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Biomedical subjects

R Benveniste

Publications and source records attributed to R Benveniste.

At least 37 records · Page 2Linked to original sources
PubMed1985

Effects of epidermal growth factor, phorbol myristate acetate, and arachidonic acid on choriogonadotropin secretion by cultured human choriocarcinoma cells.

Epidermal growth factor (EGF) binds specifically (Ka = 4 X 10(9) M-1; 1.3 X 10(11) receptors/mg cellular protein) to JEG-3 cells, which respond in the succeeding 24 h by a 400% increase in hCG secretion without a significant change in cell number. Since JEG-3 cells store less than 2% of the 24-h hCG secretion, a significant increase in hCG in the culture medium reflects increased hCG biosynthesis. It is observed that a tumor promoter, phorbol myristate acetate (PMA), binds specifically to the cells (Ka = 5 X 10(8) M-1; 3.9 X 10(11) receptors/mg), reduces (33%) the affinity but not the number of EGF receptors, and stimulates hCG to the same extent as does EGF. The relative potencies of PMA (100%), phorbol dibutyrate (25%), and nonesterified phorbol (no effect) to stimulate hCG parallel their known tumor-promoting activities. Since phospholipids and fatty acids have been implicated in the mechanism of action of EGF and PMA, the effects of arachidonic acid (AA) and inhibitors of its metabolism were studied. The addition of AA had no effect on basal or PMA-stimulated hCG secretion, but it potentiated the effect of EGF by 200%. Indomethacin (a cyclooxygenase inhibitor) had an effect similar to that of AA. Nordihydroguairetic acid (a cyclooxygenase and lipoxygenase inhibitor) reduced by 90% basal and EGF- or PMA-stimulated hCG secretion. These results indicate that the AA pathway can influence the effects of EGF and PMA on hCG secretion and suggest an intermediary role for the lipoxygenase system.

Arachidonic Acid↗
PubMed1983

Ontogenesis and characteristics of epidermal growth factor receptors in human placenta.

Epidermal growth factor promotes growth in many cell types. The role of epidermal growth factor during gestation and fetal development is unknown. This study investigates the presence and binding characteristics of epidermal growth factor receptors in placentas throughout gestation. Cell membrane preparations were obtained from first-, second-, and third-trimester placentas and hydatidiform moles. Specific epidermal growth factor receptors were observed as early as 6 weeks of gestation. Throughout gestation, Scatchard analysis of epidermal growth factor binding was curvilinear, and dissociation studies were consistent with site-to-site interaction with negative cooperativity. Affinity constants at high (Ke = 9.9 +/- 0.79 X 10(9) L/mol) and low (Kf = 3.0 +/- 0.25 X 10(9) L/mol) receptor occupancy were unchanged throughout normal gestation. However, the number of receptors per milligram of protein significantly increased with advancing normal gestation (correlation coefficient, r = 0.81). In hydatidiform moles, the number of receptors was reduced when compared to that of normal tissue of similar gestational age. Our study provides a quantitative basis for further evaluation of epidermal growth factor as a possible factor in embryogenesis and fetal development.

Cell Membrane↗
PubMed1983

Efficient isolation of endogenous rhesus retrovirus from trophoblast.

An examination of various rhesus monkey (Macaca mulatta) organs has shown a preference for type C viral antigen expression in the placenta. Separate cocultivations of isolated trophoblasts from 10 rhesus monkey placentas with cell lines from heterologous mammalian species led to rapid isolation of type C rhesus retrovirus in 4 of 10 cases. These four retrovirus isolates have been designated MMC-2 through MMC-5. Five of the remaining six sets of cocultivations grew simian foamy virus and were discontinued. Distinction of these viral isolates from the initial rhesus isolate (MMC-1) and the previous isolate from the stumptail monkey, Macaca arctoides (MAC-1), could be made by liquid DNA hybridization, although not by limited restriction endonuclease digestion. Both MAC-1 and MMC-1 were obtained in single long-term cocultivation experiments (over 7 months). The present isolates MMC-2 through MMC-5 were detected in 2 to 5 weeks. Consequently, primary trophoblast cells represent a useful differentiated cell type for isolation of infectious retrovirus from this primate species.

Animals↗
PubMed1983

Characterization of an immune suppressor from transformed human trophoblastic JEG-3 cells.

Cultured human choriocarcinoma JEG-3 cells secrete an immunosuppressor that inhibits lymphocyte proliferation stimulated by either an antigen or a mitogen. In this study, the immunosuppressive factor was characterized by three methods: ion-exchange and exclusion chromatography, partition in organic solvents, and thin-layer chromatography on silicic acid. This JEG-3 cell factor appeared to be a protein complex of about 150,000-200,000 Da that contained an immunologically active polar lipid. The structural and functional characteristics of JEG-3 cell immunosuppressor are similar if not identical to those of SIF, a suppressor lymphokine derived from T cells. These secretions from transformed trophoblastic cells may correspond to normal placental products or represent a function of malignant cells.

Cell Line↗
PubMed1981

Human chorionic gonadotropin alpha-subunit in pregnancy.

We have validated a direct radioimmunoassay (RIA) which measures the alpha-subunit of human chorionic gonadotropin (hCG) in pregnancy sera in the presence of large amounts of hCG. The levels of alpha-subunit in 124 sera from normal pregnancy women were measured and compared to the levels of hCG measured by a radioreceptor assay (RRA) which utilizes a rat testicular membrane preparation. Unlike hCG, which peaks at the end of the first trimester, the alpha-subunit continues to rise until 36 weeks of pregnancy. During the first half of pregnancy the levels of alpha-subunit remain less than 100 ng/ml whereas in the second half the levels remain less than 200 ng/ml. Three pathologic pregnancies were also studied; on the basis of the hCG determinations, they could not be differentiated from normal pregnancy. In contrast, all determinations of alpha-subunit were abnormally elevated. The concept that placental dysfunctions would cause an early and abnormally high level of alpha-subunit secretion deserves further investigation.

Adolescent↗
PubMed1981

Human chorionic gonadotrophin (HCG) and free alpha subunit secreted by cultured human choriocarcinoma (JEG-3) cells.

The cultured human choriocarcinoma JEG-3 cells secrete biologically active HCG and free HCG alpha-subunit. When compared with the alpha-subunit dissociated from HCG obtained either from pregnancy urine or JEG-3 cells, free alpha-subunit has a larger molecular weight, is more acidic and is non-functional, lacking the property to recombine with the HCG beta-subunit. The understanding of the biochemical differences observed between free alpha-subunit and alpha-subunit found in HCG is important and should help to unravel the biosynthesis of gonadotrophins. Two proteins which bind to the cell membrane, epidermal growth factor and concanavalin A, are capable of stimulating JEG-3 cell secretion. Epidermal growth factor stimulates the secretion of HCG while concanavalin A stimulates both HCG and HCG alpha-subunit secretion. Amphotericin B, an antifungal agent commonly used in tissue cultures, which also affects the cell membrane, was shown to stimulate HCG and HCG alpha-subunit secretions. The use of these agents should contribute to the understanding of membrane-related events which lead to the secretion of HCG and alpha-subunit.

Amphotericin B↗
PubMed1981

Thin-layer chromatography for separation of phospholipids in amniotic fluid.

A method using a 1-dimensional, thin-layer chromatographic technique for the separation of phospholipids in amniotic fluid is described. The phospholipids of major importance in evaluating fetal lung maturity were separated: sphingomyelin, lecithin, and, notably, phosphatidylglycerol. Preliminary clinical results correlate the increasing appearance of phosphatidylglycerol with increasing lecithin : sphingomyelin ratios.

Amniotic Fluid↗
PubMed1979

The carbohydrate moiety of human chorionic gonadotropin: lack of competition with HCG for testicular receptors and anti-HCG-serum.

A glycopeptide fraction has been prepared from human chorionic gonadotropin (HCG) by digesting the reduced, S-carboxymethylated hormone with pronase and fractionating the digest by gel exclusion chromatography. The glycopeptide fraction was estimated to contain (w/w) 29% sialic acid, 31% hexose, 23% hexosamine, and 17% amino acids and/or peptides; thus, the glycopeptide mixture is 83% carbohydrate compared to intact HCG which is about 30% carbohdyrate. There was no cross-reactivity of the glycopeptide fraction with an antiserum directed against HCG. Also, when corrected for minimal non-specific effects, the fraction failed to displace 125I-HCG from a rat testicular preparation even when tested at a 10,000-fold (w/w) excess. Thus, any model involving carbohydrate effects in gonadotropin action must include the protein moiety as a necessary component.

Binding, Competitive↗
PubMed1979

Heterogeneity of the human chorionic gonadotropin alpha-subunit secreted by cultured choriocarcinoma (JEG) cells.

The cultured human choriocarcinoma cell line, JEG-clone 3, secretes substantial quantities of both biologically active hCG and an immunoreactive alpha-subunit (JEG-alpha). This study is concerned with a comparative characterization, using RIA, of the chromatographic properties (via gel exclusion and isoelectric focusing) of JEG-alpha and standard urinary hCG-alpha. Most of the molecules comprising the JEG-alpha fraction have an apparent molecular weight greater than that of hCG-alpha. Both hCG-alpha and JEG-alpha exhibit heterogeneity on electrofocusing. However, JEG-alpha contains a major component with an isoelectric pH (pI) of 4.8; this is a minor component, if present at all, in hCG-alpha. The JEG-alpha pI 4.8 component chromatographs with an apparent molecular weight greater than hCG-alpha, while a minor JEG-alpha pI 7.0 component chromatographs with an apparent molecular weight similar to that of the standard. Heterogeneity is expected in the carbohydrate moieties of the glycoprotein hormone subunits. Results of studies on the incorporation of 14C- and 3H labeled amino acids into JEG-alpha suggest that heterogeneity also exists in the protein moiety of JEG-alpha. An interesting possibility is that the form(s) of JEG-alpha with larger apparent molecular weight represents a precursor of the alpha-subunit used to form hCG.

Biological Assay↗
PubMed1979

Circulating big human prolactin: conversion to small human prolactin by reduction of disulfide bonds.

The heterogeneity of circulating human PRL (hPRL) in sera of patients with hyperprolactinemia was studied by exclusion chromatography on Sephadex G-100. Big hPRL, which elutes between the void volume and the elution position of monomeric small hPRL, is stable upon rechromatography but is almost entirely converted into small hPRL after reduction with 0.5% mercaptoethanol. These results suggest that the existence of circulating big hPRL is dependent upon the formation of interpolypeptide disulfide bonds and does not represent a classical biosynthetic precursor of hPRL.

Adolescent↗
PubMed1979

Seventy-two hour infusions of LHRH in normal men: gonadotropin and testicular steroid responses.

Four normal male subjects received LHRH by continuous infusion for 72 hrs at 1.4 microgram/min. Mean basal LH was 7.7 +/- 1.0 mIU/ml, increased to a maximum of 120 +/- 22.7 mIU/ml, and then declined to levels between 28--34 mIU/ml for the last 30 hrs of infusion; FSH rose from 3.7 to 11.4 mIU/ml (p less than 0.05) at 16 hrs and then returned to baseline. Testosterone levels rose by 50% at 12 hrs, and remained elevated throughout with maximum values between 6.6 and 12 ng/ml. Estradiol-17beta levels were 26.2 +/- 4.7 pg/ml basally, rose to 104 +/- 8 pg/ml and while levels declined therafter, they were significantly above baseline throughout the 72 hrs. Plasma androstenedione and dehydroepiandrosterone levels also showed significant increases. By contrast, transient elevations in pregnenolone and 17-hydroxypregnenolone levels probably corresponded to the nomral morning rise in plasma levels of these steroids; 17-hydroxyprogesterone rose from 1.3 +/- 0.15 to 3.9 +/- 0.26 ng/ml at 12 hrs and remained elevated through the infusion. An increase in 17-hydroxyprogesterone:testosterone ratio was observed in all subjects. Thus, chronic LHRH infusion effected a persistent increase in endogenous LH with, in turn, prolonged stimulation of gonadal steroid secretion.

17-alpha-Hydroxypregnenolone↗
PubMed1978

An immunological approach to the study of protein conformational heterogeneity: its application to growth hormone.

The study of multiple forms of a hormone due to changes in its secondary or tertiary structure (conformational heterogeneity) has been restricted to methods of physical chemistry which require relatively large quantities of purified preparations and has precluded such studies on circulating hormones. The present report describes an immunological approach to the study of GH conformational heterogeneity based on the simultaneous use of two homologous RIAs: one which recognizes porcine (p) GH in its native conformation (pGH RIA) and a second (pB RIA) for a 55-residue S-aminoethylated cyanogen bromide fragment (pB) of pGH (corresponding to residues 126-180 of human GH). In the pGH RIA, pB was nonreactive, whereas pGH displacement of [125I]iodo-pB from anti-pB serum required about 10(3) M excess and was nonparallel to pB. Treatment of pGH with urea plus mercaptoethanol (ME), which would be expected to affect the conformation of the GH molecule, increased its immunoreactivity 68-fold in the pB RIA and its dilution curve became parallel to that of pB. Similar treatment of porcine pituitary extract increased its immunoreactivity 97-fold in pB RIA with a corresponding slope change. In contrast, urea plus ME decreased pGH and pituitary extract immunoreactivity by 84% and 92%, respectively, in the pGH RIA. The pB RIA/pGH RIA ratio of immunoreactivity for pGH and for pituitary extract increased 795-fold and 569-fold, respectively, after combined treatment, whereas lesser increases were observed after urea or ME treatment alone. In conditions where alterations in GH conformation were suspected (highly purified, aged human GH preparations which were no longer satisfactory as radioiodinated tracers for radioimmunoassay), calculation of the pB RIA/hGH RIA ratio of immunoreactivity indicated increased values. Based on these results, a model is proposed in which unfolding a native hormone, with concomitant exposure of the antigenic determinant of pB, hidden within pGH, leads in increased immunoreactivity in the pB RIA and decreased immunoreactivity in the pGH RIA. In this model, the pB RIA/pGH RIA ratio of immunoreactivity would thus serve as an index of the state of hormonal unfolding.

Animals↗
PubMed1978

Characterization of the human chorionic gonadotrophin fractions in pregnancy urine.

The human chorionic gonadotrophin (hCG) preparation (ca. 3000 IU/mg) from pooled pregnancy urine has been fractionated using anion-exchange and gel exclusion chromatography. The various fractions, many of which were heterogeneous, were assayed using radioimmunoassay (RIA) and a testicular radio-receptor assay (RRA). Three fractions were obtained with potencies between 9000 and 20 000 IU/mg); five fractions were characterized by potencies between 1000 and 6000 IU/mg; and four fractions exhibited low potencies (less than 500 IU/mg). A linear relationship between RRA and RIA potency (P) was found to exist for the various fractions: In(PRRA) = 1.14 In(PRIA) - 1.67. The major fractions, which exhibited various potencies, were further characterized by amino acid and carbohydrate analysis. Urine was collected from an individual at various times of gestation, and the hCG fraction was concentrated by benzoic acid and ethanol precipitation. Ion-exchange chromatography of the hCG fraction yielded multiple immunoreactive components throughout pregnancy; thus the observed heterogeneity in the commercial preparation does not appear to result from processing pooled urine from numerous donors. Also, heterogeneity was observed in an individual donor whose urine was not treated with the benzoic acid and alcohol fractionation scheme. It seems indisputable that individual urinary hCG is heterogenous, and this does not appear to reflect isolation artefacts.

Amino Acids↗
PubMed1978

Pituitary and adrenal function in normal women after treatment with Lergotrile mesylate.

We studied the effects of a dopaminergic agonist, 2-chloro-6-methyl-ergoline-8-beta-acetonitrile methanesulfonate (LERGOTRILE mesylate), on the pituitary and adrenal function of five normal women. No changes were observed in basal serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), growth hormone (GH) and cortisol levels, in LH and FSH levels after luteinizing hormone-releasing hormone (LH-RH) stimulation, or in GH and cortisol levels during insulin-induced hypoglycemia. Lergotrile treatment produced a fall in basal prolactin and thyrotropin (TSH) levels and there was dissociation in the responses of the women to thyrotropin-releasing hormone: attenuation of TSH release, but retention of prolactin release.

Acetonitriles↗