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PubMed1984

Enrichment of high proliferation potential colony forming cells from mouse marrow by selecting low-density cells expressing receptors for wheat germ agglutinin.

Suspensions enriched for high proliferation potential colony forming cells (HPP-CFC's) were prepared from mouse marrow by selecting low-density cells stained with fluoresceinated wheat germ agglutinin on a fluorescence activated cell sorter. HPP-CFC's were 12-fold more concentrated in the final suspensions and co-enriched with a population of primitive CFUs detected in the spleens of irradiated mice reconstituted 13 days earlier with marrow. This is consistent with previous observations suggesting that these populations are closely related. The degree of enrichment for other haemopoietic progenitors was in the order HPP-CFC greater than day 8 CFUs greater than BFUe greater than GM-CFC greater than CFUe. "Extra" colonies developed when human placental conditioned medium (HPCM) was added to cultures of enriched suspensions already containing pregnant mouse uterus extract (PMUE). HPP-CFC's probably formed most of these and we discuss why counting these extra colonies may be more reliable than the conventional "size" assay for HPP-CFC's.

Animals↗
PubMed1981

Valproic acid and several metabolites: quantitative determination in serum, urine, breast milk and tissues by gas chromatography-mass spectrometry using selected ion monitoring.

A method has been developed for the simultaneous quantitative determination of valproic acid (2-propylpentanoic acid) and its metabolites 2-propyl-2-pentenoic acid (trans), 2-propyl-3-pentenoic acid (trans), 2-propyl-4-pentenoic acid, 3-hydroxy-2-propylpentanoic acid, 4-hydroxy-2-propylpentanoic acid, 5-hydroxy-2-propylpentanoic acid, 3-oxo-2-propyl-pentanoic acid, and and 2-propylglutaric acid. All compounds were extracted at pH 5.0 with ethyl acetate. The concentrated extracts were trimethylsilylated and the resulting mixtures analyzed by a gas chromatography-mass spectrometry-computer system operated in the selected ion monitoring mode. Linear calibration curves were obtained in the concentration ranges studied (0.1-20 microgram/ml for metabolites, 0.1-150 microgram/ml for valproic acid. The recoveries of the drugs were between 92 and 97%. The relative standard deviations were between 3.9 and 8.1% (analysis of multiple 10-microliter samples of patient urine). The lower detection limits were found to be between 2.8 and 18 ng/ml using 200-microliter serum samples. The derivatized extracts were stable for at least one week. Applications of the method described include studies of placental transfer for valproic acid and metabolites in the human, the elimination of these substances by the neonate, their transfer via mother's milk, and their levels in mouse brain.

Adult↗
PubMed1990

Cellular factors involved in transcription and Tax-mediated trans-activation directed by the TGACGT motifs in human T-cell leukemia virus type I promoter.

Human T-cell leukemia virus type I (HTLV-I) encodes a 40-kDa nuclear protein, Tax, which stimulates transcription from three 21-base pair (bp) repeats in its U3 region. Tax trans-activation is mediated via cellular factors that interact with the TGACGT motifs in the 21-bp repeats. Gel mobility shift assay and UV cross-linking analysis show that two proteins of 52 and 46 kDa in size bind the 21-bp repeat specifically. Base substitutions in the TGACGT motif which abolished Tax trans-activation abrogated factor binding whereas the repeats containing mutations that did not affect Tax trans-activation supported factor binding as the wild-type repeat. The 52- and 46-kDa factors are present in human T-cell lines Jurkat and MT4 (HTLV-I transformed) and in HeLa cells but are undetectable in a human placental cell line JEG-3, which gave a reduced level of trans-activation. JEG-3 extracts contain a distinct DNA binding activity that shows analogous sequence requirements as the 52- and 46-kDa proteins in interacting with the various 21-bp repeats. c-Jun and CREB (cAMP-responsive element binding factor) can stimulate transcription from HTLV-I long terminal repeats in JEG-3 cells. At least two copies of the 21-bp repeats are required for optimal trans-activation by c-Jun and CREB. Most single point mutations in the TGACGT motif that abolished Tax trans-activation, however, did not affect c-Jun- or CREB-directed transcriptional enhancement. These data indicate that many transcription factors including c-Jun and CREB exert stimulatory effects on HTLV-I transcription although they do not directly respond to Tax. The 52- and 46-kDa cellular proteins most likely are involved directly in Tax-mediated trans-activation, and they are tentatively named Tax activation factors I and II, respectively.

Base Sequence↗
PubMed1986

Pharmacokinetic studies of the disposition of acetaminophen in the sheep maternal-placental-fetal unit.

Pharmacokinetic studies of the disposition of acetaminophen were carried out in 12 chronically catheterized pregnant sheep. Acetaminophen was infused separately into the mother and the fetus on two occasions to steady state. The transplacental clearances from both sides of the placental membrane and the nontransplacental clearances in both the mother and the fetus were then determined using an indirect method, a two-compartment open pharmacokinetic body model. The placental clearance and the nontransplacental clearances were also determined independently by direct methods by measuring the extraction ratio and urinary clearances in both the mother and fetus. Model dependent (indirect) and model independent (direct) methods yielded similar results. Neither acetaminophen glucuronide nor acetaminophen sulfate was transferred across the placenta. However, acetaminophen was transferred in both directions exclusively via a passive diffusion mechanism with an extraction ratio of 0.12 across the placenta. Neither glucuronidation nor sulfation capacity was found in the sheep placenta. However, the conjugation pathways accounted for nearly all the nontransplacental elimination of acetaminophen in the mother. Both transplacental clearance and nontransplacental clearance of acetaminophen in the fetus increased with gestational age. The glucuronidation and sulfation activities were determined in the fetus in utero and found to be 16 and 74% of the respective activities in the pregnant ewe on a body weight basis. The two conjugation clearances did not increase significantly with the gestational age of the fetus during the third trimester.

Acetaminophen↗
PubMed2003

Placental trophoblast from successful human pregnancies expresses the tolerance signaling molecule, CD200 (OX-2).

PROBLEM: Th1 cytokine-dependent abortions in the CBA x DBA/2 mouse model have been linked to down-regulation of expression of the CD200 (OX-2) 'tolerance' signal on trophoblast and in decidua prior to onset of the abortion process. Abortions could be prevented by administration of a soluble CD200. Is CD200 expressed on trophoblast in successful human pregnancy? METHOD OF STUDY: As one cannot easily obtain trophoblasts in large quantities from successful human pregnancies in the first trimester prior to the onset of the abortion process at 6 weeks gestation, we examined as a first step, trophoblast isolated from term placentae (i.e. successful pregnancies). CD9- trophoblasts were isolated by affinity column and stained for intracellular cytokeratin, and surface CD200 using PE-anti-human CD200 monoclonal antibody. mRNA was extracted from CD9+ and CD9- cells and tested by reverse transcription-polymerase chain reaction for CD200 mRNA. CD9- placental cells were separated by velocity sedimentation and test for CD200-dependent suppression of an allogeneic human mixed lymphocyte culture where cytotoxic T cell (CTL) generation, and Thl --> Th2 cytokine production shift were measured. RESULTS: CD9- but not CD9+ placental cell populations contained cells with mRNA for CD200, both a normal length transcript and a truncated transcript. Flow cytometry showed a CD200+ cytokeratin+ moderate-to-large-sized cell population compatible with trophoblasts and a smaller subset of cytokeratin- cells that expressed CD200 at normal and at high levels. The moderate-sized population proved most potent at inhibiting CTL generation and caused a Th1 --> Th2 cytokine shift. These effects were blocked by monoclonal anti-CD200. CONCLUSIONS: A subpopulation of cytokeratin+ placental trophoblasts express bioactive CD200 able to alter maternal immune responses in a favorable (Th2 > Th1) direction. Two populations of CD200+ small- and medium-small-sized cytokeratin- placental cells remain to be identified. Studies of karyotyped first trimester elective termination and spontaneous miscarriage tissues are needed.

Animals↗
PubMed1988

Pituitary factors regulating mouse placental lactogen-II secretion during the last half of pregnancy in mice.

Hypophysectomy of mice during the last half of pregnancy results in an increase in the maternal serum mouse placental lactogen-II (mPL-II) concentration. The present study was undertaken to isolate and identify the pituitary factors that regulate mPL-II. Administration of pituitary extracts from virgin and day 12 pregnant mice (five pituitaries per mouse, every 8 h, sc) and bilateral pituitary implants under the kidney capsule significantly inhibited the increase in the mPL-II concentration that occurs within 24 h after hypophysectomy on day 12 of pregnancy, which indicated that the pituitary secretes factors that regulate mPL-II. To characterize these factors biochemically, pituitaries from virgin female mice were incubated for 14 days, and the culture medium was pooled, concentrated, and then fractionated by gel exclusion chromatography on Sephadex G-100, followed by ion exchange chromatography on DEAE. The fraction from DEAE chromatography that contained predominantly mouse (m) GH as well as a very small amount of a substance with an approximate mol wt of 23K suppressed the posthypophysectomy rise in serum mPL-II. To determine whether purified PRL, GH, and the unknown 23K substance present in the active fraction from the DEAE column could suppress the posthypophysectomy rise in serum mPL-II, repeated injections and constant infusion of these substances were carried out in hypophysectomized mice. Repeated injections of purified mPRL and bovine (b) PRL and constant infusion of mPRL were without effect. Repeated injection of mGH at doses of 1 and 10 micrograms/mouse or of bGH at doses of 50, 150, and 450 micrograms/mouse were also not effective, but bGH injections at a dose of 5 mg/mouse suppressed the posthypophysectomy rise in serum mPL-II. Subsequently, two pools of mGH were prepared by ion exchange HPLC; mGH-A contained predominantly mGH as well as a very small amount of an unidentified 23K mol wt substance, and mGH-B contained only mGH. When mGH-A and mGH-B were infused at a constant rate into hypophysectomized pregnant mice, both suppressed serum mPL-II concentrations; there was no difference in activity between mGH-A and mGH-B. These results indicate that mGH, but not mPRL, regulates the serum PL-II concentration in the mouse. mPL-II secretion is under the inhibitory control of mGH, the concentration of which increases rapidly at the beginning of the last half of pregnancy.

Animals↗
PubMed1994

Quantitation of two histochemical markers in the same extract using chemiluminescent substrates.

Factors that influence early events of primary tumor development have been cumbersome to evaluate because of the need to either wait for tumor palpability after experimental manipulation or use of radiolabel to evaluate cell clearance. To facilitate these and similar analyses of cells in vivo, new methods are described that utilize histochemical marker genes to quantitate tumor cell number in a target tissue through the use of luminescent, enzymatic assays for these gene products. 3T3 Cells transfected with either human placental alkaline phosphatase or bacterial lacZ genes were injected subcutaneously into athymic nude mice. Using luminescent substrates designed for marker gene enzymes, extracts from homogenized tumor cell-bearing skins were assayed for the corresponding marker enzyme activities, which were optimized for recovery from skin extracts and correlated to cell number. The homogenization buffer used for these assays was designed to accommodate the optimal and simultaneous recovery of cytosolic beta-galactosidase and membrane-linked alkaline phosphatase from the skin, as well as from cultured cells. These assays provide an inexpensive, sensitive method for quantitatively monitoring the fate of cells genetically tagged with marker genes in various in vivo environments.

3T3 Cells↗
PubMed1996

Insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBP-1, -2 and -3) in diabetic pregnancy: relationship to macrosomia.

To evaluate the role of insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) in excessive fetal growth (macrosomia) in diabetic pregnancy, 84 insulin-treated diabetic mothers and their infants were tested for serum concentrations of IGF-I, IFG-II, and IGFBP-1, -2 and -3. These parameters were correlated with the birth weight of neonates and placental weight. IGF-I and II levels were determined by specific radioimmunoassays (RIAs) after serum samples were extracted with aid-ethanol. IGFBPs were measured by Western immunoblot with specific antibodies to the respective IGFBP species. Serum concentrations of both IGF-I and IGF-II in mothers with either IDDM or NIDDM increased with the gestational period, reached a plateau at the third trimester, and returned to non-pregnant levels within 7 days after delivery. These values were not different from those in normal mothers before and throughout pregnancy. As previously reported, IGF-I concentrations in cord serum of neonates born to diabetic mothers were (P < 0.01) higher than those of newborns of normal mothers. Likewise, cord blood IGF-II levels were 2-fold higher in babies of diabetic mothers (P <0.001). Fetal IGF-I and IGF-II correlated with each other and with maternal HbA1C, and they positively correlated with either birth weight or placental weight. Cord IGFBP-3 concentrations were significantly higher in diabetic pregnancy, but IGFBP-2 concentrations were not different from those in normal pregnancy. Cord IGFBP-1 concentrations were significantly higher only in babies of mothers with IDDM. None of these cord IGFBP concentrations correlated with birth weight or placental weight. The data suggest that fetal IGF-II, like IGF-I, is involved in fetal and placental growth in diabetic pregnancy. The role of IGFBPs remained to be determined.

Adult↗
PubMed1998

[Inhibitory effect of methanol extract of Boschniakia rossica Fedtsch. et Flerov on rat hepatic preneoplastic lesions induced by diethylnitrosamine].

OBJECTIVE: To investigate the inhibitory effect of Boschniakia rossica(BR) on hepatocarcinogenesis in rats. METHOD: Based on immunohistochemistry techniques, the expression of placental form glutathione S-transferase(GST-P), mutant p53 and p21 protein were investigated in hepatic preneoplastic lesions induced by Solt-Farber protocol in the liver of rats that had been treated with the above method, administered with BR extract and of control group. RESULT: The extract of BR(500 mg/kg) has inhibitory effect on the formation of diethylnitrosamine-induced GST-P-positive foci in F344 rat and the expression of mutant p53 and p21 protein was lower than that of hepatic preneoplastic lesions, and the increasing gamma-glutamyltranspeptidase(gamma-GT) activity in rat liver treated with Solt-Farber protocol was decreased by the extract of BR. CONCLUSION: These results indicate that BR has inhibitory effect on DEN induced hepatic preneoplastic lesions in F344 rat.

Animals↗
PubMed1988

Human placental sialidase: further purification and characterization.

An acid sialidase [EC 3.2.1.18] has been purified from human placenta by means of successive procedures including extraction, Con A-Sepharose adsorption, ammonium sulfate precipitation, activation, p-aminophenyl thio-beta-D-galactoside-CH-Sepharose (PATG-Sepharose) affinity chromatography and high-performance liquid chromatography on a Shim pack Diol 300 column. The purified enzyme liberated sialic acid residues from sialooligosaccharides, sialoglycoproteins, and gangliosides. In particular, gangliosides GM3, GD1a, and GD1b were hydrolyzed much faster than alpha (2-3) and alpha (2-6)sialyllactoses, and sialoglycoproteins by the enzyme. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme gave five protein bands with molecular weight of 78,000 (78K), 64,000 (64K), 46,000 (46K), 30,000 (30K), and 20,000 (20K). Rabbit antisera were raised against 78K and 46K proteins, and the two antibodies were specifically reactive with the respective component on immunoblot analysis. Both anti-78K protein and anti-46K protein antisera could precipitate sialidase activity. It is likely that the 78K protein and 46K protein are sub-components which are essential for sialidase activity.

Female↗
PubMed1997

Oxygen and placental villous development: origins of fetal hypoxia.

The increasing practice of preterm delivery in the fetal interest for conditions such as pre-eclampsia or intrauterine growth restriction (IUGR) has provided an opportunity to study placental structure in pregnancies with prenatal evidence of fetal compromise. These data suggest that the origin of fetal hypoxia in IUGR with absent end-diastolic flow in the umbilical arteries is due to a failure of oxygen transport from intervillous space to umbilical vein. Failure of the fetoplacental circulation to extract oxygen from the intervillous space under such circumstances means intervillous PO2 is closer to maternal arterial values than under physiological conditions. Correspondingly the placental villi are chronically exposed to a higher oxygen tension than under normal circumstances--the term ¿hyperoxia', relative to normal intraplacental oxygenation, is proposed to describe this situation. Both the trophoblast and villous core react to increased oxygen despite fetal hypoxia. These results challenge the generally accepted concept of ¿placental hypoxia' in all circumstances where fetal hypoxia might arise. Therefore three categories are proposed for the origins of fetal hypoxia: (1) preplacental hypoxia; (2) uteroplacental hypoxia; and (3) postplacental hypoxia. Examples for these three disease states are listed in this review and the structural reaction patterns of placental villi to these differences in oxygenation are discussed.

Animals↗
PubMed1984

Modulation of bovine placental prostaglandin synthesis by an endogenous inhibitor.

An endogenous, heat-labile, inhibitor of prostanoid synthesis in maternal caruncle tissue of bovine placentomes was studied. Inhibitory activity was present in caruncle extracts from days 120-250 of gestation, but was not detectable at term (260-280 days). The disappearance of inhibitory activity coincided with an increase in the secretion of prostanoids by dispersed caruncle cells in culture. Coculture of caruncle cells from placentomes of 120-day gestation with fetal cotyledon cells resulted in suppression of prostanoid synthesis by the cotyledon cells. However, this inhibition was not observed in cocultures of dispersed caruncle cells and fetal cotyledon cells from term placentomes. Our findings indicate that an endogenous inhibitor modulates bovine placental prostaglandin synthesis. A decline in the level of this inhibitor at term may be one factor triggering increased prostanoid synthesis required for parturition.

Animals↗
PubMed2003

A proinflammatory cytokine response is present in the fetal placental vasculature in placental insufficiency.

OBJECTIVE: Vascular disease in the umbilical placental circulation is associated with fetal growth restriction and adverse outcome. It may be identified antenatally by the study of umbilical artery Doppler flow velocity waveforms. The cause of this vascular disease is unknown. We have previously provided indirect evidence for endothelial cell activation and a proinflammatory cytokine response. Recently, a family of inhibitors of cytokine signaling has been identified, referred to as the suppressors of cytokine signaling (SOCS). Activation of SOCS occurs when cytokines are produced in stimulated cells. We tested the hypothesis that endothelial cell activation was present in umbilical placental vascular disease and was associated with production of proinflammatory cytokines and members of the family of SOCS. STUDY DESIGN: Placentas were collected at delivery and microvascular endothelial cells were isolated. We studied 13 normal pregnancies and 10 with umbilical placental vascular disease identified by an abnormal umbilical artery Doppler study. Placental pieces were digested with collagenase and purified by adherence to Dynabeads coated with monoclonal antibody against CD31. The RNA was extracted from isolated endothelial cells. The messenger RNA expression of cytokine production (interleukin-6 and interleukin-8) and the members of SOCS family (CIS, SOCS1, SOCS2, and SOCS3) were assessed by use of semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: In the microcirculation of the placenta, endothelial cell expression of interleukin-6 messenger RNA (2.50+/-0.60 vs 1.25+/-0.26) and interleukin-8 messenger RNA (2.83+/-0.55 vs 1.58+/-0.27) was up-regulated in umbilical placental vascular disease in comparison to normal pregnancy. The endothelial cell mRNA expression of SOCS2 (3.36+/-0.77 vs 1.76+/-0.29) and SOCS3 (2.77+/-0.60 vs 1.48+/-0.26) was enhanced in placental vascular disease. There was no significant difference in expression of CIS and SOCS1 in microvessel endothelial cells. CONCLUSION: We have demonstrated that microvessel endothelium of the fetal placental vasculature produces both the proinflammatory cytokines (interleukin-6 and interleukin-8) and members of SOCS family (SOCS2 and SOCS3) in umbilical placental vascular disease. This cytokine production may play a key role in the interaction of endothelial cells of the placenta villi with neighboring cells. The up-regulation of SOCS2 and SOCS3 indicates these are the major negative regulators in umbilical placental microvessel endothelial cell activation pathways. By its occurrence, this also confirms the presence of a proinflammatory cytokine response.

Adult↗
PubMed2004

Microvascular endothelial cell activation is present in the umbilical placental microcirculation in fetal placental vascular disease.

OBJECTIVE: Fetal growth restriction is associated with an abnormal umbilical artery Doppler study. A vascular disease is present in the fetal umbilical placental microcirculation. We hypothesized that the local production of factors that are injurious to microvessel endothelium is responsible for this vascular disease and that endothelial cell activation is a feature of this. Because the expression of the cell adhesion molecules is associated with endothelial cell activation, we isolated endothelial cells from the microvessels of the umbilical placenta and examined them for evidence of gene expression of cell adhesion molecules. STUDY DESIGN: Endothelial cells from the microcirculation of human placenta were isolated and purified with collagenase digestion and extraction with superparamagnetic beads that were coated with monoclonal antibody against CD31. Microvessel endothelial cells were isolated from the placentae of 13 women with a normal pregnancy and delivery at term and 10 placentas with umbilical placental vascular disease that was defined by abnormal umbilical artery Doppler study. Total RNA was extracted from isolated endothelial cells. The messenger RNA expressions of cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and platelet endothelial cell adhesion molecule-1) were assessed by semiquantitative reverse transcription-polymerase chain reaction. RESULTS: Microvessel endothelial cells from the fetal placentae of pregnancies that were complicated by umbilical placental vascular disease showed an enhanced expression of intercellular adhesion molecule-1 messenger RNA (2.12+/-0.45 vs 0.92+/-0.25) and platelet endothelial cell adhesion molecule-1 messenger RNA (4.29+/-0.87 vs 2.41+/-0.42) in comparison to normal pregnancies. There was no significant difference in expression of vascular cell adhesion molecule-1 messenger RNA (1.55+/-0.37 vs 1.68+/-0.38). CONCLUSION: We have shown that vascular disease in the fetal umbilical placental circulation is associated with an increase in the expression of intercellular adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 by microvessel endothelial cells. We postulate that locally released factors cause injury and activation to microvessel endothelial cells. In this regard, the process in the fetus is similar to that of atherothrombotic vascular disease of later life.

Adult↗
PubMed1996

Trans-acting factors dictate the species-specific placental expression of corticotropin-releasing factor genes in choriocarcinoma cell lines.

CRF, in addition to its role in the hypothalamus, demonstrates species-specific expression in the placentas of higher primates, but not rodents. Transient transfections of BeWo and JEG-3 choriocarcinoma cells, as models for human trophoblasts, demonstrate regulated expression of human (h) CRF-luciferase reporter genes, whereas little or no expression is detected in other lines, including CV-1 cells. The rodent choriocarcinoma cell line, Rcho-1, a model for rodent trophoblasts, is defective in the expression of transfected hCRF genes. The mouse CRF promoter behaves similarly to the corresponding hCRF construct. It is active in BeWo and inactive in Rcho-1 cells. The transcriptional response to cAMP contributes to the specific expression of CRF. Analyses of deleted or mutated hCRF promoters identify a key role for protein kinase A-dependent pathways. A major part, but not all, of this effect is mediated by the canonical cAMP response element conserved in mouse, rat, and human CRF promoters. Additional deletions of the human CRF promoter identify control regions that also contribute to the observed species-specific expression pattern, and each identified region binds factors in nuclear extracts derived from the appropriate cell line. These studies using human and rodent choriocarcinoma cell lines as models of placental trophoblasts demonstrate dominant effects of cellular trans-acting factors, rather than DNA sequence differences, in dictating the species-specific placental expression of CRF.

Animals↗
PubMed1986

Characterization of progesterone binding to nuclear receptors in rat placenta.

Exchange assays have been validated to study several forms of the progesterone receptor found to occur in nuclei of rat placenta after extraction with high salt. One form was solubilized by the extraction procedure (KCl extractable Rpn) and another form remained attached to nuclear structures (KCl resistant Rpn). Specific binding of progesterone was optimized in both forms using buffered media containing 0.01 M Tris, 30%-glycerol (v/v), 0.2 mM leupeptin, and 1 mM dithiothreitol (TDGL), pH 7.8, at 0-4 degrees C for 18-24 h. At 0-4 degrees C the nuclear receptors were stable and degradation was negligible even after 44 h of in vitro incubation. The binding reaction between progesterone and receptor demonstrated mass action principles of ligand exchange throughout this interval. Saturation analysis indicated the presence of a single binding moiety of high affinity (app Kd = 2.9-3.2 nM) for both forms of the receptor. However, the nuclear progesterone receptor was thermolabile and after a 10 min exposure to 30 degrees C no longer complexed ligand. At an intermediate incubation temperature of 22 degrees C the binding reaction was stable for about 30 min. The KCl resistant binding sites were markedly more thermolabile. Addition of 10 mM Na molybdate protected all forms of the nuclear progesterone receptor from thermal denaturation and extended the life of the complex 3-4-fold. The dissociation rate constant of progesterone-nuclear receptor complex in each preparation was 6-8 X 10(5) s-1 resulting in a half-life of about 3 h. The KCl resistant and extractable binding sites were sensitive to blockade by 1 mM N-ethylmaleimide which was reversed by co-incubation with a 2-fold molar excess of dithiothreitol. This suggested that reduced sulfhydryl groups located on or near the surface of the ligand binding domain of the receptor were necessary to bind hormone. These studies showed that the interactions between ligand and the KCl resistant and extractable receptor sites found in rat placenta were of high affinity, saturable, and heat sensitive. Thus, these binding moieties exhibited physicochemical behavior very similar to each other and to the placental receptor which has previously been partially purified from the cytosol. The conclusion is made that all of the nuclear receptor binding sites for progesterone are structurally identical. Thus, the distinctive physicochemical properties responsible for KCl resistant and extractable forms of the nuclear progesterone receptor must reside in other domains of the receptor molecule.

Animals↗
PubMed1988

Case control study of hypospadias, based on registry information.

With the aid of data in the Swedish Medical Birth Registry for infants with hypospadias and controls, a number of variables were compared. Records of infants born in 1982-1983 (188 cases and 376 matched controls) contained information on involuntary infertility, previous spontaneous and induced abortions, use of oral contraceptives or a remaining IUD at conception, smoking in early pregnancy, occupation in early pregnancy, family situation, and diagnoses given during pregnancy and at delivery, including information on caesarean section and vacuum extraction. Among all variables studied, only one group of statistically significant differences appeared: women whose sons had hypospadias more often than controls had a diagnosis of weak contractions, a higher rate of induced deliveries, and also a higher rate of caesarean sections. The finding of a higher caesarean section rate in infants with hypospadias was verified in a separate study of 1,736 hypospadic infants delivered in 1973-1981 and compared with all births in Sweden during that period. No difference in the rate of vacuum extractions was seen. This finding is interpreted as a result of an abnormality of the fetal-placental-maternal organism interaction, perhaps also disturbing the early pregnancy and increasing the risk for hypospadias.

Contraception Behavior↗
PubMed2005

Expression, localisation and hormone regulation of the human copper transporter hCTR1 in placenta and choriocarcinoma Jeg-3 cells.

Copper is an essential trace element necessary for normal growth and development. During pregnancy, copper is transported from the maternal circulation to the fetus by mechanisms which have not been clearly elucidated. The copper uptake protein, hCTR1 is predicted to play a role in copper transport in human placental cells. This study has examined the expression and localisation of hCTR1 in human placental tissue and Jeg-3 cells. In term placental tissue the hCTR1 protein was detected as a 105 kDa protein, consistent with the size of a trimer which may represent the functional protein. A 95 kDa band, possibly representing the glycosylated protein, was also detected. hCTR1 was localised within the syncytiotrophoblast layer and the fetal vascular endothelial cells in the placental villi and interestingly was found to be localised toward the basal plasma membrane. It did not co-localise with either the Menkes or the Wilson copper transporting ATPases. Using the placental cell line Jeg-3, it was shown that the 35 kDa monomer was absent in the extracts of cells exposed to insulin, estrogen or progesterone and in cells treated with estrogen an additional 65 kDa band was detected which may correspond to a dimeric form of the protein. The 95 kDa band was not detected in the cultured cells. These results provide novel insights indicating that hormones have a role in the formation of the active hCTR1 protein. Furthermore, insulin altered the intracellular localisation of hCTR1, suggesting a previously undescribed role of this hormone in regulating copper uptake through the endocytic pathway.

Cation Transport Proteins↗