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Cloning and molecular characterization of a dermatophyte cDNA clone, encoding a 70 kD heat shock protein of trichophyton rubrum
ESDR I JSID I ND Abstracts
0367
0370
ISa-MELANOCYTESTIM~TINGHORMONE(a-MSH)WVOLVEDIN HAIR GROWTHCONTROL? I, B. Ro off” V Botc ‘Inst.of Mol. Phamwology, Berlin,‘Deptof Dermatology,Charit&Humboldt-Univcrstty, Berlin,‘Dept of Pathology,Lqola UniversityMedicalCenter,Maywcod,IL, USA, ‘Deptof Dermatology,LJnivers~ty of Muastcr a-MSH, a productof proteclyticprocessingand furthermcdiftcatmmof prcqiomclanocortq is an cmiogcnousaeumpcptidcwhichis now recognizedto regulatemultiple phystologicalfunctionsin mammalianskin. In the currentstudy,we weremtcrestedin lwalizing a-MSH expressionin adolcecemmouseskin, in cxplonngany hair cyclcdepcndcnceof& expression,and in checkingwhethera-MSH exertsany &c&s cc kemtixcyte pmlifemtionm srfu. Using RP-HPLCand a spectficanUa-MSH RIA, a-MSH like unmunorcactivity(IR) was Identified wlucb eluted at the same time as the synthetic a-MSH By tmmua&istnchcmisoy, telogcnrldn ofC57BW6 mice as well as all stagesof the entiremurinchair cyclerevealed presenceof a-MSH-IR in large subcutaneousncrvcbundles,as well as m the cmxlar and longihldinalfibers of follicularneuralnetworkB, locatedaroundhair follicle @IF)isthmus. Double immunov~~ualiration of a-MSH-IR and PGP9.5IR documented rheneuronalorigin of a-MSH-IR fibers.No expressionof a-MSH-IRwas observtxiIIIthe cotdemdsand in HF cpithelium III telogcnskid However,aflcr inductionof activehair growth(anagccIII-IV), a-MSH-IR also appearedia pmumal ORS and hair matnx keratincqtcs, wheresuchIR disappearedagam in anagenVI and catqcn In addition,a-MSH-IRwas de&ted m epxiennal and follicularkeratincqes of the dcvclopingHF in neonatalmutineskin. Organcultureassays of nwmc tclagcnrcvcalcdthat a-MSH inhibitedDNA synthesisboth in epidcnnaland HF kemtinccytcsoftetw skin. However,no pmliferaticmet&t was seenin anagenII and IV skm The&data raw the posstbihtythat a:MSH of neuralor epithelialorigin-my bc involved inthcmntrolofhailgmwth
PHOSPHOL~PASEANDCYCLOOXYGENASE-2 INCREASEMNONCONKUENTH~MAN KERATINCXXTZ?S. Alice Pentland', Ray Konaer’, Rama Malavivr?, and K$swn;t Rvs-Sikoca’, ’ Dept of Dzm, Univ. Rochester, Rochester, NY and 2 ayne ughes Institute, St Paul, Mn. Phospholipase 4 (PLAJ-mediated fatty acid release is key for Prostaglandn & (PC&j-mediated cell proliferation, and contributes to bacterial and fungal killing in wounds. Thus, regulation of fatty acid metabolism in epithelial repair is of interest. Non-confluent (NC) primary human keratinocyte cultures mimic wound repair (~30% confluent). These cultores also release 10 x more PGE, than confluent cultures, doubling NC cell proliferation. The PLA, and cyclooxygenase (COX) mechanisms of increased PGE, formation were studied. Mass spectrometric analysis of sups taken from NC cultures showed quantities of arachidonic acid (AA) and linoleic acid (LA) released by NC cultures were lbfold (AA) and g-fold (LA) that of confluent cultures. However, Western blot of cPLA, indicated only a modest increase in cPLAz protein. Western blot of COX-2 in NC cells showed a 3 x increase COX-2. Exogenous AA conversion to PC& confined this. Ca++-dependent phospholipases were suggested by the 20 fold increase in PC& release observed in A23187-stimulated NC cultures over controls, The 2:l ratio of AALA release suggests the presence of both an AA-specific (cPLA,) and a non substrate-specific PLA, (sPL4) activity in NC cells, suggesting that keratinocytes migrating in a wound upregulate the activity of both cPLA, as well as non-selective phospholipase(s). COX-2 is also induced in NC cultures. This data suggests that enhanced fatty acid release and F’GE, synthesis is mediated by coordinate increases in phospholipase and COX-2.
0368
0371
p-PHENYLENED*ANrNE
IPPD)
Yo Kawakubo, Brunhilde Dermatology, University
N-ACETYLATrON
slilmeke Clinic,
IN
HUMAN
SKIN
CYTOBOL.
and Hans Merk, Department RWTH-*a&en, Oermany.
of
pd (diacetyl-PPD formation from monoacetyl-PPD), respectively. These data suggest that PPD is metabolized in human skin through Nacetylation to monoacetyl-and diacetyl-PPD and the responsible enzyme is NATI. Recent studies have shown that N?+=1gene exhibit variation
CHARACT!ZRIZATION OF A CLONING AND MOLECULAR DERMATOPHYTE CDNA CL.ONE, ENCODING A 70 KD HEAT SHOCK PROTEIN OF TRICHOPHYTON RUBRUM SassanRezaie.JosefBan. Christi Poitschek.DiviTschachler.Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, University of Vienna Medica School, Austria Tnchephytccmbmm causes90% cfall chmnic cases cfdermatophytcsis To characterize T.mbmm proteins et the molecular level, we stablished a cDNA library from poly (A)* RNAs tsdated from a 30 day culturecfthis fuogua.CompletecDNA of T rubrumwas used as the hybndtzxticnprobe for hbrary screening.Several strmgly hybridizing clones were identified end fwthm analyzed Here we describea recombmantcDNA clone of 1376 bp containinga single cpen reading t?ameof 1962 bp coding for 654 amino acids when this cDNA clone was used as a probe for Northernanalysts. it hybridued to a RNA ~pectesof’ about 3.5 kb whwh was constitctively expressedby Tmbmm cultured under standard condaicns.Analysts&be aminoactd sequenceofthe clone detectedstxq homologywth eukaryotic70 kDa heat shockprcteins @iSPs)and 84% identityto the sequenceof the two members cf the HSP 70 fkmdy of Nercspom cma and Cladosporum herbam. The presenceof theDLGlT-S-V umsesas sequenceallow us to include this new HSP famdy member m the Dnak subfamily Insummary, wehave cloned and charaderired the first member of the HSP family of demmtophyw and have shown that a IS expressed constdutivelyby Tmbmm m cell culture.
in human populations. Therefore, slow aceeylation by N*T1 enzyme may be a risk factor for individual susceptibility to PPD.
0369
0372
OXYGEN TENSIONSIN EXPERb5NTAL CONDITIONS OF WOUNDHEALING ; Demmnent ofDemmtclogyand CuttmoousSurgery,UniversityofMiarmSchwl ofluledtcine; M&i, FL. Law oxygentensionhas recentlyhen shownto showlatecellgmwih,clcmalexpansion, synthesiswd tmtuctipticnofcertain gmwththetas andextr&luler matrixcomponents Theseresultshave beenobtainedby expcsingcellculturesto a hypoxicenvircmment. However,there1slinetedinfcmmticmcmwhatthe e&al oxygentensionis at the cellsurface. Usma an oxwett probe.we studtedhow experimentalcondihcnsaffectthe oxygentension
Expression of vitamin 0 receptor (VDR) and retinoic acid receptors @AR-a,-P.-y; RXR-a,-P,?) in basal cell carcinomas. J Kamradt’, J Reich&h’. XH Zhu’. XF &, MF Holick’ Department of Dermatology, University of Saarland, Homburg, Germany; ‘Vitamin D. Skin and Bone Research Laboratory Boston University MedIcal Center, Boston, MA, U.S.A. We have analzed immunohistochemically the expression of 125.dlhydoxyvitamin D3 receptor proteins. the three retinoic acid receptor proteins @AR-a.+,-~) and rebnold X receptor-a:P,-y proteins in basal cell carcinomas (BCCs, n=15). Staining patterns of these different receptors were correlated to the labeling pattern of the proliferation marker Ki-67. Additionally we used RT-PCR to show the levels of VDR RNA in BCCs (n=7) compared to normal human epidermis (n=5). A strong lmmunoreactivity for VDR .RAR-a and RXR-a was observed in all BCCs, staining for RARy was moderate. whereas no or very weak labeling for RAR-p and RXR-P.7 protein was detectable. In contrast to RAR-y, that revealed no or only marginal differences in staining Intensities compared to adjcent uneffected epidermis, lmmunoreaciivity for RAR-a, RXRir and VDR was cons!stently stronger in the tumor cells. In general, labeling of BCCs for VDR, RAR-sly and RXR-a was pronounced in peripheral cells, whereas staining of central areas of the tumors was heterogenous Most of the tumors showed no correlation in comparing the labeling patterns of VDR, P.AR-a/-y and RXR-a with the staining pattern of KI-67. The results of the RT-PCR demonstrate higher levels of VDR-RNA in all BCCs compared to the levels in normal epidermis. Our findlngs indicate: (I) VDR, RAR4-y and RXR-a are in contrast to RAR-p strongly expressed in BCCs (II) expression of VDR, RAR-a and RXR-a is upregulated in BCCs compared to normal epidermis (Ill) Selective retlnoic ac!d metabolites and/or 1.25.dlhydroxyvltamin D. may be useful for preventive or terapeutical treatment of BCCs
at
&t&ble thec& surface. We found this dissolved oxygen tennon was dir&y related to the height ofthe mediumabwe the cellsurface(t=+.g793,p* 021),but was cmstant when
no cellswerepresentin the flask(r-0.9732, p=O.oOl).In both humandermalfibroblastsand NlH/3T3cultures,oxygentensiondecreasedlineerlyas celldensityincreased(r--0.835, pcO.ooO1, r-0.916, p+XlOl, respectively)Whenhumandermalfibmblastswereexposed to 2% 4, maximumhypoxiclevels(0 mmHg) wereachievedwithin 30 minutes,and the reccve,ytimeWBSultbin a swcilarhme tame. The additionofrctencme,an mtihtor ofcellular respition, blockedthis decreasein oxygeotensionat the cellsurface,suggestingthat ceUul.x c&sumpt~cm of oxygenISresponsiblefor the d&me. Lastly,we examinedthe cellsurface oxygentensionin uantroland ecutely-wounded humanskin equivalents@SE), wns~stingofa keretinccytelayerovere typ I ccl&en matrixccntainingfibmblaats.We foundthat oxygen tensicmdroppedsignificantly~O.ooOl) in wxaely-wounded areasofHSEoas mmparcdto unwoundedareasof HSEs and that this dropwas preventedby the additionofmitmnycinC. Theseresultsmdwatethat cell surfaceoxygentensim is indirectlypropwticnalto celldensity, andthat the amountofdetectable oxygm at the cellsurfaceis a functionof cell density, the oxyen tension tn the incubator, and increasedceUular activity, BEoccurs after injury
’
0367
0370
ISa-MELANOCYTESTIM~TINGHORMONE(a-MSH)WVOLVEDIN HAIR GROWTHCONTROL? I, B. Ro off” V Botc ‘Inst.of Mol. Phamwology, Berlin,‘Deptof Dermatology,Charit&Humboldt-Univcrstty, Berlin,‘Dept of Pathology,Lqola UniversityMedicalCenter,Maywcod,IL, USA, ‘Deptof Dermatology,LJnivers~ty of Muastcr a-MSH, a productof proteclyticprocessingand furthermcdiftcatmmof prcqiomclanocortq is an cmiogcnousaeumpcptidcwhichis now recognizedto regulatemultiple phystologicalfunctionsin mammalianskin. In the currentstudy,we weremtcrestedin lwalizing a-MSH expressionin adolcecemmouseskin, in cxplonngany hair cyclcdepcndcnceof& expression,and in checkingwhethera-MSH exertsany &c&s cc kemtixcyte pmlifemtionm srfu. Using RP-HPLCand a spectficanUa-MSH RIA, a-MSH like unmunorcactivity(IR) was Identified wlucb eluted at the same time as the synthetic a-MSH By tmmua&istnchcmisoy, telogcnrldn ofC57BW6 mice as well as all stagesof the entiremurinchair cyclerevealed presenceof a-MSH-IR in large subcutaneousncrvcbundles,as well as m the cmxlar and longihldinalfibers of follicularneuralnetworkB, locatedaroundhair follicle @IF)isthmus. Double immunov~~ualiration of a-MSH-IR and PGP9.5IR documented rheneuronalorigin of a-MSH-IR fibers.No expressionof a-MSH-IRwas observtxiIIIthe cotdemdsand in HF cpithelium III telogcnskid However,aflcr inductionof activehair growth(anagccIII-IV), a-MSH-IR also appearedia pmumal ORS and hair matnx keratincqtcs, wheresuchIR disappearedagam in anagenVI and catqcn In addition,a-MSH-IRwas de&ted m epxiennal and follicularkeratincqes of the dcvclopingHF in neonatalmutineskin. Organcultureassays of nwmc tclagcnrcvcalcdthat a-MSH inhibitedDNA synthesisboth in epidcnnaland HF kemtinccytcsoftetw skin. However,no pmliferaticmet&t was seenin anagenII and IV skm The&data raw the posstbihtythat a:MSH of neuralor epithelialorigin-my bc involved inthcmntrolofhailgmwth
PHOSPHOL~PASEANDCYCLOOXYGENASE-2 INCREASEMNONCONKUENTH~MAN KERATINCXXTZ?S. Alice Pentland', Ray Konaer’, Rama Malavivr?, and K$swn;t Rvs-Sikoca’, ’ Dept of Dzm, Univ. Rochester, Rochester, NY and 2 ayne ughes Institute, St Paul, Mn. Phospholipase 4 (PLAJ-mediated fatty acid release is key for Prostaglandn & (PC&j-mediated cell proliferation, and contributes to bacterial and fungal killing in wounds. Thus, regulation of fatty acid metabolism in epithelial repair is of interest. Non-confluent (NC) primary human keratinocyte cultures mimic wound repair (~30% confluent). These cultores also release 10 x more PGE, than confluent cultures, doubling NC cell proliferation. The PLA, and cyclooxygenase (COX) mechanisms of increased PGE, formation were studied. Mass spectrometric analysis of sups taken from NC cultures showed quantities of arachidonic acid (AA) and linoleic acid (LA) released by NC cultures were lbfold (AA) and g-fold (LA) that of confluent cultures. However, Western blot of cPLA, indicated only a modest increase in cPLAz protein. Western blot of COX-2 in NC cells showed a 3 x increase COX-2. Exogenous AA conversion to PC& confined this. Ca++-dependent phospholipases were suggested by the 20 fold increase in PC& release observed in A23187-stimulated NC cultures over controls, The 2:l ratio of AALA release suggests the presence of both an AA-specific (cPLA,) and a non substrate-specific PLA, (sPL4) activity in NC cells, suggesting that keratinocytes migrating in a wound upregulate the activity of both cPLA, as well as non-selective phospholipase(s). COX-2 is also induced in NC cultures. This data suggests that enhanced fatty acid release and F’GE, synthesis is mediated by coordinate increases in phospholipase and COX-2.
0368
0371
p-PHENYLENED*ANrNE
IPPD)
Yo Kawakubo, Brunhilde Dermatology, University
N-ACETYLATrON
slilmeke Clinic,
IN
HUMAN
SKIN
CYTOBOL.
and Hans Merk, Department RWTH-*a&en, Oermany.
of
pd (diacetyl-PPD formation from monoacetyl-PPD), respectively. These data suggest that PPD is metabolized in human skin through Nacetylation to monoacetyl-and diacetyl-PPD and the responsible enzyme is NATI. Recent studies have shown that N?+=1gene exhibit variation
CHARACT!ZRIZATION OF A CLONING AND MOLECULAR DERMATOPHYTE CDNA CL.ONE, ENCODING A 70 KD HEAT SHOCK PROTEIN OF TRICHOPHYTON RUBRUM SassanRezaie.JosefBan. Christi Poitschek.DiviTschachler.Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, University of Vienna Medica School, Austria Tnchephytccmbmm causes90% cfall chmnic cases cfdermatophytcsis To characterize T.mbmm proteins et the molecular level, we stablished a cDNA library from poly (A)* RNAs tsdated from a 30 day culturecfthis fuogua.CompletecDNA of T rubrumwas used as the hybndtzxticnprobe for hbrary screening.Several strmgly hybridizing clones were identified end fwthm analyzed Here we describea recombmantcDNA clone of 1376 bp containinga single cpen reading t?ameof 1962 bp coding for 654 amino acids when this cDNA clone was used as a probe for Northernanalysts. it hybridued to a RNA ~pectesof’ about 3.5 kb whwh was constitctively expressedby Tmbmm cultured under standard condaicns.Analysts&be aminoactd sequenceofthe clone detectedstxq homologywth eukaryotic70 kDa heat shockprcteins @iSPs)and 84% identityto the sequenceof the two members cf the HSP 70 fkmdy of Nercspom cma and Cladosporum herbam. The presenceof theDLGlT-S-V umsesas sequenceallow us to include this new HSP famdy member m the Dnak subfamily Insummary, wehave cloned and charaderired the first member of the HSP family of demmtophyw and have shown that a IS expressed constdutivelyby Tmbmm m cell culture.
in human populations. Therefore, slow aceeylation by N*T1 enzyme may be a risk factor for individual susceptibility to PPD.
0369
0372
OXYGEN TENSIONSIN EXPERb5NTAL CONDITIONS OF WOUNDHEALING ; Demmnent ofDemmtclogyand CuttmoousSurgery,UniversityofMiarmSchwl ofluledtcine; M&i, FL. Law oxygentensionhas recentlyhen shownto showlatecellgmwih,clcmalexpansion, synthesiswd tmtuctipticnofcertain gmwththetas andextr&luler matrixcomponents Theseresultshave beenobtainedby expcsingcellculturesto a hypoxicenvircmment. However,there1slinetedinfcmmticmcmwhatthe e&al oxygentensionis at the cellsurface. Usma an oxwett probe.we studtedhow experimentalcondihcnsaffectthe oxygentension
Expression of vitamin 0 receptor (VDR) and retinoic acid receptors @AR-a,-P.-y; RXR-a,-P,?) in basal cell carcinomas. J Kamradt’, J Reich&h’. XH Zhu’. XF &, MF Holick’ Department of Dermatology, University of Saarland, Homburg, Germany; ‘Vitamin D. Skin and Bone Research Laboratory Boston University MedIcal Center, Boston, MA, U.S.A. We have analzed immunohistochemically the expression of 125.dlhydoxyvitamin D3 receptor proteins. the three retinoic acid receptor proteins @AR-a.+,-~) and rebnold X receptor-a:P,-y proteins in basal cell carcinomas (BCCs, n=15). Staining patterns of these different receptors were correlated to the labeling pattern of the proliferation marker Ki-67. Additionally we used RT-PCR to show the levels of VDR RNA in BCCs (n=7) compared to normal human epidermis (n=5). A strong lmmunoreactivity for VDR .RAR-a and RXR-a was observed in all BCCs, staining for RARy was moderate. whereas no or very weak labeling for RAR-p and RXR-P.7 protein was detectable. In contrast to RAR-y, that revealed no or only marginal differences in staining Intensities compared to adjcent uneffected epidermis, lmmunoreaciivity for RAR-a, RXRir and VDR was cons!stently stronger in the tumor cells. In general, labeling of BCCs for VDR, RAR-sly and RXR-a was pronounced in peripheral cells, whereas staining of central areas of the tumors was heterogenous Most of the tumors showed no correlation in comparing the labeling patterns of VDR, P.AR-a/-y and RXR-a with the staining pattern of KI-67. The results of the RT-PCR demonstrate higher levels of VDR-RNA in all BCCs compared to the levels in normal epidermis. Our findlngs indicate: (I) VDR, RAR4-y and RXR-a are in contrast to RAR-p strongly expressed in BCCs (II) expression of VDR, RAR-a and RXR-a is upregulated in BCCs compared to normal epidermis (Ill) Selective retlnoic ac!d metabolites and/or 1.25.dlhydroxyvltamin D. may be useful for preventive or terapeutical treatment of BCCs
at
&t&ble thec& surface. We found this dissolved oxygen tennon was dir&y related to the height ofthe mediumabwe the cellsurface(t=+.g793,p* 021),but was cmstant when
no cellswerepresentin the flask(r-0.9732, p=O.oOl).In both humandermalfibroblastsand NlH/3T3cultures,oxygentensiondecreasedlineerlyas celldensityincreased(r--0.835, pcO.ooO1, r-0.916, p+XlOl, respectively)Whenhumandermalfibmblastswereexposed to 2% 4, maximumhypoxiclevels(0 mmHg) wereachievedwithin 30 minutes,and the reccve,ytimeWBSultbin a swcilarhme tame. The additionofrctencme,an mtihtor ofcellular respition, blockedthis decreasein oxygeotensionat the cellsurface,suggestingthat ceUul.x c&sumpt~cm of oxygenISresponsiblefor the d&me. Lastly,we examinedthe cellsurface oxygentensionin uantroland ecutely-wounded humanskin equivalents@SE), wns~stingofa keretinccytelayerovere typ I ccl&en matrixccntainingfibmblaats.We foundthat oxygen tensicmdroppedsignificantly~O.ooOl) in wxaely-wounded areasofHSEoas mmparcdto unwoundedareasof HSEs and that this dropwas preventedby the additionofmitmnycinC. Theseresultsmdwatethat cell surfaceoxygentensim is indirectlypropwticnalto celldensity, andthat the amountofdetectable oxygm at the cellsurfaceis a functionof cell density, the oxyen tension tn the incubator, and increasedceUular activity, BEoccurs after injury
’