Combined Chemical and Electron Microscopic Studies of Pheomelanosomes in Human Red Hair

Combined Chemical and Electron Microscopic Studies of Pheomelanosomes in Human Red Hair

506 Vol. 81, No.6 KLEMP AND STABERG 21. Huff SE, T aylor HL: Obse rvation s on peripheral circulation in pso riasis. Arch Dermatol Syphilol 68:385-...

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21. Huff SE, T aylor HL: Obse rvation s on peripheral circulation in pso riasis. Arch Dermatol Syphilol 68:385- 388, 1953 22. Huff SE: Obse rvations on peripheral circulation in various dermatoses. Arch Dermatol Syphilol 71:575- 578, 1955 23 . Heden stam CG: Digita l plethysmography in psoriasis. Acta Derm Vene real (Stockh) 39:41- 46, 1959 24. Thune P : P lethysmographic recordings of skin pulses. Acta Derm Venereal (Stockh) 50:263- 269, 1970 25. H errmann F, Kanof NB: The fluorescein pattern of derma toses. J

Invest Dermatol 8:421- 432, 194 7 26. Ferguso n EH, Epstein WL: Clearance of I'"' injected in t ralesionally in patients with pso ri as is. J Invest Dermatol 37:441- 445, 1961 27. DiLorenzo PA, Brown DW, Walker SH, Dern PL, Go ltz RW: Technetium -99 m pertec hnetate disappearance studi es in normal and pso riat ic skin. J Invest Dermatol 56:39- 43, 1971 28. Kristense n JK, Henrikse n 0: Excess cumulative blood flow a nd repayment durin g reactive hyperemia in hum an cutan eous tissue. Ac ta Physiol Scand 108:1- 6, 1980

0022-202X/83/8106-0506$02.00/0 TH E JOURN AL OF INVESTIGATIVE DERMATOLOGY,

81:506- 511 1983

Vol. 81, No. 6

Copyright © 1983 by The Williams & Wilkins Co.

Printed in U.S.A.

Combined Chemical and Electron Microscopic Studies of Pheomelanosomes in Human Red Hair KOWICHI JIMBOW, M.D., PH.D., 0SAMU ISHIDA, M.D., SHOSUKE ITO, PH.D., YOSHIAKl HORI, M.D., PH.D., CARL J. WITKOP, JR., D.D.S., M.S., AND RICHARD A. KING, M.D ., PH.D.

Sapporo M edical College (KJ, 01), Sapporo, Fujita-Gakuen Unive rsity (S/J, Toyoahe, Yamanashi Medical College (YH), Yamano shi, Japan ond University of Minnesota (CJW, RAK), Minneapolis, Minnesota, U.S.A.

This study clarified the fine structure of pheomelanosomes in human red hair by quantifying the contents of pheomelanin and eumelanin and by identifying the fine structure of melanocytes and melanosomes based on their melanogenesis type in follicles. Out of 5 red-haired subjects, 3 were found to exhibit pheomelanogenesis in follicles, while the remaining 2 were found to have a mixed type melanogenesis of pheomelanin and eumelanin. Melanocytes in the pheomelanic follicles contained spherical melanosomes which revealed sequences of development identical to those seen in the pheomelanosomes of mice and guinea pigs. In contrast, the follicles of mixed type melanogenesis contained 2 different populations of melanocytes, i.e., one with synthesis of spherical melanosomes such as seen in the pheomelanic follicles and the other with synthesis of ellipsoidal-lamellar (filamentous) granules of eumelanosome form. It was concluded that (a) visual differentiation of hair color does not always reflect the melanogenesis type in human red hair, (b) chemical analysis of melanogenesis type corresponds well to the fine structural differentiation of eumelanosomes and pheomelanosomes, and (c) human pheomelanosomes are spherical granules with microvesicular (vesiculoglobular) and proteinaceous matrices on which melanin deposition is spotty and granular.

morphogen esis is well understood in mice a nd guinea pigs, and t he key structura l feature of pheomelan ogenesis is t he sy nt hesis of spherical melanosomes with a granular and microvesicu lar internal structure [1] . In huma ns, pheomelanogenesis is best seen in red hair. However, t he fine structure of pheomelanosomes in hum a n red hair has been unsettled. Earlier, Birbeck and Barn icot [2] observed t hat t he pigment gran ules in the melanocytes of human red hair are round and slightly oval a nd that they are composed of a loose aggregation of sma ll dense particles. Mahrle and Orfanos [3] a lso observed the presence of rat her sp herica l granules, the fine structure of which was different from typ ical eume lanosomes. Later, however, Mottaz and Zelickson (4], and Stanka [5] showed t hat t he melanocytes of human red hair co ntain ellipsoidal -lamellar (filamentous) melanoso mes simil ar to eume lanosomes of stages II and III. Furthermore, it has recently been reported t hat, in the keratin ocytes of red hair bulbs, melanosomes with a granular in ternal structure outnumbe r ellipsoida l melanosomes, and the s uggestio n has been made that the melanosomes cha nge their shape from ellipsoidal to sp herical when they are tra nsferred to keratinocytes [6]. This study clarifies the fine structure of pheomelanosomes in huma n red ha ir by quantifying the contents of pheomel a nin and eumela nin and by iden t ifying the fine structure of melanocytes and mela nosomes based on their melanogenesis type in folli cles. MATERIALS AND METHODS

Two basic classes of melanin pi gment are synthesized in ma mmals, i.e., brown -blac k eumelanin and yellow to reddi s hbrown pheome la nin . Th e cytophysiology of pheomela nin sy nt hesis in terms of regulation of hair color and melanosome Manuscript received May 24, 1983; accepted for publication August 15, 1983. Re prin t requests to: Kowichi Jimbow, M.D ., Department of Dermato logy, Sapporo Medi cal College, Min am i 1, Nishi 16, Sapporo 060, Japan. Abbreviations: APH: am ino- hyd roxyp henyl ala nin e HI: hydrioclic acid HPLC: hi gh-pe rform ance liqu id chromatography PTCA: pyrrole-2,3,5-tri ca rboxylic ac id

The red hair (fire reel a nd light red) of 5 Caucasian subjects was processed in this study. For electron microscopic study, hair folli cles were plucked, fixed in Karnovsky's solu t ion conta ining 2% para for maldeh yde a nd 2% glu ta ra ldehyde [7], a nd mailed to J apan. Hair bulbs in the a nagen phase were t hen selected under a dissecting microscope. These were fi xed wi t h 1% os mium tetroxide, stained en bloc with 1.5% uranyl acetate, dehydrated by alco hol solut ions, mounted in epoxy resin, and examined with a Hitachi HS-8 electron mi croscope 18]. For chemi cal a nal ys is of eumelanin and pheo melanin contents, a bundle of hair folli cles from eac h subject was cut with scissors and mail ed to Japan. The ha ir folli cles were then processed to chemi cal ana lysis using high-performan ce liqu id chromatography (HPLC) followin g our previously reported met hod [9]. Briefly, eumelanin co ntent in follicl es was estimated by multiplyin g t he amount of py rrole-2,3,5-trica rboxyli c ac id (PTCA) , a major pyrroli c product or eum ela nin , with a facto r of 50

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Subject

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I. Estimation of contents of eumelanin and pheo m.elanin and type of melanogenesis in human red hair

Age

1 (KN) 2 (JB) 3 (JB) 4 (JB)

23 8 10

5 (JH)

19

11

Hair color Fire red Fire red Fire red Light red Fire red

Melanin co ntentsa

Ratio of pheo melani n/

Type of

% wt/ wl

eu melanin

melanogenesisb

0.051 0.17 0.12 0.18 0.025

1.3 7.4 5.2 4.0 0.6

Mixed Pheomelani c Pheomelanic Pheomelanic Mi xed

Eume lanin % wt/wt

Pheomelanin

0.038 0.023 0.023 0.045 0.045

" Based on t he co ntents of PTCA (eumelanin indicato r; multiplied by a factor of 50) and AHP (pheomelanin indicato r; mul t iplied by a factor o f 5) in the folli cles. b Based on the rat io of AHP / PTCA contents in the follicl es.

FIG J. Fine stru cture of melanocytes a nd mela nosomes in hum an red hair specified as pheo melan ic by chem ical analysis. a, Four melanocytes (MC) are noted in t he hair bulb. They a re actively engaged in sy nt hes is of melanoso mes with va ri ou, developmenta l stages. b, The cytoplasm of t he mela nocyte is full of mel a noso mes, mitoc hondri a, rou gh a nd smooth endoplasmic reticulum , Go lgi vesicles, and free ribosom es. MS 11 and III = pheo melanosomes at stages ll a nd lll of development. c, A high-power view of the stage Il pheo mela nosome (MS If) shown in b (box). d, Fine structure of pheomelanosomes at stages I (MS I) and III (MS l!J). e, Fine stru cture of stage III pheomelanosomes (MS lll) and "mosaic" m elanoso mes. The mosaic melanosomes (MMS) reveal in compl ete lamellae and spotty melanin deposition. [, F ine structure of stage IV pheomela nosomes (MS I V).

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afte r permanganate ox idation of mela nin. A hair sa mple, approximate ly 30 mg, was homoge ni zed in 5 ml of K 2 C0 3 a nd, to th is, 3% KMn0 4 was added until t he purple color of KMn0 4 persisted. Solid NazS0 3 was added to decompose the residual KMn0 4 and the mixture was centrifuged. T he supern atant was ac idified to pH 1 with 6 M HCI and was extracted 5 times with et her. T he co mbined ether extract was d ried over MgSO, and evaporated. The residue was dissolved in water and ce ntrifuged. The supernatan t was injected into HPLC for a nalysis of PTCA. Pheomelan ic co ntent was esti mated by multiplying t he amoun t of amin o- hydroxyphenylala nine (AHP), a specific indicator of pheomelanin, wi t h a fac to r of 5 after hydriodic ac id (H I) hydro lysis of mela nin. A sa mp le (app roximately 30 mg) was suspended in 5 ml of 57% HI co nta ining 100 mg of H 3 P0 3 and was heated under reflu x. The hydro lysate was evapo rated to dryness a nd t he res idue was taken up in 0.1 M HCl and was cent rifu ged. T he supern ata nt was injected into HPLC for t he a na lys is of AHP.

RESULTS Chem ical a nalysis indicated that t he red hair fo llicles of 3 of 5 subjects were primari ly p heomelanic. T he ratios of pheomela nin (AHP x 5)/eumelanin (PTCA X 50) in t he follicles were 7.4, 5.2, a nd 4.0 (Table I). In all t he follicles specified as pheomela nic by HPLC analysis, t he melanocytes of t he hair bu lb were actively engaged in melanogenesis and possessed well -deve loped de ndritic processes (Fig 1a). T hey were fu ll of me lanosomes t hat were mostly at melanized stages (stages III and IV ). T he cytoplasm also contained a fai rly large amount of mitochondria, rough and smooth endoplasmic reticulum , Golgi complexes, and ribosomes (Fig 1b ). Each melanosome was spherical and limited by a membrane (Fig 1c-f), and grouping of mela nosci mes with in autophagic vacuoles was not encou nte red. In these pheomelanic melanocytes, stage I melanosomes appeared as large spherical vacuoles (0.8- 1.0 11m in diameter),

Vol. 81, No.6 contammg microvesicles (vesiculoglobu lar bodies) [10,11], about 40 nm in diameter, and amorphous proteinaceous materials (Fig 1d). Some of t hese proteinaceous materia ls were inco mplete filaments or lamellae. Stage II melanosomes were also spherical and contained more numerous microvesicles and proteinaceous material s t han stage I melanosomes (Fig 1c). In stage III melanosomes, melanin deposition was noted in t he microvesicles as well as on the proteinaceous materia ls (Fig 1d,e). However, melanin was noted only on certain portions of t he granu les, and thus deposit ion was microgranular and spotty. Stage IV melanosomes were fu lly melanized granules which were highly electron-de nse, sp herical, and limited by a membrane (Fig 1(). In t hese pheomelanic melanocytes, ellipsoida l melanosomes with typical lamellar or filamentous matrices such as seen in eumelanosomes were not seen. In certa in melanosomes of stages II and III, incomplete fi laments or lamellae were noted together wit h spotty deposition of melanin. T hese melanosomes were, however, sp herical, a nd t hus t heir fine struc ture was entirely different from t hat of typical eumelanosomes. Another form of "mosaic" melanosomes were those t hat were somewh at ellipsoidal and contained some filamentous materials. In t hese melanosomes, melanization again was spotty and none of t he lamellae or filaments wit h t he regular striation such as seen in eume lanosomes were noted. These findings clearly indicate that t he melanocytes specified as pheomelanic by chemical analysis synthesized t he melanosomes, t he fine structure of which was identical to that of pheomelanosomes seen in mice a nd guinea pigs [8,12- 14). In t he pheomelanic follicles, melanosomes were tra nsfe rred to t he keratinocytes and were degraded markedly within t hem. T hese tra nsferred melanosomes were distributed eit her singly or in groups. The latter were irregu lar on t he outer contour

F rc 2. Fi ne stru ctu re of pheo mela nosomes transfe rred to t he kerati nocytes. a, Low-power view of a mel anocyte and melanocytic dendri tes (MD). The kerati nocytes (KC) do not co ntain a ny ellipsoida l-lamellar eum ela nosomes. b, The pheomelanosomes a re di stributed sin gly or in groups. TF = tonoJi lame nts of kera tinocyte. c, The pheomelanosomes t rans ferred to keratinocytes revea l a marked degradation . A hi gh -powe r ,.. vi ew of the box in b.

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FI G 3. Fine structure of ha ir bulb specified as mixed type melanoge nes is by chemical ana lys is. a, Pheo melanic (PMC) and eumelani c (EMC) melanocytes a re see n among t he keratinocytes (KC). Insets I and 2 indicate a hi gh-power view of mela nosomes specified by arrows I and 2 in a pheo mela ni c mela nocyte. b, A pheomela nocytic (PMC ) dendri te is located next to t he cytoplas m of a eum elanocyte (EMC) . c, A hi gh-power view of mosaic gra nules t hat are ellipsoidal ( EM S ). None of t he lamellar or fi lamentous structures such as see n in eumelanoso mes a re noted within t he granul es (see d) . d, The cytoplasm of t he eumela nocyte is full of melanoso mes at stages II (M S II) and III (MS III) . e, T he melanosomes t ransferred to the keratin ocytes in t he follicles of mix ed type melanoge nes is. The arrows indicate t he prese nce of ellipsoidal eumelanosomes. DP = dendri t ic processes of melanocytes.

(Fig 2). Again , no ellipsoidal mela nosomes of eumelanosome form were see n in keratin ocytes of the pheo melanic follicles. The foll icles of t he remaining 2 subjects were spec ified as "mi xed" type melanogenesis by chemical analysis, the ratios of pheomelanin /eumela nin being 1.3 and 0.6. In these subjects, 2 di fferent cell populations of melanocytes were noted in t he same ha ir bulb (Fig 3a.) . One type (approxim ately 80- 90 %) produced spherical mela nosomes of pheomelanic form (Fig 3b,c). Th e other (approximately 10- 20%) possessed, in cont rast, ellipso idal melanosomes of typical eumela nic form (Fig 3b,d) . Similar to typica l pheomelanic melanocytes, t he former types of mela nocyte pmduced numerous spherical melanosomes, t he size and fin e structure of which were ident ical to

t hose seen in Figs 1 and 2. Again , t hese pheomelanic melanocytes revea led several mosa ic melanosomes which were rather ellipsoidal and contained some lamellar structure. In t hese melanosomes, melanin deposit ion was spotty a nd micrograml lar, a nd no la mellae wit h t he regular stri ation of eumelanosomes were see n (Fig 3c). In cont rast, t he melanocytes of eumelanic type produced ellipsoidal-la mell a r mela noso mes such as seen in typical eumela nosomes of stages II and III . T he number of eumelanic mela nocytes was far smaller t ha n t hat of pheomela nic melanocytes. T o t he follicul ar keratinocytes of mi xed type melanogenesis, eumelanic and pheomelanic melanosomes were t ra nsferred at stage IV of development . T hus, both eumelanosomes and ph eomelanosomes were occasio na lly

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seen in one keratinocyte (Fig 3d) . Reflect ing t he numerical difference of pheomelanic and eumelanic melanocytes, most of the melanosomes transfe rred to t he keratinocytes were sp herica l and were markedly degraded (Fig 3e). Ellipsoidal eumelanoso mes were sin gly distributed, while spherical pheomelanoso mes were seen as either single or grouped, complex uni ts. DISCUSSION The fine structure of pheomelanosomes is identical in mice and gu inea pigs [8, 12- 14]. In t hese animal s, t he stage l pheomelanosome is a spherical vacuole containing proteinaceous material and a few microvesicles (vesiculoglobular bodies ). At stage II , t he vesiculoglobu lar bodies become more numerous. At stage III, melani zation begins within t he bodies as well as on proteinaceous matrix, and thus t he melanin deposited is spotty and granular. The stage IV pheomela nosome remains sph erica l and co nta ins a completely melanized matrix without showing the electron lucency t hat is seen in t he eumelanosome [7 ,8]. In contrast, t he fi ne structure of human pheomelanoso mes has been unsettled and , tentatively, has been considered to be, similar to eum ela nosomes, ellipsoidal an d lamell ar (or fi la mento us), though spotty mel anin deposition occurs within t he melanosomes (4,5] and t hese melanosomes become spherical, showing a marked degradation when transferred to t he keratinocytes [6]. Previous studies, however, have been based on "red" hai r that was ass umed to be pheomelanic only by visual differentiation. Previously, we have shown that the tissue contents of PTCA a nd AHP correspond we ll to the types of melanogenesis in mouse hair, thus providing a method to estimate t he pheomelanin and eumelanin contents in tissues [9]. By using this tec hnique, we found in the present study that t he follicles in 3 out of 5 red- haired subjects were primarily involved in pheome lanoge nesis a nd that t he red fo llicles of t he remaining 2 subjects were engaged in mi xed type melanogenesis. Rorsma n et al [15], Prota et al [16,17] and Sealy eta! [18] indicated t hat many natural mela nins a re copolymers (mixed type melanin) of eume la nin and pheomelanin , and that "pure" eu melanin a nd pheomelanin se ldom, if ever, exist in mammals. In t he present st udy, a simil ar result was obta ined, and 3 subjects with pheome la ni c fo llicles showed a sma ll a mount of eumelanin (0.0230.045 , wt/ wt). However, in our previous analysis of ye llow mouse hair (C57BL, Av), which is considered typical pheomela ni c hair, a sma ll amount of eumelanin (0.018%, wt/wt) was also noted. T he ratio of pheomelanin/eumela nin content was 7.0 a nd even 5-S-cyste inyldopa-mela nin , a sy nt hetic pheomela nin , yielded a trace a mount of PTCA [9]. Thus, the fo llicles of ou r 3 subjects (the ratio of pheomelanin/eumelanin content being 4.0- 7.4) ca n safely be specifi ed as pheomelanic. In t he hair bulb of t hese ph eomelanic follicles, only one population of melanocytes was seen which synt hesized spherical melanosomes with sequences of development identical to those of pheomela nosomes in mice a nd guinea pigs. On t he other ha nd, t he follicles of mi xed type melanogenesi s (the ratio of pheomelanin/ eumelanin content being 0.6- 1.3) reveal e~ 2 different popu lations of me la nocytes, i.e., one wit h synthesis of ellipsoidal-la mella r eumela nosome-like gra nules and t he other with synthesis of spherical melanosomes such as seen in t he pheomelanic follicles. ln t he hair of agouti mice, where a switch from eumelanogenesis to ph eomelanogenesis occurs during t he natural growt h of follicl es, pheomel anic a nd eumelanic m e l a~ nosomes are see n in one melanocyte during t he transit ion phase [14,19 ]. In t he present study, however, t he follicl es of mixed mela noge nesis did not show such coexistence of 2 different types of melanosomes in one melanocyte. In t he present st udy, pheomelanocytes revealed several mosaic mela noso mes. However, a small number of such mosa ic gra nules were also noted in the pheome lanocytes of ye llow mice (20]. Furthermore, t hese mosaic melanosomes showed several features different from "typica l" eumelanosomes: (a) they never

revealed lamellar structures with a regular striation such as seen in eumelanosomes; (b) melan ization in these melanosomes, i.e., spotty and microgranular, was similar to t hat in typical pheomelanosomes; and (c) stage IV melanosomes in pheomelanic melanocytes with such mosaic melanosomes were a lways spherical a nd revealed no electron- lucent bodies such as seen in typical stage IV eumela nosomes. These mosaic melanoso mes thus appear to take the same struc ture as t hat of typical ph eomelanosomes at stage IV of development. However, whether or not t he presence of such mosaic mela nosomes is related to t he fact t hat a small content of eumelanin (PTCA) was noted in the pheomelanic follicles remains to be clarified by furth er study. In conclusion, visual differentiation of hair color does not always reflect the melanogenesis type in human red hair. Chemical analysis of rel ative contents ofpheomelanin and eumela nin in folli cles corresponds well with t he fine structural differentia tion of pheomelanosomes and eumelanosomes in t he hair bulb. Human pheo melanosomes are spherical granules with microvesicula r (vesicu loglobular) a nd proteinaceous matrices on which melanin deposit ion is spotty and granular. Thus, t he previously held controversy as to t he true structure of pheomelanosomes in huma n red hair derives from t he fact t hat previous studies determined melanoge nesis type only th rough visual differentiation , not t hrough chem ical analysis as has been done in t he present study. REFERENCES 1. Quevedo WC Jr, Fleisc hma nn RD , Holstein TJ: Pheo mel anogenesis in the mouse: a review of its ge netic and developmental features, Pigment Cell 1981. Edited by M Seiji. Tokyo, Univ of Tokyo P ress, 1981, pp 129- 137 2. Birbec k MSC, Barnicot NA: E lectron mi croscope studies on pigment formation in hum an ha ir follicles, Pigment Cell Biology. Edited by M Gordon. New York, Academic Press, 1959, pp 549561 3. Mahrle G, Orfanos CE: Haarfarbe und H aa rpi gment: E lektronenmikros kopisc he Untersuchungen a m natiirlichen und gebleichte n Haa rpi gment. Arch Dermatol Forsc h 248: 109- 122, 1973 4. Mottaz JH , Zelickso n AS: Ult rastructure of hair pigment, Adva nces in Biology of Skin , vol 9. Edited by W Montagna, RL Dobson. New Yor k, Pergamon Press, 1969, pp 471- 489 5. Stank a P: U ltrastructura l study of pigment cel ls of hum an red hair. Ce ll T issue Res 150:167- 178, 1974 6. Toda K, Matsumoto J, Fitzpatri ck TB: Structure of melanosomes in hum an red hair bulbs, Biology a nd Disease of the Ha ir. Edited by T Kobo ri, W Montagna (consultin g editors) ; K Toda, Y Ishibashi, Y Hori , F Morikawa. Tokyo, Univ of Tokyo Press, 1976, pp 217- 228 7. Ka rnovsky MJ : Formaldehyde-gluta raldehyde fix ation of high osmolarity for use in electron microscopy. J Cell Bioi 27:137a, 1965 8. Jimbow K, Oikawa 0, Sugiyama S, T a keuchi T: Comparison of eumela noge nesis a nd pheo mel anogenesis in retina l and follicul a r melanocytes: role of vesiculo -globular bodies in mela noso me differentiation. J In vest Dermatol 73:278- 284, 1979 9. Ito S, Jimbow K: Qua nti tative a na lys is of eumelanin a nd pheomela nin in ha ir and melanomas. J Invest Dermatol 80:268- 272, 1983 10. Jimbow K, Kukita A: Fine structure of pigment gra nul es in t he human hair bulb: ultrastructure of pigment granules, Biology of Norm a l and Abnormal Mela nocytes. Edited by T Kawamura, TB Fitzpatri ck, M Seiji. Tokyo, Univ of To kyo Press, 1970, pp 279- 302 11. Jim bow K, Fitzpatrick TB: Characte ri zation of a new melanosomal stru ctural co mponent- t he vesiculo-globul ar body- by co nvent ional tra nsmission, hi gh voltage, and sca nnin g electron microscopy. J Ultrastruct Res 48:269- 283, 1974 12. Moye r FH : Genetic variations i"n the fine structure and ontoge ny of mouse melanin granules. Am Zool 6:43- 66, 1966 13. P a ra kkal PF: The fin e structure of anage n hair follicl e of the mouse, Adva nces in B iology of Ski n, vol 9. Edited by W Montagna, RL Dobson. New York, P ergamon Press, 1969, pp 441469 14 . Jimbow K, T akeuchi T : U ltrastructural co mparison of pheo- a nd eumelanoge nsis in animals, Pigment Cell, vol 4. Edi ted by SN Klaus. Basel, Ka rge r, 1979, pp 308- 317 15. Rorsma n H , Agrup G, Ha nsson C, Rose ngren A-M, Rosengre n E: Detection of phaeo mela nins, Pigment Cell, vol 4. Edited by SN Kl aus. Basel, Ka rge r, 1979, pp 244- 252 16. Prota G, Rorsman H , Rosengren A-M, Rose ngren E: Phaeo mel ani c

D ec. 1983 pigments from a huma n mela noma. E xperi entia 32:970-971, 1976 17. Prota G: Rece nt advances in the chemistry of mela noge nesis in mammals. J Invest Derm atol 75:122- 127, 1980 18. Sealy RC, Hyde JS, Felix CC, Menon IA, Prota G, Swartz HM, Persad S, H aberman HF: Nove l free radicals in sy nt hetic and natural pheomela nins: distiction between dopa mela nins and cystein yldopa mela nins by ESR spectroscopy. Proc Nat! Acad

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Sci USA 79:2885-2889, 1982 19. Sakurai T, Ochiai H , Takeuchi T: Ult rastructu ral change of mel anosomes associated wit h agouti pattern formatio n in mouse hair. Dev Bioi 47:466-471, 1975 20. Granholm NH, Duimstra JR: Ult rastructural analys is of pheomelanogenesis in hair-bulb melanocytes of lethal yellow (C57BL/ 6J-A "/a) mice (abstr) . Am Zoo! 21: 1033, 1981

0022 ·202X/83/8105-05 11$02.00/ 0 THE JOUilNAL OF I NVESTIGATIVE DEnMATOLOG Y, 81 :511-5 13, 1983 Copyright © 1983 by The Williams & Wilkins Co.

Vol. 81, No.6 Printed in U.S.A.

Correlation Between Content of Viral DNA and Evidence of Mature Virus Particles in HPV -1, HPV -4, and HPV -6 Induced Virus Acanthomata ELKE -INGRID GRUSSENDORF-CONEN, M.D. , LUTZ GISSMANN, PH.D., AND JOCHEN HbLTERS Abteilung Dermatologie der M edizinischen Fokultiit an der RWTH Aachen, Aachen, and lnstitut fur Virologie im Zentrum. fii.r Hygiene, Uniue rsitiit Freiburg, Freibu.rg, F.R.G.

In HPV -1 and HPV -4 induced warts as well as in HPV6 positive condylomata acuminata the quantity of viral DNA encapsulated into virus particles was determined and compared to the total amount of viral DNA present in the papillomas. As shown by filter h ybridization using 1 ' H-labeled viral DNA molecularly cloned in Escherichia coli, the amount of total viral DNA found in HPV-1 or HPV -4 induced skin warts is similar. HPV -4 DNA, howevet·, is encapsulated into virus particles with less efficiency . HPV-6 DNA can be detected only at minute amounts in condylomata acuminata and the percentage of DNA recovered from virions is extremely low.

We have as ked whether HPV-4 and HPY- 6 induced proliferations wi t h only low particle production contain a lower a mount of vira l DNA t ha n t he particle-rich HPY-1 warts. To a nswer this question we purified t he virus particles of single warts a nd determined t heir DNA content. The amount of nonencapsulated virus DNA in t he ce ll was measured in parallel. MATERIALS AND METHODS Six ty sk in warts a nd co ndylomata acum inata from 7 different pat ients were analyzed. Purification of H uman Wart Virus

It is well established that hum an warts are virally induced fibroepit helial proliferations wit h different morphologic and histologic features. For some time they were thought to be induced by one and the sa me virus. Recent application of biochemical techniques, however, has led to the discovery of 16 different papilloma virus types in humans [1 - 7; H zur Hausen a nd L Gissmann, unpublished data] (see Table 1). The number of virions produced by each wa rt tends to ref1ect the type of viru s [8). HPY -1 is commonly found in skin warts wit h high particle yields, HPV -4 predominates in morphologically iden t ical skin warts with rather moderate particle production [4,9 ). Anoge nital wa rts (condylomata acuminata) induced by HPV -6 contain on ly minute quantities of virus particles. Manusc ript received February 13, 1983; accepted for publication May 17, 1983. This work was supported by t he Deutsche Forsc hun gsge meinschaft, No. Gr 641/3. Reprin t requests to: Dr. Elke-lngrid Grusse ndorf-Co nen, Abt. Dermato logie, Medical Faculty, Aac hen University, Goethestrasse 27-29, D-5100 Aac hen, F.R.G. Abbreviations: 1 x TE: buffer- 10 mM Tris-H Cl, 1 mM EDTA, pH 8.1 pBR 322: bacte rial plasmid ca rrying t he a mpi cillin a nd tetracycline genes PBS: phosphate-buffered sa line SDS: sodium dodecyl sulfate SSC: 1 X sse co ntains 0.15 M NaCl and 0.015 sodium citrate

The biopsy material was cut in to small pieces, ground with sterilized sea sa nd, a nd resuspended wi th phosphate-buffered saline (PBS). The suspension was incuba ted for 1 h at 37"C with 0.01 % trypsin a nd clea red by low-speed centrifugation. The sediment was stored for the extraction of cellular DNA (see below). T he supernatant was adjusted with solid cesium chloride (CsCl) to a density of 1.34 g/ml and centrifu ged in a Spinco SW55 rotor for 24 h at 35,000 rpm and 20"C. The visible vi rus band was collected by puncturin g the tube with a sterile needle and it was dialyzed aga inst 10 mM Tris-HCl, 1 mM EDTA, pH 8.1 (1 X TE) . Purification of Viral DNA

The viral proteins were solubili zed by 30-min incubation with 1% sodium dodecyl sulfate (SDS) at 37"C a nd t hen precipitated with 1 M CsCl by low-speed ce ntrifugation for 1 h at o·c. The supernatant was di alyzed aga inst 1 x TE and t he co ntent of DNA was determined by optical measurement at 260 nm . Extraction of Cellular DNA from Warts

The cellula r DNA as well as the nonencapsulated viral DNA was extracted from t he same warts that had been used for virus particle purification (see above) . The sediment of the homogenized material was resuspended in 2-5 ml 1 x TE. Labeling of Viral DNA

The DNA of HPV -1, HPV -4, and H PV-6, molecularly cloned in pBR 322 (bacterial plasmid ca rryi ng the ampicillin and tetracyc line ge nes) [10], was labeled with ["H]th ymidine t riph osp hate by the ni cktran slation procedure as described earlier [11].