Interaction of human placental ribonuclease with placental ribonuclease inhibitor

The interactions of human placental ribonuclease inhibitor (PRI) with bovine pancreatic ribonuclease (RNase) A and human angiogenin, a plasma protein that induces blood vessel formation, have been characterized in detail in earlier studies. However, studies on the interaction of PRI with the RNase(s) indigenous to placenta have not been performed previously, nor have any placental RNases been identified. In the present work, the major human placental RNase (PR) was purified to homogeneity by a five-step procedure and was obtained in a yield of 110 micrograms/kg of tissue. The placental content of angiogenin was also examined and was found to be at least 10-fold lower than that of PR. On the basis of its amino acid composition, amino-terminal sequence, and catalytic properties, PR appears to be identical with an RNase previously isolated from eosinophils (eosinophil-derived neurotoxin), liver, and urine. The apparent second-order rate constant of association for the PR.PRI complex, measured by examining the competition between PR and angiogenin for PRI, is 1.9 X 10(8) M-1 s-1. The rate constant for dissociation of the complex, determined by HPLC measurement of the rate of release of PR from its complex with PRI in the presence of a scavenger for free PRI, is 1.8 X 10(-7) s-1. Thus the Ki value for the PR.PRI complex is 9 X 10(-16) M, similar to that obtained with angiogenin, and 40-fold lower than that measured with RNase A. Complex formation causes a small red shift in the protein fluorescence emission spectrum, with no significant change in overall intensity. The fluorescence quantum yield of PR and the Stern-Volmer constant for fluorescence quenching by acrylamide are both high, possibly due to the presence of an unusual posttranslationally modified tryptophan residue at position 7 in the primary sequence.     

Evolution of placental proteases

The placenta is a critical organ in mammals required for the transport of nutrients from the mother to the fetus during gestation. Other critical functions of the placenta include hormone regulation and immune regulation. The origin of the mammals and early placenta is relatively recent in evolutionary terms, and consequently there are few placenta-specific genes. In two separate branches of mammalian evolution, gene duplications have given rise to two large families of protease genes that are expressed only by placental tissues. A family of aspartic protease genes is expressed only in artiodactyls, and a family of cysteine protease genes is expressed only in rodents. These genes have probably evolved to perform specific functions in the placenta that are carried out by broader specificity proteases in mammalian species that do not express these proteases.     

Proteins-markers of placental insufficiency

The proteomic analysis of amniotic fluids of women with physiological pregnancy and pregnancy, complicated by placental insufficiency has been carried out on the second and the third trimesters. The differences in protein patterns between physiological pregnancy and pregnancy complicated by placental insufficiency seen during these gestation periods include: i) the absence of peroxiredoxin 2, epidermal fatty acid binding protein, and haptoglobin in placental insufficiency; ii) appearance of several proteins absent in physiological pregnancy: hippocalcin-like protein 1, CDC37-like protein, NKG2D ligand 2 (II trimester), CDC37-like protein, NKG2D ligand 2 (III trimester). These differences in the amniotic fluid proteome, obviously, have pathogenetic importance for the development of the placental insufficiency. The revealed proteins of distinction may serve as markers of this obstetrical pathology.     

Nontrophoblastic placental tumors

The aim of the study was to investigate potential influence of placental tumors on fetal outcome. The study comprised 10 cases of placental tumors. The analysis included the sonographic assessment of the tumor, signs of fetal anemia, as well as signs of hemodynamic disturbances or heart failure, and intrauterine treatment. The fetal hemodynamic was examined on the basis of Doppler blood flow in the umbilical artery and vein, middle cerebral artery, and ductus venous. The evaluation of fetal heart included the measurement of heart size, blood flow through cardiac valves and the assessment of fetal heart function based on cardiovascular score. The fetal outcome was also assessed according to birthweight, gestational age at delivery, pH, Ap score at 5th minute, abnormal neurological development and the need of intrauterine therapy. Ten cases of placental tumors were prenatally detected from 1999 to 2011. Among them 7 cases of hypoechogenic, non-vascularized cysts were identified and these neither effected the hemodynamics nor complicated fetal outcome. The vascularized tumors (chorioangioma) were the cause of severe anemia and hemodynamic disturbances and these led to fetal cardiac heart failure. In all cases of vascularized tumors from 2-3 intrauterine transfusion were performed. Rich vascularized tumors (chorioangioma) may cause hemodynamic disturbances and fetal heart failure. This may require intrauterine treatment and may result in abnormal fetal outcome and neurological development.     

Control of placental protein production by retinoic acid in cultured placental cells

Summary The synthesis of human chorionic gonadotropin (HCG), the subunit of HCG (HCGα), and pregnancy-specific β₁-glycoprotein (PSβG) was studied in temperature senstive (ts), simian virus 40 (SV40)tsA mutant-transformed human first trimenster placental (SPA255-26) cells. Retinoic acid increased the production of HCG and PSβG but inhibited the production of HCGα in these cells. Passage ofSPA255-26 placental cells in medium containing retinoic acid induced a stable altered phenotype characterized by elevated levels of HCG and PSβG and a reduced level of HCGα. The retinoic acid induced phenotypic changes in these placental cells were reversible; removal of retinoic acid immediately decreased the production of HCG and PSβG while increasing the production of HCGα. The ratio of HCG to HCGα in controlSPA255-26 cells was approximately 0.1; this ratio in creased to 4.8 in cells maintained in medium containing retinoic acid. Similarly, the HCG-to-HCGα ratio increased in choriocarcinoma cells maintained in retinoic acid containing medium. Our data suggest that retinoic acid may be needed to maintain a blanced production of HCG, HCGα, and PSβG in placental cells in vitro. Retinoic acid may also play a role in modulating placental protein production during pregnancy.     

Restriction of placental growth in sheep enhances placental metabolism of fetal beta-endorphin-like immunoreactivity

The opioid polypeptide beta-endorphin is present in fetal blood but it is not clear whether its source is the fetus or the placenta. We therefore measured beta-endorphin in extracts of fetal femoral arterial and umbilical venous blood plasma in sheep by radioimmunoassay to determine whether the fetus or the placenta is the major source of beta-endorphin in the fetal circulation. Chromatographic analysis of extracts of fetal arterial plasma showed that beta-lipotropin and other precursors of beta-endorphin made only a minor contribution to the immunoreactivity detected. Concentrations of immunoreactive beta-endorphin were higher in the femoral artery than in the umbilical vein in fetal sheep between 113 and 128 days of pregnancy. Therefore the placenta removes beta-endorphin or a closely related polypeptide of fetal origin from the umbilical circulation in sheep at this stage of gestation. Acute hypoxaemia and hypoglycaemia increase the concentrations of immunoassayable beta-endorphin in blood plasma of adult and fetal sheep, but little is known about the effects of chronic hypoxaemia or hypoglycaemia on the circulating levels of beta-endorphin and related polypeptides in the fetus. Therefore we also measured immunoreactive beta-endorphin in blood plasma from fetal sheep in which growth retardation in association with restricted placental growth was produced by removal of endometrial caruncles before mating. Intra-uterine growth retardation was accompanied by chronic hypoglycaemia and chronic hypoxaemia in the fetuses. This was not associated with higher concentrations of beta-endorphin-like immunoreactivity in fetal arterial or umbilical venous plasma, but was accompanied by significantly increased placental extraction of fetal immunoreactive beta-endorphin from the umbilical circulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Progesterone maintenance of the placental progesterone receptor and placental growth in ovariectomized rats

These studies examine the trophic effects of progesterone (P) on the progesterone receptor (Rp) and growth of the decidua basalis (DB) and junctional zone (JZ) in the rat placenta. Pregnant rats were ovariectomized (Ovx) in mid-pregnancy and received steroid replacement therapy consisting of implantation of P pellets (25 mg) and injections of estradiol (E), 2 micrograms s.c., daily. Placental protein synthesis, measured by 3H-leucine incorporation in vitro, decreased more than 99% within 24 h of Ovx. However, treatment with P immediately after castration maintained control levels of synthesis. Delay of P treatment for 4 h caused a 60% decline in protein production measured 20 h later (p less than 0.01). Intraperitoneal implantation of a 50-mg pellet of the antiprogestin, RU-38486, in intact pregnant rats decreased protein synthesis by 50% within 6 h and by more than 90% 12 h and 24 h post-implantation (p less than 0.01). Growth of DB and JZ in Ovx rats treated for 48 h with P and/or E was studied both histologically and by changes in protein and DNA content. Rp binding activity was also measured by exchange assay under equilibrium conditions. Only P was able to reverse the effects of Ovx on growth of the DB and JZ. P also maintained Rp levels in the DB above those observed in Ovx and Ovx + E-treated groups (p less than 0.01). The Rp may be a constitutive product in the JZ since binding activity was not altered by Ovx or by steroid treatments. This study shows that P is clearly a trophic hormone of the maternal and chorioallantoic placenta and is essential for placental growth, cellular differentiation, and histological integrity.     

Expression of placental lactogen and cytokeratin in bovine placental binucleate cells in culture

Binucleate cells are present in ruminant placenta and play an endocrine role in the production of many hormones during pregnancy. We isolated and cultured binucleate cells from bovine placenta at middle to late gestation and characterized these cells using immunofluorescence techniques. Enriched preparations of binucleate cells were obtained using Percoll density gradient centrifugation following collagenase digestion. Binucleate cells in culture preferentially attached to collagen-coated dishes rather than to noncoated plastic dishes. The cells gradually extended their edges on collagen substrata, and finally assumed a flattened morphology. Antibodies to placental lactogen (PL) and pregnancy-associated glycoprotein-1 (PAG-1) specifically stained the majority of round binucleate cells, but not the flat cells. We found that PL-positive binucleate cells were consistently devoid of cytokeratin. In contrast, cytokeratin was expressed in PL-negative binucleate cells as well as mononuclear epithelial cells. Furthermore, the PL-negative flat binucleate cells also developed intense cytokeratin networks in the cytoplasm. These results indicate that cytokeratin expression is inversely proportionate to that of PL in cultured binucleate cells. We conclude that downregulation of cytokeratin in binucleate cells is a function of the state of cellular differentiation.     

Synthesis of first trimester placental alkaline phosphatase in cultured human term placental cells

Alkaline phosphatase activity in human placental cells transformed by a tsA mutant of simian virus 40 (SV40) can be greatly induced by growing these cells at 40 degrees C, the temperature at which the tsA transformants regain their nontransformed phenotype. The induction of alkaline phosphatase in these cells requires the synthesis of both RNA and protein. The induced alkaline phosphatase from a SV40 tsA30 mutant-transformed term placental cell line (TPA30-1) was purified, characterized, and compared with alkaline phosphatase from term placenta and first trimester placenta. The form of alkaline phosphatase found in TPA30-1 cells differs from the phosphatase of term placenta in physiochemical and immunological properties. The TPA30-1 phosphatase is, however, indistinguishable from the alkaline phosphatase of human first trimester placenta by several criteria, including electrophoretic mobility, apparent molecular weight (Mr = 165,000), size of monomeric subunit (Mr = 77,000), heat lability, and sensitivity to inhibition by amino acids and EDTA. In addition, alkaline phosphatase from both TPA30-1 cells and first trimester placenta can be inactivated by antiserum to liver alkaline phosphatase but not by antiserum to term placental alkaline phosphatase. The induction of first trimester phosphatase in cells derived from term placenta provides a system for the study of alkaline phosphatase gene regulation in human placenta.