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103 A keratin K10 gene mutation in epidermolytic hyperkeratosis (EH)
JSID Abstracts
103 A KERATIN KERATOSIS
199
106 KlO GENE MUTATION (EH). X.Mctt~‘,
K.Tamai. YMitsubasbi.
IN EPIDERMOLYTIC
K.Nomma
I. Hashimoto.
HYPER-
K.Umcki. D.Sawamura.
Dcpamwt
of Dcrmatom,
Hirosaki University School of Medicine, Hifoaaki, Japan. EHiscawcdbyanabena&noftbekcmtininmmudiamfilamentsand mccntstudicsbaveiadic&dcauaalmumtioosintbckaatinK10 gene.Inrhissa3dy.wtwraminedLrratinKlOaadKlgencmutationin spomticcascofEHwithscvcmgmc4iazdscalcandblistming
and Kl a at binh.
usinggeaomic pQ1WCampmidDNA
focusing 011the gems coding the NandCtmminioftberod~oftbeK10andK1. The amplified ~~~wacsequmaddireclly.‘IbcpptienthadaGroAtraasition at the sccutwl position of don 156 in the kmwin KlO gent, which resulted edlAdomaininKlO.Thc lnaArg+Hissubzlitntlonatthehighly-wnzerv mutationwasfutthawnfumed
bythclossofAciIsiteintbePCRpmduct
‘bus our multz reveal4 a muwion in the helix inidation pep&% of KlO.
EXPRESSION OF CARBONIC ANHYDRASE LIKE PROTEIN IN THE EPIDERMIS. M. Takahashi * and T. Tezuka, Department of Dermatology, Kinki University School of Medicine, Osaka, Japan. Monoclonal anfihodies against epidermal proteins reacted with hovinc carbonic anhydrase (CA). This findings suggesled that CA or CA like protein mighr be expressed in the epidermis. A polyclonal antibody (PoAb) againsl bovine CA, which was purified by 2-dimensional electrophoresis, was developed 10 injecl it into rahbhs. The anti-bovine CA PoAb was used for immunohistochemical examination and immunohlotting analysis for characterizalion of CA or CA like protein in the epidermis. The positive reaction m was revealed in the upper slratum granulosum and the lower stratum corneum. The about 50 kDa protein in human epidennal extract of IO M urea solution containing ii% 2. mercaptoethanol and S% NP-40 was reacted positively with anti-bovine CA PoAb. This protein nacled slightly with anti-human CA II PoAh bul not with anti-human CA I PoAb. These results indicate that CA or CA like protein is expressed in the normal human epidermis.
104
107
BIOCHENICALAND ll4NUNOHISTOCHEMlChL ANALYSESOF KERATIN EXPRESSION IN NERKBLCKLL CAUCINONA.K. Yoshikawa. Y. Katazata, Y. Hozumi and S. Kondo. Department of Deraatology. Yaaagata University School of Medicine. Yaaagata. Japan. There have been few biochemical analyses of keratin in Merkel cell carcinoaa although numerous iaaunohistccherical studies have been reported. So. two cases of Nerkel ccl I carcinolla were analyzed for keratin expression by biocbeaical and imnunohistcchemical a&hods. In biochemical analysis by two-diaensional gel electrw phoresis. simple epithelia-type keratins (K8. K18 and K20), stratified epithelia-type keratins (K5 and K14) and hyperproliferstive-associated keratins (K6 and K16) were detected. lamunohistochemical and inaunoblot results using several specific anti-keratin monoclonal antibodies agreed approxiaately with those of two-dimensional gel electrophoresis. Electron microscopic examination showed dense-core granules and perinuclear intermediate filaments in tbe cytoplasm of the tuaor cells. Neurofilaaents were also investigated by ianunohistocheaical and iamunoblot techniques. Intermediate filaaent expression in Merkel cell carcinoma was discussed.
lMMlJNOLOCALIZATION OF CUTANEOUS FATTY ACIDBINDING PROTEIN IN RAT SKIN. R. andM.paxtment of Dermatology, Niigata University School of Medicine, Niigata, Japan. Cutaneous fatty acid-binding protein (C-FABP) has been purified from rat skin. Since there was little information about Ibe physiological role of C-FABP in the skin, we investigated the immunolocalization of C-FABP in normal rat skin using immunohistochemical technique. In epidermis. C-FABP staining was strongly expressed in the upper prickle and tbc granular cell layers. In sebaceous glands, C-FABP was expressed in petipbed and differentiating cells. Since epidermis and sebaceous glands am active sites of fatty acid synthesis, tbesc results suggest that C-FABP may have impottant roles in the transport and synthesis of fatty acids.
1OS CHARACTERIZATION OF DEIMINATED PROTEINS PRESENT IN NONBORN MOUSE EPIDERMIS. T. Senshu’ and K. Akiyama. w of Call Chemistry, Tokyo Metropolii Institute of (krontobgy. Tokyo. Japen We charactsrized deiminated proteins present in newborn mouse epidermis. Epidermal materials scraped from fresh frozen skbt wara dlfferentlally extracted to obtain soluble (A), ureasolubla (B). and urea-men2@oeWanol-soluble (C) fractiins for WWem blotting analyses of filaggrin. keratins, and deimtnated prWina. B oontainad multiple charged isomers of deiminated flm However, both multiple units of filaggrin and keratins in Bold poorly deiminated. C showed a major band migrating ahbd of Kl and a minor band co-migrating wlh KlO after one dimensbW separation. The former was resolved into a major and minor series of charged isomers after two-dimensional sepadbn. They were characterized to be partially degraded prOduc@of Kl abouf 30- and 46-kDa smaller than Kl, respectively. Tha data suggest preferential deimination of Kl crosslinked with intenolecular disuifiie bonds.
HUMAN KERATIONOCYIES EXPRESS THE C-TERMINAL DOMAIN OFDYSTROPHlNINTHEDFSMOSQhfALAlTACHMENTPLAQ~. N.Ohtake”, S.Shimada’, M.Fujimoto*, M.Furucz and K.Tamaki*. Departments of Dermatology, lYsmanashi Medical University, Yamanashi, *Faculty of Medicine, University of Tokyo, Tokyo, Japan. A number of dystropbin isoforms have been dctcctcd io various tissues. We have studied the expression of dystrophin in human skin using a monoclonal antibody against the C-terminal domain of dystrophin by immunofluorcscencc and immunoclcctron microswpies. The antibody reacted to the intercellular portions of all epidcrmal kcratiocytes in immunoflu orcacence microscopy. The reaction products were localized in the dcsmosomal attachment plaques of kcrationocytcs in immunoclectron microscopy. ‘I&se results indicetc that the C-terminal domain of dystrophin is expressed in the desmosomal attachment plaques, although we cao not
103 A KERATIN KERATOSIS
199
106 KlO GENE MUTATION (EH). X.Mctt~‘,
K.Tamai. YMitsubasbi.
IN EPIDERMOLYTIC
K.Nomma
I. Hashimoto.
HYPER-
K.Umcki. D.Sawamura.
Dcpamwt
of Dcrmatom,
Hirosaki University School of Medicine, Hifoaaki, Japan. EHiscawcdbyanabena&noftbekcmtininmmudiamfilamentsand mccntstudicsbaveiadic&dcauaalmumtioosintbckaatinK10 gene.Inrhissa3dy.wtwraminedLrratinKlOaadKlgencmutationin spomticcascofEHwithscvcmgmc4iazdscalcandblistming
and Kl a at binh.
usinggeaomic pQ1WCampmidDNA
focusing 011the gems coding the NandCtmminioftberod~oftbeK10andK1. The amplified ~~~wacsequmaddireclly.‘IbcpptienthadaGroAtraasition at the sccutwl position of don 156 in the kmwin KlO gent, which resulted edlAdomaininKlO.Thc lnaArg+Hissubzlitntlonatthehighly-wnzerv mutationwasfutthawnfumed
bythclossofAciIsiteintbePCRpmduct
‘bus our multz reveal4 a muwion in the helix inidation pep&% of KlO.
EXPRESSION OF CARBONIC ANHYDRASE LIKE PROTEIN IN THE EPIDERMIS. M. Takahashi * and T. Tezuka, Department of Dermatology, Kinki University School of Medicine, Osaka, Japan. Monoclonal anfihodies against epidermal proteins reacted with hovinc carbonic anhydrase (CA). This findings suggesled that CA or CA like protein mighr be expressed in the epidermis. A polyclonal antibody (PoAb) againsl bovine CA, which was purified by 2-dimensional electrophoresis, was developed 10 injecl it into rahbhs. The anti-bovine CA PoAb was used for immunohistochemical examination and immunohlotting analysis for characterizalion of CA or CA like protein in the epidermis. The positive reaction m was revealed in the upper slratum granulosum and the lower stratum corneum. The about 50 kDa protein in human epidennal extract of IO M urea solution containing ii% 2. mercaptoethanol and S% NP-40 was reacted positively with anti-bovine CA PoAb. This protein nacled slightly with anti-human CA II PoAh bul not with anti-human CA I PoAb. These results indicate that CA or CA like protein is expressed in the normal human epidermis.
104
107
BIOCHENICALAND ll4NUNOHISTOCHEMlChL ANALYSESOF KERATIN EXPRESSION IN NERKBLCKLL CAUCINONA.K. Yoshikawa. Y. Katazata, Y. Hozumi and S. Kondo. Department of Deraatology. Yaaagata University School of Medicine. Yaaagata. Japan. There have been few biochemical analyses of keratin in Merkel cell carcinoaa although numerous iaaunohistccherical studies have been reported. So. two cases of Nerkel ccl I carcinolla were analyzed for keratin expression by biocbeaical and imnunohistcchemical a&hods. In biochemical analysis by two-diaensional gel electrw phoresis. simple epithelia-type keratins (K8. K18 and K20), stratified epithelia-type keratins (K5 and K14) and hyperproliferstive-associated keratins (K6 and K16) were detected. lamunohistochemical and inaunoblot results using several specific anti-keratin monoclonal antibodies agreed approxiaately with those of two-dimensional gel electrophoresis. Electron microscopic examination showed dense-core granules and perinuclear intermediate filaments in tbe cytoplasm of the tuaor cells. Neurofilaaents were also investigated by ianunohistocheaical and iamunoblot techniques. Intermediate filaaent expression in Merkel cell carcinoma was discussed.
lMMlJNOLOCALIZATION OF CUTANEOUS FATTY ACIDBINDING PROTEIN IN RAT SKIN. R. andM.paxtment of Dermatology, Niigata University School of Medicine, Niigata, Japan. Cutaneous fatty acid-binding protein (C-FABP) has been purified from rat skin. Since there was little information about Ibe physiological role of C-FABP in the skin, we investigated the immunolocalization of C-FABP in normal rat skin using immunohistochemical technique. In epidermis. C-FABP staining was strongly expressed in the upper prickle and tbc granular cell layers. In sebaceous glands, C-FABP was expressed in petipbed and differentiating cells. Since epidermis and sebaceous glands am active sites of fatty acid synthesis, tbesc results suggest that C-FABP may have impottant roles in the transport and synthesis of fatty acids.
1OS CHARACTERIZATION OF DEIMINATED PROTEINS PRESENT IN NONBORN MOUSE EPIDERMIS. T. Senshu’ and K. Akiyama. w of Call Chemistry, Tokyo Metropolii Institute of (krontobgy. Tokyo. Japen We charactsrized deiminated proteins present in newborn mouse epidermis. Epidermal materials scraped from fresh frozen skbt wara dlfferentlally extracted to obtain soluble (A), ureasolubla (B). and urea-men2@oeWanol-soluble (C) fractiins for WWem blotting analyses of filaggrin. keratins, and deimtnated prWina. B oontainad multiple charged isomers of deiminated flm However, both multiple units of filaggrin and keratins in Bold poorly deiminated. C showed a major band migrating ahbd of Kl and a minor band co-migrating wlh KlO after one dimensbW separation. The former was resolved into a major and minor series of charged isomers after two-dimensional sepadbn. They were characterized to be partially degraded prOduc@of Kl abouf 30- and 46-kDa smaller than Kl, respectively. Tha data suggest preferential deimination of Kl crosslinked with intenolecular disuifiie bonds.
HUMAN KERATIONOCYIES EXPRESS THE C-TERMINAL DOMAIN OFDYSTROPHlNINTHEDFSMOSQhfALAlTACHMENTPLAQ~. N.Ohtake”, S.Shimada’, M.Fujimoto*, M.Furucz and K.Tamaki*. Departments of Dermatology, lYsmanashi Medical University, Yamanashi, *Faculty of Medicine, University of Tokyo, Tokyo, Japan. A number of dystropbin isoforms have been dctcctcd io various tissues. We have studied the expression of dystrophin in human skin using a monoclonal antibody against the C-terminal domain of dystrophin by immunofluorcscencc and immunoclcctron microswpies. The antibody reacted to the intercellular portions of all epidcrmal kcratiocytes in immunoflu orcacence microscopy. The reaction products were localized in the dcsmosomal attachment plaques of kcrationocytcs in immunoclectron microscopy. ‘I&se results indicetc that the C-terminal domain of dystrophin is expressed in the desmosomal attachment plaques, although we cao not