Production of monoclonal antibody to a high molecular weight desmosomal plaque protein and its cDNA cloning

388

PRODUCTION OF MONOCLONAL ANTIBODY TO A HIGH MOLECULAR WEIGHT DESMOSOMAL PLAQUE PROTEIN AND ITS cDNA CLONING TAKASHI HASWIMOTO, MASAYUKI AMAGAI, YOSHIO INOKUCHI~~,JUN KUDOHd, NOBUYOSHI SHIMIZU~~AND TAKEJI NISHIKAWA. Departments of DennatoloRy and Molecular Biology/i,Keio University School of Medicine, Tokyo. We obtained a mouse monoclonal antibody agaist a mass protein of approximately 680kDa, which reacted with attachment plaque of desmosome. We isolated 9 positive cDNA clones by immunoscreeningusing the monoclonal antibody as a probe. A clone with the largest insert "as further characterizedby Northern blotting and DNA sequencing.

THEPGRIPICATION ANDSOM BICCHMICAL PROPERTIES OF HIGHNOLKCDLAR WEIGlfl’ FWl’RASE(PRCEWGM) PRon RAT’SSKIN YASUSHI SUGA,KENJITAKAHORI, HIDEOKIIXANA Degtnent of Dermatology, Juntendo Univ. School of Medicine, The purification and properties of proteasome from rat’s skin was investigated. Skin proteasome was purified by successive (NH1j2S04 fractionation, Wenyl se&arose CL-48,and HPLCgel filtration chromatographles. The molecular weight was estimated to be 750kd by gel filtration. With regard to substrate specifity, SLLVl-MCA demonstrated the highest activity. 0.03% SDSstimulated enzyme activity to 2.000 % of the initial activity. Activity was inhibited by DPP, chymostatin, NIX, IA and leupeptin. These results show that both serine and cystein residues are related to the enzyme activity of proteasome. Total and specific enzyme activities of the epidermis were 150 % and 600 X, respectively, of the activity of the dermis. The imohistochemical study with anti-HMP-rabbit-&G-antibody by the ABCmethod revealed that the enzyme was distributed in the epidernis.

CELL?oCELL~A~ANDCWC6~

ADAPTATION

SHINICHI INOHARA, YASUSHI TANAKA, YUKOTATSI.iW, HAJIME NATSUURA, YUKIO KITAiW, SEIMIRO SAGAMI. Department of Dznatolcgy, Hycqo College of Medicine, Hyogo

Dept. of Dermatol..Kyoto Univ. Recently. we have reported a newly

SYSTFX? IN THE KERATINWYIFS ERm ZINC DEFICIEWF HUMANANTIRAT.

Imnunohistochemicaldistribution of desnoscnal proteins (desnnplakin,desrrcqlein)and cytoskeletal proteins (actin, keratin, tublin) in the involved epidermis fran zinc deficient hwan and rat were examined canparing with the epidermis fran normal human and rat. As .the results, whereas linear distribution of desnwscnal proteins and actin along the cell margin was oixerved in the all epidermal cells except horny layer fran noma human and art, this linear distribution could not be observed in the lower epidermis from zinc deficient human and rat. Cur previous report (Arch DerrnatolRes, 282:210-212, 1990) showed that actin might have a role in desmosane fornntion of the epidermal cells. Thus, the present report suggests that disoraanizationof actin misht tx relevant to disorganizationof desmosciw in the involved epidetis due to zinc deficiency.

OF A MURlNE VASCULAR ENDOTHELIOMA CELL LINEW-21‘l-0 SERUM FREE CUIXIRE CONDI-ITONS KEN-ICHI TODA, YURIYA MARUGUCHI. KAORU TSUJIOKA. CHIUNGSHAN CHEN. YUJI HORIGUCHI. YOSHIKI MIYACHI. AND SADAO

IMAMURA establIshed muI‘fne tumorigenic cell lineIF-2) preserving morphological as well as functional propertIes of vascular endotheltal cells~EC).(Cancer Res.. in press) To further characterize F-2 cells, we have developed serum free culture system of the cells with Cosmedium0xnobio Co.). In this system. F-2 cells slowly Droliferated and reached a ulateau ohase of erowth with resultant ionnatIon of tubule-like st&ure anii honeycobb appesrence, which was sknilar to the structure that was also accomplIshed under the serum containing regular culture condltlons with basement membrane comwnents. Matrigel. This !zems to swxest that the current serum free cult& system &ces the dlfferencIa& of F-2 cells to mimick the In vhro vascular tissue structures. The culture supematant of F-2 cells under the regular culture conditkms slgnlf@mtly facW.ated the growth of human EC. compared with that under the serum free culture conditions or reaular culture medium onlv. These obsewatlons show that the regular &dture system may InduG activated phase of F-2 cells. whereas the serum free system does rather resting and dftrerentiated phase of the cells. These two different culture systems may be useful for learning the biology of EC.

LOCALIZATION OF TYPES TISSUES

BY IN

KYU-SIJK LEE

I

AND

AND JOON-YOUNG

of extracellular

Ths

tissues

grains

III

analysis

showed

reprcssntstivs

cONA-mRNA with

KEIRYUNG

hybridization

expression ksloid

COLLAGEN

mRNAs

technique matrix dense

was mRNAs

on the

cultured

ksloid I and

genes.

results

that

are

actively

various

producing

fibrotic

suggest

For

skin

in situ

identification different diseases.

types

types

Northern

of types

tool

from

tissues.

of nunerous

labelsd

fibroblssts.

to detect

in ksloid

over-expression

Thsse

RNA

applied

localization

of radioactively

hybrids total

UNIVERSITY

demonstrated

is s valuable

IN KELOIO

SONG

OEPARTNENT OF OERIIATOLOGY, TAEGU, KOREA An in situ

III

SITU HYRRIOIZATION

I and

transfer

fibroblssts

III collagen

hybridization

of fibroblssts of collsgens

which in