minutes.

8. The supernatant was then removed and the pellet allowed to air dry. The pellet was

resuspended in 1 ml of TE pH 7.5 and the DNA in solution was quantitated using a

a

UV spectrophotometer.

Phenol/Chi

1. Tothet

wasadc

room t6

 

2. The tub
remove
phase.

3. The tul
aqueou
was ad

chilled

 

 

 

208

Phenol/Chloroform extraction protocol to remove proteins139

1.

To the volume of sample to be extracted, an equal volume of phenol and chloroform
was added. The tube was inverted until an emulsion formed, after which it was left at
room temperature for 10 minutes.

The tube was then centrifuged at 13000 rpm for 10 minutes. The aqueous phase was
removed to a clean tube and ‘/4 of the volume in chloroform was added to the aqueous
phase.

The tube contents were mixed and centrifuged at 13000 rpm for 10 minutes. The
aqueous phase was once again removed and placed into a clean tube. 10 M NH4OAC
was added to the tube at 1/5 the volume of the aqueous phase along with 1 ml of
chilled 95% ETOH. The contents of the tube were mixed thoroughly and placed at -

20°C for 2 to 3 hours.

The tube was centrifuged at 13000 rpm for 15 minutes after which the supernatant

was decanted off and the pellet allowed to air dry.

Depending on whether the sample was RNA or DNA, it was either resuspended in

DEPC treated DDW or TE, pH 7.5. RNA samples also had 2 pl RNAse Inhibitor

added to them prior to being stored at -20° C.

Isolation of total RNA from frozen tissues with TRIzol reagentIn

1. Approximately 300 mg of frozen tissue was placed in 4 m1 of TRIzol reagent in a 50

ml P01ypropylene tube. The tissue was then homogenized with a Tekmar Tissumizerfr

and allowed to incubate at room temperature for 5 minutes. In between uses the bit

for the Tissumizer was rinsed with DEPC treated DDW.

 

2. Afieri

 

inverte
temper

minute

 

3. Oncet
divider
isoproy
roomt

4. The tu'
supem
vortex

5- lhe tu‘
SUpem

6- Oncet

reSUSpt

 

DNAsetrt
l. 20 inc
2' 41110i
3' ltlloi
4‘ There

at 37°(

 

 

 

,_“_:‘v.vlg.-— q ‘1V.W-1.bvv

209

2. After incubation, 0.4 ml of chloroform was added to the tube and it was capped and
inverted several times to mix the contents. The sample was then incubated at room
temperature an additional 2 minutes prior to being centrifuged at 12,000 x g for 15
minutes at 4°C.

3. Once the tube was centrifuged, the aqueous layer was removed with a pipette and
divided into 0.5 ml aliquots placed in 1.5 ml eppendorf tubes. One half an ml of
isopropanol was added to each of the aliquots and they were shaken and incubated at
room temperature for 10 minutes.

4. The tubes were then centrifuged at 12,000 x g for 10 minutes at 4° C. The
supernatant was decanted off and the pellet was washed with 75% ETOH by gently
vortexing the tubes.

5. The tubes were then centrifuged again at 7500 x g for 5 minutes at 4°C. The
supernatant was decanted off the pellet and it was allowed to air dry.

6. Once the pellet was dry, 0.5 ml of DEPC treated DDW was added to the pellet to
resuspend it. The samples were then stored at -20°C.

DNAse treatment of total RNA preparations

1. 20 ul of total RNA sample was thawed on ice.

2. 4 ul of RQ l RNase-Free DNase“ was added to the RNA.

3. 1 ul of RNase Inhibitor°

4. The contents of the tube were mixed throughly and centrifuged breifly, the incubated

at 37°C overnight.

 

 

iAZNa
integrity
genomic
6. If there
sample '
Reverse tn
Molonej
produce a c
oligos.
1. On ice t
a. 2.5

hip

for an
3~ Oncet

added

 

 

210

5. A 2 pl aliquot of the treated RNA was then run on a 1% agarose TBE gel to check the
integrity of the RNA samples, via the ribosomal bands, and for the presence of
genomic DNA.

6. If there was no genomic DNA present and the RNA sample appeared undegraded, the
sample was phenol/chloroform extracted to remove the DNase enzyme.

Reverse transcriptase of total RNA preparations to produce cDNA

Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT)m was used to
produce a complementary strand of cDNA to ssRNA with random priming hexamer
oligos.

1. On ice the following reaction mix was made with the reagents being added in order.

a. 2.5 pl of 1 mM dithiothreitol (DTT)
b. 1 pl of 0.6 pg/pl of hexamer (N)6°
c. 4 p1 of 2 mM dNTPs

d. 1 pl RNAse Inhibitor°

e. 8.5 pl of DEPC treated DDW

f. 4 pl of DNAse treated RNA

2. The contents were spun briefly and heated to 70° C for 5 minutes, then cooled on ice

for an additional 5 minutes.

3. Once the reaction mix had been cooled 6 pl of the following enzyme solution was
added to it.

a. 1 pl of M-MLV enzyme

b. 5p

 

Mr

4. The tut
for 10 r

5. Afterw

' Polymers
All the

The PCR 1
volume. I
1. The fol

a5

b5

 

d.1

2' After

aliqut
3- Thet

 

 

211

b. 5 pl of 5 x first strand buffer“1 (250 mM Tris-HCL pH 8.3; 375 mM KCl; 15 mM
MgC12)

4. The tube was then spun briefly and incubated at 37°C for 2 hours then heated to 90°C

for 10 minutes to kill the enzyme.
5. Afterwards the cDNA product was stored at -20° C.
' Polymerase chain reactions

All the non-radioactive PCR reactions were run in either 50 pl or 25 pl total volumes.

The PCR reactions that used the incorporation of a or-32P dCTP had a 29.5 pl total
volume. However, the remaining reagents used in both types of reactions were similar.
1. The following reagents per reaction were mixed on ice in order for a 50 pl.

a. 5 pl 10 x PCR buffer"1 (50 mM MgC12; 200 mM Tris-HCI, pH 8.4; 500 mM

KCl)

b. 5 pl 20 mM concentration of both primers

0. 5 pl 2 mM dNTPs (2 mM dATP; 2 mM dCTP; 2 mM dTTP; 2 mM dGTP)
for the PCR reactions that were run with (rt-”P dCTP”, a 1 mM concentration of

dCTP was in the dNTPs mix, and 1 pl of (1-32P dCTP per reaction was added.

(1. 1 pl T aq DNA Polymerasem per reaction.

e. 24 pl DDW

2. After the master mix for the number of reactions to be run was mixed, it was
aliquoted in the appropriate volumes to 0.5 ml eppendorf tubes, that were on ice.

3. The tubes then had the calculated amount of DNA template added to each one.

 

 

 

 

4. A drop
them 1:
Ertractior
PCR pr
the gels by
they were
were excis
not exceed
The follow
l- T0 the
suspen
2~ The on
dissolv

3- The tul

 

212

4. A drop of mineral oil was placed in the reaction tubes to prevent evaporation prior to

them being placed in a therrnocycler.

Extraction of DNA bands from agarose gels

PCR products that were run on 1 x TAE agarose gels, had their DNA extracted from
the gels by use of a Qiaex kit‘. After the gels had been stained with ethidium bromide,
they were placed on a UV light box and the individual bands that were to be extracted
were excised from the gel and placed in a 1.5 ml eppendorf tube. The gel plug sizes did
not exceed 300 mg in weight.
The following protocol was then used to purify the DNA bands from the agarose.
1. To the agarose plug, 1 m1 of QX 1 buffer was added along with 20 pl of Qiaex Il bead
suspension.
2. The contents of the tube were incubated in a water bath at 55°C until the agarose plug
dissolved. During the incubation, the tube was gently shaken intermittently.
3. The tube was then centrifuged at 13,000 rpm for 30 seconds at 4°C. The supernatant
was decanted off the precipitate and 500 pl of QX 1 buffer was used to resuspend the

pellet.

4. The tube was spun again with the same parameters as above and the supernatant was

decanted off.

~. The pellet was resuspended in 500 pl of Buffer PE with ETOH, and centrifuged for

10 minutes at 13,000 rpm at 4°C.

The supernatant was decanted off and the pellet was allowed to air dry.

 

 

 

7. Oncet
sit atr
8. The St
superr
9. TheD
Radiolab‘
Primer
Polynucle
the labele.

1. The fr

2| The tr
heatec
3. Itche

30p].

 

213

. Once the pellet was dried, it was resuspended in 25 pl of TE, pH 8.0 and allowed to

sit at room temperature for several hours.

The suSpension was then centrifuged for 15 minutes at 13,000 rpm and the

supernatant was removed to a clean tube.

9. The DNA quality was checked by electrophoresing a small amount on an agarose gel.

Radiolabeling primers for DNA sequencing

Primers were radiolabeled with y—33P dATPr for sequencing reactions, using T4

Polynucleotide Kinasem . Both the Sequenase‘ and A Taq Cycle Sequencing kitt required

the labeled primers.

1. The following reagents were mixed together in order.

a.

b.

C.

6.5 pl of DDW

1 pl of 20 pM primer

2 pl of 5 x forward reaction bufferm (350 mM Tris-HCl, pH 7.6; 50 mM MgClz;
500 mM KCl; 5 mm 2-mercaptoethanol)

1 pl of y-33P dATP

0.5 p1 of T4 Polynucleotide kinase enzymem

2. The tube was then spun briefly and placed in a therrnocycler at 37°C for 2 hours then

heated to 80°C for 10 minutes.

3. If the labeled primers were going to used for cycle sequencing, they were diluted with

30 pl of DDW.

 

 

 

 

 

 

 

The inot
plate‘. A p
placed on t
placed in a
After 7 mir
the plate w
incorporatj
Sequencin

Three 3
determine
Sequenase
dATP lab
Terminato
3 Seqt
1. An am

dry ice
at 7 it
b. 2,}

In]

 

214

The incorporation of 33P into the primers was checked on thin-layer chromatography
plate‘. A piece of plate was cut out and a small drop of the labeled primer solution was
placed on the bottom. The dr0p was dried onto the plate and the bottom edge was then
placed in a 0.8 M Sodium Phosphate solution. Care was taken not to submerge the dr0p.
After 7 minutes the solution migrated up the plate and it was removed and dried. Then
the plate was exposed to film“ for 15 minutes and developed. Primers that had 50%
incorporation of 33P or better were subsequently used in sequencing.

Sequencing PCR products
Three separate commercially produced DNA sequencing methods were used to
determine the nucleotide sequence of the coding region of CRIP. Two of the kits
Sequenace Version 2.0 DNA Sequencing kit‘ and ATaq Cycle Sequencing kitt used y—3’P
dATP labeled primers. The third method used or-ddNTP 33P in a Thermo Sequenase
Terminator Cycle Sequencing kit’.
2 Sequenase Version 2.0 DNA Sequencing kit protocol is as follows:
1. An annealing mixture was mixed on ice and boiled for 2 minutes then snap cooled on
dry ice.
a. 7 pl of DNA (PCR product)
b. 2. pl of 5 x Sequencing buffer (200 mM Tris-HCl, pH 7.5; 100 mM MgC12; 250
mM NaCl)
c. 1 pl of y-33P dATP labeled primer.
w After the annealing mixture was snap cooled, it was thawed and 6.5 pl of enzyme

solution was added to it. The enzyme solution contained the following:

 

 

 

 

a. 65

b. It

50

 

 

 

 

i iStd
conch
(160y
‘NaCU
50nd
ddTT
dTTP

4.Then
Four
was:

5- The:

1. Pfin]
dflut
2-Ama
21.2]

M

 

 

 

 

215

a. 6.5 pl of enzyme dilution buffer (10 mM Tris-HCl, pH 7.5; 5 mM DTT; 0.5 mg/ml
BSA)

b. 1 pl of T7 DNA polymerase (20 mM KPO4 pH 7.4; 1 mM DTT; 0.1 mM EDTA;
50% Glycerol; 13 units/ pl Polymerase)

3. 3.5 pl of the above combined solutions was then added to a set of four tubes
containing 3 pl of ddNTPs preheated to 37 °C. The termination mixes used were ddG
(160 pM dITP; 80 pM dATP; 80 pM dCTP; 80pM dTTP; 1.6 pM ddGTP; 50 mM
NaCl), ddA (80 pM dITP; 80 pM dATP; 80 pM dCTP; 80 pM dTTP; 8 pM ddATP;
50 mM NaCl), ddT (80 pM dITP; 80 pM dATP; 80 pM dCTP; 80 pM dTTP; 8 pM
ddTTP; 50 mM NaCl); ddC (80 pM dITP; 80 pM dATP; 80 pM dCTP; 80 pM

dTTP; 8 pM ddCTP; 50 mM NaCl).

 

 

4. The tubes were then incubated at 37 °C for 5 minutes, then 4 pl of stop solution (95 %
Forrnamide; 20 mM EDTA; 0.05% Bromophenol Blue; 0.05% Xylene Cyanol FF)
was added immediately to each tube.

5. The samples were then stored at -20°C until they were run on a gel.

2 A Taq Cycle Sequencing kit
1. Primers that are radiolabeled with y-dATP 33P with the above listed protocol were
diluted with 30 pl of DDW.
2. A master mix composed of the following reagents was mixed on ice.
a. 2 pl of y- 33P dATP labeled primer.

b. 2 pl of reaction buffer ( 260 mM Tris-HCI, pH 9.5; 65 mM MgC12)

 

 

 

 

c. 2p]
Dilr

TW

(1. 9y
3. The In
4. Thec
four
dth
each
dCTl
deal:

5. Onec
therr
94°t
55°t

72°t

Stort

1- The

 

216

c. 2 pl of a 1:8 ATaq DNA Polymerase enzyme 32 U/ p1: A Taq DNA Polymerase
Dilution buffer (10 mM Tris-HCl, pH 8.0; 1 mM 2-mercaptoethanol; 0.5%

Tween-20®; 0.5% Nonidet P40)

 

d. 9 pl DDW

3. The master mix was then added to a tube containing 2 pl of DNA template.

4. The combined master mix and DNA template was added in 4 pl amounts to a set of
four tubes containing the termination analogs. The termination mixes used were
ddG (15pM each dATP, dCTP, 7-deaza-dGTP, dTTP; 22.5 pM ddGTP); ddA (15pM
each dATP, dCTP, 7-deaza-dGTP, dTTP; 300 pM ddATP); ddT (15pM each dATP,

dCTP, 7-deaza-dGTP, dTTP; 450 pM ddTTP); ddC (15pM each dATP, dCTP, 7-

 

deaza-dGTP, dTTP; 150 pM ddCTP).
5. One drop of mineral oil was then placed in each tube and they were put in a
thermocycler and run at the following temperatures.

94°C 30 seconds

55 °C 5 seconds
72°C 1 minute 30 seconds
Repeated 30 cycles

6. 4 pl of stop solution ( 95% F ormarnide; 20 mM EDTA; 0.05% Bromophenol Blue;
0.05% Xylene Cyanol FF) was then added to each of the tubes. Reactions were
stored at -20°C.

3 Thermo Sequenase Radiolabeled Terminator Cycle Sequencing kit

1. The following reagents were mixed on ice to produce a reaction mixture.

 

 

 

 

 

2. A tern
needet
a. 2 l

b. 0.:

L4»)
:‘=-
(J‘-

H

217

a. 2 pl of reaction buffer (260 mM Tris-HCl, pH 9.5 ; 65 mM MgC12)

b. 50 to 500 ng of DNA template

c. 0.5 to 2.5 pmol of unlabeled primer (1 pl of 20 pmol primer in 39 pl of DDW
makes 0.5 pmol)

d. 13 pl of DDW

e. 2 pl of Thermo Sequenase Polymerase (4 U/pl Thermoplasma acidophilum; 5 0

mM Tris-HCl, pH 8.0; 1 mM DTT; 0.1 mM EDTA; 0.5% Tween-20; 0.5%
Nonidet P-40; 50% Glycerol)
2. A termination mix for each ddNTP was combined on ice for the number of reactions
needed. Then 2.5 pl of termination mix was put into one tube of a set of four ddNTPs.

a. 2 pl of dGTP termination master mix (7.5 pM dATP, dCTP, dGTP, dTTP)

b. 0.5 pl of ddNTP labeled with (rt-33R The ddNTPs contained the following
ingredients: ddGTP (0.3 pM a-33P ddGTP, 11.25 pCi); ddATP (0.3 pM or-33P
ddATP, 11.25 pCi); ddCTP (0.3 pM or-33P ddCTP, 11.25 pCi); ddTTP (0.3 pM
a-33P ddTTP, 11.25 pCi).

3. 4.5 pl of the reaction mix was then added to each of the four termination mix tubes
and a drop of mineral oil placed in each tube.
4. The tubes were then put in a therrnocycler with the following program

95°C 30 seconds

60°C 30 seconds

72°C 1 minute 30 seconds

Repeated for 30 cycles

 

 

 

 

5. At the
0.05%
tube.

6. The re
acrylar

Silver stat
SDS P.

stainedus

DNA ban.

Stained in

Dittipitati

1. Gelsr
Staine
a. 4.
b. 4
c. 2
d. 1

2- Thef
and3
DDW

3- TheI

218

5. At the end of the cycles, 4 pl of stop solution (95% F ormamide; 20 mM EDTA;
0.05% Bromophenol Blue; 0.05% Xylene Cyanol FF) was added to each reaction
tube.

6. The reactions were then stored at -20°C until they were run on a glycerol tolerant
acrylamide gel.

Silver staining acrylamide gels
SDS PAGE gels that were not loaded with radiolabeled PCR reaction products, were

stained using a Silver Staining kit". The fixative enhancer in the kit was also used to fix

DNA bands in gels that were used for autoradiography. The dish that the gels were

stained in was washed thoroughly and all soap rinsed out in order to prevent additional

precipitate and blotching from occurring on the gel.

1. Gels were first soaked in a F ixative Enhancer solution for 20 minutes prior to being
stained. The fixative solution contained the following for a 400 ml volume.

a. 40 ml of Fixative Enhancer for a final concentration of 10%.
b. 40 ml of acetic acid for a final concentration of 10%.
c. 200 ml of methanol for a final concentration of 50%.

d. 120 ml of DDW.

2. The fixative enhancer solution was then aspirated off the gels with a 60 ml syringe
and 300 ml of DDW was poured over the gels to rinse them for 20 nrinutes. The
DDW was refreshed once at 10 minutes.

3- The DDW was aspirated off the gels with a syringe and the silver stain solution was

immediately poured over the gels. The dish containing the gels was then placed on a

 

 

 

swivel
over tl

ingrec

w
4- When
gels a
subse.

5- The g
Prepari
A Prt
stacking
btheen
Would n

1- The

219

swivel platform with a piece of white paper under it and the solution slowly swirled

over the gels until they were stained sufficiently. The following silver stain solution

ingredients were mixed in a clean flask with a teflon stirring rod in order.

a. 5.0 ml Silver Complex Solution

b. 5.0 ml Reduction Moderator Solution

c. 5.0 ml Image Developer Reagent

d. 50 ml Developer Accelerator Solution was immediately added to the above
ingredients before use. The concentration of the developer accelerator solution
was 5%.

4. When the gels were stained sufficiently, the silver stain solution was aspirated off the
gels and a 5% acetic acid solution was poured over the gels for 20 minutes. This was
subsequently decanted off and DDW was poured over the gels.

5. The gels were then placed on a x-ray viewing box and photographed.

Preparing SDS PAGE stacking gels

A Protean II xi Vertical Electrophoresis" system was used to pour two SDS PAGE
stacking gels at the same time. After the plates were positioned with a 1.0 mm spacer
between them, they had a paraffin strip placed at the bottom to insure that the acrylamide
would not leak out. The following acrylamide gel solutions were used to pour two gels.
1. The lower 10% separating gel.

a. 15 ml of 40% bisacrylarnide (38% acrylamide; 2% bisacrylamide)

b. 15 ml of4 x lower gel buffer (1.5 M Tris; 0.4% SDS, pH 8.8)

c. 30 ml DDW

d. 50

e. 25

2. The ab

The to

 

 

 

 

 

3. A sma
thatth
4. Aftert
placed
5. The 11]

were i

 

 

 

220

d. 500 pl of 10% Ammonium Persulfate

e. 25 pl of N,N,N’,N’-Tetramethyl-ethylenediamine (TEMED)

. The above solution was poured into a 60 ml syringe with a thin tube attached to it.
The tube was placed in between the glass plates and the acrylamide was allowed to
nm in until the plates were 3A full.

. A small amount of hydrated butanol was injected on top of the acrylamide to ensure
that the edge would be level.

. After the gels had polymerized, the butanol was poured off. Fifteen well combs were
placed in between the glass plates.

. The upper stacking gel was then mixed with the following ingredients and the plates
were filled as previously described.

a. 3 ml of 30% bisacrylamide (30% acrylamide; 0.8% bisacrylamide)

b. 7 ml of 4 x upper gel buffer (0.5 M Tris; 0.4% SDS, pH 6.8)

c. 20 ml of DDW

d. 50 pl of 10% Ammonium Persulfate

.0

30 pl of TEMED

. Once the gels polymerized, they were locked into the self cooling electrophoresis cell

and submerged in 1 x running buffer (1.5M Tris; 7.2% Glycine; 0.05% SDS), which

was also placed in the upper wells of the plates too.

. The gels were then run for 8 to 10 hours at 200 volts, 40 watts and 35 mAmm.

 

 

6% Acrylamide go
The 6% acrylam
1. Two glass plate
with 95% ETO
2. The smaller of
3. The two plates
sequencing tap
large alligator
4. A 6% acrylam
a. 60 ml of t
b. 1 ml 10%
C- 15 pl of T
5- The solution .
Order to load
0m Corner as
tipped back e
filled. Once
deep.
6- Once the pla
rernOved.
7' The gel Was

3- txrna w,

 

 

221

6% Acrylamide gels

8.

The 6% acrylamide gels were primarily used for running sequencing reactions.
Two glass plates were prepared by washing them with DDW, then wiping them down
with 95% ETOH until all visible dirt was removed.
The smaller of the two glass plates was then evenly coated with Sigmacoatg.
The two plates had a spacer placed between them and the sides were taped with
sequencing tape“. After the sides were taped, they were also clamped together with
large alligator clips.
A 6% acrylamide gel solution was prepared by mixing the following.
a. 60 ml of 6% acrylamide or 6% glycerol tolerant gel solution
b. 1 ml 10% Ammonium Persulfate
c. 15 pl of TEMED
The solution was poured into a 60 ml syringe and the plunger inserted into it. In
order to load the plates without forming bubbles, the plates were tilted up and onto
one comer as they were being loaded. When they were half full, the plates were
tipped back evenly, but still on a 45° angle, which was steadily decreased as the plates
filled. Once full, the flat edge of a comb was inserted between the plated about ‘/2 cm
deep.
Once the plates polymerized, the clamps and tape on the bottom edge of the gel were
removed.
The gel was then locked into the vertical sequencing gel electr0phoresis system”.

1 x TBE was placed one bottom well and 0.6 x TBE in the upper well.

 

 

 

 

 

 

 

9. After the gels we

mounted on chro

 

 

 

 

222

9. After the gels were electophoresised, the plates were separated and the gels were

mounted on chromatography paper“. The gels were then dried in gel drier.

 

 

 

 

 

 

 

Protocols for Cli:
Deficiency:

Synchronization of
The donor cows \
days apart. Follow
injections twice a do
of PSH shots the do:
synchronized with l
donors were then ar
they demonstrated 5
Embryo transfer I
The donor cows
Catheter“ Was threa
PhOSphate buffered
W and 1% Per
P at ovary transre:
individual flush es .
Viability and grade
The embryos “

and then directly i:
insemtltation gun:

were then pregnar

 

‘1— “.3"

223

Protocols for Clinical Investigation of Bovine Hereditary Zinc
Deficiency:

Synchronization of donor and recipient cows
The donor cows were synchronized with two intramuscular injections of lutalyse 11

days apart. Followed 13 days latter by a series of follicle stimulating hormone (F SH)

 

injections twice a day for 5 days to induce superovulation. On the forth day of the series
of FSH shots the donors had their lutalyse injections repeated. The recipients were .

synchronized with lutalyse on the third day the donors received their FSH shots. Both

 

donors were then artificially inseminated with semen from the affected BHZD bull when
they demonstrated standing heat.

Embryo transfer protocol

 

The donor cows were flushed 7 days after their insemination date. A 22 gauge Folley
catheter"h was threaded into the uterine horn of the donor cow and 35 ml of Dulbecco’s
phosphate buffered saline 0.1 micron filtered“1 with 1.5% heat inactivated bovine calf
serumii and 1% Penstripm solution added to it was flushed into the horn and then collected
in an ovary transfer petri dish. Each horn was flushed six times and each of the
individual flushes were collected in separate petri dishes. The embryos were graded for
viability and grades 1 through 3 were immediately implanted into viable recipients.

The embryos were aspirated into ‘A cc Cassou strawsij with 10% bovine calf serumii
and then directly implanted into the uterine horn with a ‘A cc Cassou artificial
insemination gun ipsilateral to the ovary with the corpus luteum. The recipient cows

were then pregnancies checked at 35 days post-implantation.

 

 

 

 

 

 

 

 

 

 

 

 

 

APPENDIX C

 

 

 

 

 

Product and M:

a. Smith Kline Be
1. Scourg

2. Escher

h. Schering-Plou;
1. Bo-Se

°~ Purina Mills 11
1. Nurse

2. Calf S

3. Calf C

‘1 Merck&C0.
1. Iverm.

e- Becton Dicky]

1- AcidC

2- Trace

f~ Thermo Jarret

1- Poly:

 

 

 

APPENDIX C

Product and Manufactures References:

Smith Kline Beecham, West Chester, Pennsylvania
1. Scourguard 3K and Calfguard (Rota and Corona Virus Vaccine)
2. Escherichia coli Vaccine
Schering-Plough Animal Health, Kenilworth, New Jersey
1. Bo-Se (Vitamin E 50mg/ml and Seliniurn 1mg/ml intramuscular injection)
Purina Mills Inc., St. Louis, Missouri
1. Nurse Chow #200
2. Calf Startena [Course] Medicated OTC 20
3. Calf Growena
Merck & CO. Inc.
1. Ivermectin
Becton Dickinson Vacutainer Systems 6526, Ruthford, New Jersey
1. Acid Citrate Dextrose (ACD) blood collection vacutainer tubes
2. Trace element free blood collection vacutainer tubes
Thermo Jarrell Ash Corp., Franklin, Massachusetts

1. Poly Scan 61B

224

 

 

 

 

g. Sigma Chemica
1. Thin-L

2. ZincA

3. Sigma

h ABIDWhth
1. Appliet

i. Corning,lnc.,t
l. 50mlcl<

2. lSrnlclt

l Rmmhhmn
l. UV/VIS

k- Shimadzu Cor]
Division, Kyor

1. UV1601

l- Ambion, 1m,
l-RNAmJ
mLifeTechnoloi
1- TRlzol

2- M-ML\
3'5Xfin

4. Taq DIN

5- 10xPC

 

Sigma Chemical Company, PO Box 14508, St. Louis, Missouri

. Thin-Layer Chromatography Plate
2. Zinc Acetate

3. Sigmacote

ABI Division, Perkin-Ehner

1. Applied Biosystems 394 DNA/RNA Synthesizer

Corning, Inc., Coming, New York 14831

1. 50 ml clear polypropylene centrifirge tubes

2. 15 ml clear polypropylene centrifuge tubes

Perkin-Elmer and Company, GmbH

1.

Shimadzu Corp., Spectrophotometric Instruments Plant Analytical Instruments

UV/VIS Spectrometer Lambda 2 Model No. VDE0871/B

Division, Kyoto, Japan

1. UV160U Spectrometer

Ambion, Inc., 2130 Woodward St. #200, Austin, Texas

1. RNAse Zap

. Life Technologies, Inc. , Gibco BRL, Grand Island, New York

1.

2.

TRIzol Reagent Cat No. 5596UA1UB
M-MLV Reverse Transcriptase Kit

5 x First Strand Buffer

T aq DNA Polymerase

10 x PCR buffer

 

 

 

 

6. T4 P01

7. 5me

8. 100 bp

9. Termir

10. Dulbet
11.1%Pe

n. Promega Cor]
1. RQl‘

2. RNA.

0. Boehringerlv
1. RNA:

2- Randt

p. Perkiltlihner
1. DNA

‘1 MJ Research
1. Pro

l- Amersham/L
l. (1.32}
M331)

3~ Ther

s. Qiagefldric,

1~ Qiar

6. T4 Polynucleotide Kinase
7. 5 x Forward Reaction Buffer
8. 100 bp Ladder
9. Terminal Deoxynucleotidyl Transferase
10. Dulbecco’s Phosphate Buffered Saline 0.1 micron filtered
11. 1% Penstrip solution
11. Promega Corp, 2800 Woods Hollow Rd., Madison, Wisconsin
1. RQl RNAse-Free DNAse
2. RNAse One
0. Boehringer Mannheim, 9115 Hague Rd., PO Box 50414, Indianapolis, Indiana
1. RNAse Inhibitor
2. Random priming hexamer oligos
p. Perkin-Elmer Instruments Division Norwalk, Connecticut
1. DNA Thennocycler Model No. P7340
q. MJ Research, Inc. , Watertown Massachusetts
1. FTC-100 Programmable Thermal Controller
r. Amersham/Lifesciences, 26111 Miles Rd., Cleveland, Ohio
1. (X-32P dCTP
2. y-33P dATP
3. Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit
8. Qiagen, Inc., 9259 Eton Ave., Chatsworth, California

1. Qiax DNA Extraction Kit

 

 

 

 

 

 

 

 

 

 

 

 

t. United States]
1. Scout:
2. A Taq
u. Fisher Scienti
1. Electrt
v. Polaroid Corp
1. Polaro
w. Life Technolt
1. Sequ
2. Mod:
x. BioRad,200C
1. Prote
2. Silve
3. Anal
l- Hamilton Co]
1. Ham
2. Heto Lab eq

1. Heto

aa. Heofer Scier

1‘ Dry:
bb‘ Whattnan Int

1. 3m

 

 

t. United States Biochemical (U SB), PO Box 22400, Cleveland, Ohio
1. Sequenase Version 2.0 DNA Sequencing Kit
2. A Taq Cycle Sequencing kit
11. Fisher Scientific, Pittsburgh, Pennsylvania
1. Electrophoresis System Photo-Documentation Camera
v. Polaroid Corporation, Cambridge, Massachusetts
1. Polaroid 3000/667 film
w. Life Technologies, Inc., Gaithersburg, Maryland
1. Sequencing Gel Electrophoresis System
2. Model SA Adjustable Sequencing Gel Electrophoresis System
x. BioRad, 2000 Alfied Nobel Dr., Hercules, California
1. Protean II xi Vertical Electrophoresis Cell
2. Silver Stain kit
3. Analytical Grade Mixed Resin Beads AG® 501-X8(D) Resin
y. Hamilton Company, PO Box 10030, Reno, Nevada
1. Hamilton Syringe Model No. 1203118
2. Heto Lab Equipment, Denmark
1. Heto Dry CD-l Gel Drier
aa. Heofer Scientific Instruments, San Francisco, California
1. Drygel Sr.
bb. Whatrnan International Ltd., England

1. 3 mm Chromatography Paper

 

 

 

 

 

 

 

 

 

 

 

 

2. No.11

cc. Eastman Kodal
1. X-OM

dd. Automated Mi
1. Ambis

ee. WashingtonB
1. Genom

ff. Tekmar Comp
1. Tekrr

gg. 3M, Saint Pa
1. SCOtt

hh. CR. Bard, Int
1. 22g

11- Hyclone Labs
1. 1.59

11" IMV lnternC
1. 1/40(

2~ l/4Ct

kk Jorgtnsen La

1- lo:

228
2. No. 1 filter paper

cc. Eastman Kodak Company, Rochester, New York
1. X-OMAR AR and BioMax MR
dd. Automated Microbiology Systems, Inc., 3939 Rultin Rd., San Diego, California
1. Ambis Radioanalytic Imaging System
ee. Washington Biotechnology, Inc. , 6917 Arlington Rd, Bethesda, Maryland
1. Genomix DNA extraction kit Cat. No. 605
ff. Tekmar Company, PO Box 371856, Cincinnati, Ohio
1. Tekmar Tissumizer
gg. 3 M, Saint Paul, Minnesota
1. Scotch Brand Electrical Tape
hh. C.R. Bard, Inc. Covington, Georgia
1. 22g Folley catheter
ii. Hyclone Labs, Logan, Utah
1. 1.5% Heat Inactivated Bovine Calf Serum
j j. IMV Intern Corp., Minneapolis, Minnesota
1. 1A cc Cassou straw
2. ‘A cc Cassou artificial insemination gun
kk. Jorgensen Laboratories, Loveland, Colorado

1. 1 oz Clear Gelatin Capsules

 

 

 

m—urzm ;-y~,-v,.—\-.-.;-—_—v Vfi' ~ -— — -—-—

 

 

BIBLIOGRAPHY

 

 

 

 

 

[Q

U)

Raulin, J. 1

Parisi, A.F.

Vallee, Bl
enzymes as

Chesters, J

Prasad, A,:
experiment

Shay. Nr.
2111C defiCit

Mills, Q1;
1909. Met;

COUSins, F
SPeCial ref

Whanger, ]
Accumma

‘ Brenner;

Rev of Nu

 

 

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3. Vallee, BL. and Auld, D.S. 1990. Zinc coordination, function and structure of zinc
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4. Chesters, J.K. 1992. Trace element-gene interactions. Nutr Rev 50:217-223.

5. Prasad, AS. 1991. Discovery of human zinc deficiency and studies in an
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6. Shay, NE and Cousins, R.J. 1993. Cloning of rat intestinal mRNAs affected by
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7. Mills, C.F., Quarterrnan, J ., Chesters, J .K., Williams, RB. and Dalgarno, AC.
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lactating c

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but nontor
Holstein c

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and the ab

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Rodin, A.
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enteropafl

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entel'Opat]

 

 

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acrodenn:

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. Jubb, T.F

epidermal

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872725-72

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Med 2821:

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in cattle.

Bmmmer:
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Microbiol

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Nature 11

Blackmo:
ruminant

Brumrner
in cattle.

Weisman
in cattle, t
(Stockhol

Miller, W
deficiency
behavior,
48:1329-1

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Calves fed

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Med 11 3:

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CalVes, J TS

 

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53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

Mills, C.F.
deficiency

Price, J. at
Friesian 0:

Bauer, R.,
Reduced 1
relative to
62Zn. AP]

Birkenme
encodes a
immunog

Hempe, J
relatinshj

Miller, \l
metabolic
extended

0estreicl
Zinc absr

Wedekin
Zinc bioe
Oxide. J ,

Van Can
Of Ora] d
Nun 104

Cousins1
Am J or

 

53.

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