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Analysis of melanin control mechanisms by epidermal organ culture system
130 STUDY OF CHARGE-SELECTIVE EPIDERWAL JUNCTION
TO:lPARISON OF I’ATER-HDI.DISG CAPACITY OF THE STRATIJ!,I CDHNEU# BETI(EEN Sl,RFACE LAYER AKD DEEP SEATED LAYER IY VIlRO.
OK.Hashimoto and ti.Tagami i)epar.tment of Dermatology,Tohoku
T.Kazama, University Lniversity
layer
of
stratum
K.Og”ro and Y.Sato, Department School of Medicine, Nligata.
:N THE DERHO
of Dermatology,
Nllgata
By our- Qrevlous tracer experiments performed vlth eQlderna1 sheet (ES), chase Iamlna densa (LD) was exposed on the dermal surface hy removing dermis aiter treatment with 10 mN dithlathreltol, the chaige-selective perneablllty (CSP) of the derma-epldermal To study what substances form junction (DEJ) was demonstrated. anionic sites (AS) on the LD of the DEJ. the ES was digested with heparltinase (H-ase) or chondroltinase ABC (CH-ase).and then, stained with polyethylenelmine. In addition, to study what suhstances play an important role in the CSP, tracer esperlrents “sing native ferrltln (NF) were Qsrfurmad with the ES after dlgestion with H-ase, CH-ase, neuraminldase (N-ase) or hyaluronldase (HY-ase). Although H-ase dIgestion completely removed AS, CH-ase dIgestIon did not. Af!ar dIgestIon h’lth A-ase or N-ass, the permeability of the Lll of the DEJ against NF was slgniflcantly Increased. while dleestlon with CH-ase or HY-&se had no effect on it. Thesk data indicate that AS are aalnly composed of heparan suifate. and that slalic acid as we!1 as heparan sulfate Play an mporta;t role xn the CSP.
!‘e compared the Iuater-holding capacity of the beteeen surface layer and deep stratum corneuni seated layer in vitro using a simulation model for :,leasurements rwre in viva stratum corneunl. performed after placing the simulation models in various relative humlidities, the environment \!ith \jlth lO* trvpsin. tape stripping or treatment -&&one/ether. After these lipid extraction with treatments the conductance of surface layer vas hirher than that of deep seated layer of stratum There ras ,no significant difference in corneum. transepidernai Qatar loss. These results suggest formed that the uatcr-holding capacity of nwiy is not superior to that of the stratum corneum superficial
PERKEABILITY
corneum.
HEMATOXYLIN STAINABLE PROTEIN OF NEWBORN RAT EPIDERMIS -BIOLOGICAL FUNCTIONAS TRANSGLUTAMINASE SUBSTRATEM. Takahashi and T. Tezuka, School of Med., Osaka.
Department
of Dermatology,
Kinki
Univ., ANALYSIS OF MELANIN CONTROL EPIDERMAL ORGAN CULTURE SYSTEM
We have ourified a hematoxvlin stainable protein(HSP), which locates in the cell membrane kegion of the stratum corneum of newborn rat and is rich in glutamic acid(14.3%) and lysine(13.7%), from J-day-old rat epidermis using preparative isoelectric focusing, Suoerose 12 column and Mono 0 anion-exchange column chromatographies. To'examine a biological function of HSP as-transglutaminase(iGa&e) substrate, exper'ments of an incorporation of '4C-putrescine, an h 51-HSP itself and a change of the antigenicity of aggregation of HSP by immunofluorescent technique using a polyclonal antibody against HSP were carried out under the incubations of a commercial Isotope labelled putrescine was aggregated and formed the high molecular weight pro&in and the antigenicity was changed in viva In contrast, these results under the active condition of the TGase. were not observed under the inactive condition. Therefore, these findings suggest that HSP from newborn rat epidermis may be a substrate of TGase and may play a role of the formation of cornified envelope.
1
I Mot&. M. Okuda, G Imokawa tion. Tochigl.
Tochigl
MECHANISMS
Research Laboratory,
BY Kao Corpora-
Melanin control mechanisms have been elucidated mainly in cultured pigment cells In the present study, we have attemped to develop epidermal organ culture system whxh responds well to melanogenic stunulations to locrease melanin content Keratotome shce of uppermost skin (0 4mm tluck) was put between two rnlll1pore fllters (0 22 Pm) and then cultured at 37°C in an open culture tube coniazning serum-free Eagle MEM ( + or - Ing/ml EGF and 0.4 )-’g/ml Hydrocortisone) under the lxgh oxygen atmosphere (50% Oz. 45% N?. 5% COz). The culture tubes were revolved at 15rpm. Melanogenx activity was measured by the lncorporatlon of “C-2-thiouracil (2-TU) as well as 3HzO-release from 3,5- ‘H-tyrosine after 3-7 culture days. The present organ culture system demonstrated hyperpigmentation accompanied by uxreased melanocyte population and melanin content. This reaction was also assocmted with increased 3HzO-release as well as high 2-TU Incorporation as compared wth those at 4°C culture Phenylthlourea and skin extracts markedly inhilxted ‘H+release and 2-TU incorporation conconutant with suppressed plgmentatxm. whereas PGEz and db-CAMP stxnulated these melanogenic
actlvitles
HISTOCHEMICALOBSERVATIONOF KERATINOCYTE DIFFERENTIATION IN EPIDEBMOLYTIC HYPEBKEBATOSIS: MATURATION OF PLASMA MEMBRANE.
STUDY ON CELL CYCLE AND
S.Nakano, H.Hachisuka and Y.Sasai, Department School of Medicine, Kurume, 830, Japan
of Dermatology
We previously reported the presence of glycoconjugate at the plasma membrane or intercellular space of cornified cells in epidermoIn this study, we examined the distribution of lytic hyperkeratosis. soluble involucrin and S-S containing protein, major components of envelope protein, to observe the maturation of plasma membrane. In addition, we calculated DNA content. PAP method and DACMstain were employeed for hisrochemistry. For calculation of DNA content, cell suspension was prepared using 2OmM EDTA and 0.25% Tripsin-Hanks soluAs differentiation progresses in skin, involucrin was observed tion. in cytoplasm from lower spinous to granular cells and deposition to cell periphery occurred from upper spinous cells. Week reactivity was remained in cornified cell cytoplasm. In contrast, S-S containing protein appeared rather abruptly from granular to cornified cells in Electron microscopic examination showed cytoplasm and cell membrane. These findings the translucent marginal band in cornified cells. indicate that the chemically resistant plasma membrane is formed progressively from granular cells by enzymatic reaction and fading gradually with terminal cell differeniiaion by endogenous proteolysis. cell cycle calculated YPS almost identical with normal epidermis.
ALTERNATION OF ENVIRONYENTALFACTERS SURROUNDING MELANOCYTESIN YlTlLlGO Department
LEIONS.
of Deroatology,Kobe
0
T. Horikawa,
University
School
Y. Misbima. of Medicine.
It is well known that melanocytes lose the ability of pre aelanosome formation and finally disappear in the vitiligo lesion. Ye have been investigating the cellular environment which may play essential role to the loss of melanocyte in vitiligo lesion. Debara,Tagami and others reported that de -layed type hypersensitivity response decreases in the viti -1igo lesion. In the present study, we examined the effect of PUVA and topical steroid on the IL-1 activities of vitili -ginous epidernis which was subse uently grafted by normal skin using suction blister method 9 SBT). It has been found that low IL-1 activities of vitiliginous epidermis in the curable lesions by SBT were often seen, on the other hands IL-1 activi -ties were in normal range in the incurable lesions.
TO:lPARISON OF I’ATER-HDI.DISG CAPACITY OF THE STRATIJ!,I CDHNEU# BETI(EEN Sl,RFACE LAYER AKD DEEP SEATED LAYER IY VIlRO.
OK.Hashimoto and ti.Tagami i)epar.tment of Dermatology,Tohoku
T.Kazama, University Lniversity
layer
of
stratum
K.Og”ro and Y.Sato, Department School of Medicine, Nligata.
:N THE DERHO
of Dermatology,
Nllgata
By our- Qrevlous tracer experiments performed vlth eQlderna1 sheet (ES), chase Iamlna densa (LD) was exposed on the dermal surface hy removing dermis aiter treatment with 10 mN dithlathreltol, the chaige-selective perneablllty (CSP) of the derma-epldermal To study what substances form junction (DEJ) was demonstrated. anionic sites (AS) on the LD of the DEJ. the ES was digested with heparltinase (H-ase) or chondroltinase ABC (CH-ase).and then, stained with polyethylenelmine. In addition, to study what suhstances play an important role in the CSP, tracer esperlrents “sing native ferrltln (NF) were Qsrfurmad with the ES after dlgestion with H-ase, CH-ase, neuraminldase (N-ase) or hyaluronldase (HY-ase). Although H-ase dIgestion completely removed AS, CH-ase dIgestIon did not. Af!ar dIgestIon h’lth A-ase or N-ass, the permeability of the Lll of the DEJ against NF was slgniflcantly Increased. while dleestlon with CH-ase or HY-&se had no effect on it. Thesk data indicate that AS are aalnly composed of heparan suifate. and that slalic acid as we!1 as heparan sulfate Play an mporta;t role xn the CSP.
!‘e compared the Iuater-holding capacity of the beteeen surface layer and deep stratum corneuni seated layer in vitro using a simulation model for :,leasurements rwre in viva stratum corneunl. performed after placing the simulation models in various relative humlidities, the environment \!ith \jlth lO* trvpsin. tape stripping or treatment -&&one/ether. After these lipid extraction with treatments the conductance of surface layer vas hirher than that of deep seated layer of stratum There ras ,no significant difference in corneum. transepidernai Qatar loss. These results suggest formed that the uatcr-holding capacity of nwiy is not superior to that of the stratum corneum superficial
PERKEABILITY
corneum.
HEMATOXYLIN STAINABLE PROTEIN OF NEWBORN RAT EPIDERMIS -BIOLOGICAL FUNCTIONAS TRANSGLUTAMINASE SUBSTRATEM. Takahashi and T. Tezuka, School of Med., Osaka.
Department
of Dermatology,
Kinki
Univ., ANALYSIS OF MELANIN CONTROL EPIDERMAL ORGAN CULTURE SYSTEM
We have ourified a hematoxvlin stainable protein(HSP), which locates in the cell membrane kegion of the stratum corneum of newborn rat and is rich in glutamic acid(14.3%) and lysine(13.7%), from J-day-old rat epidermis using preparative isoelectric focusing, Suoerose 12 column and Mono 0 anion-exchange column chromatographies. To'examine a biological function of HSP as-transglutaminase(iGa&e) substrate, exper'ments of an incorporation of '4C-putrescine, an h 51-HSP itself and a change of the antigenicity of aggregation of HSP by immunofluorescent technique using a polyclonal antibody against HSP were carried out under the incubations of a commercial Isotope labelled putrescine was aggregated and formed the high molecular weight pro&in and the antigenicity was changed in viva In contrast, these results under the active condition of the TGase. were not observed under the inactive condition. Therefore, these findings suggest that HSP from newborn rat epidermis may be a substrate of TGase and may play a role of the formation of cornified envelope.
1
I Mot&. M. Okuda, G Imokawa tion. Tochigl.
Tochigl
MECHANISMS
Research Laboratory,
BY Kao Corpora-
Melanin control mechanisms have been elucidated mainly in cultured pigment cells In the present study, we have attemped to develop epidermal organ culture system whxh responds well to melanogenic stunulations to locrease melanin content Keratotome shce of uppermost skin (0 4mm tluck) was put between two rnlll1pore fllters (0 22 Pm) and then cultured at 37°C in an open culture tube coniazning serum-free Eagle MEM ( + or - Ing/ml EGF and 0.4 )-’g/ml Hydrocortisone) under the lxgh oxygen atmosphere (50% Oz. 45% N?. 5% COz). The culture tubes were revolved at 15rpm. Melanogenx activity was measured by the lncorporatlon of “C-2-thiouracil (2-TU) as well as 3HzO-release from 3,5- ‘H-tyrosine after 3-7 culture days. The present organ culture system demonstrated hyperpigmentation accompanied by uxreased melanocyte population and melanin content. This reaction was also assocmted with increased 3HzO-release as well as high 2-TU Incorporation as compared wth those at 4°C culture Phenylthlourea and skin extracts markedly inhilxted ‘H+release and 2-TU incorporation conconutant with suppressed plgmentatxm. whereas PGEz and db-CAMP stxnulated these melanogenic
actlvitles
HISTOCHEMICALOBSERVATIONOF KERATINOCYTE DIFFERENTIATION IN EPIDEBMOLYTIC HYPEBKEBATOSIS: MATURATION OF PLASMA MEMBRANE.
STUDY ON CELL CYCLE AND
S.Nakano, H.Hachisuka and Y.Sasai, Department School of Medicine, Kurume, 830, Japan
of Dermatology
We previously reported the presence of glycoconjugate at the plasma membrane or intercellular space of cornified cells in epidermoIn this study, we examined the distribution of lytic hyperkeratosis. soluble involucrin and S-S containing protein, major components of envelope protein, to observe the maturation of plasma membrane. In addition, we calculated DNA content. PAP method and DACMstain were employeed for hisrochemistry. For calculation of DNA content, cell suspension was prepared using 2OmM EDTA and 0.25% Tripsin-Hanks soluAs differentiation progresses in skin, involucrin was observed tion. in cytoplasm from lower spinous to granular cells and deposition to cell periphery occurred from upper spinous cells. Week reactivity was remained in cornified cell cytoplasm. In contrast, S-S containing protein appeared rather abruptly from granular to cornified cells in Electron microscopic examination showed cytoplasm and cell membrane. These findings the translucent marginal band in cornified cells. indicate that the chemically resistant plasma membrane is formed progressively from granular cells by enzymatic reaction and fading gradually with terminal cell differeniiaion by endogenous proteolysis. cell cycle calculated YPS almost identical with normal epidermis.
ALTERNATION OF ENVIRONYENTALFACTERS SURROUNDING MELANOCYTESIN YlTlLlGO Department
LEIONS.
of Deroatology,Kobe
0
T. Horikawa,
University
School
Y. Misbima. of Medicine.
It is well known that melanocytes lose the ability of pre aelanosome formation and finally disappear in the vitiligo lesion. Ye have been investigating the cellular environment which may play essential role to the loss of melanocyte in vitiligo lesion. Debara,Tagami and others reported that de -layed type hypersensitivity response decreases in the viti -1igo lesion. In the present study, we examined the effect of PUVA and topical steroid on the IL-1 activities of vitili -ginous epidernis which was subse uently grafted by normal skin using suction blister method 9 SBT). It has been found that low IL-1 activities of vitiliginous epidermis in the curable lesions by SBT were often seen, on the other hands IL-1 activi -ties were in normal range in the incurable lesions.