Proteinase inhibitors in amniotic membrane

A.56 LIPOSOME-MEDIATED TRANSFECTION OF HUMAN PRIMARY CYTO-TROPHOBLASTS FROM TERM PLACENTA: A COMPARATIVE STUDY. R. Vasicek’, L. Saleh’, M.F. Wolschek’, P. Husslein’ and M. Knhfler’, Department of Obstetrics and Gynecology’, Department of Internal Medicine IV’, Faculty of Medicine, Vienna, Austria In the past, regulatory mechanisms controlling trophoblast-specific expression of hormone genes were mainly investigated in cytotrophoblastic choriocarcinoma cells cells due to the efficient uptake of DNA into these tumor cells. Primary cytotrophoblasts, however, differ from choriocarcinomas with respect to growth control/hormone expression and might use distinct mechanisms to establish trophoblast-specific gene expression throughout placental development. Although transfection of these cells has been described by some authors, no data are currently available which compare/optimize different methods of DNA delivery. Efficient transfection of these non-proliferating cells, howevdr, is crucial when low-expressing genes are studied. We compared six commercially available cationic liposome compounds and the classical CaPO,-precipitate with respect to uptake of the human trophoblast-specific ahCG promoter by term trophoblast cells, Percoll gradient centrifugation and isolated by immunopuritication. Methods included PCR and cloning of the proximal ahCG promoter into luciferase reporter constructs, different transfection reagents/conditions and uptake of promoter constructs and CMV4Gal plasmids (transfection control) into differentiating cytotrophoblasts. Our data indicate that compared to other substances, the non-toxic Fu GENETM 6 Transfection Reagent (B&ringer Mannheim GmbH, Austria) enhances GaVLuciferase expression 10 to 15 fold and therefore provides a suitable tool for rapid and efficient transfection. Beside these facts, we demonstate for the first time that the ahCG gene promoter is functional in primary trophoblasts undergoing syncytia formation.

ECTOPIC EXPRESSION OF OSTEOCALCIN AND OSTEOPONTIN MIGHT BE INDUCED BY VITAMIN D IN THE HUMAN PLACENTAL VILLI. H.Fukuoka, T. Fujikawa, S. Hayakawa’, K. Satoh’, Dept. of Developmantal Medical Sciences, the University of Tokyo, Dept. of Obstet. Gynecol, Nihon University’ Osteocalcin (OC), an abundant y -carboxylated protein, is produced uniquely by mature osteblast and is climcally used as a metabolic marker of bone formation. &topic synthesis by the plastelets, megacaryocyte and memacaryoblastic cell sarcoma was found. We analyzed OC(n#nl;mean+ SD) during pregnancy. The serum level of matemal OC had not changed during pregnancy. In oarturition, OC in the umbilical vein-(31.1 & 5.2) was high& than in the artery (24.01 9.6)(p
Placenta

HORMONAL REGULATION OF GELATINASE B AND CYCLOOXYGENASE 2 PRODUCTION BY PROGESTERONE AND ESTRADIOL-17~ IN UTERINE CELLS. T. Sato, H. Michizu, K. Imada, Y. Mori, and A. Ito, Department of Biochemistry, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan. Object - Degradation of extracellular matrix is a critical event in parturition. Prostaglandins (PGs) also play an important role in the initiation and maintenance of labor such as myometrial contraction, cervical ripening and fetal membrane rupture. We herein investigated the regulation of progelatinase Blpromatrix metalloproteinase 9 (proMMP-9) and PGE, production in pregnant rabbit uterine cervical fibroblasts by gelatin-zymography, - _ _ Western and Northern blotting, alid ra‘hio immunoassay. Results - The nroduction of nroMMP-9 in uregnant rabbit uterine cer%x was relativiiy higher than’ thit in nonpregnant one. When the cer&al-fibroblasts were treated with interleukin 1 (IL-l) (1 ne/mI), the production of proMMP-9 was increased ‘(iS-foldj.. The’ increased proMMP-9 production was transcriptionally suppressed by progesterone (1 FM). PGE, production was also augmented by IL-1 and the production was suppressed by progesterone and estmdiol-17s in a dose dependent manner. The decrease of PGE, production by both hormones was due to the suppression of COX-2 production in rabbit uterine cervical fibroblasts. Conclusion - Progesterone and estradiol-l7f3 substantially participates in maintaining the function of uterus by preventing the MMPs-dependent degradation of extracellular matrix in uterus and the PGl$initiated labor.

Proteinase

Inhibitors

in Amnlotlc

Membrane

(( J.S.

Kim, B.K.

Na, J.H., Hwang’, E.J. Shin, C.Y. Song, J.M. Jeong’. and J.C. Kim’ )) Department of Biology, Chung-Ang University, Department of Biology, Suwon University’, Department of Ophthalmology, Chung-Ang Universi$, Seoul, Korea To investigate the therapeutic effects of human membrane on keratitis, we analyzed the human membrane components as proteinase inhibiiors. Methods, Inhibitory effects of human amniotic membrane extracts on various proteinases were investigated using in vitro azocaseine assay and zymography. Proteinase inhibitors in human amniotic membrane were analyzed with anti-protelnase inhibitors antibodies. Results, Human amniotic membrane extracts showed inhibitory effects on various proteinases such as protelnases of Acanthamoeba, P. aerughwsa, A. fumigatus, S. aureus, trypsin, plasmin, cathepsln G, and collagenase. Western blot analysis revealed that existence of al-antitrypsin, a2-macroglobulin, inter-a-antkrypsin, a2-plasmin inhibitor, and a P-antichymobypsin in human amniotic membrane. Conclusions, Human amniotic membrane is known to be useful to treatment of keratitis. Human amniotic membrane extract showed inhibitory effects on various proteinases and had various proteinase inhibitors. This suggests that the therapeutic effeds of human amniotic membrane may be due to protelnase inhibitors. Supported by a grant of Goof Health RND project(HMP-97-M-5-0055), Ministry of Health and Welfare, R.O.K. Purpose,

amniotic amniotic

(1998),

Vol. 19