METOCi-{ONBRIM STUDIES OF HUMAN SKéN CELL CUBE-RES HQFECTEfi WSTH VERRUC‘Q VIRUS Thesis for the Degree 0f M. S. WES-113.13,?! SM’E‘E UNIVERSIYY PETER {3!CUHE WAMKE 2957 LIBRARY “'3 Michigan 3 care University ad E‘._Ik 1', AB STRACT HITOCHONDBIAL STUDIES 01’ HUMAN SKIN CELL CULTURES INFECTED WITH VEBRUCA VIRUS by Potor Gicuhi Uaiyoki Thin otudy wao dolignod to invoctigoto tho ottoctn of vomca vino on tho mitochondria of human akin colic and particularly tho total mbor or litochondria in tho colic. Tao colic wot-o prodncod in two typo: of lodia but tho com mtriont no tho canoe who oourco of vol-moo viz-no no from wart than and tioouo stored in 50 por cont phon- phato httorod glycol-inc. Morphological and cytological . Itndioo "mitochondria from infoctod toot cons and unintoctod couo woro donoo Conn woro hcuogonizod and thou fractionatod icing difforontial contrimgation nothodo. i‘ho mitochondria woro idontitiod by thoir ability to stain with low ccncontra- tiono of Janus grooa B. Comparioon of tho aorphology of mitochondria iooiatod tron tho con- and thooo noon in tho intact col].- appoarod oinilar. Mitochondria from inroctod cont uoro Mini: tilanontou. olongatod or nightly much. Mitochondria tro- uninfoctod «u- ooro mainly rodliko. Intoctod colic. roux-dioc- or nodiun. containod uoro mitochon- dria than tho unintoctod coils. MITOCHONDBIAL STUDIES OF HUMAN SKIN CELL CULTURES INFECTED WITH VEITBUCA VIRUS 3! Potor Gicuhi Uaiyaki A THESIS mbnittod to Michigan stato Uhivoroity in partial fulfillment of tho roquiroaonto for tho dogroo or MASTER OF SCIENCE DcpaMont of Microbioloy and Public Hoalth 19 67 F. ‘——-— c/‘\ DEICATION mic I'ork in dodicatod to my parents. 11 ACKNOWLEDGE! ENT 8 no author will" to oxtond hia hoartfolt approoiation to Dr. Haltor N. hack uhoao guidanco. pationt nndoratanding and con-taut intoroat aado thio invoatisation pouiblo. Thank- aro orprooaod to Dr. Virginia hall-anti and Dr. Richard mocha for holpi‘nl moationa. Sincoro thanka aro orprouod to follow andtato atudonta for thoir participation in atinnlating dimooiona in tho tiold of microbiology. ill INTRODUCTION 0 o a o o REVIEW 0? LITERATURE o MATERIALS AND MEIEODS. BENLTS. a o a DIWSSION o o SUMMARY. a o . BIBLIOGRLPHI .- O 0 TABLE OF CONTENTS iv Pogo 26 35 51 57 58 Tablo l. 2. 3. a. 5. 6. LIST OF TABLES Pago lworago mmbor or aitochondria par gran of m calla, nnintoctod or intoctod with from wart viruo. Cultnroa woro maintainod in yoaat oxtraot nodinnande‘calraorma............. ”5 Averago nnnbor of mitochondria por gram of AD colla, nninfoctod or infoctod with glycorinatod wart viz-no. Culturoa woro naintainod in yoaat oxtract nodinn and 2% 0.1! “Mo o o o o o o o o #6 Aworago nnnbor of mitochondria por gran of AH calla, nnintoctod or infoctod with from wart virus. Culturoa woro naintainod in Eagloc' baaalnodinnandZScaltoom. I ooo ooooo 1*? Avorago nnnbor of mitochondria por gram of AD calla, uninroctod or infoctod with glycorinatod wart wirno. Culturoo woro naintainod in Eagloa' basal nodiun and 25¢ calf worm. . . . . . 1&8 Aworago nnnbor of mitochondria por gran or AU colla aoodod with glyoorinatod wart natorial hoatod to 56°C for 1} hr. colla woro grown on basal nodimn (Eagloa) and naintainod in 2% oalfoorun.................... 1‘9 Aworago nnmbor of mitochondria par gram of AU colla aoodod with glycorinatod wart natorial hoatcd to 56°C for 5 hr. calla woro grown on yoaat oxtract nodima and maintainod with 2,1 caltaorun.................... 5° LIST OF FIGURES Figaro Pogo 1. Normal AU calla stainod with honotoxylin and oooin. leJanoooooooooooooooo 36 2. Mitochondria in normal control colla vitally wtainod with Janna 313.011 Ba XIIOOa o o o o o o 37 3. Mitochondria in infoctod cello vitally otained with Janna groan B, 5 daya poat inoculation . . 38 vi INTRODUCTION Tho human wart virus fros vorruca mlgaris, a asabor of tho papova group. is rolatod to polyoma and papilloaa virusos. Tho virus is of consddorablo intorost as it is tho only virus known to produco tunors in nan. mo tumors aomotinos booomo malignant. Tho vorruca virus is structurally similar to tho polyoaa virus which producos malignant disoasos in animal» 'i‘ho viral causation of nalignant tuners in non is at ' prossnt a thoory and roots only on indiroct swidonco. no but basis for this boliof lies in tho undisputod rolo of wiruaos in malignancios of aniaals. It is thsrsfors difficult to ooncoivo that tho human opocios would to so uniquo as to oscapo intootion by yirusos which produoo tumors. Infarction of a call with a virus is mifostod in many ways. Tho offoct nay bo obsoryod in tho coll. tho organ and tho oonploto animal. Tho aost froquont aioroscopio rosult of viral infootion is nscrosis and dogonoration. howowor. this nay not always to obssrwod. Also. noorosis and dogsnoration aro tho final romlts of infoction. Infoo- tion of a call by a virus producos changos. sono of which aro not oasily dotorainod and nay roquiro spacial nothods for thoir dotoction. than virusos ponotrato mitablo calls thoy wultiply at tho orponss of tho colls. Sinoo virusos lack tho full physiological and biochooical sndownsnt of colls. thoy aro unablo to gonsrato tho snorgy roquirod for thoir aultiplioation. consoqusntly thoy 41on on tho ansyno l 2 syncs of tho infootsd oollo to oupply tho onorgy. rho living call is a vary coaplax unit. it is tho fun- domontol unit of anion all living amino aro mado. i'horo aro many typos of calls and tho calls of tho brain or tin tisno aro difforant in aorphology as wall as in function. Dospito tho diffomooa all calls havo aovoral things in ooooon. i‘hoy havo a call mombrano. a cytopla- containing various ormallas and a control nmolaos. In addition to having soao dofinito otruotnros. aolls havo a aumbor of fan-- tiaool oapaoitios in oomsao. no protopla- of living calls has too. studiod ozton- aivoly tut nah romains to ho andorstood. that was originally ooaaidorod a otrootoro composod of am «tonal mubrono. a aytopla- and a control mmcloas has boon m to ba difforon- tiatod into organsllos adaptod to carry on tho many divorsa prooossos of lifo. With tho aid of tho alootm microscopo. ooll biologisto havo bogun to disoorn tho molooular activitias of tho parts of tho systu. In roasnt yoara, thoir work has “You“ with that of tho bioohuista who havo traood oomo of tho pathways by which tho coll oarrios out tho bioohasioal roootioas ohioh undarlia tho prooosaos of lifo. Ono of tho oost important componanta of tho coll aro tho aitoohondrio. rhoy aro tho sitos of oridativo raaotions that provids tho call with tho onsrgy it noods for ito ootabalio and synthatia roootiona. rho aitochondria aro pros-at in all living calls. both animal and rogotablo. Thoy aro tho “mar. planto' of all lira. Each mitochondrion is adaptod to its 3 function by a fine structuro. Thay attract anargy from tho ohomical bonds in tho nutrionts of tho call by oxidation and rospiration. Tho anorgy is gonorotod in tho form of tho aompound adoncaino triphcsyhato (ATP). ll‘ho ATP in taro storos tho snargy produood in tho mitochondria and applios it to tho coll whoa noodod. It is also known that tho mitochondria aro an outta-cl! sonsitiva indicator of call injury. It was thorofcro daoidod to study tho offocts of varruca virus on tho hast-mall mitochon- dria. particularly as tho virus affoats tho total motor of oitochondria in skin ti sooo collo ondor various uporiaontal conditions. Othor studios inalodod tho cytological ad acrphological aharaotsriaatioa of tho mitochondria ban: in tho intact calls and in tho isclotad stats. REVIEW 0! LITERATURE no u t w i a Common warts nay bo soparatad into four voriotios (l). l. Var-moo vulgar-i a-tho roisad warts froquontly obsorvod on tho hands. 2. Vorruoa plana Jwvonilis which aro anally found on tho hands and toes of childran. 3. Vorrmoa aouminato which aro pointod filiform and caaoo gonital losions which nay bocoao aalignant. b. hmgoal papillomata. warts may occur also on lips. in tho nostrils. in tho auditory noatua or on tho odgas of oyolids. root (2) dosoribos tho oomaon wart as a boaign tumcr which is anally aoopoaad of a motor of papillao comprising horny soalas and clastorod in patchoa. It may to flat and tho papillaa inconspicuous or it may rossmblo a tuft of hair- liko proioctions clustorod on a stalk. It may havo a oanliflowar- lira complarity. rho papillaa may. in coma instsaaoa. to fusad into a hornliko proooss. attaining a contimotor or maro in longth and diamotor and projoot from a ouppod doprosoion in tho skin. warts diffor from aalignant oanaors in that thoy grow outward froo tho urfaoo of origin. whila consors invariably grow inward (3. h). warts aro oxtrooaly hard duo to axoassivo taratinisation of tho opidoroal calls. havo a moth margin and aro soaatimas mirroundad by an orythsmatouo halo. It is in thoaa typo of warts that hunting at al.. (5) roportod that tho wart virus is ooamonly found. I} 5 It is not know man tho infootiva naturo of warts was first disoovarod. For many yoars pooplo havo known hast warts can sproad by contact and it is furtha‘r known that warts can disappoar upon soggostiou or simply by boing winod away avon though no soimtifio ovioouca oxists toniggost that this ispossiblo- rho first orporiaonts ropcrtiag tho infoatioos naturo of warts woro tho-a of Jadasuhn (6). his uporiaonts. boson in 18” consistad of a. soriaa of,iutrodormol iniootiono. of ground up wart aatarial or wort tisua fronoats into both himsslf and his assistants. tarts roultod in 31 out of 7! sitos of inoculation. m. domonstrating that warts woro tron-isoiblo froa man to non. no ~mado‘no att-pt to oaplaia tho otiologioal factor. rho viral oaturo of tho agont involvad was d-ustratod. also ciuffa (7) produoad warts on both his hnda and thoso of tho patimtslfru whom tho warts had originally boa rucvod. rho nonstald l filtrotos of utrootod wort tiono wars and and oftor 5 months incubation poriod. warts appoarad on both sabiocts. Ciuffc's findings not only aonfirusd thcso of Jadasoohu but also doooastratad that a aoll-frao filtrata of wart matarial was isfaotious. aorra. in 1908 confirmad tho filtorability of mo wart agoot. ihoso findings woro farthor continual! by Vila and Kingory (8) who woro ablo to iaduoa warts by tho introduction of coll-froa filtrotoa. gingory (9) roaovod an arporioomtolly produood wort and aftor filtoring tho attract. roinoculatad anothar voluntaar. 6 phenom osaih a not moth.‘ Goldsohaidt and aim (10) ropsatod thoso pi-ooodnros sod woro unstooossfol in producing mt. nth thoir taunt-c In all tho trunnion motion” in man hoinss'. a long incubation pol-ion. 5 aonths or ooro. no wind hoforo tho appoamoo of' a wart moth- In host ihotuoos tho Isa-ts roarossod shortly aftor thou oppooi-oooo. h 1953. aims (u) roooood o not fun his fine». mouatoa a tooth ooopohoioo of tho hooosooilod "Moons notoriol onto tho ohorioauoatoio outrun of 10 day old ohioh m. Hhitim “poatll moths apputod ca tho oho- rioonohtoio ooohmoo aftor oovorol on. imhotiom This «an. m mooosmny trust-m. in ocso with Iatoi'ial filtorod W o Balaton tonal» no ooaoludoa tho out tins mood tho aaooivo proliforatioo ooo 'poari' for-anon. , and (12) found that tho “out isolatod by livias us a straia of a oohory Mill. sisal ooo Nooy (1)) woro mausito noooosfuny onl- tivato tho viral aunt mm "moo “Maris ma ooodylooa ooo-imam in ton on. ohorioauutoio ammo of tho ohiok utoyo on! in tisuo suitor" of Hons sad aoolzoy hidnoy oons. sis“). and hovy's findings confinoo that of man: (it). fold!» (1t) scold not inlato tho virus hy tho iaooolatioo of tho ohiok onontoio nonbmo with ostoriol froa ootanooos losions. V ‘ rho first apart of tho mound isolation of tho u‘rt wont no in 1961 by haidson and nignaa (15). i‘ho 7 honan wart vii-as us noossofhlly isolator! in nonhsy kidnoy timo oulmro oollu Inooolation of honor: volnntoors oith oolhfroo nutriont fluids frol- tho onltnroo prodnood wart growth in tho okin of tho voluntoors. .In tho mo you. Hoyaohi (16) in on! lahorotory roportod tho isolation ond oorisl pmoaatiuofthooortasohtinonltnrosofnoml hush Irina this osont III nontrolixod by undilntod homol- ogous patio-It's sol-on ond by honan s-na globulin. stronss ot ol.- (17, 18) mind oonoontrotod oxtraots of out tisoooo moor tho olootron niorosoopo. 'crystsllino' viroo-liko partiolos fro- okin papillonas woro sharootorihoo by utmolou inolnsion hodios. 'iho om out and nous). akin propantions am not outnin unifon partiolos hut aoroly ”who“ ooottoroa claps of nottor. oolloson fihors on! ophorioal oioos of varying disnotor. 'iho partioloo in tho oryotauins an-oy owns“ 5! n' in am. as find- inns of Strauss ot ol. ooi-o not oonfimd ty Biocol (12). no lattor. ooins a tofinod oonoontration «ma. fun! a lot-so may of partiolo oiloou u out of tho pnporations. unifon 16 an tins-lito partiolos non ohsoi'ooo ond Siscol hypothosiood that thus night to tho wort scoot. Tho olootm nioriosoopo hao playod a najor rolo in oooooroh dooinod to intostisoto tho dovolopnontof tho tins in art tiomoo Alaoida ot ol. (19) found that tho virus is fouod in ossooiation with flu nooloi of oolls of tho strata opinosnn. Vino partiolos gradually sproad tin-Mont tho moloi of tho «lit in tho strum moon sod porsist as 8 oloso-pookod nggrogntos on'ooddod in tho mhstonoo of tho otutun ooo-non. This oomlatoo to tho devolopnsnt of hambilio intramoloni' inclusions sun with on. light nioroooopo. ‘ Elootron niorosoopy otudios oloo stoma thst. tho oosino- philio intmoloot inolnsions dosorihod by atmss st do (20) woro tho product of annual horotinization thot ooonrs innotoolloondworoinnotoyrolatodtothotims. rho otsomtions of Lipsohutn in l9”. hlont ot ab (21) and Alwyn (22) all uhotutiatod thoso «mito- - ‘ mam st .1. (19) upon-a um um. portioloo can only“ oo‘oninthooytoplanoftorthoyoororoloasodhon tho melons by dimption of tho nooloai- onto-ooo. no soon-not of dovolopaont of tho hum tort virus was sinilar to tho dovolopnont of empo- min-u virus in rabbit papillo- noon forth”. no opooifio portions ooold to dotootod in proliforsting oollo that woro moi-going connotation ooo noox'ooio. It is not m mothor tho vino ooo prooont in thopooliforotingoollsinsono fonthatoooldnothoroo- ognizod in tho olooti-on aiorosoopo or mthor tho notion of no virus in uniting proliforotion too on indiroot “I“ 31001! ofi am (233‘ found oollo infootod with tho hm, wort. vino oontoinod a grootor wt of dooxyrihomoloio aoid (Du) thon aorta). gm «11o. Tho narkod intrmoloar synthosisofnmoosfoonatohoginoorly inthoooni'soof infootion of coils of 10"! stratum oalpighii. In tho oorly phaoo of infootion. on ooidOphilio intronnoloar inclusion 9 body was magnizoblo. i‘ho inclusion body ond nuclouo than onlorgod and was folloood by pmgroosivc disorganization of tho nuclear structure. Tho inclusion body booms baoophilio End otoinod- with tho fonlgon otoin. Finally tho nuolouo diocppcarod leaving tho inclusion body in tho coll romant. n . rslotimy lato otogo. oll tho oollnlor mm woo rolocotod in tho inclusion body and did not significantly incroaoo in onount thoronftor- , willimno ct o1. (2t) undortook studios dooignod to chor- octorino tho wort vix'uo morphologically. Tho mcloi of nous coll: in tho gronulnr loyor contninod uniforn portiolos of noon diamotor 46 nu. Host of tho porticloo oonoiotod of dense can of dionotor 35 no surroundod by a loss donoo lnyoi- of width obmt 6 on. In oono. tho control rogion no icon donoo than tho poriphory. it no tino woro virus particles noon in tho cytoplonn of any of tho collo. Tho atoll oopsid of tho partials was found to consist of oz «poo-mo orrangod in o 5:332 axial ”not". no nontox- ond ormgonont of tho copoonoroo on tho tort virus was ostablimod to be tho sono no that found on tho polyona vino (23). It is of intoroot thot tho wart Virus mien couoso bsnign tumors in m is structurolly similar to tho polyono vino‘llhich produces a lids opocti'un of nolimiont tumors in oovorol opooioo of oninols. ‘williamc (25) uood tho ooridino crongo toohniquo to dononstroto DNA nod ribonucloio noid (mm) in vol-moo vulgaris. Tho DNA linkod to virol protcin woo rooiotont to dooxyribonu- cloooo (Orinoco) toast-ooo DNA in nonal oollo no roodily lo dogtndod. In tho human wort tho epidermal cello containing muooo resistant nuclei appeared to bo oituotod in areas in which vii-no particles woro found. - This lod to tho conclusion that one ml in tho intronucloor inclusion bodioo of human opidomol collo infoctod with tho wart virus it o nuclco-r protoin. Magic (26) in invostigntions cmiod out in our laboratory. infootod human thin coll culturoo (A0) with tori-non Virus. Bo:- rooultn mowod that within 21) to 158 houro in colic infoctod with yorruoa viruo contained intrnnuolocr inclusiono of on» The inclusions resistod moval by DNiaso unlooo they woro first digested with popsin. ~ it no tino woro nich inclusions monoptiblo to ribonuoloooc digestion. Buncoll (27) in our laboratory found conplmont fixing antibodioo specific for tho wort ogont in potionts’ corn. Undo: thcoo circunotmcoo tho wort agont and antibodioa oppooi'od in tho some individual. Almcido ond Goffo (28) dotootod antibody to hunon wort virus by immio oloctron nicroocopy or ogmgol procipitin tests using antigon cztrnotod from torts. Antibody one dotcctod in about half a series of #2 pntilhtt filth torts. Boycohi (16) could propogcto tho wort agent only by coll to coll tronofcr. Dilution of corn or globulin did not provont infoction by tho wort vii-no indicating that large soil-onto of antibodioc woro nccoooory to provont infoction of nonol coils by tho out agont. Attonpto to ostoblioh tho sgont in nonksyo, rabbits, hunters. mics, and chicken woro not oucoosoful. By oloctrcn microscopy ophoricol particles ll are observed in extracts from wart ti 55:19 and infected cul- tures which woro not proaent in normal akin cells. The oizo and range of thoso particles varied from 50 to 75 mu. Tho particles woro not uniform and no particloo of any one also woro predominant. Lyell our! 111ch {29) found oooinophilio. intraoytOplaaio ond intramzclcar inclumon bodice: in some wan-to. They foumi both type i wart and typo 3 wart and mooted tho nono “Von-ooo. wilgaria' bo confined to typo 33' warts and that 15pr 1 be Wed 'Mymooia.‘ Cram!!! and croutord (30) studied the nucloio aoida or polyona. 310p. rabbit papillma, bovine yapilloma. oanino papillono and human papilloma vimooo. with olootmn mioroo- cap: and malytiool contrimgxtion. . who polyma vino diffuod in axe and moloio acid proportion: from tho papiiimm Virus“. be four papillm vii-ooo: woro similar to each other in mm: roopooto but differed significantly in tho base composition of their moloio acids as indicatod by different gmnina- oytooino ratios. By oioctron microscopy Noyes (31) studied tractiono of tho human tort virus from We obtained by sucrose density grodiont contrimgntion. iiotorial obtainod from hand 3 consisted of intact particles with roll prooorvod structure. ‘mo hollow tubolar oopmoroo that studded tho our-taco of tho portioioo moored about 802 in diamotor and projoctod mm 56-50:; In addition, bands 3 and. It contained some bizarre. olongatod, enlarged dumb boll fomo whose mrt’aoo 12 no compoud or ocpoonoroo typical of thooo cmring tho porticulato {on of tho wart vino. It ooo manna that tho call tingliko “motor“ woro dmlopoutal tono of tho human wart vino. w A Boy“ (32) studied tho oytOpathio ofl'oct of tho vino. no primary bman lam-tonic m: M aooclo coll almo- Appmxiutoly 107 to 108 mm: m vimopartioloo woro roqnirod to initioto tho oollular chanson-a addod to a coll monoloyor in a Loigaton taboo Spootonoono rocroooioo or actually «curios anon onto oooally ooouro at poborty m. It hao not boon dotoraiuod moth“- thio io duo to antibody romtion or othor oonooo ouch oo local timo rooiotonoo. auto (33) M01306 Winona to oxploro tho “too-quoted that: at char-ins ooo-ta ovoid no toundno tool mom thot aagio or motion boo any ortoot on oat-ton no opocnlatod that dronotic coroo ooootiaoo roportod woro probably too roult of» tho nagio ritual coincid- ing oith tho out! of tho ooo-to natural nu. 0nd o i‘ho mono canonization ohondriooo oabraooo all m granular (mitochondria). filaontouo or rodmopod (choc- _ driooonto). cytoplanio bodioo that aro round in all animal «11o. both tortobuto and invortobroto. and in all plant collo. According to county (31!) tho toninoloa appliod to aitoohondrio io rathor ooaplicatod. at looot so ditroront moo havo boon mood for thio organollo. In tho Anglo-8am 13 oountrioo tho none mitochondria la and. both for tho ontu'o group of orsaoonoo. ohondriooomo to ouch ono of tho: ond tho moo mtoohondria ma ohondrtooont to tho groanin- out! fllononmo toms reopootzvoly (35). 10:1on Loon (36) “ported that tho mitochondria: were oloo oollod photoomn. plot-notation. phmoxon, pox-autumn. oaorooonon Moo'ond (flaunts. - ‘ A mitochondria no: bo morphologically donut .- o coll orgonollo hounded by o nacho-ooo within which oro ooobronouo otmoturoo (oz-iota) otthot ammo or "donor. o around “bow or annular matrix ond monououy. don-o m1.- ttono. mummy. ottoohondrio no: bo doflnod oo bioohon- xoaz mohlnoo man not oo-tho Won-o at o oou (3?). 73": oro oharaotoruod by 1: ”not of. phyuooohuion M nono and otolnlng proportlooo' m tho noro important prop- orttoo aro. thou- nobility in Into. thon- mountol color- tioo with staino ouch o-o Jomo soon a. tho moat: to: opootol toohntqooo for nation for thou- prooomtton. thou upoputou mpoutuu. thou Wino ultrootmtnro one thou- my: *moootrotioo of carton: «you (35). In 1921. color! (31») upon-a thot nacho-anal m- m to quolitotho. mutual” ooo/or topomphtoolo alias to tho can on: at tho “woman (mm 0.21: to 0.3m 1mm 0.35: to 0.5o) and mo 1mm of tho rooolntton mo: of tho light “ammo. «ontotuo aha-goo on only to «mm in option otoroooopoo to o for oritorto. nth tho odmt of tho olootron oloroooopo, 1t hoo mono manual: lb easier to obsorve the mitochondria. and mitochondrial ohongoo non be donned in o more diversified ond rolioblo man. The mitochondria are ubiquitous in living dolls and “to: some ezrperienoo they can oaailybo recognizod niorooooP- loony. The examination of mitochondria in living oollo oflorl certain dittioultioo owing to thou-low rotmtivo indox. thoy m clearly visiblo with dork riold illumination ond phooo contrast. Vital omination io- extremely inpoflant in tho study of the variations of tho oitoohondrio with tioo out! undoi- the action or different otinulio mo oitoohondrio oro no”: no conspicuous as tho melons. moy our: grootly in size..mapo and position (3h. 36. 38). In tho living coll. thooo bodies aro oontirmoudly changing also, ohapo and pool.- tiom Louis and Lewis (36) oboemd on am no 20 ditrorant up“ exhibited by a singlo nitoohondrion within o poi-lad of is nimtoo. Qualitativo ohongoo (39) mt only involvo vario- tiono in size and mnpo of tho wholo nitoohondrion but oloo ito ultrooti'uotui-o. Robot-til ot a1" (35) ototo thot on. mitochondria hon too difforont typoo or motion. ono of ositotion ad no othor of displacement tron ono port of tho ooll to author. studioo of mm oollo by ooono or moving picturoo tokon oith tho phooo nioroooopo lion thot mitochondrial nomonto ooo duo to oytoplamio anti-onto. Tho mitochondrio mo oonotontly during intorphaoo but ouch novomont boomoo dininimod during coll division. hodoriok (ha) using phooo controot ulcroooOpy ond niommmtomphy in tho in vitro otudy of oulturoo of 15 fibroblooto. woo oblo to occurotoly follow tho novonoht of tho nitochondrio in tho rooting oo ooll oo in tho dividing «no. During tho dioplocooont. tho nitochondrio ooy ottoch to o oortoin oito on tho nucloor ooobmo. Mitochondria tilononto frog-oat into gronuloo ond thooo aro in turn unitod togothor (36). It hoo oloc boon oboorvod thot oitoohondrio inoroooo in oioo in hypotonic oolutiono ond dininim in hyportonio oolutiono. Zoninoor (kl). nor-on (t2), Rocuagol ond moood (b3) ond Cloudo (M) oboorvod thot ioolotod oitochondrio hohovo oo o-oootoro choosing in .180 with tho tonioity of tho nodiun. noir ooouino oorvoo no on only oign ct coll danaoo. Bouillor (39) boliovoo thot ohilo thio orplanotion io corroct in oooo ooooo. it oonnot hocmo o oonorol rulo. i‘ho oitochondrio oro vory lobilo otruoturoo ond con ho oltorod by tho ootion of voriouo ogonto. “no ond Lolio (36) found that olkolio. monio goo ond oodiuo hydroxido ooooo tho oitochondrio to soil without ony oin or vioccoity. Chloro- rora. xylol. othor ond oootic ooid diooolvo tho oitochondrio, Jonuo noon B unlooo uood in loo conoontrotiooo or kimono... 13200.000 io toxic to tho collo ond oouooo tho mitochondrio to divido into gromloo. 'ihooo roulto lod mio ond novio to moot thot tho nitochondrio woro oonooinod with tho phyoiolosy of tho coll ond woro prchobly involvod in roopiro- tin. mio mggootion hoo boon oonfinod by morono cytol- ocioto ond biochooioto. 16 Moloy ond Johnoon (80) found thot tho milling of mitochon- drio io quiokonod ond/or ocoontuotod by pH rioo or oddition to tho oodio of voriouo oubotoncoo ouch oo colciuo. potoooiuo. phoophotoo ond mocinotoo. Strolling of oitochondrio con bo oloood by odding odonooino triphoophoto (ATP). citroto. oognooiun. fluorido ond dinitrophonol. ‘i‘huo. tho o-otic phonooonon connot Iholly orploin tho ooolling or oitochondrio. Bouillor (39) ouggootod thot oitochondrio ovoll Ihon thoy looo oooo ATP oo tho lottor prooorvo tho oitoohondriol otruoturoo by fixation of tho ooloiun ion. Thoro oro oony othor oitochondriol chongoo. out of ohich oro irrovoroiblo. arch chongoo orroct: 1. ho oitochon— driol oonbrono thot bocoooo oinglo or io dioruptod. 2. ho criotoo. which through vooiculotion brook up into gronulotiono ond diooppoor. 3. 'i‘ho ootrix mich oomo o roughly grou- ulor. filooontouo oppooronco or oloo io convortod into o-iophilio oooooo. Vitol ond ouprovitol otoining io tocilitotod by coloro- tion with o diluto oolution or Jonuo groon B. In thio otoin tho oitochondrio bocooo groonioh bloc in color. Chooboro (#5) otudiod thio rooction ond concludod thot it ooo not duo to o chooicol coobinotion. Ho oloo round thot Jonuo groom B hod o cooguloting ottoot. Ho ouggootod thot tho otoin ohould not to uood oo o oolo ooono or idontifying oitochondrio. Hormr. Looio and Looio (36) uoing Jonuo groon B otudiod tho oitcchon- drio in living oollo ond found thot ohorootoriotio oitochon- driol mopoo voro prooorvod. but thot tho Jonuo groon B bocooo l7 toxic to tho oollo ortor o poriod of 90 oinutoo. ’ Lozorov ond Cooporotoin (#6) otudiod tho onxyootic booio forthoJonnogroonBotoiningrooctionondi‘oundthotm' roootion woo oxygon dopondnt. thot tho dyo would to roduood by tho lactic dohydrogonooo onoyoo oyotoo. Tho oitcohoodrio toho on o groonioi bluo color Ihioh io lotor‘roduood to rod ond rinolly docolorizotion occuro.‘ hitcchondrio con oiloo ho - oolorod in coll culturo with oothylono bluo but thio color.- tion I" found to ho 130001031!“ by Mflt illninotion (36)- fixo cclorotion with otoino. oich oo croql violot ond toluidono bluo. ooy bo rolotod to no oxidotion-roduotioo potontiol of tho coll. lt hoo long boon moon thot oitoohondrio oro lobilo otruotui'oo ond diolntogroto 'lhon oxpoood to fixotivoo (3|. 35. 36). Pcrthio rooooo. oll tho nothodo uood for thoir rirotion oro boood on tho otobiliootion of tho otruoturoo w tho prolongod ootia of oxidizing oguto sch oo o-io ooid ond potooliun biohronotoo mi! and Mil (36) found tho! tho limo .2)!th oxtroordinory divoroity of i‘oro ottoo io tho noo propontion. ovon in odioining collo of tho oooo typo. tho dittoroot oitoohond'riol up" w to loooliood in “throat pm. of tho cytoplon or ooy bo oixod togothoro Dittorooooo bottom oollo of tho oooo typo oro unolly oboorood during tho voriooo otogoo of oitooio. it tho otogo tho oollo ooy ho oboorvod to hovo oinilorly mopod oitoohondrio wilo in onothor port proo- ticolly oll tho oollo ooy hovo quito difrorontly mopod 18 mitochondria. In em propmtion on gradations in also and mayo ”: bo round. Louis on! Louis (36) morphologically choolflod tho utoohondna at anon gromlos. dub-boll mod gremlins. ”indie-moped granules. redo. throodo. loops. rings and notmrko. Robcrtlo ot a]... (35) contmd that the map. of tho mitochondria. «nu. “flout. 1! bottom: filmmtono or granular. In. no «no. oopoctony dorms functional) otagoo. othor toms thou tho tnouohtouo ~- or granular torn may to non. Thuo. o ohmdrloooht‘ noun c ot mo md to oomo o club ton. or honooo ont ot m Inn to toko tho tom of o tonnu rookot. Tho utoohondrio. at othor thooo no: bocouo "unlu- In oppoorohoo with o control oloor 20110:» no uzo or utoohmdrto 1o oloo ton-tool» In moot «11o tho rodvdxapod mitochondno aro 0.511 to 1:: in Motor om! of Mono longth. rooohtng o mm of 7m 'rho also and Capo or mitochondrto dopond oloo on tho pH of tho nodmo In one! pa thoy boom "noun. Homing w) W {not and otnnod proponttoho of utochondrio out! roportod that thoy ooo: to undorgo Moor: radon. Ho round thot on incl-onto in dzo of utoohondrio. could "out from o cooloo- two of tn or ooro utoohohdno. iho diotnhutlon or utochondrio 1o in xenon). unifor- but thoro oro um moptlono to this rulo (35. 36, 39, t7, #8); In no can thoy no: to oomlotod proforonttou: about tho mclouo or in poriphorol ”tapioca During condo: “not” tho ntoohondrlo toad to oggrogoto about tho oplndlo 19 and upon division of the cell aro diatnmtcd in approxinotoly oquol quantity among tho daughtor cello. Tho distribution of mitochondria within tho cytoplaan should bc considorcd in rolation to thoir function. co morgy supplioro. ~ In ‘oooo oollo they movo olovly but in others thoy'havo a pomonoht location near tho region of tho coll puamably cuppiiod nth «orgy. In oomo collo no mitochondria provido adorinito criohtotinm In tho cylindrical'opitholicl collo thoy aro gonoroliy oriohtod in tho haoocpicol dirocticn' pat-allol to tho main axis (35h Poliiotor (‘09) nuggootod that tho orionto- tion mitochondria cacao. may dopohd upon tho dirootion of tho currents of dimoion within tho oollo and would to intinatoly linkod to tho oubnicroocopio otmtturo of tho ground oytoplaan. Tho mitochondrial oont’oht'varioo with tho-coll typo and {notional atago of tho coll. In-tho moo livor. it hco boon ootimctod that tho aitochondria account for 30 to 35 put out of tho total nitrog‘on. In tho kidnoy it ropro’ooato 17 por cont of tho dry night and 20 yo: cont of tho total nitrogoh at tho wholo tiamo (35). that tho other hand. in lymphoid ticaxo valuoc aro ouch loo’or (50. 51). ‘ In nouoo livor homog- one-too mm 8.7 2: 101° mitochondria po‘r gram or man tioauo havo boon conntod (52). In 9. noml livor coll thoro aro approximately 2500 mitochondria. Homvor. cmoor collo contain fml' mitochondria than nomal colloc In tumor collo. on storago or 786 if 222 mitochondria. por coll woro found (53. 5h). Kmtoon and Son (55) round that in senior!“ hopatm thoro woro rotor mitochondria than in normal cell» Bach coil 20 contained an average of 600 mitochondria. Allard at a]... (51!) suggested that tho anal]. number of mltodiondria in tumor cells could be related to scarcity of organized ergaatmplcnnio structures. Tao intimate relation- cup between mitochondria and tho ergaotroplam is striking and magenta come sort of dependence of one upon tho othoro Accordingly a reason for tho relative scarcity of mitochon- dria in malignant cells may be that they are dependent on tho coexistence of organized crgastroploomic structures and thcro are relatively few of thooo in tumor cello. Do Bobcat-til ot a)... (35) attrimted the fewer mitochon- dria in tumor collo to the fact that there is on incrooco or amorobio glycolyaio with docrcaao in oxidationo in cancer. Junqueira (55) round a direct oorrolntion botwoon number of mitochondria and secretory activity in salivary gland calls. The advances nndo in tho loot twenty year: in thou-standing tho mitochondria have. boon duo mainly to rotinod biochmioal techniques, electron microscopy. phnco contract associated with or without microcinonatography and difforonticl contrimgaticm. such methodn havo: .1. mafia it poosiblo to probe deeper into the ultrantructurnl complexity of the mitochondrial. 2. The biochemical nnturo. particularly its myme content and the role it plays in cellular metabolism. 3. Its mobility within the cytOplaon. 1}. Ito max-o in the tomction of various cytoplasmic components. Ultrastmotural morphological changes involve bioohcmioal variations. Thus, than is a corrolntion between morpholOgy and function. 21 Electron microsecozpy makee the study of quantitative chanzeo more reliable since through pictures of the mitochondrial: it precludes any confusion with other cytOplamic organelles. The concept of quantity doco not only apply to 1 change. in number of mitochondria but also to its components (increaoo or decrease in orietae) and chemical composition. Phase . microscozay and microcinmtography havomado it possible to. study mitochondrial motion and variations in cell shape in relation to physiological and pathological otate of the cello. Claude and Fallen (5?) studied the morphology of ioeletod mitochondria using the electron microscope. The mitochondria that they studied were obtained tron a lymphoeercome of the rat and were isolated by differential centrifugation. In purified tom the mitochondria appeared eo opherical bodies (0.6 - 1.3!: diemotor). Alec mall olononto 80-10011 in dicnetor could be dieting-aimed within the body of certain mitochono' dria. The isolated mitochondria retained their individuality outside their normal cytOplemic environment» They poroiotod eo diocrete bodice in aqueous media as long on the preper tonicity of the media was maintained. : Host of the'lcnowlcdge related to mitochondrial ultra- otmcturo has been contributed by raledo (58, 59. 60) and SJootrmd ct elo, (61, 62, 63).- In 1952 Palade demonstrated that mitochondria have a limiting membrane. a system of ridgoo or criatae in 3 layers protruding from the inside air-face or the limiting membrane and a matrix. In 1953 Siootrond (61), SJoetrand and modin (6h) specified that mitochondria aro 22 boondod by dunno onto:- oontinuono nonbrono within which no innor mitochondria unbronoo thot torn oopto which sprood oorooo practically tho wholo unbrono vidth. century to Polodo'o intorprototion. ssootrond ond hio «aorta-o boliovo thot thoro in o noro topographical oonnootion botwoon tho onto: and innor mitochondria nonbranoo. Polodo (59) ototod thot tho onto:- no-bruo io doublo and tho oriotoo oro toido of tho innor nonbrono mot; Thio gonorol pottorn or organiza- tion no found in «u. of vortohrotoo. inurtobrotoo. protozoa and plonto. Elkholn om! Monti-and (65) ototod thot oriotoo III" on intogrol port of tho innor nonbnno. tron mom. work- (63. 66. 67. 68) it any to omludod thot o nitoohondrion in 1. Bounded by o continua“ nonhmo omnotins or tn don-o ioyoro dividod by o ion donoo onto: noo'brono. rho urtooo nonbrono in to ho oonoidorod oo tho limiting lulu-no Ihilo tho oriotoo aro omootod to tho innor matron. (59. 66). 2. Inoido tho nitoohondrion it o minotod oyston. nanny ot right ongioo to tho noin oxio of tho oou organollo ond toned '01‘1 otoo nitoohondrioloo' ond 'innor nonbronoo' by Poiodo and SJooti-ond roopootivoly. )- Tho matrix oontoino opaqno gromloo of uriouo oizoo. h. Tho obovo gonoroJ. pottorn hoo boon found in on oono otudiod. oninol or vogotobloo Although .11 utoohondrio oxhibit tho mo sword. pottorn of organizotion. ultrootmoturoliy thoy vory unord- ins to opooioo. tiunoo and organs. criotoo vary in oizo, 23 mapo, nunboro and arrongonoht within tho ooll organolloo Thoy aro not stablo stmoturoo and aro oapoblo or changing thoir morphology (39). Ditroronooo in mmbor, arrangonont and map. or oriotao must oorvo a uootnl'pnrpooo oinoo tho noubrano including oriotao oorvo ao oarrioro of a largo nunbor of mm" involvod in oonplox bioohonioal'roaotiono (69). Palado (58) notod a correlation botwoon abandon” or oriotoo and tho quantity ot‘onxynoot Chemically. nitoohondrio oonoiot aainly of onzynoo and lipido (70h Sohnoidor (50) notod tho hoary phoopholipid oontont or nitoohondria. no oomod that ito Iain purpooo ooo that of o. otruotnrol oomponontr snootrand (61. 63) odnittod that tho doublo Ionbronoo woro protoin layoro whilo Bolton and Folix (67) omjootnrod that tho oniophilio otato of tho nombrnnoo no duo to a oooponont lipoidal in natoro. Dittorontial oontrifusation (50) Iakoo it poooiblo to iooloto mitochondria tron o hrokon ooll noponoiono Tho honogonato nay ho dividod into four traction“ moloar. mitochondrial. niorooomol and mpornatont fluid or ooluhlo traotion. Bioohomiool analysis or oooh traction dotinoo noro aoonratoly tho ohonioal oonpo‘tion and tho function or oooh ooll constit- uont. Tho moot striking proporty of tho nitoohondria io thoir ouzyno system rolatod to tho roopiratory activity of tho coll. It io in tho mitochondria that oynthooio of ATP takoo plaoo. owing to its high onorgy phoophoto bonds, tho ATP holdo a proninont rolo in oollular notabolian an a souroo of onorgy for oynthotio roootiono. 2h Mitochondrial alterationo in cancer cello aro omen but are noithcr constant nor specific (71). Howatoon and Ram (55) found that in Notikotf'o hepatma, at pH 7.3.. tho diameter of tho mitochondria woo 0.2m: to l.2u Ihile in nonal cello it one 0.3511 to 0.7%. Elongatod for-no of mitochondria present in normal cello were about in tumor cello. The. cri otae in normal cello were proninont but not as prominent co in tzmor cello. The trannoroo nonbraneo in the 1:1th dria or tumor cello appeared to branch and anaotonooo more than thooo of tho nitoohmdria oi‘ nonal liver cello. Degen- orating mitochondria were larger. had breaho in limiting nembranoo and rovealod only reunanto or transferee, nombronoo. The inside of dogonorating nitochondria mowed a great deal of granular material arranged in irregular olunpo suggesting incroaao in mitochondrial substance. Increase in mitochondria in Bone Sarcoma no roported by Bomhard ot a1" (72) and in bone tumor giant cello by Oberling (71). £1th and Han (55) reported a docroaoo in oizo of mitochondria in havikoft'o hopatona. Pancett (73) obeervod tho name in Lucko'e renal adenocaroinoma. Mannheiller and Bernhard (7‘!) also observed the me in hamster kidney experimental tunor. Mitochondrial dogoneration was demonstrated in Novikoff'o hepatoma by Howatoon and Han (55). in tho hopatoma of man by Oberling (71) and in chorioallantoio non- braneo infected with fowl pox, vaccinial viruses and herpee simplex virus by Morgan et a1... (75, 76). Dnochovaki (77) reported that there may be a decroaoe in tho nmnber of criotao 25 no in mouse nannary cancer or there may be an increase in the number or crietao as in Novikorr'o hepatoma (55). ' Bonadotti ot a1..‘(78) reported that thore io on intranitochondrial localization ot'viruo-L-like particleo in avian orythroblaatoaio. ickornann and Kurt: (79) using contrifugation tochniquoo for separation of otruoture unite of‘oollo or liver tissue {mind 16 par cont of the herpes virus bound to the mitochondria by an intimate attaohnont. Further evidence was obtained that the mitochondria underwont oelcctive deterioration when infected with the virus- The concept was advanced that the organelles function in the development or the viruo. changeo in enzymatic activitioo associated with mitochondria woro oboorved after infection with herpes oinplox vine. i‘hio oboorvation led iokernonn and Kurtz to suggest that the mitochondria were a oito of herpeo simplex viruo oyntheoio. MATERIALS AND METHODS All reagento were node with too cycle. all glaoo diotillod water. Solutiono ueod ao growth and maintenance modia were made with two cycle, all glen diotillod water. At tiaeo tho compooition of the media were altered in order to meet the opeoifio requirononto of each experiment. £29.62!!! - m g' balanced a; oolutigg m2}. W.w00ntained 0.7 gm calcium chloridegin 100 I]. double distilled watch, ‘ W.-Cmtained the following reagents in 550 ll of double distilled 1'8“?- Grams Gm Glucooo 5 KHzPou 0.3 Nacl ' no - _ weapon-2320 0.3 K01 2 Phenol Rod 0.1 31330137320 1 gran ‘ The two oolutiono were autoclavod ooparatoly at 10 lbe preooure (115°C) for 10 minnteo, cooled to room temperature and then omblnodm The pH no adjusted to 7.0-7.2 with eterilo oolution of 7‘ NaHCO 3., WW» . 10x It so - on noel 3‘0 oran- KCl 2.0 gram: _NaH2POu‘HZO 26 ' 0.70 gram (continuod on next 9880) 27 10 "too uti c t uod CaClz 1.0 gram thOh'flizo 1.0 gram Glucose 5.0 grams Phenol Bod 0.1 gran The reagento were'dieoolved in 500 ml of double diotilled water. sterilized by seitz filtration and the pH adjueted to 7.0-7.2 with eterile 71 Rondo}. One ml of each mixture of vitamine and anino aoide microbiological iooooiatoe. lno.. Bethesda. haryland.) were added Just before uee. tion of the vitamin and amino acid mixturee. each dieeolved in a liter of double dietillod water wae ao followe: The composi- v tam . W . Biotin 0.1 gram Arginino 2.1 grams Choline 0.1 gram Cyotino 1.2 grams Polio acid 0.1 gram Hiotidine 0.8 gran Nicotinanido 0.1 gran Iooloucine 2.6 grane Pantothonio acid 0.1 gran Leucino 2.6 gram Pyridoxal 0.1 gran lyeine 2.6 grane Thiamin 0.1 gram hethionine 0.8 gran Riboflavin 0.01 gran Phenylalanine 1.6 greme i-Inoaitol‘ZHZO 0.18 gm 'i'hreonine 2.8 grane * i‘ryptophan 0.1! gram Tyrooino 1.8 grano Valine 2.u grame Glutanine 30.0 grams 28 This medium consisted of 0.15 yeaet extract and 0.35% glucose in Hanka' balanced oalt eolution. WW Penicillin (100 unite) and dihydroatroptomyoin (100 ug) per ml wore added to all nutrient media. Tissue mpleo were treated by placing into a oolution containing 500 unite of penicillin and 500 ug of dihydroetreptonycin. Ocoaoionally. oxytetracyoline (Torramycin) was added to the mcdia, 5 ug per ml or loco. man 'rrypein (1:250. Difco) wae prepared by dieoolving 0.25 gm in 100 ml calciun free Hanke' balanced ealt oolution. The trypoin eolution wao etorililed by Belt: filtration. The pH was adjuoted to 7.0-4.2 by addition of eterilo 7% Rance? Coll monolayore wore expoeed to trypein for periode of i to 1 minute. time trypoin was then removed with a pipette. W The hiotory of the A!) cell line need for thie etudy wae adequately deocribed by Wheeler et al.. (81). originally the epidermal calls were obtained from a 17 year old boy (AU). m. akin tiome was trypeinized and placed in growth medium which oonai otcd of W poolod human eerum. 5% chick embryo extract and 55;! Hanko' balanced ealt solution. One hundred unite of penicillin and 100 ng of dihydroatreptmycin per ml were used in all modia. 29 The AD cello were grown in 150 ml capacity milk dilution bottloo or Leighton culture tubeo. The cello adhered to the glaoo own-face and produced a confluent ol-xeot of epithelial-like cello in from two to five dayo. mien originally received in thio laboratory the cello required human com for growth. Iid'lecellcwere adapted to . growth in 20% calf serum. After a eheot of cello develOped. the cultureo were tranoforrod by treating the cell monolayer with 0.25% trypoin (13250, 1311’») and tranoforring the cello to two bottles. The eorum concentration in the medium woo reduced to.2% to maintain the monolayer. The cell line wao not homogeneouo. hoot and eometimeo all the cello in a monolayor were epitheliod (Pig. 1) but at other timeo many of the cello had fibroblaotio-like appear- once. Leighton tubeo containing ooverlipo were oeeded with one m1 of growth‘medium containing approximately 500,000 cello and were stationary incubated at 37°C. After a confluent monolayer was obtained mall wart. fragnente were added. Growth medium conoioted of m oupplcmonted with 20% heat inactivated calf oorum and 0.5% lactalbunin hydrolyaate, (LAH) (Difoo). ' After a confluent monolayer of cello wae formed and before it was infected with the wart fragmento. growth medium wao replaced by maintenance medium which consisted of m supplemented with 2% inactivated calf oorum. 3O Sampleo of cello were removed at ‘ opecific intervalo and either vitally otained with Januo green B or eloe fired for etaining with acid fuchoin or henntorylin. ' The wart tioonee were obtained from a dermatologiet'e office in Detroit. Michigan. they were renoved by total _ enuncleation (85). The tioneowere immediately placed in ’ vialo containing , eterile 50$ phoophate buffered glycerine. Upon arrival to the laboratory they were kept refrigerated at 13°C. Before uoe they were waotud with Banko' balanced oalt eolution and then treated with heavy doeeo of the antibiotic oolution for "171118 Wide of 2b to 48 honro. mey were minced with ocioooro and/or ecalpele and the null . fragmento cf wart tioouee were added to All cell monolayero in tioeue culture glaoe bottlee or Leighton tubeo. In one experimento the wart tioeue fragmente were firet heated to 56°C for 30 ninuteo. cooled to rooa temperature and then added to w monolayers. ' ‘ ‘ Fred: wart tiooueo were removed from individuale in the laboratory. They were treated immediately with the antibiotic eolution and held at 33° 0. Cell free extracte were obtained by grinding the wart tieoue in a nortar and pestle. Hanko‘ balanced oelt eolution containing penicillin and otreptonycin wao need ao diluent. A 55 ouopenoion of wart material wae centrifuged in a horioon- tal position at 795 x g for 10 ninuteo to remove the den 31 debrio and unbroken cells. One ml of the supernatant fluid was added to AU cello. er t eote Sterility teoto for bacterial contamination were no. of all culture naterialo in brain heart infusion broth (Difco). They were considered free of viable bacteria if there no no turbidity after 72 hr incubation at 37°c. Some culturee were tested for the presence of pleuropnomnonia-like organieno by oeeding cello and oampleo of cell cultureo into pleuropneunonia-like organise nediuu- (Difco). ' e U o The procedure used for fractionation wao that of Bentley and Hoerr (86) with ainor aodification borrowed mainly from the procedure of Hageboon et al.. (87). After cytOpathogenic effect (OPE) wee detected. infected cell nonolayero and uninfected cultureo were mechanically leooened from the glaoo wall of the bottle. removed, and euopended in 3 to 5 ml of Hanko' balanced oalt eolution. The infected and uninfected cell ouepenoione were pipetted into each of two weighed culture tubeo. The tubeo. were centrifuged for 5 ninuteo in the horizontal pooition at 795 x g and the eolution wao removed with a pipette. The wet packed cello were weighed uoing a Chriotian Becker analytical balance. Each cell oanple wae then ouopended in 2 al of 0.88 h merooe eolution (Merck reagent grade) at 32 p3 7.0«7.2. The infected cells and control cells were placed into two different all-glass tissue microgrinders. Grinders were cooled by placing the tubes in ice-bathe and grinding produced a homogenised paste in l to 2 ninutee of grinding. care was taken to keep the opecimeno cool (Om-4°C) in the subsequent steps of separation. Each homogenate was diluted to a total volume of 15 ml. t e]. , of 's The unbroken cells, nuclei and cell debris were removed from the euopenoion by centrifugation. The homogenate wee centrifuged at 795 x g for three ninutee .c 13% (International aultiopeed centrifuge). The supernatant fluidwao moved from the sediment and the fluid wae centrifuged again for three ninuteo as above. A third centrifugation wee dme resulting in slightly opaleecent nopenoicn. The eedinente were diecarded. One ml of 0.88 on moroee wee placed in a conical tube and 5 ml of the suspension was carefully layered over the oucroee solution foraing a two-phaoe system. The mixture was undisturbed for 10 ninuteo at 11°C. The nixture was centrifuged (795 x g) in a horizontal pooition for 1. ninuteo at too and then the upper phase of the mixture was carefully removed. Examination of the sediment after this treatment. by staining with Janus green 3 mowed that the nethod rmoved only occasional mitochondria. Examination of the upper phaee aupematant fluid contained nainly free mitochondria and some cell masses. 33 Isolation of flitochondgia The suspension containing free mitochondria plus other elements derived from the cytOplaanof wcello was con— trifuged for 30 minutes at 17.8:00 x g to sediment the mitochon- dria. The firmly packed, opaque sediment of mitochondria was suspended in 0.88 M sucrose solution... In order tofree the 7 mitochondria preparation from the soluble substances in the original homogenate the mitochondria were resedimented at 17.1300 x g and remopended again in 0.88 1! sucrose. The supernatant fluid was examined for presence of mitochondria. Hone was found. The final sediment was yellowidi in color and after dilution with sucrose solution constituted the mitochondrial suspension. gitochondgig goungg Mitochondria counts were made using the method of Harman (88) with minor modifications. A known volume of suitably diluted mitochondria preparation was spread over a measured area on a microscOpe slide. The near was fixed in t1 foraalin or exposed to osmium tetroxide tunes (21 omic acid) at room temperature and stained with acid fuch sin. The stained mitochondria were counted with a Banach and Lamb (Apochrooiatic) microscOpe fitted with 10x ocular and 90}: (oil) objectives. ‘ For each dilution. at least three slides were prepared and in each slide twenty fields were counted. The total number of nitochondria per gram of wet AU cells was calculated from the known volume of suspension, the area occupied. the total number of fields per unit area and the weight of the cells. 3h 0 o f h HierOphotographe were taken with a Banach and Lamb camera using Kodak Pauatomic—x meet film and Panatomio-BX meet film. The film was developed in DK50 or Microdol-x (Kodak) and fixed in sodium thiomlfate (hypo). I. RESULTS 0, e c s ell at o he found by Hayashi (l6) and Magic (26) AU cell cultures inoculated with shall fragments of recently removed warts were infected with the wart virus. The evidence for this infection was a progressive CPE. Host of the cells in the monolayers became round. The rest of the cells became angular and occasionally formed nicrovilli. ‘l'ne cytOplasn of the cells flowed increasing granulation. is degeneration continued cells began to aggregate and detached from the walls of the bottles or Leighton tubes.*usually by five days post inoculation. Uninfected cultures did not show such an effect. Cultures which were inoculated with heated wart framents did not show any CPE. Heavy suspensions of infected cells from these cul- tures were'used for serial passage of the wart agent. Infected monolayers again demonstrated the cytOpathogenic effect as described above. Degeneration was evident 7 to 10 days post inoculation. The time required for degenera- tion to occur varied between 5 to 10 days. ‘Uninfected cultures did not show any CPE. . The above cultures were grown on YEN medium containp ing 2oz calf serum and maintained on.2% serum. When.AU cell cultures were grown on.Eagles' basal median plus serum. 1 to 3 additional days of incubation were required before OPE todk place. 35 H's 1e Home). as cells stained with heaatoxylin and seeds. Minna” X2”. Pig. 2. niteehe-dria in sexual control cells vitally stained with Jesus as. I. unification mOOe Pig. 3. nitoohondria in infected cells. 5 days post inoculation. nagnification X1100. IIe 39 Occasionally, areas of ”resistant” cells were found in the monolayers. Attempts to grow these cells in large quantities for fractionation were unsuccessful. 912;; Qgctionatigg In preliminary studies it was found that distilled water or isotonic saline solution did not satisfy the cytological criteria of integrity so necessary for the identification of mitochondria. The two media caused extensive swelling of the mitochondria and mitochondria freed in either water or saline solution were spherical. In an isotonic solution. the mitochondria were refrac- tive whereas in water they were larger and pale in color. In both media they stained weakly or not at all with Janus green B. MitochOndris isolated in isotonic saline solution tended to agglutinate and resulted in a prohi- bitive loss when attempts were made to isolate the mitochon- dria free from nuclei and unbroken cells. a 0.88 14 sucrose solution was satisfactory for the centrifugal fractionation of All cells. The mitochondria varied greatly in length when isolated in 0.88 H morose solution. the majority of then p.113; l-lm in length and the remainder either granules or slightly elongated rods and filaments. They were readily stained with a solution of Janus green B in 0.88 n sucrose solution (dye concentra- tion 1310.000-13 30.000) introduced under the coverslip. The staining reaction was faint when a dye concentration to of l:h0,000 was used. The morphological. cytological characteristics and the ability to stain with Janus green B were the criteria used for identification of mitochondria. ’ men uninfected cellsgrown on ccverslips in Leighton tubes were stained with danns green B. it was found that the mitochondria decolonized rapidly upon exPosure to bright illumination. In uninfected cells. most of the mitochondria were mart rode, others were coma-maped and the rest were spherical granules (Pig. 2). This was generally true regardless of the sodium need for growth. Infected cells stained poorly with Janus green 3 and decolomized rapidly upon exposure to bright illumina- tion. The mitochondria in infected cells (Fig. 3) became filanentous resulting in a stringy network. i'hie was generally true regardless of the aediun need. Occasionally. unbroken AU cells in the homogenates were stained with Janus green 3. i In uninfected cells. the intracellular mitochondria were predominantly red- like. other mitochondria were cma-shaped and the rest spherical. In infected cells the intracellular mitochon— dria were mainly filamentous. The morphology of mitochon- dria isolated from the cells and those seen in the intact cells appeared similar. However. mitochondria isolated from infected cells appeared either elongated or slightly swollen. Unlike some cells (87),. the isolated mitochon- dria from: the All cells did not require a high concentra- tion of dye to be stained suitably. IIIe 141 non 0.83 M. sucrose homogenates of‘AU cells were fixed by addition of an equal volume of 2% aqueous solution of 0331111“; tetroxido and the mixture allowed to stand 9 to 12 hours at 16°C. users were suitable for staining with standard cytological techniques. However. fixation and wearing distorted some of th. elongated mitochondria. The fixed mitochondria were deeply stained by acid fuchsia, hmatoxylin and basic dyes such as safrsnine and crystal violet. When fresh wart fragments were seeded onto monolayers of Missile there was. in every instance. an increase in number of mitochondria found in infected cells (Table 1)“ When the original passage was followed by serial passages of the infected cells. there were always wore mitochondria found in infected cells then in uninfected cells. The mitochondria count on uninfected cells. original and passages, varied from b.30 x, 108 to 5.51 x 10 with an average of 15.98 r 105 mitochondria. 1. count of the .nitochondria from wart infected cells ranged fro! 1.65 x 1010 to 2.22 r 1010 giving a 1.99 x 101.0 average number of mitochondria per gram of wet as cells. There was approximately a fortrfold more :1th in infected cells than in uninfected cells (Table l). Counts of mitochondria from passage to passage varied slightly in both infected and uninfected cultures. 8 There was no noticeable trend apparent of increase or decrease in numbers as passages were continued. 1:2 When glycerineted wart fragments were need as infecting materiel. there were more nitoohondria in infected cells than in uninfected cells (Table 2). In uninfected cells the neuter of mitochondria varied .frn 11.82 x 103 to 5.7? x 108 per gre- of wet In cells with an average of 5.15 x lo8 mitochondria. i count of the nitccheudrie in can mluers seeded with gln- erineted m fregsents varied fro- )...“ 3 101° to 2 2.30 1 101° no: an average coast .‘r nos 3 191° per gram of wet in cells. ‘ more as approximately a thirty-eight fold acre mitochondria in infected cells than in uninfected cells (Table 2). the nitcchendrie counts in cells that were infected with glycerineted wart truncate were eespersble to those counts that were chained so. fredn wart frea- sents were used as infecting latex-lei (fable 1). when uninfected cells were um on a coupler eed couplets sodiu- there was an increase in mitochondria count. cells gram & lsgles’ sodiu- (fable 3) contained sore sitoehendria the: cells grown on sn'inouplete sediun not: as m (mi. 1). no .imam. count in infected cells. original and passage varied free 1.33 e: 1012 to 1.77 r it12 with an average count of 1.55 x 1912 per gre- of wet it cells. In the ease of uninfected cells. the aitechondria count ranged free loh91109to2.”2109withensversgeeountef 2.09 r 10’ per gr— of m «11- (mm 3). {in in nu. 1. 43 there was no noticeable trend of increase or decrease in numbers of mitochondria as passages were continued. It was also found that infected cells had a seven hundred and forty fold increase in umber of mitochon- ~dria when compared with tho number found ln'nninfeoted «n... Uninfeoted cells can in Bagles' basal sedim contained a 5.2 fold increase in mister of sitoohndrie as compared to uninfected sells min In! (fables l. 3). Mitochondria counts in infected cells (fable 3) grave in negles' ”dice. while exhibiting seas variation. mowed no noticeable trad in meters es peenges were continued. Uninfeeted cells grown in Miss. ledin (Tables 3. It) contained sore mitochondria per grew of NI cells compared with uninfected eells‘grown in m (Tables 1. 2). the eitoehocdria count (ratio It) in uninfected cells ranged fsoe 1.27 r in? h 2.71. x 109 with on «4.2.3. counter 2.2sxlo’mmofwetAUcells. Inca. ;. infected cells (tablet) the sitechccdris count varies from 1.20 r it)"2 to 1.91 :10“ with an average count of 1.59 r 1912 per gm of wet in cells. infected cells had mm mm ma lore Iitochondrie then uninfected cells. ‘ ‘ . _‘ on. reults cf .1th counts in cells than)... ted with heated wart eateriel are max-iced.“ tables 5* and 6. no separate controls were done for the heated wart eateriel but the controls as due in rabies l. z, .. -- a“. '1‘ M 3 and 1} were used for comparison. Cells grown in the more completo basal medium (Table 5) contained more mitochondria per gram or cells than cells grown in the incomplete yeast extract median (Tables 1. 2 and 6). The remlts in Tables 5 and 6 also indicate that there was no appreciable lucraaso or decrease in aitoohoedira count in cells that were inoculated with heated wart rngnonte. The original passage was followed by serial passages of tho unheated calla. 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