Oxidative stress promotes the increase of matrix metalloproteinases-2 and -9 activities in the feto-placental unit of diabetic rats

Maternal diabetes increases the risk of congenital malformations, placental dysfunction and diseases in both the neonate and the offspring's later life. Oxidative stress has been involved in the etiology of these abnormalities. Matrix metalloproteases (MMPs), involved in multiple developmental pathways, are increased in the fetus and placenta from diabetic experimental models. As oxidants could be involved in the activation of latent MMPs, we investigated a putative relationship between MMPs activities and oxidative stress in the feto-placental unit of diabetic rats at midgestation. We found that H₂O₂ enhanced and that superoxide dismutase (SOD) reduced MMPs activities in the maternal side of the placenta and in the fetuses from control and diabetic rats. MMPs were not modified by oxidative status in the fetal side of the placenta. Lipid peroxidation was enhanced in the maternal and fetal sides of the placenta and in the fetus from diabetic rats when compared to controls, and gradually decreased from the maternal placental side to the fetus in diabetic animals. The activities of the antioxidant enzymes SOD and catalase were decreased in the maternal placental side, catalase activity was enhanced in the fetal placental side and both enzymes were increased in the fetuses from diabetic rats when compared to controls. Our data demonstrate changes in the oxidative balance and capability of oxidants to upregulate MMPs activity in the feto-placental unit from diabetic rats, a basis to elucidate links between oxidative stress and alterations in the developmental pathways in which MMPs are involved.     

Expression of an isoform of the testis-specific estrogen sulfotransferase in the murine placenta during the late gestational period

Cytosolic sulfotransferases play essential roles in regulating the activities and transfer of steroids. To evaluate their biological significance in the murine uterus and placenta during the course of gestation, we determined their activities with several steroids as substrates. Activated estrogen sulfotransferase (EST) was found in the placenta and uterus during the late gestational period. Reverse-transcribed cDNA of murine placental EST (mpEST) was isolated from mouse placenta at 18 days of gestation and its expression in the tissue coincided with a change in its enzyme activity. The open-reading frame of mpEST encodes a protein composed of 296 amino acids with a predicted molecular mass of 35.5 kDa and was revealed to be an isoform of the murine testis-specific EST gene (99.7%). Also, the amino acid sequence of mpEST showed 49.6 and 77.9% homology with human placental and endometrial EST, respectively, showing that it corresponds to human endometrial EST. COS-7 cells transfected with mpEST exhibited sulfotransferase activity with the phenolic hydroxy groups of steroids and artificial substrates. The best acceptor substrate was estrogen.     

Placental calcium and phosphorus transfer in the guinea pig: lack of effect of modulators of Ca-ATPase and alkaline phosphatase activity

The effects of modulators of Ca-ATPase and alkaline phosphatase (AP) activity on placental calcium and phosphorus transfer were studied using the in situ perfused guinea pig placenta. The diuretics ethacrynic acid and furosemide had no significant effect on placental calcium and phosphorus transfer when injected into the mother (1.0 or 10.0 mg X kg-1) or added to the solution perfusing the fetal side of the placenta (0.25 or 2.0 mM). These two drugs have previously been shown to inhibit placental Ca-ATPase and enhance AP activity in vitro. D-Penicillamine, which inhibits placental AP but not Ca-ATPase activity in vitro, also had no significant effect on net calcium and phosphorus transfer from mother to fetus either when given to the mother (50 mg X kg-1) or added to the placental perfusion solution (0.25 or 2.0 mM). These results suggest that placental transfer of calcium and phosphorus in the guinea pig may not be directly related to placental Ca-ATPase and AP activities.     

Oxidative stress promotes the increase of matrix metalloproteinases-2 and -9 activities in the feto-placental unit of diabetic rats

Maternal diabetes increases the risk of congenital malformations, placental dysfunction and diseases in both the neonate and the offspring's later life. Oxidative stress has been involved in the etiology of these abnormalities. Matrix metalloproteases (MMPs), involved in multiple developmental pathways, are increased in the fetus and placenta from diabetic experimental models. As oxidants could be involved in the activation of latent MMPs, we investigated a putative relationship between MMPs activities and oxidative stress in the feto-placental unit of diabetic rats at midgestation. We found that H₂O₂ enhanced and that superoxide dismutase (SOD) reduced MMPs activities in the maternal side of the placenta and in the fetuses from control and diabetic rats. MMPs were not modified by oxidative status in the fetal side of the placenta. Lipid peroxidation was enhanced in the maternal and fetal sides of the placenta and in the fetus from diabetic rats when compared to controls, and gradually decreased from the maternal placental side to the fetus in diabetic animals. The activities of the antioxidant enzymes SOD and catalase were decreased in the maternal placental side, catalase activity was enhanced in the fetal placental side and both enzymes were increased in the fetuses from diabetic rats when compared to controls. Our data demonstrate changes in the oxidative balance and capability of oxidants to upregulate MMPs activity in the feto-placental unit from diabetic rats, a basis to elucidate links between oxidative stress and alterations in the developmental pathways in which MMPs are involved.     

Dehydrogenase and oxoreductase activities of porcine placental 11beta-hydroxysteroid dehydrogenase

Dehydrogenase (cortisol to cortisone) and oxoreductase (cortisone to cortisol) activities of porcine placental 11beta-hydroxysteroid dehydrogenase (11beta-HSD) were measured in tissue fragment cultures on day 75 of gestation. Dehydrogenase activity was over fivefold greater than oxoreductase activity (p < .001). There were positive linear associations (p < .01) between net dehydrogenase activity (dehydrogenase minus oxoreductase) and fetal weight, fetal length, and placental weight. These data indicate a predominance of placental 11beta-HSD dehydrogenase activity at this gestational stage that would insure a net conversion of cortisol to cortisone as it traverses the placenta. The data further suggest that 11beta-HSD activities may provide an optimal glucocorticoid environment that is supportive of enhanced fetal and placental growth.     

羊胎盘提取液及其组分(SPIF-Ⅰ)的免疫活性研究

对羊胎盘提取液(Sheep p lacental extract,SPE)及其组分免疫调节活性因子-Ⅰ(Sheep p lacental immunoreglating ac-tivity factor-Ⅰ,SPIF-Ⅰ)进行高效液相色谱(HPLC)分析并通过E玫瑰花环试验进行免疫生物活性评价。结果表明:在羊胎盘提取液中质量分数为11.94%的SPIF-Ⅰ同羊胎盘提取液一样能显著促进人外周血淋巴细胞E玫瑰花环形成,具有明显的剂量-效应依赖关系。SPIF-Ⅰ是羊胎盘提取液中一种具有免疫调节的小分子活性多肽。     

Subcellular distribution of isocitrate dehydrogenase in early and term human placenta.

The activities of NAD-specific and NADP-specific isocitrate dehydrogenases were measured in early and term human placenta. In both tissues the activity of NADP-specific isocitrate dehydrogenase was severalfold higher than that of the NAD-dependent enzyme. Subcellular distribution of these two enzymes in the placental tissue was estimated. About 60% of the total NADP-specific isocitrate dehydrogenase activity was found in the mitochondrial fraction and about 40% in the cytosol fraction. Insignificant amounts of the total activity were bound to the microsomal fraction. The whole of the NAD-specific isocitrate dehydrogenase activity was localized in the mitochondrial fraction. The total mitochondrial NADP-specific isocitrate dehydrogenase activity in both early and term placenta was also estimated from the mitochondrial specific activity of this enzyme and the amount of mitochondrial protein in wet tissue, calculated from the activities of citrate synthase or cytochrome c oxidase assayed in the isolated mitochondrial fraction and in the tissue of early and term human placenta.     

羊胎盘提取液及其组分(SPIF-Ⅰ)的免疫活性研究

对羊胎盘提取液(Sheep p lacental extract,SPE)及其组分免疫调节活性因子-Ⅰ(Sheep p lacental immunoreglating ac-tivity factor-Ⅰ,SPIF-Ⅰ)进行高效液相色谱(HPLC)分析并通过E玫瑰花环试验进行免疫生物活性评价。结果表明:在羊胎盘提取液中质量分数为11.94%的SPIF-Ⅰ同羊胎盘提取液一样能显著促进人外周血淋巴细胞E玫瑰花环形成,具有明显的剂量-效应依赖关系。SPIF-Ⅰ是羊胎盘提取液中一种具有免疫调节的小分子活性多肽。     

Purification and characterization of lysosomal β-glucuronidase from human placenta

1. Subcellular fractions of human placenta were prepared by nitrogen-bomb homogenization and differential centrifugation. 2. beta-Glucuronidase from placental lysosomes was purified 2100-fold on a protein basis. 3. The lysosomal enzyme, at different stages of purification, was characterized by using 4-methylumbelliferyl beta-d-glucuronide and phenolphthalein beta-d-glucuronide as substrates. 4. Only one isoenzyme of beta-glucuronidase was found in placenta; the enzyme in the endoplasmic reticulum appeared to be the same as the lysosomal enzyme. 5. The isoenzyme contained in normal plasma was different from that of the placenta. 6. The elevated beta-glucuronidase activity found in plasma obtained during pregnancy was due to increased activity of the normal plasma isoenzyme; no contribution was made by placental isoenzyme. 7. Plasma contained a heat-stable, non-diffusible activator of placental beta-glucuronidase. 8. A heat-stable competitive inhibitor of placental and plasma beta-glucuronidase was also present in plasma.     

PHosphate metabolism and foetal growth in the rat. I. Net transfer of 31P and 32P phosphate from the maternal plasma to the normal placenta

Net transfer of 31P and 32P inorganic phosphate form the maternal plasma to the rat chorio-allantoic placenta has been studied after intraperitoneal injection of [32P] ortho-phosphate in primigravid females at the 12th day or late stages of gestation. The concentration and label uptake per unit weight of placenta of the inorganic phosphate (Pi), organic-bound acid-soluble phosphate (POAS) and organic-bound acid-insoluble phosphate (POAIS) fractions are negatively correlated with increasing placental weight, whereas their specific activities are independent of placental weight. The amount and label uptake per whole placenta of the Pi, POAS and POAIS fractions are positively correlated with increasing placental weight. The placental concentrations of inorganic phosphate and calcium are positively related without, however, any marked accumulation of calcium. The growing placenta is thus shown to reduce progressively, on a unit weight basis, both the inorganic phosphate uptake from the maternal plasma and its further incorporation into organic-bound fractions. There is no evidence of a control by the foetal weight, acting per se, on these placental activities.     

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.     

Comparison of arylsulfatase C and steroid sulfatase from human placenta and liver

Human placental and hepatic arylsulfatase C (ASC) were purified to homogeneity and about 1,000-fold, respectively. Placental ASC hydrolyzed sterol sulfates at the same active site, whereas the major hepatic ASC did not. This major hepatic ASC isozyme was more thermolabile than placental ASC and steroid sulfatase from both placenta and liver. It was not precipitated by anti-bovine ASC IgG which quantitatively precipitated both placental ASC and steroid sulfatase activities from placenta and liver. A minor hepatic ASC isozyme with similar electrophoretic mobility to the placental enzyme copurified with the major hepatic ASC and is likely responsible for the steroid sulfatase activity in this organ. Hence, placental ASC and steroid sulfatase are biochemically and antigenically identical to hepatic steroid sulfatase. In contrast, the major hepatic ASC is a distinct protein whose catalytic and structural properties differ from all the above enzymes.     

In vitro degradation of the Neb-Trypsin modulating oostatic factor (Neb-TMOF) in gut luminal content and hemolymph of the grey fleshfly, Neobellieria bullata

The unblocked hexapeptidic Trypsin Modulating Oostatic Factor of the fleshfly, an inhibitor of both trypsin and ecdysone biosynthesis, resists very well proteolytic breakdown by enzymes present in the lumen of the gut of previtellogenic fleshflies. However, when incubated in hemolymph of adult flies, females and males, its half-life time is a mere 0.5 min. In hemolymph of last instar larvae, this value increases to about 1.5 min. Whereas PMSF, a potent inhibitor of serine proteases has no effect, captopril and lisinopril, both known to be specific inhibitors of mammalian angiotensin I converting enzyme (ACE), effectively inhibit TMOF breakdown in fly hemolymph. Digestion of Neb-TMOF by recombinant Drosophila AnCE on itself results in identical degradation products as with total hemolymph. In both cases ESI-Qq-oa-Tof mass spectrometry demonstrated the appearance of peptide fragments with the sequences NPTN, LH and NP. These observations not only confirm the reported presence of circulating ACE-like activity in flies but also strongly suggest that in flies this hemolymph ACE-like activity might be involved in the regulation of the oostatic activity as exerted by Neb-TMOF.     

System beta and system A amino acid transporters in the feline endotheliochorial placenta

There is no knowledge of the transport mechanisms by which solutes cross the cat placenta or any other endotheliochorial placenta. Here, we investigated whether the amino acid transport systems beta and A are present in the cat placenta using a placental fragment uptake technique. Data were compared with studies in the human placenta, in which the presence of these two transport systems has been well established. A time course of [(3)H]taurine (substrate for system beta) and [(14)C]MeAIB (nonmetabolizable substrate for system A) uptake was determined in the term cat and human placental fragments in the presence and absence (choline substituted) of Na(+), and further studies were carried out over 15 min. Taurine uptake into both cat and human placenta fragments was found to be Na(+) and Cl(-) dependent, and Na(+)-dependent taurine uptake was blocked by excess beta-alanine. MeAIB uptake was found to be Na(+) dependent, and Na(+)-dependent MeAIB uptake was blocked by excess MeAIB or glycine. Western blotting and immunohistochemistry performed on cat and human placenta showed expression of TAUT and ATA2 (SNAT2), proteins associated with system beta and system A activity, respectively. This study therefore provides the first evidence of the presence of amino acid transport systems beta and A in the cat placenta.     

A specific antibody to a new peptide growth factor from human placenta: immunocytochemical studies on its location and biosynthesis

Recently, we isolated a new peptide growth factor of Mr 34 000 from synctial membranes of human placenta. In its polypeptide molecular weight and receptor binding specificity it is unlike several known growth factors. In this paper we described immunocytochemical studies on its cellular location and biosynthesis. A rabbit antiserum was raised against a homogeneous preparation of the placental peptide. The specificity of the antibody was established by immunoprecipitation and immunoblot analyses. The antibody recognized both the native and denatured 34-kilodalton (kDa) peptide but showed no binding to a variety of other growth factors and hormones tested. The antibody was used to investigate the genesis and location of the 34-kDa membranous mitogen. Immunoperoxidase staining of placental tissue slices revealed a restricted localization of the antigen in the cytoplasmic organelles of cytotrophoblasts and in the brush border membranes of syncytiotrophoblasts. No other placental structures contained the antigen. A developmentally regulated appearance of the mitogen was suggested by the fact that first trimester placenta consistently stained far more strongly than term placenta. These studies show that the 34-kDa mitogenic protein originates in placenta from embryo-derived cellular structures and suggest that in its strategic location it may influence trophoblastic growth in an autocrine manner. In other studies we investigated the presence and biosynthesis of the 34-kDa peptide in the A431 vulval carcinoma cell line, which was shown earlier to contain a membrane-associated 34-kDa growth factor. The studies demonstrate that this cell line, as well as some other human carcinomas of breast and bladder origin, actively expresses this peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the cellular basis for the inhibition of cytolytic effector cells by murine placenta

Direct suppression of cytolytic effector cell function by cells of the placenta may represent one mechanism that protects the "fetal allograft" from rejection by maternal transplantation immunity. Collagenase disaggregated murine placental cells block target cell lysis by natural killer, lymphokine-activated killer, and (CTL)-type killer cells. This inhibition is reversible and noncompetitive, similar to a previously described inhibitor of CTL found in spleens of mice undergoing an acute graft vs host (GVH) response. Velocity sedimentation separation of placental cells shows that the inhibitory activity is primarily associated with cells that cosediment with nucleated fetal erythrocytes. When these erythrocytes were lysed, an increased number of non-erythrocytic cells could be separated and under this circumstance, inhibitory activity was seen in association with either small white cells or fetal erythrocytes and with large white cells. There may be several cell populations in murine placenta that can inhibit cytolytic effector cells. The possible relevance of direct placental inhibition of cytolytic effectors to protection of the "fetal allograft" is discussed.     

Preserved placental oxygenation and development during severe systemic hypoxia

Local tissue oxygenation profoundly influences placental development. To elucidate the impact of hypoxia on cellular and molecular adaptation in vivo, pregnant mice at embryonic days 7.5-11.5 were exposed to reduced environmental oxygen (6-7% O2) for various periods of time. Hypoxia-inducible factor (HIF)-1alpha mRNA was highly expressed in the placenta, whereas HIF-2alpha was predominantly found in the decidua, indicating that HIF-1 is a relevant oxygen-dependent factor involved in placental development. During severe hypoxia, HIF-1alpha protein was strongly induced in the periphery but, however, not in the labyrinth layer of the placenta. Accordingly, no indication for tissue hypoxia in this central area was detected with 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide staining and VEGF expression as hypoxic markers. The absence of significant tissue hypoxia was reflected by preserved placental architecture and trophoblast differentiation. In the search for mechanisms preventing local hypoxia, we found upregulation of endothelial nitric oxide synthase (NOS) expression in the labyrinth layer. Inhibition of NOS activity by N(omega)-nitro-L-arginine methyl ester application resulted in ubiquitous placental tissue hypoxia. Our results show that placental oxygenation is preserved even during severe systemic hypoxia and imply that NOS-mediated mechanisms are involved to protect the placenta from maternal hypoxia.     

Immunoaffinity Purification of Human Choline Acetyltransferase: Comparison of the Brain and Placental Enzymes

A rapid and efficient immunoaffinity purification procedure has been developed for human placental choline acetyltransferase (ChAT). Using this procedure, human placental ChAT was purified to homogeneity with high recovery of enzyme activity (50-60%). Purified ChAT was used to raise a monospecific anti-human ChAT polyclonal antibody in rabbits. A comparison of the physical properties of ChAT was made between the enzymes purified from human brain and human placenta. Only one form of the enzyme exists in either tissue, having identical molecular weights of 68,000 and a single apparent pI of 8.1. A more detailed comparison of the two enzymes using peptide mapping and epitope mapping indicates identity between the brain and placental enzymes.