AMTQYEC ACTNETY AND {RESPIRATfC-N 5 5 THE EXCESS!) PEA RG07 MERENEM Thesls for the Degree 0‘ pk. D. MICHEGAN STATE UNIVERSETY Paul A. Van Dreal 19-61 This is to certify that the thesis entitled MITOTIC ACTIVITY AND RESPIRATION IN THE EXCISED PEA ROOT MERISTEM presented by Paul A. Van Dreal has been accepted towards fulfillment of the requirements for __Pi..2_._degree in_Bata.n¥_ and Plant Pathology w/zm Major professor Date ////‘?/é/ 0-169 LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE APR 0 5 2003 231E493 6/01 c:/ClRC/DateDue.p65-p. 15 ABSTRACT MITOTIC ACTIVITY AND RESPIRATION IN THE EXCISED PEA ROOT MERISTEM by Paul A. Van Dreal The purpose of this study was to determine if respiration and mitosis are related functions in the primary root meristem of Pisum sativum \_r_a_i_'_. Alaska. . Respiratory inhibitors potassium cyanide and 2, 4-dinitrophenol inhibited respiration and mitotic activity. The mitotic inhibitor actidione had no effect on respiration. Colchicine affected the mitotic Spindle, but also had no effect on respiration. The carbon source fructose stimulated oxygen uptake and mitotic activity over the glucose control. DL-glyceraldehyde and ribose did not stimulate oxygen uptake or mitotic activity to the same extent as the glucose control. A radioactive experiment showed that C14-l-g1ucose did not con» tribute as heavily as C14-6-g1ucose to C1402 during the first 6 hours. The reverse is true during the 6 to 12 hour period. The meristem then requires a minimal respiratory activity before it can support a maximal mitotic rate. MITOTIC ACTIVITY AND RESPIRATION IN THE - EXCISED PEA ROOT MERISTEM BY Paul A": Van Dreal A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCT OR OF PHILOSOPHY Department of Botany and Plant Pathology 1961 I. t) , ~ x i l \ \ ACKNOWLEDGMENTS My thanks to the cytology group at Michigan State University for their aid during this investigation. My special thanks to Dr. G. B. Wilson for passing on the principles of biological research and his excellent guidance during this period of study. The special assistance of Dr. Alan Cockerline and Mrs. Natalie Knobloch is especially appreciated. - I also wish to thank the Public Health Service for their financial support during the last year of this work. Finally, I wish to express my appreciation to my wife, Mary, for her patience and aid in typing the manuscript. ************* ii TABLE OF CONTENTS Page INTRODUCTION .......................... 1 LITERATURE REVIEW ...................... 4 I. Respiration and Mitotic Activity ............. 4 II. Cyanide and 2, 4-dinitrophenol .............. 9 III. Actidione and Colchicine ................. 10 MATERIALS AND METHODS . . ................. 12 RESULTS ........... . ................. 17 I. Preliminary EXperiments ..... . .......... 17 II. Respiratory and Mitotic Response to Carbon Sources . . 20 III. Respiratory and Mitotic Response to Inhibitors ..... 30 DISCUSSION ............................ 38 SUMMARY ............................. 45 LITERATURE CITED . ...................... 46 iii TABLE I. II. III . IV. VI. LIST OF TABLES . Page Comparison of mitotic activity in the warburg vessels with that in the tank. Mitotic Index is the number of dividing cells per 1, 000 cells as percent of the intact control. . . ........................ Oxygen uptake per‘mg. wet weight per hour, measured during the run down period and during the 1% glucose response. 0 O ......... O O O 0 Variation in oxygen uptake (111 per mg. dry weight per hour) during the rundown and after addition of 1% glu- cose (at 8 hours) contrasted with the excised control. Respiratory Quotient are also included .......... Oxygen uptake (ul per mg. wet weight per hour) response of excised 2 mm. root tips to 1% glucose given 2, 4, and6 hours after excision. . . . . . . . . . Cumulated averages and their standard deviation of re8piratory and mitotic values found during the 1% glu- cose response of six representative experiments. . . . Counts per minute (CPM) of BaCl4O3 isolated after incubation of 2 mm. excised root tips of 6-12 hours in CM-l-glucose and CM-é-glucose. 1. 5 no of radioactive glucose was added to 1. 5 m1. of 0. 25% unlabeled glu- cose in each vessel. Time is in hours after glucose addition . . . . . . ._- ................... iv 18 19 20 21 42 FIGURE 10 LIST OF FIGURES Generalized picture of the response curves in the excised pea root system ............ . . . . Page Standardized curves showing the overall reSponse of the system to 1% glucose. . ............. The oxygen uptake and the mitotic response to O. 5% D-fructose .............. . ..... The oxygen uptake and the mitotic response to O. 05% DL-glyceraldehyde. . .......... . ...... The oxygen uptake and the mitotic response to O. 05% D-ribose .......... . ..... . ..... The oxygen uptake and the mitotic response to 1% glucose in the presence of potassium cyanide ..... The oxygen uptake and the mitotic reSponse to 1% glucose in the presence of 3 PPM 2, 4-dinitrophenol . . The oxygen uptake and the mitotic response to 5 PPM actidione and 75 PPM colchicine ............ The mitotic cycle .................. .- 22 24 26 28 31 33 36 41 INTRODUCTION ResPiration and mitotic activity have engendered a considerable interest in recent years, as evidenced by the recent controversy between Bullough (1955) and Gelfant (1960), as to whether or not reSpiratory energy is necessary for antephase cells to pass into mitosis. In considering the work of N.- Knobloch (unpublished), it occurred to me that excised roots might provide a good system by which to study the relationship of mitotic activity to respiration. The research problem can best be stated after a brief consideration of previous findings regarding mitotic activity in this system. The mitotic activity of the intact pen root meristem grown in Hoagland's solution is fairly constant at about 70 dividing cells per 1, 000 cells. This rate is maintained from one day after germination to some time, six or seven days later, when the secondary roots are produced. Thus over the experimental period, the intact roots have 7% of their meristematic cells in active division (mitosis). If the roots are excised at the 1. 5 centimeter level and suspended in aerated Hoagland's solution, the mitotic activity drops to about 15% of the intact root tip by six hours. If during the six to eight hour period a carbon source is supplied, the mitotic activity increases. When the number of mitosing cells per 1, 000 meristematic cells (mitotic index) is plotted against time, the curves differ depending on which carbon source is used. The following graph shows a generalized picture of some selected reSponse curves. I noted that these carbon sources are all in the normal pathways for carbohydrate metabolism, i. e. , glucose, fructose, and glyceraldehyde in the Embden-Meyerhof pathway (Fruton and Simmonds, 1958) and ribose .HOHHGOU pomfioxm ”mm “outwflopfimpoorm Aim §mo.o ”Q “omen: om; ”D momousaw 05H um ”encuuduw $3.10 ”4 .Eoumcnm noon mom pomwuxo may 2..“ wok/p.90 uncommon 0:» mo 0.250.?” ponflduodou .A mudmwrm mpsom ON A: NH w a... . 11 a q i A \\ \ i. I l | I / I H // / I \‘III 6 oos\‘oooos§aslc.. . o ..o \ .xcooooooooooo . o . o \ § 90 o \ Q o u s 0 s 0 fi 0 o o o . . s. o o x o.. . coco \ CaQO “Q Q \ U 00 no \ .vom o o x 06 oo \ oo \ \ \ \ m \ \ r OOH AitAtioV otioitw mag .tad in the Hexose Monophosphate Shunt. It seemed likely that there might be some corresponding changes in oxygen uptake and/or respiratory quotient of .the root tips during this twelve hour period. The problem was designed to give answers to the following questions: 1. Is there any relation between the mitotic response to a carbon source and the oxygen uptake or respiratory quotient of the pea root meristem? 2. Is it possible to demonstrate the separation of respiration and mitotic activity by the use of chemical inhibitors ? 3. If so, can this excised root system be used conveniently as a means of assessing the affect of various chemicals on cell function? LIT ERAT UR E. R EVIE W I. Respiration and Mitotic Activity The whole area of respiration and mitotic activity is very compliu cated and is nowhere near a final solution today. The complications were nicely pointed out by Erickson (1947) in one of the first studies suggest» ing the possibility that oxygen uptake may vary as the mitotic cycle varies. . Erickson worked with the anthers of Lillium longiflorum and found a drop in oxygen uptake as the anther went through meiosis and the succeeding mitosis. He pointed out that in any complex tissue individual cells in division could have a respiratory fluctuation and that this fluctuation .. could be hidden by the reSpiration of the surrounding cells which were not in division. I believe that this caution could be profitably noted by some of the later investigators. Stern (1948) confirmed Erickson's results by using a modified dif» ferential respirometer to measure oxygen uptake on 265 single anthers of Trillium erectum. He found a rising oxygen uptake during the pres- mitotic stages followed by a sharp drop immediately preceding and during active division. Stern then suggested that active division may be associ-» ated with anaerobic behaviour and that the rapid oxygen uptake is due to premitotic deve10pment. Sch‘olander e1; a_._l. (1952), also using the reference diver technique, found small cyclic changes in respiration during the first 1 three cleavages of echinoderm eggs. These respiratory changes reflected a sharp increase in oxygen uptake. He also found that cleavage will prom ceed without oxygen uptake and therefore concluded that cytoplasmic cleavage requires energy in the form of adenosine triphosphate (ATP). He further suggested that cleavage proceeds using stored ATP and that the oxygen uptake is used to replace the stored ATP. Bullough (1952) reviewed the work on the energyrelations of mitotic activity to that date. 7 He suggested that the magnitude of respiratory increase’during cell divisionmight be masked by the fact that normal cell metabolism is in abeyance during cell division and the normal energy supply for cell metabolism might be shunted toward cell division. . He. then reviewed the work in mammalian tissues, particularly his own work on mouse ear epidermis studied in litre. He pointed out that oxygen is necessary both for cell division and cell movements and, in particular, that the mitotic activity of epidermal cells is in direct prOportion to the oxygen tension of the culture medium. He also suggested that the important utilization of the oxygen does not take place during division but in a period just before visible prophase. This period he called antephase. If glucose is supplied in an oxygen atmosPher e, the rate of epidermal, _ mitoses increaSes. Bullough explains this on the basis that the respiration during antephase stores energy which is then used during the process of mitosis. Therefore mitosis is an all or none reaction. This is true to a certain extent as we shall see later. In 1955 Bullough carried these _ ideas a step further and proposed that the controlling mechanism was the gluocokinase reaction and that the effect of hormones was to stimulate or inhibit this reaction. Swann (1954), 'writing in the Colston papers, pointed out that sea _ urchin eggs do not enter division when exposed to cyanide, azide, carbon monoxide or oxygen tensions below 0. 4%. All of these chemicals inhibit the cytochrome system and therefore stop the production of stored energy, i. e. , ATP. Sea urchin eggs are particularly sensitive to the above chemicals because their power of glycolysis is slight. He also noted that 2, 4-dinitrophenol inhibits: division. From this fact he ruled out the {lack of diphosphopyridene nucleotide (DPN+) as being a limiting substance and again concluded that it was ATP that was limiting. . He also noted only a very small fluctuation in oxygen uptake with this tissue during cleavage. He concluded that either energy for division is required in a practically continuous flow or that it absorbs so small a part of the total energy output of the cells as to be scarcely detectable. Swann proposed that there are differing priorities for the disposal of the energy supply of the cell. Maintenance of the cell comes first, then cell growth, and finally cell division. The oxidative energy for cell division goes to fill a reservoir. This reservoir empties out all at once just before division, i. e. , during antephase. The energy thus emptied out is the total supply needed for the cell to complete mitosis. . Meanwhile, the reservoir slowly begins to fill again and the next division doesn't take place until the reservoir is full. Swann uses this hypothesis to explain why sea urchin eggs take longer for the first division than they do for the subsequent divisions. He also showed that before a critical period ether treatment delays the first division for a period of time equal to the duraa tion of the treatment but after the critical period it delays the subsequent division. Warburg (1955) considered the above data and an observation of his own that there is a relationship between cell proliferation and glycolysis, to propose that a cancer cell divides rapidly because of an impairment to its respiration and a consequent increase in fermentation. Weinhouse (1956) suggested rather that the impairment of reSpiration is not the cause but the result of tumor formation and the consequent poor oxygen Supply. This in turn is denied by Warburg's followers and so the battle. rages. Stern (1956) observed that in plant microspores there is a steep decline in oxygen'consumption coincident with the development of prophase. In connection with this findings, phase microscoPic studies show that the mitochondria are arrested in function, i. e. , they lose their normal movement and shape. He also noted that removal of oxygen can inhibit cytoplasmic cleavage. Stern goes on to caution that generalizations on the nature of mitotic metabolism based on the behaviour of developing eggs have been overdone. .In early stages of cleavage, on which most of the studies are done, the nuclear-cytoplasmic relation is far different than in other dividing cells. On the other hand he says it is difficult to believe that the energetic requirements of mitosis are greater than those of other cellular activities as held by Bullough (1952). He also cautions that the metabolism of the cell may be designated as 'mitotic only insofar as it provides substances or energy necessary for division. In closing, Stern suggested that any autonomous element can act as a source of control for cell division, providing that its activities become limiting. Therefore, it is extremely unlikely that there is a single control mechanism. Morrison (1958) worked with monoiodoacetic acid and showed that. in small doses it prohibits kinetochore cleavage. He then suggested that because of monoiodoacetic acid's action on triosephosphate dehydrou genase there is a relation between glycolysis and the chromosome move- ment cycle. To carry this a step further, Biesele (1958) listed the followu ing things that must be supplied or synthesized before a cell can divide. 1) Hereditary material (DNA); 2) Chromosome histone; 3) Lipids (mitotic cells are highin lipid content); 4) Mitotic apparatus (spindle); 5) A critical substance (unknown); 6) Energy via carbohydrate metabolism; 7) Carrier for high energy bonds; 8) Ribonucleic acid. He realized that many (of these things are synthesized all during the intermitotic period but stressed that a lag in synthesis of any of these components could delay or block mitosis. Later Swann (1958) extended the above and his own previous ideas by proposing that there is no on-off mechanism, but rather a steering of the synthetic mechanism. He suggested that this steering substance acted on the mic rosomal particles and directed them to synthesize material for differentiation or for cell division. He said that the mechanisms which form the specialized mitotic protein plus the division mechanisms plus the basic respiratory systems, take up so much space in a dividing cell that there is little room for other differentiating mechanisms. Therefore the cancer cell can be said to be a machine specialized to divide. This is indicated by the fact that cancer cells use their reSpiratory and glycolytic machinery to the limit of their capacity as shown by the fact that 2, 4wdiu nitrophenol gives a poor oxygen uptake stimulation in cancer cells. Wilson and Morrison (1958) described the mitotic cycle particularly as it related to the pea root ‘meristem and showed that the process of mitosis is a sensitive index of chemical effect. In the process of their study they tested the effect of various Kreb‘s cycle inhibitors (malonate, sodium arsenite and potassium cyanide). They found that at the concen« tration tried (1 x 10‘4M) no marked effect on mitosis was found, even when treated for a considerable length of time. This study might be criticized on the basis that this low a concentration wouldn't penetrate the intact root tips effectively. Later Wilson, Morrison, and Knobloch (1958) published a paper describing the use of the excised pea root meristem to study the control of mitotic activity. They showed that their system would respond mitotically to various carbon sources (sucrose, glucose, fructose and DL-glyceraldehyde). They also showed that the mitotic reSponse to glucose was successfully inhibited by potassium cyanide, malonic acid and 2, 4- dinitrophenol. They concluded that because these inhibitors, in suitable concentrations, inhibit the onset of cell division, therefore some level of operation of the Kreb's cycle is essential for commission of cells into mitosis. The above observations are consistent with the idea that there is a shift from reliance on aerobic to anaerobic respiration between antephase and active mitosis. The current state of affairs is about as cloudy as it was in 1947 when Erickson first noted a small cycling of respiration during cleavage. Zeuthen (1960) pointed out that the error inherent in using the differential respirometer is about 5%. This error just about accounts for the observed cycling reported in previous papers. Gelfant (1960) worked with mouse ear epidermis and completely contradicted Bullough's (1958) views. He gave evidence that excision of the mouse ear epidermis was the mitogenetic factor, not the respira— tory energy from glucose. He believed that glucokinase was not rate limiting and that hormones do not stimulate its activity in controlling F 91 energy production and mitosis. He suggested that glucose is not an energy metabolite but rather has some unknown endogenous effect on the process 1 of cell division. . ’ Amoore (1961a) has shown that anaerobiosis inhibits the mitotic I cycle but that the division stages are the least sensitive. Mazia (1960) suggested that the mitotic apparatus is itself the energy reservoir, i. e. , energy is used to make the system and once made its function proceeds spontaneously. We can best conclude along with Mazia (1960) that because the division process itself is insensitive to a great many inhibitors of cell function, the price in energy is paid before the visible onset of division. There are evidently many paths in the preparation to divide; a block in any one will delay or block division. II. Cyanide and 2, 4-Dinitrophenol The general biochemical action of cyanide is given in Fruton and Simmonds (1958). It is thought that cyanide combines reversibly with the iron-porphyrin compounds of which the cytochromes are good examples. Thus cyanide inhibits the flow of electrons down the cytochrome chain to oxygen. In the presence of cyanide, oxygen uptake is greatly reduced as well as oxidative phosPhorylation or ATP production. 10 Wilson, Morrison, and Knobloch (1958) have shown that 1 x 10‘4M potassium cyanide will inhibit completely the mitotic rise due to 1% glucose. They inoteithat at this concentration there is no evidence of 9 toxicity. Amoore (19616) has shown that in excised pea roots, 1 x 10‘“ZM sodium cyanide will halt the stages of mitosis, while 1 x IOQBM sodium cyanide only inhibits the onset of division. . . Clowes and Krahl (1957) show the biological effect of the nitro— phenols on arbacia eggs. They found that 2‘, 4-dinitrophenol causes a reversible block to cleavage, i. e. , long term continuous treatments found the effects wearing off toward the end. Hamburger and Zeuthen (1957), working with synchronously dividing cells of Tetrahymea pyriformis, noted‘that as the concentration of 2, 4-dinitrophenol increased oxygen up— take also increaSed to a high point and then) dropped. At the point of greatest uptake they found cell division inhibited. Fruton and Simmonds (1958) point out that the nitrophenols can be considered to uncouple electron flow from oxidative phosphorylation. Thus oxygen is reduced to water but the system derives no stored energy, in the form of ATP, from the process. Miihling, e_t al. (1960 have shown the effects of the nitro- phenols on the mitotic picture in pea root meristem. They point out that, at the proper doses, mitosis is inhibited without any other apparent toxic effect. III. Actidione and Colchicine The Merck Index (1960) gives the following structure for actidione (cycloheximide): CH3 1‘} CH, zoom 0 I ‘h_"' 3n -n m ~.- 1.. CE 12"”.‘11 11 It is an‘antibiotic substance with a powerful effect on dividing cells. Hadder and Wilson (1958) point out that it exhibits the following specific effects. Actidione inhibits the onset of cell division and causes the pro- duction of a devient late prophase configuration characterized by over- contractionof the chromosomes and the gradual assumption of a telophase morphology. Colchicine, on the other hand, has a greatly different effect as pointed out by Hadder and Wilson (1958).; They show that it does not inhibit the onset of cell division but it does produce scattered and clumped chromosomes. This effect is considered to be due to the action of col- chicine on the- mitotic spindle. Thus the scatters represent a partial effect of colchicine while the clumps indicate a full effect. Colchicine is a plant alkaloid with the following structure (Merck Index, 1960): Wilson and Morrison (1958) in discussing prophase poison and c-mitotic agents, state that actidione and colchicine do not act directly on cellular energetics, but must act either on the utilization of energy or directly on critical structures. Part of our problem will be concerned with using these substances to test the discriminatory power of the excised root system as an indexcf chemical effect. ”a”? ,l MAT ERIALS AND METHODS The material used in this study was the excised primary root of Pisum sativum \_ra_r. Alaska. The peas were donated by the Ferry Morse Seed Company and were selected for genetic constancy and freedom from chemical treatments. The experimental peas were washed in three changes of glass dis-» tilled deionized water and were soaked in the same for four hours. Then the peas were rolled in brown paper towels which were inverted in 400 ml. beakers containing 1-2 inches of glass distilled deionized water. Each roll was surrounded with a layer of wax paper such that the wax paper extended 2-3 inches above the roll. The beakers were placed in a Cenco incubator, set at 250C. , for 42-48 hours. I selected seedlings 2%... 312- cm. long and excised the distal 117 cm. These excised roots were placed in plastic vials into which extended a glass bubbler, which was attached to a constant flow of washed, charcoal filtered air. The vials and roots were placed in 250 m1. beakers containing Hoagland's nutrient solution. The beakers were set on a grid in a running water bath which kept the temperature at 22. 50C. After eight hours some of the roots had 2 mm. distal segments excised. These segments were put into microwarburg vessels, twenty to a vessel. The remaining roots in vials were transferred to the various treatment solutions for 12 hours. . Cytological samples of four roots per treatment were taken every two hours. The sampled tips were placed in methanol, chloroform and pro- pionic acid (6:3:2) fixative and were evacuated for ten minutes. The process of fixation was speeded up by placing the fixative vials in an oven at 60°C. for twenty minutes. The fixative was decanted and the tips were placed in hot 2. 7 N hydrochloric acid (HCl), in an oven at 60°C. , 12 Mir.':dfi-?L'_. . 1.6 - m .. r -m-fl—I-fi *r . _ ' . o __.. 13 for twenty minutes. The HCl was poured off and Schiff's base (leucobasic fuchsin) added. Whenthe meristem assumed a deep purple c010r, it was excised and put into a drop of 0. 1% fast green (in 45% acetic acid) on a slide. The meristem was macerated with a glass rod, a cover slip added and heated gently in an alcohol flame. The cover slip was pressed firmly between some brown towels. The material on the slide was dehydrated in.90% tertiary butyl alcohol and 10% ethanol (100%) overnight and made and" l -} .J permanent in diaphane. The plastic vials were obtained from a local drug store and were prepared for use by punching four sets of holes equally spaced around .co'jh‘t-u' xmfiet 4 fi - " their perimeter. Each set of holes extended from the top to the bottom, including the top and bottom surfaces. The holes allowed full circulation of the treatment solutions into and out of the vials as the air ascended from the bubbler. The Hoagland’s solution had the following chemicals added to 500 ml. of distilled deionized water: Calcium Nitrate (4 H20) 0. 950 gms Ammonium Nitrate l. 290 gms Magnesium Sulfate (7 H20) 1.800 gms Potassium Monobasic Phosphate 1. 335 gms Potassium Dibasic Phosphate 0.070 gms The stock solution was made up of equal parts of the above five chemicals. 15. 6 ml. of this stock solution was diluted to 250 ml. for use. The pH of the final solution ranged from 5. 4-5. 6. During the experiment the pH of the Hoagland's solution didn't vary by more than 0. 1 of a unit. The chemicals used were obtained from the following companies: glucose, fructose, ribose, and DL-glyceraldehyde--Nutritiona1 Biochemical Corporation; actidione--Upjohn Company; colchicine--Lights Limited; potassium cyanide--Ma11inckrodt Chemical Works; 2, 4-dinitr0pheno1-.. Eastman Chemical Company. All chemicals were made up in Hoagland's solution at the concentrations indicated. One solution was made up and used for all replicate vessels. 14 The micro-warburg vessels, supplied by Gilson Medical Elec- tronics, had a total volume of about eight ml. and held 1. 5 ml. of treat» ment solution plus 0. 1 ml. in the centerwell. Three replicate vessels of 1% Glucose were run with each experiment and compared with five replicate vessels of the treatment solution. - In'all cases the gas phase was air and the centerwell contained 20% KOH. The Gilson Refrigerated Warburg used was set at 120 strokes per minute with a 2 cm. stroke and a temperature of 22. 50C. This rate was shown to handle all quantities of tissue used (Umbreit, Burris and Stauffer, 1959). r In all runs at least two thermobarometers were used. The solution in these vessels was contaminated with pea root tip washings and was made up to 1% glucose. A bacteriological study showed a consistent kind of contamination in all vessels. The amount seems to be slightly more in the vessels with root tips, but the difference doesn't appear to be significant as far as the oxygen uptake is concerned. The pH in the warburg vessels was checked before and after with Hydrion pH paper and showed no change during the 12. hour run. All respiratory measurements were corrected for the dry weight of the root tips. The weights were determined in the following way. The tips were removed from the vessels and placed in methanol, chloro- form, propionic acid (6:3:2) fixative and evacuated for ten minutes. They were then put in a refrigerator for at least 12 hours. A At that time the fixative was decanted and the tips transferred to weighed 5 ml. beakers, which were put in an oven at 950C. for 4 hours. The beakers were equilibrated to room temperature in a dessicator for an hour and then weighed. A colorimetric study of the fixative showed that lipid was the major extracted material along with a small amount of protein and carbo- hydrate. This extracted dry weight method gave fairly consistent results and at least as reliable as cell counts. The wet weights were found to be too variable . 15 The sugar solution in the bath did show a pH change from 5. 5 down to about 3. 5 during the 12 hour run. This change could probably be accounted for by bacterial contamination and respired C03. In connection with this we compared the mitotic index in the bath with the index in the warburg vessels. Table I shows these results. Table I. Comparison of mitotic activity in the warburg vessels with that in the tank. Mitotic Index is the number of dividing cells per 1, 000 cells as percent of the intact control. Mitotic Index Mitotic Index Hours in warburg in tank 2 10 10 4 27 15 6 32 29 8 50 52 10 55 56 12 44 55 In a radioactive experiment (to be reported later) I checked on the per cent C1402 produced in a contaminated vessel which had 0. 25% glucose plus 10 uc of Cu-U-Glucose without root tips. The respired C1402 was compared with the same vessel with root tips. The contaminated vessel showed 1. 5% of the root tips activity after 12 hours of incubation. Twenty 2 mm. root tips in each vessel were allowed to respire glucose-l-Cl‘ and glucose-6-C” (2 vessels each) for 6 hours. Another replicate set was allowed to respire for the full 12 hours. At the end of the requisite time, 0. 2 ml. of 10 N H2804 were tipped into each vessel and 20 minutes allowed for the CO; to equilibrate. The 20% KOH was washed quantitatively from the center wells into 5 ml. beakers.- 16 Saturatedammonium sulfate (0. 2 ml.) was added to each beaker and the carbon dioxide present was precipitated as barium carbonate by adding 0. 5 ml. of barium chloride solution. The barium carbonate slurry was filtered onto a Special filter paper pad. The pad was then washed with distilled water and finally with 50% ethanol. The pad was removed, dried in a dessicator over night and counted in a Nuclear-Chicago gas- flow geiger counter for five minutes. Two five minute counts were made on each pad and the results averaged and divided by five to give counts per minute. 1. EW‘ RESULTS I . Preliminary Experiments Wilson, Morrison and Knobloch (1958) in their experiments on the excised system adopted the mitotic response of 1% glucose as their ”4-?" ‘ control (Figure 1, page 2). Because I am using their basic system I adopted 1% glucose as the reference curve in all the subsequent experi-u ments. In their experiments they grew 1. 5 cm. excised roots and removed the meristem just before the slides were made. Their mitotic index then is a reflection of the physiological state of all tissue present. In order to get a more meaningful correlation between mitotic activity and respiration, I felt it would be better to use segments that were as close to the whole meristem as possible. The pea root is ideal in this regard because it has a very small root cap (Esau, 1953). Thus the first 2 mm. of the root tip contains 90 -100% of the meristem. This can be seen in Fuelgen stained sections in which the deepest color appears in the 2 mm. distal section. Table II shows the comparison of the average oxygen uptake per milligram wet weight per hour for 2 mm. tips, 1. 5 cm. tips and 1. 5 cm. tips minus the 2 mm. distal fragment (without meristem). Note that the oxygen uptake is higher in the tips than in the 1. 5 cm. roots. Contrary to this, Jenson (1955), indicated that the region of elongation has the highest respiratory activity on a per cell basis. I believe that this dis- crepancy can be resolved by considering that we are measuring oxygen uptake on a per weight basis and not per cell. - Erickson and Goddard (1951) have shown that the 1-2 mm. segment in corn roots has by far the 17 18 Table 11. Oxygen uptake per mg. wet wt. per hour, measured during the run down period (0-8 hrs.) and during the 1% glucose re8ponse (8-16 hrs.). Time is in hours after excision. W Time 2 mm. Tips 15 mm. Tips 15 mm. Tips (no meristem) 3-8 hrs. 1.344 0.512 0.464 [ft-l "a 8-16 hrs. 2.052 0.672 0.536 . Increase 53% 31% 16% S [—I-_m~ ‘ .54 ’21 '- largest number of cells. This I think explains the greater respiratory sensitivity of our roots. Note also in Table II that respiration increases \ in all segments in the presence of 1% glucose, but that the 2 mm. tips ‘ show the greatest increase. Because of these results I decided to run 1 27mm. tips as our standard method. 1 ( Next I wondered whether or not the mitotic index was the same in the 2 mm. tips as in the larger 1.5 cm. roots. Table I shows that the two systems are mitotically comparable. ‘ I also checked to see if the oxygen uptake varied in the same manner as didrmitotic index. in our system. Table III shows the average oxygen uptake with and without glucose. The time in hours is the time from excision and the figures represent oxygen uptake per milligram wet weight per hour. . The Table shows that the oxygen uptake pattern is consistent with the mitotic pattern (see Figure 1). Note also the change in oxygen uptake and respiratory quotient at 14 hours. This might indi- cate a change in metabolic pathways. The excised control also shows this change and the time corresponds, as we shall see later, to the break in the cumulative oxygen uptake curves of the excised control (see Figure 2). The respiratory quotient (RQ) values are consistent with the idea | 19 Table III. Variation in oxygen uptake (111 per mg. dry wt. per hr.) during the rundown and after addition of 1% glucose (at 8 hrs.) contrasted with the excised control. , Respiratory Quotients are also included. Time in hrs. after excision. Time 1% Glucose Excised Control Oxygen R.Q. Oxygen R.Q. 2 1.59 0.96 1.27 0.96 4 1L07 0.96 1.17 0.96 6 1.20 0.86 1.08 0.86 8 1.06 0.86 0.95 0.86 10 1.22 0.94 1.04 0.85 12 1.10 0.99 0.89 0.87 14 1.10 1.28 0.77 0.97 16 1.18 1.08 0.75 0.86 18 1.27 1.15 0.65 0.93 that at least some of the glucose is being utilized via the respiratory pathways. The data of Table III suggested checking to see if the excised system would respond via oxygen uptake to the presence of glucose before the 8 hour run down. Table IV shows that the system will respond at 6 hours but not before. Because of this we can speculate and suggest that perhaps the cells must use a certain amount of endogenous carbon source before an outside source can be used. The effect of 1% glucose on the mitotic index after the same run down intervals was shown by N. Knobloch (unpublished) to give the same results as the respiratory data. The above data indicate that there is a fair amount of variability inherent in these systems. This situation was improved by correcting . H .Ifi‘m'w: 412.; i — . ‘r'T‘iT-‘TEIE .. . .n’r 20 Table IV. Oxygen uptake (ul per mg. wet weight per hour) response of excised 2 mm. root tips to 1% glucose given 2, 4 and 6 hours after excision. Hours after excision 2 4 6 8 10 12 1% glucose added afterZhours 1.27 1.10 1.17 1.02 1.19 1.12 1% glucose added after4hours 1.27 1.19 1.23 1.13 1.31 1.26 1% glucose added after 6 hours 1.27 1.19 1.26 1.28 1.28 1.26 the resPiratory values with the extracted dry weight. Table V gives the corrected average values and their standard deviations for the excised and 1% glucose controls of the last six experiments. The values for oxygen are cumulated in microliters of oxygen per mg. dry weight per hour and the mitotic figures are the cumulated number of dividing cells per 1, 000 cells averaged over four thousand cells per hour per experiment. The 5(- (average) values are plotted cumulatively in Figure 2. Study of this figure gives us the overall response of our system. II. The ReSpiratory and Mitotic Response to Carbon Sources Figure 3 (0. 5% fructose),,Figure 4 (0. 5% DL-glyceraldehyde) and Figure 5 (0. 5% ribose) show the mitotic and respiratory responses to the above three sugars. . In particular, Figure 3 shows that oxygen uptake of the fructose was as good as the 1% glucose or even slightly better. The DL-glyceraldehyde on the other hand did not produce as good a response as glucose, fructose or ribose, either mitotically or in respiration over the period of the experiment. We can then say generally I... ' .aal' 21 Table V. Cumulated averages and the standard deviation of the respira- tory and mitotic values found during the 1% glucose response of six representative experiments. Cumulative Cumulated number of O uptake per mg. dividing cells per dry weight 1 hour 1, 000 cells Hours after 1% 1% glucose Excised 1% glucose Excised glucose added control control )7 0" )‘E 0" )7 a" 5(— f 1 8 1. 3 9 1. 2 - .. .. .. 2 16, 1.8 18 1.6 27 4.4 35 8.9 3 25 1.7 27 1.7 - - - .. 4 34 1.7 35 1.7 47 8.2 46 10.7 5 43 2.2 43 ' 1.9 - - - - ‘1 6 52 2.5 51 1.8 79 6.4 58 11.2 7 61 2.8 58 2.2 - - - - l 8 70 3.4 66 2.4 135 9.6 68 15.6 9 79 3. 3 73 3.0 - - - - 10 88 3.8 80 3.0 196 15.4 81 18.9 11 96 4. 3 86 3.5 - - - - 12 105 4.7 92 3.7 251 22.0 94 20.2 22 Figure 2. Standard curves showing the overall reSponse of the system to 1% glucose (. 56 M). The letters with prime marks represent the cumulative mitotic index curves. A is 1% glucose B is Excised control 23 x62: 638:. 23.35 I ,d ’I 4. Hours Figure 2 0 5 75 '11, n . m 1 oxaana nomaxo w>uuaazsso 24 Figure 3. The oxygen uptake and mitotic response to 0.05% D-fructose (0. 28 M). The letters with primc marks represent the cumulative mitotic index curves. A is 1% glucose B is Excised control C is 0.05% fructose 25 100 75 - .300 52: 633:. 23.3.8 - 0 5 . 9.3.5 .3996 333335 100 26 Figure 4. The oxygen uptake and the mitotic response to 0.05% D,L-g1ycera1dehyde (5.6 M). The letters with prime marks represent the cumulative mitotic index curves. A is 1% glucose B is Excised control D is 0. 05% D, Loglyceraldehyde 100 F 75 - Cumulative Oxygen Uptake 8 I IN? a. . T Figure 4 27 A ,Q I d’ ’ p I O I .' ’I .0. O I’D "C ’ :3 300 I .' J o’ .0' I .0 ' I’ ." ‘ IE I .' ’ I 5) ’ U o I I, 0.. ” I :3 ’ I’ .00 ” in .3 }3 I a. I i, d: ’ 1 200 I .' ’ ' ’ g. ” D O I " ’ o I I 5’ I ’ .. ’ I, " I U 0.. d I 06 I, I.’ I ’I; I I . I D o. ’ ’.. ” ... 100 .. Hours Cumulative Mitotic Index 28 Figure 5. The oxygen uptake and the mitotic response to 0.05% D-ribose (0. 35 M). The letters with prime marks represent the cumulative mitotic index curves. A is 1% glucose B is Excised control E is 0.05% ribose 29 Figure 5 100 75 .82: 633:. 233.38 013.5 nonunowozuuglso 25 Hour: 30 that the oxygen uptake seems to be high when the mitotic activity is high andlow when themitotic activity is low. This raises the interesting question of whether or not respiration and mitosis are directly linked. III. The Respiratory and Mitotic Response to Inhibitors In order to determine if respiration and mitosis are linked I used two general types of inhibitors, respiratory poisons and mitotic poisons. Potassium cyanide (KCN) and 2, 4-dinitrophenol (DNP) were used as the respiratory poisons. Figure 6 shows the effects of 1 x 10‘“4 M and 5 x 10" M KCN on oxygen uptake and mitotic activity. 4 In this experi- ment we had to run the mitotic index material in the warburg vessels so that we could maintain the HCN atmosphere necessary for the concentra- tions used (Robbie, '1946). Note that the oxygen uptake is differentially inhibited by those two concentrations but that mitotic activity is not. This might indicate that below a certain respiratory level the mitotic activity is not maintained at a maximal level by the meristem. The fact that the mitotic activity is a little higher than that of the excised control is not inconsistent with the idea that glucose may be used for some function other than resPiration. ‘ Figure 7 shows the response of our system to 2,4-dinitrophenol. Note that oxygen uptake is stimulated only slightly beyond the 1% glucose level. This respiratory level probably represents the maximal electron flow capacity of our system '(Immers and Runnstrom, 1960). The mitotic curve shows the recovery phenomenon often reported with the phenols (Hamberger and Zeuthen, 1957).(Muh1ing e_t 341. , 1960). We can conclude then that our respiratory inhibitors did prevent the 1% glucose mitotic response. , These data are consistent with the direct coupling theoI‘Y- 31 Figure 6. The oxygen uptake and the mitotic response to 1% glucose in the presence of Potassium Cyanide. The letters with prime marks represent the cumu- lative mitotic index curves. A is 1% glucose B is Excised control F is 1 x10‘4 M KCN + 1% glucose (3 is 5 x10‘4 M KCN + 1% glucose 100 75 Cumulative Oxygen Uptake U1 0 25 Figure 6 32 300 Hours Cumulative Mitotic Index 33 Figure 7. The oxygen uptake and the mitotic response to 1% glucose in the presence of 3 PPM 2, 4-dinitro- phenol (1.6 X 10'5 M). The letters with prime marks represent the cumulative mitotic index curves. A is 1% glucose B is Excised control H is 2, 4-dinitrophenol + 1% glucose 34 Figure 7 BE: 636:: 23.35 F n 0 5 33.5 8936 333256 100 Hours 35 In Figure 8 we see the effects of actidione and colchicine. The actidione, which is a prophase poison as we have seen, does inhibit mitotic activity below that of the excised control but has no effect on respiration; if anything there is a slight stimulation. Colchicine has no effect on the onset or rate of divisions as can be seen by the close correspondence of the colchicine and 1% glucose mitotic curves. Colchicine does effect very definitely the morphological characteristics of division but has no effect on the oxygen uptake. Therefore we can affect mitosis during its course or inhibit its onset without effecting oxygen uptake. This is inconsistent with the direct coupling theory. 36 Figure 8. The oxygen uptake and mitotic response to 1% glucose in the presence of 5 PPM actidione (1.7 x10"5 M) and 75 PPM colchicine (1.9 x10”4 M). The letters with prime marks represent the cumu- lative mitotic index curves. A is 1% glucose B is Excised control I is 5 PPM actidione + 1% glucose J is 75 PPM colchicine + 1% glucose Figure 8 100 75i- Cunulative Oxygen Uptake 8 .1 37 I I I 1; I (I ./ II 00000000000.000000O'OO’OO'OOOQQanoaoc —::i_ ~4L---------J 4 8 abuts 300 8 Cumulative Mitotic Index 100 DISCUSSION The observations on the experiments can be summarized as follows: the preliminary work shows that the mitotic response to 1% glucose is accompanied by a small but definite increase in oxygen uptake. , The resPiratory quotient accompanying the above response indicates that some of the glucose is utilized by way of common respiratory pathways. The work also indicates that an exogenous carbon source is probably not utilized to a large degree until six hours or more after excision. There is also'a suggestion that the root tips might have a shift in metabolic paths about six hours after glucose addition (14 hours after excision). The carbon source experiments show that the oxygen uptake in the presence of various sugars is in the same relation to the 1% glucose oxygen uptake as the mitotic response is to the 1% glucose mitotic response. For example, the fructose (Figure 3) had a higher oxygen uptake than did the 1% glucose as did the mitotic response. . On the other hand, DL-glyu- ceraldehyde and ribose (Figures 4 and 5) gave mitotic responses less than that of the 1% glucose as well as a lower oxygen uptake. The inhibitor experiments show that inhibition of mitosis using two different concentrations gave oxygen uptake responses at two different levels of KCN (Figure 6),. but the mitotic response was about the same for both concentrations. . This might indicate that below a certain respira- tory level mitosis cannot take place at its maximum rate. . But it also indicates that the 1% glucose can allow mitosis to proceed at a level higher than the excised control. Thus glucose itself must be used for some other purpose than as a respiratory substrate. Glucose could be used to synthesize a key substrate whichis necessary to maintain mitosis at a maximal rate. .1 The 2, 4-dinitrophenol (DNP) experiment shows a maximal oxygen uptake but mitotically an excised control response up to 38 39 eight hours, This can be explained by its uncoupling action; that is electron flow takes place but ATP isn't generated. These results might be explained in that the KCN treatment would be accompanied by increas» ing concentrations of reduced diphosphopyridene nucleotide (DPNH+H+) while the DNP would allow DPNH+H+ to become oxidized to DPN+. It: is interesting to note in this regard that DPNH+H+ is necessary for syn- thetic pathways in many systems. The DNP response after 8 hours is due to the self recovery of the system to DNP (Miihling e_t all. , 1960). In order to better understand these results, I feel it would be wise to look at our pea root meristem. The 2 mm. root tip contains about 125, 000 cells. . This figure was obtained by hydrolyzing a number of tips, homogenizing them in a Kontes glass homogenizer and counting the number of cells in a hemocytometer. These results agree very well with those published in the literature. For example, Erickson and Goddard (1951) found 150, 000 in the 2 mm. tip of a corn root. However, not all of these cells are actively concerned with cell division. If we take 70 as the average mitotic index of the pea root and say that this is the number of cells in mitosis during a three hour period (duration of mitosis), then because the average time between divisions is 12 hours, there are 70 x—13E- = 280 cells per 1, 000 that are part of the mitotic cycle. Van't Hof, Wilson and Colon (1960) have shown that only one-half of the cells that are produced by mitosis go around and divide again. This is evident from the fact that the meristem remains fairly constant in size. Therefore, of all the cells in the meristem, 28% are part of the mitotic cycle, i. e. , they divide, go through processes and divide again. Another 28% are the daughters of the dividing cells and are therefore in the process of "differentiating. " This accounts for about 60% of the total meristematic tissue. The remaining cells are probably scattered through» out the meristem and are contributing to the control of cell division. 40 This is indicated by the fact that divisions occur in patches through the meristem) (L. V- Leak, unpublished). The rate of division then is a function of the physiological state of the meristematic tissue which is in turn interrelated with the function of the other tissues present. Respiratory measurements also are the algebraic sum of the physio- 10gical activity of every living cell present in our root tips. If we regard the cell cycle as given by Wilson and Morrison (1959) (Figure 9), we see that antephase is the period just before active division. Bullough (1959) said this period required energy and Gelfant (1960) disagreed. Wilson, Morrison and Knobloch (1958) feel that most of the cells which respond to a carbon source in the excised root system are *mitotically competent and thus are primarily in antephase. They do show that complete cycling does take place in the excised system about thirty hours after excision. This was done by growing the tips in col- chicine from the 8 hour rundown and noting when the tetraploid population appeared. The carbon source increases the amount of cells cycling and the rate, i. e. , polyploid population appears 26 hours after excision. The rate of cycling is less than the rate in the intact roots. My inhibitor studies indicate that there is a minimal respiratory level which is necessary for the meristem to maintain maximal mitotic activity. Below this level the rate of cellular turnover through mitosis is minimal. Above this level the rate becomes dependent on other limit- ing factors. . Thus we can inhibit mitosis without effecting respiration but cannot limit respiration without effecting mitosis. The response to sugars suggests that the meristem can better utilize certain carbon sources than others. This is reflected in the mitotic response curves. , Osmotic affects were ruled out because the system did not respond mitotically to galactose and mannose (N. Knobloch, unpub- lished). The fact that fructose gave a greater response than glucose might 41 ”I" uncle Figure 9 The mitotic cycle. 42 indicate that exogenous glucose is converted to fructose in the cell and that the enzyme,~ mutase, is slightly limiting. DL-glyceraldehyde and ribose, on the other hand, do not give as good a response during the first 12 hours. This might suggest that the major utilization of carbon source is not via the classical Embden-Meyerhof pathway. Considering this possibility and the fact that RQ measurements fluctuate about 6 hours after glucose addition, I performed the following experiment. ~ I incubated twenty 21 mm. excised rundown tips in each of eight vessels. - In two vessels were tips plus Cu-l-glucose for six hours and another two had Cu-6-g1ucose for six hours. The remaining four vessels had the same substrates but incubated for 12 hours. The respired C1402 was trapped in KOH, isOlated as barium carbonate and counted in a Nuclear-Chicago gas-flow counter. Table VI gives these results. Table VI. . Counts per minute of BaC14O3 isolated after incubation of 2 mm. excised root tips for 6 and 12 hrs. in CM-l-glucose and C14-6-g1ucose. Time is in hrs. after glucose addition. Time C14-1-g1ucose C14-6-g1ucose 6 360 l, 000 12 2,220 1,913 These results might be interpreted as demonstrating a difference in meta- bolic pathways between the first 6 hours and the 6-12 hour period. The Cu-l—glucose is not contributing to CO; formation as fast as the C14-6- glucose during the first 6 hours, but during the last 6 hours the CM-l- glucose is contributing more than the C14-6-glucose. At first glance it would seem that the Cl/C6 ratio is a clear indi- cation of the primary respiratory pathway. For example, if the ratio were 43 one, then glycolysis and the Kreb's cycle would be implicated, i. e., the C-l-carbons and the C-6-carbons are equilibrated in the reaction involving triose isomerase. . The C140; should appear at about the same time from both radioactive glucos es. . If the ratio were greater than one, the hexose-monOphosphate shunt would be implicated. The C-l-carbon of glucose is oxidized to CO; in the first few steps and thus CMOZ would be produced. sooner if the shunt is the major pathway. Tempting as this point of view may be, it should be noted that Katz and Wood (1960) have shown that the Cl/C6 ratio is not quite so simple, but instead is very complex. They indicate that the following things must be considered in interpreting these ratios: 1) Rate of utilization by the Embden-Meyerhof pathway; 2) Rate of utilization by the Pentose Cycle; also the amount of cycling; 3) The utilization of glucose in other pathways; 4) The amount of carbon-three of triose phosphate which is oxidized. The above considerations, with the exception of number 3, do not account for the low activity in the C14-1-g1ucose during the first 6 hours. This might be explained if during the first 6 hours much of the dihydroxy- acetone formed by the fructoaldolase reaction were shunted into the synthesis of some cell building material such as glycerol. You will remember that Biesele (1958) indicated that the meristem has a large amount of lipid present. It is interesting to note that both Katz and Wood (1960) and Tepperman and Tepperman (1961) found radioactive glycerol formed in tissue given CM- 1-glucose. . The increase in C1402 produced by the CM- 1-glucose vessels during the last 6 hours could be explained by an increase in the hexose mono- phosphate shunt. Although. Katz and Wood's precautions must be kept in mind, I think we can safely say that this experiment does indicate a shift in metabolic paths somewhere around 6 hours after glucose addition. In conclusion I believe that our data puts us somewhere between the views of Bullough (1955) and Gelfant (1960). . Our system is conparable 44 to the mouse ear epidermis in that it consists primarily of a mitotically competent population of cells. In our system, I believe that at least a part of the antephase cells require energy plus some other series of sub- stances before they can proceed into mitosis. SUMMARY 1.. Preliminary experiments Show that the mitotic response to 1% glucose in 2 mm. root tips is accompanied by a small increase in oxygen uptake and a respiratory quotient of one or greater. They also show that exogenous carbon source is not utilized to a large degree until six or more hours after excision. 2.- Experiments in which various carbOn sources were tested against the 1% glucose control, showed that fructose is utilized as well as glucose or better, both mitotically and in oxygen uptake. Ribose and DL-glyceraldehyde, on the other hand, did not give as great a response as the glucose control. 3. Respiratory inhibitor studies demonstrated that potassium cyanide and 2, 4-dinitr0phenol inhibit both respiration and mitotic activity. Two concentrations of potassium cyanide showed a differential effect on respiration but a common effect on mitosis. 4. Mitotic inhibitors Show a mitotic effect but no effect on respira- tion. Actdione inhibits the onset of prophase but does not inhibit oxygen uptake. . Colchicine causes abnormal mitotic figures in that the spindle is effected. . Colchicine does not inhibit onset of prophase or oxygen uptake. 5. An experiment using CM-l-glucose and CM-6- glucose showed that there is fluctuation in pathways about six hours after glucose addition. - It is speculated that during the first six hours dihydroxyacetone is shunted into glycerol synthesis. In the last six hours the hexose monophosphate shunt seems to be the primary metabolic pathway. 6.. In conclusion, our data seem to show that the meristem requires a certainlevel of oxygen uptake before mitosis can take place maximally. In addition, inhibition of mitosis does not effect oxygen uptake. 45 LIT ERATURE CIT ED Amoore, J. E. 1961 a. - Dependence of mitosis and respiration in roots upon oxygen tension. Proc. of the Royal Soc. B 145: 109-129. Amoore, J. E. 1961 b. Arrest of mitoses in roots by oxygennlack or cyanide. Proc. of thepRoyal Soc. B 154: 95-108. Biesele, J. J. 1958. . Mitotic poisons and the cancer problem. Elsevier Publishing Co. New York, N. Y. Bullough, W. S. 1952. The energy relations of mitotic activity. Biol. Rev. 31: 133-168. Bullough, W. S. 1955. Hormones and mitotic activity. Vitamins and hormones 13: 262. Clowes, G. H. A. and M. E. Krahl. 1937. Studies on cell metabolism and cell division I. On the relation between molecular structures, chemical properties and biological activities of the nitrophenols. Jr. of Gen. Physiology _2_0_: 145-171. Erickson, R- O. 1947. Respiration of developing anthers. Nature 159: 175—276. Erickson, R. O. and D. R. Goddard. 1951. An analysis of root growth in cellular and biochemical terms. Growth Symposium-)3: 89-116. Esau, K. 1953. Plant anatomy. John Wiley and Sons Inc. New York, N. Y. Fruton, J. S. and S. Simmonds. 1958. General Biochemistry. John Wiley and Sons Inc. . New York, N. Y. Gelfant, S. 1960. The energy requirements for mitosis. Annals of the New York Acad. of Sciences 105 536-549. Hadder, J. C. and G. B. Wilson. 1958. . Cytological assay of C-mitotic and prophase poison actions. Chromosome 2: 91-104. Hamburger, K. and E. Zeuthen. 1957. Synchronous divisions in Tetrahlmena pyriformis as studied in an inorganic medium (the effect of 2, 4—dinitrophenol). Exp. Cell Res.\ _1_3_: 443-453. 46 47 Immers, J. and J. Rummstriim. 1960. Release of respiratory control by 2, 4-dinitrophenol in different stages of sea urchin development. Dev. Biology _2_: 90-104. Jensen, W. A. 1955. A morphological and biochemical analysis of the early phases of cellular growth in the root tip of Vicia faba. Exp. Cell Res. _8_: 506. Katz, J. and H. G. Wood. 1960. The use of glucose-C14 for the evalu- ation of the pathways of glucose metabolism. Jr. Biol. Chem. 235: 2165-2177. Mazia, D. 1960. The analysis of cell reproduction. "Second Conference on the Mechanisms of Cell Division. " Annals of New York Acad. of Sciences 22.: 455-468. . Morrison, J. H. 1958. The effect of monoiodoacetic acid on cell division. Xth International Congress of Genetics. Vol. II. Merck Index of Chemicals and Drugs. 1960. 7th edition. . Merck and Company Inc. Rahway, New Jersey. Miihling, G. N., J. Van't Hof, G. B. Wilson and B. H. Grigsby. 1960. Cytological effects of herbicidal substituted phenols. Weeds _8_: 173-181. Robbie, W. A. 1946. The quantitative control of cyanide in mano- metric experimentation. Jr. Cell. and Comp. Physiology 21: 181. Scholander, P- F. , C. L. Chaff, S. L. Sveensson and S. J. Scholander. 1952. Respiratory studies of single cells 111. Oxygen consumption during cell division. Biol. Bull. 102: 185-199. Stern, H. 1956. The physiology of cell division. Ann. Rev. of Plant Physiology 1: 910114. - Stern, H. , and P.- L. Kirk. 1948. The oxygen consumption of the microspores of Trilium in relation to the mitotic cycle. Jr. Gen. Physiology _3_1_: 243. Swann, M.. M. 1954. The control of cell division. Recent Develop- ments in Cell Physiology. Vol. 7 of the Colston Papers. Academic Press Inc. New York, N. Y. Swann, M.. M. 1958. The control of cell division. A review of special mechanisms. . Cancer Research _1_8_: 1118-1160. 48 Tepperman, J. and H. M. Tepperman. 1961. ,Metabolism of glucose- 1-C“ and glucose-6-C” by liver slices of refed rats. Am. Jr. of Physiology 200: 1069n1073. Umbreit, W. W.,. R..H. Burris and J. F. Stauffer. 1957. Manometric Techniques. Burgess Publishing Co. Minneapolis, Minn. Van't Hof, J. , G. B. Wilson and A. Colon. 1960. Studies on the control of mitotic activity. The use of colchicine in the tagging of a syn- chronous population of cells in the meristem of Pisum sativum. Chromosome 11: 313-321. Warburg, O. 1955. On the origin of cancer cells. Science 123: 309. Weinhouse, S. 1956. On reSpiratory impairment in cancer cells. Science 124: 267-269. Wilson, G. B., J. H. Morrison and N. Knobloch. 1958. Studies on the control of mitotic activity in excised roots I. The experimental system. Jr. of Biophysical and Biochemical Cytology 5: 411-420. Wilson, G. B. and J. H..Morrison. 1958. Mitotic activity and behaviour as an index of chemical effect. The Nucleus 1: 45-56. Wilson, G. B. and J. H.. Morrison. 1959. .The mitotic cycle and the ontogeny of neoplastic growth. Cytologia _2_4: 43-49. Zeuthen, E. 1960. Cycling in oxygen consumption in cleaning eggs. Exp. Cell Res. _1_9_: 1-6. 3,1) [ESE 1,111.1 um Jet—44853— “11111111111111'1111115