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En bloc preembedding immunogold electron microscopy: Advantages and a limitation for an immunolabelling of skin antigens
404
ROLE OF PROTEIN DEPHOSPHORYLATION IN GROWTH AND OF Bl6
MELANOGENESIS
MURINE MELANOMA CELLS
AKTRA ITOH, MASAMITSU ICHIHASHI, Department of Dermatology, Kobe
NON-INVASlVEME?E!OD1oMFASUREMET~ICACMVITYOFHIJMW SKIN
AND YUTAKA MISHIMA.
University School of Medicine 5-l Kusunoki-cho 7-chome, Chuo-ku Kobe 650, Japan.
Shi=ido research
Protein phosphorylationis recognized as a major posttranslational regulatory mechanism. A steady-state level of protein phosphorylationdepends on the balanceof the activitiesof proteinkinases (PK) and protein phosphatases (PPs). Several reports suggested the involvement of protein kinases in the regulation of melanin biosynthesis. However, the role of PP in melanogenesis has not been clarified. In this study, type 2A PP, one of four major serinelthreonine protein phosphatases, has been found to be present in B16 murine melanoma cells in substantial amounts. It is thought that type 2A PP may play a role of melanogenesis through dephosphorylationof proteins posphorylated by the action of PK-C and PK-A in pigment cells. In order to elucidate such regulation, the effect of okadaic acid, recently found inhibitor of type 1 and 2A PPs, on cell growth and melanogenesis will be discussed.
previously that transcutaneous pC02 was a we reported useful parameterto evaluatemetabolicactivityof guineapig skin (The xfth meeting of JSID,1987).We nw report improvementsto the techniqueto apply for human skin usingNovAMEpIutranscutarteous@2/pco2 monitor.At first po2 and pa32 weremeasured onventralforwnn site at temperature44-C. Then the changes in these two psranvaters were observedwith tims for about 3 minutes wder cuff occlusion.It was sh.wn that the decreasingrate of @2 was higher in younger than in older subjectsand the increasingrate of PO32 indicatedthe same result as that in @2. In another experimentboth rates were increasedby tape-strippingstimulation.Fran these results it was suggestedthat the rates of change in @2 and pC02 were effectiveparametersto measure metabolicactivityof human skin in viva.
RESISTANCE
OF MELANIZED
CELLS
TO KILLING
BY W-B
AND W-C IRRADIATION. N.KOBAYASHI, T.MURAMATSU, H.TADA, T.SHIRAI, M.IHARA*, AND T.OHNISHI*. Departments of Dermatology and Biology*, Nara Medical Universitv. Kashihara. Nara. Japan In order to define t& biological role-of _
melanization on cell killing by ultraviolet (UV) radiation, survival and DNA damage after UV irradiation were examined in melanized melanoma cell line and non-melanized cell lines (normal and XP-A fibroblasts). Compared with non-melanized cell lines, the highly melanized cell line showed a significant degree of resistance to killing by UV-B and UV-C. In addition, the formation of avrimidine dimers bv UV-B and WV-C was more $ominent in non-meianized cell lines. These results suggest that melanization protects cells from UV-B and UV-C in vitro and the mechanism -.w might be associated with direct shielding of DNA by melanin.
PREEMBEDDINClMMUN&OLD ELIXTHON MICROSCOPY: ADVANTAGI3 AND A I.1M1TATION FOR AN 1MMUNOLABELLlNG OF SKIN ANTIGENS
ISN 81&
HIRGStll SHINIZU~ TAKEJI NISHIKAWA, ROBIN AJ EADW : Department of Dermatology, Keio University School of Medicine, Tokyo Japan and Department of Gall Pathology, Institute of Dermatology! London, U.K. In preemtwlding immunogoldelectron microscopy(W), limiyg;;dy penetration into the spwimen is an inevitable problem. however, we succeeded in demonstrating epidernolysis bullosa ’ acquisita .&igen with this technique. In this study we tried to clarify further the advantages and a limitation of this method. Very small pieces of normal humanskin (Clmm ) were incubated in inst different sites of epldermal IN2 antigens at 4 C, antibodies followed byB nm and/or 15nmgold conjugated to secondary antibodies. The tissueswerepost fixedand embeddedin &on. Ultrathin sections vere cut from very surface of the specimen and observed under EM. Collagenow part of type VII collagen was’inmunolocalized 011 the central banded portion of anchoring fibrils. In double immunostaining using 5 and 15 nm immunogold,type IV collagen and carboxy terminus of type VII collagen were immunolwalized at the saw sites including under-surface of the lamina densa and anchoring plaque like structures. Bullous pewhigoid antigen was failed to be immunolabelled. We conclude that this immunogoldEMmethod is siwle and useful for the immunolabslling of come of extracellular skin antigens, which lomlim below the lamina densa.
Laboratories,
YOKOHAM, JAPAN
THE HANDSTAMP METHOD FOR EVALUATING ANTIMICROBIAL AGENTS. SETSUKO NISHIJIMA,KENNETHJ.McGINLEY * AND JAMES J.LEYDEN* Department of Dermatology,KansaiMedical University Osaka, Univ. of Pennsylvania,Philadelphia,PA* We used the handstamp method for evaluating antimicrobial agents. Five kinds of Japanese antimicrobial agents, 4% chlorhexidine gluconate(CHG),chlorbenzarconium(CBC), 10% povidone iodine(PVI).0.3%triclsan(TRI),nonmedicated detergent(NMD)were examined by 3 min hand scrub. After 3 min hand scrub,we determined bacterial colony of the handsurface by the handstamp method and subunqual bacteria. CHG,CBC and PVI had excellent antimicrobial agent against handsurface bacteria. On the other hand, 0.3% TRI hardly eradicated handsurface bacteria. Subungual bacteria were hardly eradicated by all 5 antimicrobial agents. The subungual was most difficult region to eradicate bacteria.
AMINOACIDCWE0SITIoNS OF FIBFWS PROIERi~ m HUMANh0RM?GNAILSEPARATFDANDF'URIFIDDWI'IRTyJc>-DIMENSIONAL~RFSIS SA'TWHIDKKIO, AND JQJI JIWI y_mtof Dermatology,ShimsneMsdicalUniversity, We sepaxated,usingti-d.imansionalpolyacrylamide gel ele&rophoresis,crude S-carboqi+zhylated(SCM)fibrous proteins (FPs)frcxnhunannormalnail intoeightmnponents. meeight~entswereextractedfrcmthe~dirclension gel. The amino acid ccqxxitions of the eight SCM FP preparationsthus purifiedware thendetermiwd. The SCM cysteine (6.7%)and glycine (7.5%)contentsof the crude SCM Fppreparationcharacteristically high andlcw, respaccysteine (0.7-2.0%)and glycine (22.0tively. fkxewr, SCM 35.0%)contentsof the eight electrophxeticallyseparated andpurifiedFP ccmponentswere all not as high admtas lcw, respactively. These results suggestthat, with respect to cysteineandglycine contents,the characterof the FP canponentsof hunanxxrmalnailis notsodifferentfran, but rather fairly similarto that of the stratumcorneum.
ROLE OF PROTEIN DEPHOSPHORYLATION IN GROWTH AND OF Bl6
MELANOGENESIS
MURINE MELANOMA CELLS
AKTRA ITOH, MASAMITSU ICHIHASHI, Department of Dermatology, Kobe
NON-INVASlVEME?E!OD1oMFASUREMET~ICACMVITYOFHIJMW SKIN
AND YUTAKA MISHIMA.
University School of Medicine 5-l Kusunoki-cho 7-chome, Chuo-ku Kobe 650, Japan.
Shi=ido research
Protein phosphorylationis recognized as a major posttranslational regulatory mechanism. A steady-state level of protein phosphorylationdepends on the balanceof the activitiesof proteinkinases (PK) and protein phosphatases (PPs). Several reports suggested the involvement of protein kinases in the regulation of melanin biosynthesis. However, the role of PP in melanogenesis has not been clarified. In this study, type 2A PP, one of four major serinelthreonine protein phosphatases, has been found to be present in B16 murine melanoma cells in substantial amounts. It is thought that type 2A PP may play a role of melanogenesis through dephosphorylationof proteins posphorylated by the action of PK-C and PK-A in pigment cells. In order to elucidate such regulation, the effect of okadaic acid, recently found inhibitor of type 1 and 2A PPs, on cell growth and melanogenesis will be discussed.
previously that transcutaneous pC02 was a we reported useful parameterto evaluatemetabolicactivityof guineapig skin (The xfth meeting of JSID,1987).We nw report improvementsto the techniqueto apply for human skin usingNovAMEpIutranscutarteous@2/pco2 monitor.At first po2 and pa32 weremeasured onventralforwnn site at temperature44-C. Then the changes in these two psranvaters were observedwith tims for about 3 minutes wder cuff occlusion.It was sh.wn that the decreasingrate of @2 was higher in younger than in older subjectsand the increasingrate of PO32 indicatedthe same result as that in @2. In another experimentboth rates were increasedby tape-strippingstimulation.Fran these results it was suggestedthat the rates of change in @2 and pC02 were effectiveparametersto measure metabolicactivityof human skin in viva.
RESISTANCE
OF MELANIZED
CELLS
TO KILLING
BY W-B
AND W-C IRRADIATION. N.KOBAYASHI, T.MURAMATSU, H.TADA, T.SHIRAI, M.IHARA*, AND T.OHNISHI*. Departments of Dermatology and Biology*, Nara Medical Universitv. Kashihara. Nara. Japan In order to define t& biological role-of _
melanization on cell killing by ultraviolet (UV) radiation, survival and DNA damage after UV irradiation were examined in melanized melanoma cell line and non-melanized cell lines (normal and XP-A fibroblasts). Compared with non-melanized cell lines, the highly melanized cell line showed a significant degree of resistance to killing by UV-B and UV-C. In addition, the formation of avrimidine dimers bv UV-B and WV-C was more $ominent in non-meianized cell lines. These results suggest that melanization protects cells from UV-B and UV-C in vitro and the mechanism -.w might be associated with direct shielding of DNA by melanin.
PREEMBEDDINClMMUN&OLD ELIXTHON MICROSCOPY: ADVANTAGI3 AND A I.1M1TATION FOR AN 1MMUNOLABELLlNG OF SKIN ANTIGENS
ISN 81&
HIRGStll SHINIZU~ TAKEJI NISHIKAWA, ROBIN AJ EADW : Department of Dermatology, Keio University School of Medicine, Tokyo Japan and Department of Gall Pathology, Institute of Dermatology! London, U.K. In preemtwlding immunogoldelectron microscopy(W), limiyg;;dy penetration into the spwimen is an inevitable problem. however, we succeeded in demonstrating epidernolysis bullosa ’ acquisita .&igen with this technique. In this study we tried to clarify further the advantages and a limitation of this method. Very small pieces of normal humanskin (Clmm ) were incubated in inst different sites of epldermal IN2 antigens at 4 C, antibodies followed byB nm and/or 15nmgold conjugated to secondary antibodies. The tissueswerepost fixedand embeddedin &on. Ultrathin sections vere cut from very surface of the specimen and observed under EM. Collagenow part of type VII collagen was’inmunolocalized 011 the central banded portion of anchoring fibrils. In double immunostaining using 5 and 15 nm immunogold,type IV collagen and carboxy terminus of type VII collagen were immunolwalized at the saw sites including under-surface of the lamina densa and anchoring plaque like structures. Bullous pewhigoid antigen was failed to be immunolabelled. We conclude that this immunogoldEMmethod is siwle and useful for the immunolabslling of come of extracellular skin antigens, which lomlim below the lamina densa.
Laboratories,
YOKOHAM, JAPAN
THE HANDSTAMP METHOD FOR EVALUATING ANTIMICROBIAL AGENTS. SETSUKO NISHIJIMA,KENNETHJ.McGINLEY * AND JAMES J.LEYDEN* Department of Dermatology,KansaiMedical University Osaka, Univ. of Pennsylvania,Philadelphia,PA* We used the handstamp method for evaluating antimicrobial agents. Five kinds of Japanese antimicrobial agents, 4% chlorhexidine gluconate(CHG),chlorbenzarconium(CBC), 10% povidone iodine(PVI).0.3%triclsan(TRI),nonmedicated detergent(NMD)were examined by 3 min hand scrub. After 3 min hand scrub,we determined bacterial colony of the handsurface by the handstamp method and subunqual bacteria. CHG,CBC and PVI had excellent antimicrobial agent against handsurface bacteria. On the other hand, 0.3% TRI hardly eradicated handsurface bacteria. Subungual bacteria were hardly eradicated by all 5 antimicrobial agents. The subungual was most difficult region to eradicate bacteria.
AMINOACIDCWE0SITIoNS OF FIBFWS PROIERi~ m HUMANh0RM?GNAILSEPARATFDANDF'URIFIDDWI'IRTyJc>-DIMENSIONAL~RFSIS SA'TWHIDKKIO, AND JQJI JIWI y_mtof Dermatology,ShimsneMsdicalUniversity, We sepaxated,usingti-d.imansionalpolyacrylamide gel ele&rophoresis,crude S-carboqi+zhylated(SCM)fibrous proteins (FPs)frcxnhunannormalnail intoeightmnponents. meeight~entswereextractedfrcmthe~dirclension gel. The amino acid ccqxxitions of the eight SCM FP preparationsthus purifiedware thendetermiwd. The SCM cysteine (6.7%)and glycine (7.5%)contentsof the crude SCM Fppreparationcharacteristically high andlcw, respaccysteine (0.7-2.0%)and glycine (22.0tively. fkxewr, SCM 35.0%)contentsof the eight electrophxeticallyseparated andpurifiedFP ccmponentswere all not as high admtas lcw, respactively. These results suggestthat, with respect to cysteineandglycine contents,the characterof the FP canponentsof hunanxxrmalnailis notsodifferentfran, but rather fairly similarto that of the stratumcorneum.