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P7 The role of calnexin in folding and maturation of tyrosinase: Major protein of melanogenesis
90
Abstracts JSIDjCSID
Joint Mrrting
P3
P6
P4
P7 THE ROLE OF CALNEXIN IN FOLDING AND MATUP.ATlON OF TYROSINASE, MAJOR PROTEIN OF MELANOGENESIS, K. K&ml&w Division of Dermatology, University of Alberta, Edmonton, Canada. We have succeeded in cloning p90 cDNA from SK-MEL-23 cDNA library using immunoscreening method. The nucleotide sequence of p90cDNA has 100% homology to human calnexin cDNA. Calnexin is a new molecular chaperone which transiently binds nascent glycopmteins synthesized on the endoplasmic reticulum (ER) ribosome, which helps maturation of these proteins and inhibits the transportation of the misfolded or mutant protein from expressing on the correct place. Many investigators has reported that calnexin does influence maturation of secretory glycoproteins: MHC class I, II, Tcell receptor, immunoglobuline, integrine, and P-glycopmtein. We assume that calnexin might be involved in melanogenesls cascade. Tyrosmase is an unque glycoprotein which is located only on the melanosome and plays the most unportant role in melanogenes~s cascade. In order to examine the interaction between calnexin and wild-type tyrosinase, CO’S7 cells were transfected with calnexin cDNA alone or calnexin plus human wild-type tyrosinase cDNA. The co-transfectant revealed higher tyrosinase activity than the single on the dopa assay. On melanin assay the cotransfectant synthesized more melanin than the single transfectant. On immunoprecipitation calnexin showed the binding with tymsinase. Castanospemune, a glycosidax inhibitor, prevented tyrosinase from maturation. With these results, we conclude that calnexin binds tymsmase in ER. Furthemvxe. we propose that calnexin might be involved in inhibition of the transponatmn of the mutant tyrosinase to melanosome by retaining in the ER.
P8 ASSEMBLY OF HAlR KERATINS IN TRANSFECTED CULTURED CELLS. M. Mizoguchi’. IA. Nia. M. Manabe. H. Ogawa. Department of Dermatology, Juntendo UniVersitv School of Medicine. Tokyo. Japan. Mouse hai;kerafins am present I” adult mice pdwadly in the cortex epithelii cells of hair follicles. A cDNA expression vector which drives an acidic hair bratin. mHa-1, was transfected in various cell hnes includmg BHK cells. COS-1 cells and HK-I cells e&her singly or in combtnation tih a cDNA expresslo” veClor whiti drives a basic hair kerafin. mHb-4. Expression of these keratins were identifiid by a monoclonal antibody or a monospecific anti-peptide antibody. The immunoflluorescan, staining showed that the expression of mHa-1 alone or coexpression of mHa-1 and m&f&4 in those cells results in mHa-I no, only being megrated into the f~lamentous network of pre-exisflng so” kerafins but that if is also accumulated m cyioplasmic aggregates a, a focal area near the rudeus. Cur results indicated that the acidic hau keraf\n was no, !n,e@ed preferentially info th bask hair kerntin. rather that I, was capable of cqmlynwization with pm-existing soft keratins. Furthermore. the presenca of mHa-, withhvithou, mHb-4 a, a focal pxlantiw she indicated a possible interaction wti nuclear lamin.
RAPID INDVCTION CFAPOPTDSlS aU HUMAN SWAMWS CELL CARCINOMA CELL LINE BY PHOTODYNAMiC THERAPY. Y. Mhts”m~,a~Y.Tamada’~,,.Mer~ MI oha& Dep, of Dermatology, Nagoya University School of Medicine. Nagoya *Dept. of Dermatology, Alchi Medwal Unw. Aichi, Japan. Photodynamic therapy (PDT) using a hematoporphyrin derwawe ss the photosensitizer and light IS used for treatment of various malignant tumors. We ~nvertigated if ard how PDT induced apopfosis in human squamous cell carcmma cells in vitro. Human squamouscell carcinoma cslf line (HSC-2) was cultured in MEM containing f 0% calf swum. HSC-2 cells were treated ~8th 3.0 rg/ml Photofrin If by 2.25 J/cm’ of WAirradiatmn. Tfwywere also treated with BO pglml of cisplatin. Total DNA was extracted ;nd analyzed electrophoretically on al% agarose gel. Nuclear matrix protein 4117 (NMP, was assayed by an enzyme-linked immunoabsorben, assay. PDT induced MUAfragmentations wlthin 2 hr after PDT, whereas cisplatin did 118 hr after treatment. Release Of NMP from HSC-2 was delected 1 hr after PDT, ard increased gradually. Cn the other hand. no increase in release of NMP wasobservedwithin 12 hr in the caseof cisplatln. It was clearly demonstrated that apoptosis of “SC-2 was induced rapidly after treatment with PDT, compared with that by cisplafin The rapid induction of apoptoris in HSC-2 might be characteristic for PDT-induced apopfose If was suggested that PDT might act a, a fate slage in the pathway of apoptosis. ;nd mduce apoptosis rapidly. The dellled mechanism in the rapid induction of apoptosis !n HSC-2 by PDT wil I be discussed.
Abstracts JSIDjCSID
Joint Mrrting
P3
P6
P4
P7 THE ROLE OF CALNEXIN IN FOLDING AND MATUP.ATlON OF TYROSINASE, MAJOR PROTEIN OF MELANOGENESIS, K. K&ml&w Division of Dermatology, University of Alberta, Edmonton, Canada. We have succeeded in cloning p90 cDNA from SK-MEL-23 cDNA library using immunoscreening method. The nucleotide sequence of p90cDNA has 100% homology to human calnexin cDNA. Calnexin is a new molecular chaperone which transiently binds nascent glycopmteins synthesized on the endoplasmic reticulum (ER) ribosome, which helps maturation of these proteins and inhibits the transportation of the misfolded or mutant protein from expressing on the correct place. Many investigators has reported that calnexin does influence maturation of secretory glycoproteins: MHC class I, II, Tcell receptor, immunoglobuline, integrine, and P-glycopmtein. We assume that calnexin might be involved in melanogenesls cascade. Tyrosmase is an unque glycoprotein which is located only on the melanosome and plays the most unportant role in melanogenes~s cascade. In order to examine the interaction between calnexin and wild-type tyrosinase, CO’S7 cells were transfected with calnexin cDNA alone or calnexin plus human wild-type tyrosinase cDNA. The co-transfectant revealed higher tyrosinase activity than the single on the dopa assay. On melanin assay the cotransfectant synthesized more melanin than the single transfectant. On immunoprecipitation calnexin showed the binding with tymsinase. Castanospemune, a glycosidax inhibitor, prevented tyrosinase from maturation. With these results, we conclude that calnexin binds tymsmase in ER. Furthemvxe. we propose that calnexin might be involved in inhibition of the transponatmn of the mutant tyrosinase to melanosome by retaining in the ER.
P8 ASSEMBLY OF HAlR KERATINS IN TRANSFECTED CULTURED CELLS. M. Mizoguchi’. IA. Nia. M. Manabe. H. Ogawa. Department of Dermatology, Juntendo UniVersitv School of Medicine. Tokyo. Japan. Mouse hai;kerafins am present I” adult mice pdwadly in the cortex epithelii cells of hair follicles. A cDNA expression vector which drives an acidic hair bratin. mHa-1, was transfected in various cell hnes includmg BHK cells. COS-1 cells and HK-I cells e&her singly or in combtnation tih a cDNA expresslo” veClor whiti drives a basic hair kerafin. mHb-4. Expression of these keratins were identifiid by a monoclonal antibody or a monospecific anti-peptide antibody. The immunoflluorescan, staining showed that the expression of mHa-1 alone or coexpression of mHa-1 and m&f&4 in those cells results in mHa-I no, only being megrated into the f~lamentous network of pre-exisflng so” kerafins but that if is also accumulated m cyioplasmic aggregates a, a focal area near the rudeus. Cur results indicated that the acidic hau keraf\n was no, !n,e@ed preferentially info th bask hair kerntin. rather that I, was capable of cqmlynwization with pm-existing soft keratins. Furthermore. the presenca of mHa-, withhvithou, mHb-4 a, a focal pxlantiw she indicated a possible interaction wti nuclear lamin.
RAPID INDVCTION CFAPOPTDSlS aU HUMAN SWAMWS CELL CARCINOMA CELL LINE BY PHOTODYNAMiC THERAPY. Y. Mhts”m~,a~Y.Tamada’~,,.Mer~ MI oha& Dep, of Dermatology, Nagoya University School of Medicine. Nagoya *Dept. of Dermatology, Alchi Medwal Unw. Aichi, Japan. Photodynamic therapy (PDT) using a hematoporphyrin derwawe ss the photosensitizer and light IS used for treatment of various malignant tumors. We ~nvertigated if ard how PDT induced apopfosis in human squamous cell carcmma cells in vitro. Human squamouscell carcinoma cslf line (HSC-2) was cultured in MEM containing f 0% calf swum. HSC-2 cells were treated ~8th 3.0 rg/ml Photofrin If by 2.25 J/cm’ of WAirradiatmn. Tfwywere also treated with BO pglml of cisplatin. Total DNA was extracted ;nd analyzed electrophoretically on al% agarose gel. Nuclear matrix protein 4117 (NMP, was assayed by an enzyme-linked immunoabsorben, assay. PDT induced MUAfragmentations wlthin 2 hr after PDT, whereas cisplatin did 118 hr after treatment. Release Of NMP from HSC-2 was delected 1 hr after PDT, ard increased gradually. Cn the other hand. no increase in release of NMP wasobservedwithin 12 hr in the caseof cisplatln. It was clearly demonstrated that apoptosis of “SC-2 was induced rapidly after treatment with PDT, compared with that by cisplafin The rapid induction of apoptoris in HSC-2 might be characteristic for PDT-induced apopfose If was suggested that PDT might act a, a fate slage in the pathway of apoptosis. ;nd mduce apoptosis rapidly. The dellled mechanism in the rapid induction of apoptosis !n HSC-2 by PDT wil I be discussed.