1
405 PRESENCE AND LOCALIZATION OF PROTEINS IMMUNOLOGI- ras ~21 EXPRESSION IN PROLIFERATING SKIN DISEASES CALLY RELATED TO ERYTHROCYTE SPECTRIN AND PROTEIN - 4.1 IN SKIN TADAMICHI SHIMIZU, HIROKO KOIZUMI, AND AKIRA OHKAWARA. Department of Dermatology, Hokkaido University School of Medicine, Sapporo Analogues of human erythrocyte spectrin and pro- tein 4.1 have been examined in the skin by immuno- chemical techniques using anti-human erythrocyte spectrin, anti- pig brain 8-fodrin and anti-human erythrosyte protein 4.1 antibodies. Immunoblot analysis revealed that pig epidermis contains spec- trin-like proteins of 240kDa and 235kDa as well as 4.1-like protein of 68kDa. In addition, pig epider- mis also contains higher molecular weight proteins. Immunofluorescence microscopy using these anti- bodies revealed that both spectrin-like proteins and 4.1-like proteins localize at the cell perip- heral cytoplasma of the basal cells and eccrine sweat gland cells. These results suggest the presence of membrane skeletal protein lattice in these cells. ARATA KIKUCAI,MASAYUKI AMAGAI*, KOICHI SAKURAOKA,AND TAKEJINISHIRAWA* Departments of Dermatology, Saiseikai Yokohamashi Nanbu Hosp., Yokohama,*Keio University Schoolof Medicine, Tokyo We reportedthe localization of ras oncogeneproduct~21 (p21ras) PositiveCells in various-&n tumorsand indicated that p21rasmay play s role in the differentiation of cells in variousskin conditions previously. In this study,we have examinedp2lrsspositivecells not only in skin carcinomas but also in other proliferating skin diseasessuch as psoriasis wlgaris, lichenplanus,verruca vulgaris, vsrrucaplanaejuvenilisand verruca senilisto elucidate the role of p2lrss in the epidermis. p2lrss positivecells were detectedby anti-rasp21 monoclonal antibodyNCC-RAS-001. We concludxhat in both tumorousand inflammatory skin conditions p21rasmay play a role in the differentiation of epidermal cells. FIBRONECTION AND FIBRIN IN EXPERIMENTAL MOUSE SKIN STAPHYLOCOCCUS AUREUS INFECTION HISANORIAKIYAMA, YOSHIKOABE, AKIKO KANAMOTO,HIROKO granularpattern in the dermis and musculuscutaneus layerat b hours after the inoculation and increased with time. No KANZAKI, JIROARATA Departmentof Dermatology, Okayama Univ.Medical School,Okayama Fibronectin (FN)wss studiedby immunohistochemistry in experimental staphylococcus sureus (SA)skin infection in cyclophosphamide-treated mice. FN began to depositin a DETMITION OF ORNITHINE DEXXRBOXYL?!SE GRdE EXPRESSION IN MCUSESKIN'IWA~WITHPH9REOLFSTER'IUMORPFX2GTERAND UL'll7AVIOLEX LIGHT USING IN SITU HYPRIDIZATICN. MXXIO FQKUDA, TAKESHI KC&O, MASAhlITSU ISHII, NOBLMJKI MIZUiW, ISPD MXI'SIJI-YUASA#, SWZO OU?NI#, AND ?DSHIO HAMADA. Departments of Dermatologyand Bicchemistry#, Osaka City University MedicalSchool,Osaka lccalizaticnof OCC gene expression in the mouse skin treated with ohotil ester /TPA)and ultraviolet liaht (WL) Ornithinedecarbcxylase(ODC),a rate-limiting enzyme in polytine biosynthesis, is a well-established marker of skin tumor pramticn. In the present study, we examined the depositof FN was observedin s linear patternuntil 7 days after the inoculation,By electronmicroscopy, SA was observedin edematous dermal tissueassociated looselywith fibrinand collagenfiber until 24 hours, encircled by fibrin during2 to 6 days, and began to conglomerate in fibrinmass at 7 days after the inoculation.These resultsdemonstrate that solubleFN is main FN in experimental mouse ski" SA infection, and indicatethat both FN and fibrin play a" importantrole in the developement and elimination of ski" SA infection, using in situ-hybridization.techniw. >. In the skin treated with TPA, the rum&r of positivecells (containing mxe than 5 grains) increased by 52-foldin the follicular epidermis, by 19-fold in the interfollicular epidermis,and by I-fold in the dermis, as camparedwith controls.In the skin treatedwith UVL, each increased by 25-fold, by la-fold,and by 3.5-fold,respectively. These results indicate that TPA and UVL increase ODC gene expression in the -se epidermis, especially in the follicular region. A NEW METHOD FOR QUANTIFYING KERATINOCYTE DIFFERENTIATION USING IMMUNOFLUORESCENT STAINING OF INVPLL'CRIN AND CYTOFLUOROGRAPHY. EFFECTS OF BUTYLATED PHENOLIC ANTIOXIDANTS ON EPIDERMAL OIWITHINE DECARESJW AmIVITY AND ITS GENE EXPRESSION INCUCEDBYpHoRBoL Es~lvMxPRC#YrER. HIDENOBUOKUMURA*,**,KOJI HASHIMOTO*,KUNIO MATSUMOTO*, KUNIHIKO YOSHIF.AWA*. *OSAKA UNIVERSITY SCHOOL OF MEDICINE,**NOEVIRCo.Ltd.,SHIGA INVOLUCRIN IS A PRECURSOR OF DETERGENT-INSOLUBLE CORNIFIED ENVELOPE AND A MARKER OF TERMINAL DIFFERENTIATIONOF EPIDERMAL KERATINOCYTES.TO QUANTIFY DIFFERENTIATIONOF CULTURED HUMAN KERATINOCYTES,THE POPULATION OF INVOLUCRIN- POSITIVE CELLS WAS ESTIMATED BY IMMUNOFLUORESCENT STAINING USING ANTI-INVOLUCRINANTIBODY AND FLOW CYTOMETRY. NORMAL HUMAN KERATINOCYTESWERE CULTURED UNDER THREE CONDITIONS FOR INDUCTION OF DIFFERENTIATION:O.Iti CaZ+,l.&nM CaZ+,AND l'.'BmM Ca2+ WITH 10% FETAL CALF SERUM. THE POPULATION OF sHc*TITANIm, T YUASA#, 9?S HI KCNO, NCWYUKI MIZIJN3, 1S.W MATSUI- sHu20oTAN1, AND 'ICSHIO HAMADn. Departmentsof Dermstolcxqy and Biochemist@, Osaka City University MedicalSchool,Osaka It has been reportedthat free-radical scavengers, such as butylated hydroxyanisole (BHA) or hydroxytoluene (BFT), suppress skin tumor promotion. In the present study, we examined the effect of BHA and BHT on the activity of ornithine decxboxylase (One, a well-established indicator of tumor promotion) and its gene expression induced by phorholester tumor prater (TPA)in the -se skin. TPA-induced ODC activity was inhibited remarkably by topical application of 55 pmol of BHA (inhibition rate; about SO%, measured at 6h). In Northern and dot-blot analysis,.TPA-caused ODC mRNA level was also markedly inhibit4 hy the sas~e dose of BH?+(inhibition rate; a!xut 60%, measured at 4h). On the other hand, BHT failed to <..&<b.<C _A ^~.^I IIuIWILrrn-cnus& ODC induction and its gerie expression. m___ ____,L_ xggest the involvwent of the decrease in .rL____on level in the mechanism of ODC-inhibitory IHA. INVOLUCRIN-POSITIVE CELLS INCREASED FROM 8.0% TO 22.1%(2.8 FOLD) BY ELEVATING Ca2+ CONCENTRATION,AND TO 37.1%(4.6 FOLD) BY ADDITION OF FCS. THE CYTOFLUOROGRAPHIC ANALYSIS OF INVOLUCRINEXPRESSION MADE IT POSSIBLE TO DETERMINE THE NUMBER OF DIFFERENTIATEDCELLS IN / 4 LARGE NUMBER OF SAMPLES PRECISELY AND RELIABLY. THUS, IT IS A USEFUL METHOD FOR m gene -,-mci, effectof E QUANTIFYING KERATINOCYTE DIFFERENTIATION.

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Page 1: Fibronection and fibrin in experimental mouse skin staphylococcus aureus infection

405

PRESENCE AND LOCALIZATION OF PROTEINS IMMUNOLOGI- ras ~21 EXPRESSION IN PROLIFERATING SKIN DISEASES CALLY RELATED TO ERYTHROCYTE SPECTRIN AND PROTEIN

-

4.1 IN SKIN

TADAMICHI SHIMIZU, HIROKO KOIZUMI, AND AKIRA OHKAWARA. Department of Dermatology, Hokkaido University School of Medicine, Sapporo Analogues of human erythrocyte spectrin and pro-

tein 4.1 have been examined in the skin by immuno- chemical techniques using anti-human erythrocyte spectrin, anti- pig brain 8-fodrin and anti-human erythrosyte protein 4.1 antibodies. Immunoblot analysis revealed that pig epidermis contains spec- trin-like proteins of 240kDa and 235kDa as well as 4.1-like protein of 68kDa. In addition, pig epider- mis also contains higher molecular weight proteins.

Immunofluorescence microscopy using these anti- bodies revealed that both spectrin-like proteins and 4.1-like proteins localize at the cell perip- heral cytoplasma of the basal cells and eccrine sweat gland cells.

These results suggest the presence of membrane skeletal protein lattice in these cells.

ARATA KIKUCAI, MASAYUKI AMAGAI*, KOICHI SAKURAOKA, AND TAKEJI NISHIRAWA* Departments of Dermatology, Saiseikai Yokohamashi Nanbu Hosp., Yokohama, *Keio University School of Medicine, Tokyo We reported the localization of ras oncogene product ~21

(p21ras) Positive Cells in various-&n tumors and indicated that p21ras may play s role in the differentiation of cells in various skin conditions previously. In this study, we have examined p2lrss positive cells not

only in skin carcinomas but also in other proliferating skin diseases such as psoriasis wlgaris, lichen planus, verruca vulgaris, vsrruca planae juvenilis and verruca senilis to elucidate the role of p2lrss in the epidermis. p2lrss positive cells were detected by anti-ras p21 monoclonal antibody NCC-RAS-001. We concludxhat in both tumorous and inflammatory skin conditions p21ras may play a role in the differentiation of epidermal cells.

FIBRONECTION AND FIBRIN IN EXPERIMENTAL MOUSE SKIN STAPHYLOCOCCUS AUREUS INFECTION

HISANORI AKIYAMA, YOSHIKO ABE, AKIKO KANAMOTO, HIROKO

granular pattern in the dermis and musculus cutaneus layer at b hours after the inoculation and increased with time. No

KANZAKI, JIROARATA Department of Dermatology, Okayama Univ. Medical School, Okayama Fibronectin (FN) wss studied by immunohistochemistry in experimental staphylococcus sureus (SA) skin infection in cyclophosphamide-treated mice. FN began to deposit in a

DETMITION OF ORNITHINE DEXXRBOXYL?!SE GRdE EXPRESSION IN MCUSESKIN'IWA~WITHPH9REOLFSTER'IUMORPFX2GTERAND UL'll7AVIOLEX LIGHT USING IN SITU HYPRIDIZATICN. MXXIO FQKUDA, TAKESHI KC&O, MASAhlITSU ISHII, NOBLMJKI MIZUiW, ISPD MXI'SIJI-YUASA#, SWZO OU?NI#, AND ?DSHIO HAMADA. Departments of Dermatology and Bicchemistry#, Osaka City University Medical School, Osaka

lccalizaticn of OCC gene expression in the mouse skin treated with ohotil ester /TPA) and ultraviolet liaht (WL)

Ornithine decarbcxylase (ODC), a rate-limiting enzyme in polytine biosynthesis, is a well-established marker of skin tumor pramticn. In the present study, we examined the

deposit of FN was observed in s linear pattern until 7 days after the inoculation, By electronmicroscopy, SA was observed in edematous dermal tissue associated loosely with fibrin and collagen fiber until 24 hours, encircled by fibrin during 2 to 6 days, and began to conglomerate in fibrin mass at 7 days after the inoculation. These results demonstrate that soluble FN is main FN in experimental mouse ski" SA infection, and indicate that both FN and fibrin play a" important role in the developement and elimination of ski" SA infection,

using in situ-hybridization.techniw. >.

In the skin treated with TPA, the rum&r of positive cells (containing mxe than 5 grains) increased by 52-fold in the follicular epidermis, by 19-fold in the interfollicular epidermis, and by I-fold in the dermis, as campared with controls. In the skin treated with UVL, each increased by 25-fold, by la-fold, and by 3.5-fold, respectively. These results indicate that TPA and UVL increase ODC gene expression in the -se epidermis, especially in the follicular region.

A NEW METHOD FOR QUANTIFYING KERATINOCYTE DIFFERENTIATION USING IMMUNOFLUORESCENT STAINING OF INVPLL'CRIN AND CYTOFLUOROGRAPHY.

EFFECTS OF BUTYLATED PHENOLIC ANTIOXIDANTS ON EPIDERMAL OIWITHINE DECARESJW AmIVITY AND ITS GENE EXPRESSION INCUCEDBYpHoRBoL Es~lvMxPRC#YrER.

HIDENOBU OKUMURA*,**,KOJI HASHIMOTO*,KUNIO MATSUMOTO*, KUNIHIKO YOSHIF.AWA*. *OSAKA UNIVERSITY SCHOOL OF MEDICINE,**NOEVIR Co.Ltd.,SHIGA

INVOLUCRIN IS A PRECURSOR OF DETERGENT-INSOLUBLE CORNIFIED ENVELOPE AND A MARKER OF TERMINAL DIFFERENTIATION OF EPIDERMAL KERATINOCYTES. TO QUANTIFY DIFFERENTIATION OF CULTURED HUMAN KERATINOCYTES, THE POPULATION OF INVOLUCRIN- POSITIVE CELLS WAS ESTIMATED BY IMMUNOFLUORESCENT STAINING USING ANTI-INVOLUCRIN ANTIBODY AND FLOW CYTOMETRY. NORMAL HUMAN KERATINOCYTES WERE CULTURED UNDER THREE CONDITIONS FOR INDUCTION OF DIFFERENTIATION:O.Iti CaZ+,l.&nM CaZ+,AND l'.'BmM Ca2+ WITH 10% FETAL CALF SERUM. THE POPULATION OF

sHc*TITANIm, T YUASA#, 9?S

HI KCNO, NCWYUKI MIZIJN3, 1S.W MATSUI- sHu20oTAN1, AND 'ICSHIO HAMADn.

Departments of Dermstolcxqy and Biochemist@, Osaka City University Medical School, Osaka It has been reported that free-radical scavengers, such as

butylated hydroxyanisole (BHA) or hydroxytoluene (BFT), suppress skin tumor promotion. In the present study, we examined the effect of BHA and BHT on the activity of ornithine decxboxylase (One, a well-established indicator of tumor promotion) and its gene expression induced by phorhol ester tumor prater (TPA) in the -se skin. TPA-induced ODC activity was inhibited remarkably by

topical application of 55 pmol of BHA (inhibition rate; about SO%, measured at 6h). In Northern and dot-blot analysis,.TPA-caused ODC mRNA level was also markedly inhibit4 hy the sas~e dose of BH?+ (inhibition rate; a!xut 60%, measured at 4h). On the other hand, BHT failed to <..&<b.<C _A ^~.^I IIuIWIL rrn-cnus& ODC induction and its gerie expression. m___ ____,L_ xggest the involvwent of the decrease in

.rL____on level in the mechanism of ODC-inhibitory IHA.

INVOLUCRIN-POSITIVE CELLS INCREASED FROM 8.0% TO 22.1%(2.8 FOLD) BY ELEVATING Ca2+ CONCENTRATION, AND TO 37.1%(4.6 FOLD) BY ADDITION OF FCS. THE CYTOFLUOROGRAPHIC ANALYSIS OF INVOLUCRIN EXPRESSION MADE IT POSSIBLE TO DETERMINE THE NUMBER OF DIFFERENTIATED CELLS IN / 4 LARGE NUMBER OF SAMPLES PRECISELY AND RELIABLY. THUS, IT IS A USEFUL METHOD FOR

m gene -,-mci, effect of E

QUANTIFYING KERATINOCYTE DIFFERENTIATION.