Nucleotides do not modulate rat luteocyte human chorionic gonadotropin responsiveness by inhibiting human chorionic gonadotropin binding

Nucleotide inhibition of ¹²⁵I-labeled human chorionic gonadotropin binding to luteocyte receptor was studied by investigating effects of nucleotides on the apparent equilibrium association constant (K_a) and number of binding sites (B_max), and on rate constants for association (k₊₁) and dissociation (k_?1, k_?2). K_aandB_max were determined by various analyses of equilibrium binding data using washed 2000g pellet of an ovarian homogenate from rats 7 days after pregnant mare's serum gonadotropin-human chorionic gonadotropin priming. Adenyl and guanyl nucleotides, as well as other nucleotides, lowered the K_a of ¹²⁵I-labeled human chorionic gonadotropin binding to luteocyte receptor without affecting B_max. The degree of inhibition was dose related at nucleotide concentrations greater than 10^?3 m. GTP and guanyl-5′-ylimidodiphosphate inhibitions were similar in the presence or absence of EDTA (1.25 × 10^?3 m). ATP and GTP lowered K_a by slowing the rate of association. Inhibition of binding could not be demonstrated at lower nucleotide concentrations even when luteocyte membranes were purified partially by sucrose density gradient ultracentrifugation. In light of the high nucleotide concentrations required to inhibit ¹²⁵I-labeled human chorionic gonadotropin binding and the inhibition by Mg²⁺ and PP₁ at similar concentrations, the effect appears to be a nonspecific ionic effect. Therefore, in contrast to the glucagon-hepatocyte system, luteocyte human chorionic gonadotropin responsiveness does not appear to be modulated by nucleotide inhibition of human chorionic gonadotropin-receptor interaction.     

[125I] gonadotropin binding to the ovary of an Indian major carp,Catla catla,at different stages of reproductive cycle

At different stages of the annual reproductive cycle ofCatla catla, a major Indian carp, specific binding of gonadotropic hormone to the plasma membrane receptors was demonstrated. Maximum specific binding of [¹²⁵I]Catla gonadotropic hormone was obtained at 30‡C and pH 7.5 during 2 h of incubation.Catla gonadotropic hormone binding was saturable with high affinity. Competitive inhibition experiment showed that binding site was specifically occupied by piscine gonadotropic hormone,Catla gonadotropic hormone and murrel gonadotropic hormone, human chorionic gonadotropin was a weak competitor while bovine thyroid stimulating hormone, bovine prolactin and ovine follicle stimulating hormone had no effect. Scatchard analysis ofCatla gonadotropic hormone binding to the plasma membrane preparation from the carp oocytes of different reproductive stages showed that the range of dissociation constant(K _d ) varied from 0.78 to 0.97 x 10^-10 M. However, maximum binding capacity (B-max) varied remarkably between the different stages of reproductive cycle, it was 6.11 ± 0.36 fmol/mg protein in the preparatory stage which increased to about three-fold in prespawning stage of reproductive cycle (17.0 ± 0.29 fmol/mg protein) and spawning (18.7 ± 0.17 fmol/mg protein) and lowest in postspawning stage of reproductive cycle (5.28 ± 0.28 fmol/mg protein). Fluctuation in the number of gonadotropic hormone binding site at different stages of annual reproductive cycle was found to be coincided well with the pattern of ovarian steroidogenesis in response toCatla gonadotropic hormone as determined by the formation of progesterone from pregnenolone.     

Determination of kinetic parameters of epitope-paratope interaction based on solid phase binding: An inexpensive alternate to biospecific interaction analysis

A method has been developed for biospecific interaction analysis between antigen and antibody using solid phase binding approach. Real time kinetics between monoclonal antibody and human chorionic gonadotropin have been studied. Kinetic constants of the bimolecular reaction are determined. Affinity constants measured by several independent methods have been found to be relatively consistent. Convenient and simple procedures to determine affinity constant, K_onand K_off of monoclonal antibody-human chorionic gonadotropin interaction using binding of [¹²⁵I]hCG to immobilized monoclonal antibody are presented. Values obtained compare well with those obtained using surface plasmon resonance technology, making this method a viable alternative.     

Studies on regulation of chorionic gonadotropin secretion in primates

The regulation of secretion of chorionic gonadotropin in primates has been studied using bothin vivo andin vitro models.In vivo studies using the pregnant bonnet monkey revealed that at the doses tested, the administration of progesterone or estradiol 17Β in combination or alone did not result in any appreciable change in the duration or magnitude of serum chorionic gonadotropin levels. However, administration of lutropin-releasing hormone by intravenous route resulted in significant increase in chorionic gonadotropin levels within 30–60 min and the extent of stimulation seemed to depend on the state of pregnancy. Forin vitro studies, explants or cells prepared from first trimester human placenta has been used. The functional integrity of these cells has been established by demonstrating the binding of [¹²⁵I]-labelled human chorionic gonadotropin antibody to the cells as well as the synthesis of [³H]-labelled human chorionic gonadotropin.In vitro studies using the cells revealed that addition of lutropin-releasing hormone caused a significant increase in chorionic gonadotropin and estradiol 17Β secreted into the medium. Thus bothin vivo andin vitro results suggest that lutropin-releasing hormone could be one of the factors involved in regulation of chorionic gonadotropin secretion in primates.     

Gonadal receptors I. Evidence for irreversibility in the binding of human chorionic gonadotropin and human luteinizing hormone

The binding of human chorionic gonadotropin and human luteinizing hormone to particulate receptors of rat testes has generally been assumed to follow an equilibrium model similar to that proposed for many enzyme systems. Our work shows that equilibrium dissociation constant (K_d) and number of hormone binding sites (B_max) are highly sensitive to changes in hormone and/ or receptor concentration and to treatment received by tissue or receptor preparation prior to the assay. The results of binding assays obtained using receptor preparation pretreated with hormone (labeled as well as unlabeled) indicated that the binding reaction between hormone and receptor was irreversible and that pretreatment of the tissue with hormone greatly alters the number of high affinity gonadotropin binding sites in the testicular homogenate. Data from studies involving increasing receptor concentrations revealed that increasing the mass of particulate receptors in the binding assays leads to higher K_d as well as B_max values. These findings are incompatible with a binding model based upon occupancy of receptor sites and the state of equilibrium implied. The incompatibilities are analyzed and an alternate model advanced (Bhalla, V.K., Trowbridge, C.G., Chen, C.J.H., Lindeman, J.G. and Rojas, F.J. (1979) Biochim. Biophys. Acta 584, 436–453).     

High-affinity and specific recognition of human thyroid stimulating hormone (hTSH) by in vitro-selected 2'-amino-modified RNA.

RNA sequences containing 2'-amino pyrimidines that bind with high-affinity to human thyroid stimulating hormone (hTSH) were isolated from a random sequence library by an in vitro selection-amplification procedure. A representative RNA ligand (T-15) has an equilibrium dissociation constant (Kd) of 2.5 nM for its interaction with hTSH and can discriminate between other members of the glycohormone family; no detectable binding was observed at low micromolar concentrations of hCG (human chorionic gonadotropin), while measured Kd values for the interactions with hLH (human leutinizing hormone) and hFSH (human follicle stimulating hormone) were > 1 microM and approximately 0.2 microM, respectively. The detection of hTSH in a dot blot assay with radiolabeled T-15 RNA was demonstrated.     

Production of antibodies specific to human chorionic gonadotropin in mice immunized against its chemical analogs

Mice immunized against DS₅-hCG-Β and DS₆-hCG-Β, chemical analogs of Β-subunit of human choriogonadotropin (hCG-Β) in which 5 and 6 disulphide bonds respectively were reduced and alkylated, were found to produce antibodies specific to hCG without significant crossreactivity with human lutropin (hLH) as tested in a radioimmunoassay. In contrast, mice immunized against the native hCG-Β subunit produced hLH crossreacting antibodies. While the anti-DS5, DS6-hCG-Β serum was capable of selectively blocking the binding of [¹²⁵I]-hCG to rat testicular LH/hCG receptors, it failed to inhibit the binding of [₁₂₅I]-hLH to the same receptors. The radioimmunoassay for hCG using the mouse anti-DS₅, DS₆-hCG-Β serum was not as sensitive as that employing rabbit anti-DS₅, DS₆-hCG-Β serum. The minimal detection limit was 5 ng/ml for the mouse antibody as compared to 1 ng/ml for the rabbit antibody. Supported by U.S. Public Health Service Grant HD 08766 to OPB. An erratum to this article is available at .     

Effects of Chorionic Gonadotropin on Differentiation of Thymocytes in the Presence of Epithelial Thymus Cells

We studied the effects of the main placental hormone, chorionic gonadotropin, on differentiation of human thymocytes in vitro in the presence of thymic epithelial cells. It was shown that the hormone at a high dose (100 IU/ml) enhanced the epithelium-induced phenotypic maturation of thymocytes, which is registered by an increased expression of the membrane marker CD3 and transition of CD4⁺8⁺ thymocytes in the cells with CD4⁺8^– and CD4^–8⁺ phenotypes. In addition, gonadotropin enhanced the proliferative response of thymocytes to the mitogen during their cultivation with the epithelium. The stimulating effect of the hormone on the epithelium-induced differentiation of thymocytes is mediated by the humoral factors of epithelial cells. In addition, gonadotropin at this dose exerts its own differentiating activity with respect to thymocytes and stimulates their phenotypic and functional maturation in a monoculture.     

The structure and function of glycoprotein hormone receptors: ganglioside interactions with human chorionic gonadotropin.

Gangliosides inhibit the binding of ¹²⁵I-labeled human chorionic gonadotropin to rat testes membranes. The inhibition is the result of an interaction between the hormone and the ganglioside rather than the membrane and ganglioside, and the interaction with the ganglioside can be detected by fluorescence spectroscopy. In both the binding inhibition and fluorescence studies, human chorionic gonadotropin recognizes an oligosaccharide sequence on the ganglioside molecule distinct from the sequence recognized by thyrotropin.     

Boron nitride cages from B12N12 to B36N36: square–hexagon alternants vs boron nitride tubes

The structures and stabilities of square–hexagon alternant boron nitrides (B _x N _x , x=12–36) vs their tube isomers containing octagons, decagons and dodecagons have been computed at the B3LYP density functional level of theory with the correlation-consistent cc-pVDZ basis set of Dunning. It is found that octagonal B₂₀N₂₀ and B₂₄N₂₄ tube structures are more stable than their square–hexagon alternants by 18.6 and 2.4 kcal mol⁻¹, respectively, while the square–hexagon alternants of other cages are more stable. Trends in stability as a function of cluster size are discussed.Figure The octagonal B₂₀N₂₀ and B₂₄N₂₄ tube structures are more stable than their square-hexagon alternant cagesDedicated to Professor Dr. Paul von Ragué Schleyer on the occasion of his 75th birthday     

Theoretical prediction of antigenic sites in the Β-subunits of human choriogonadotropin and luteinizing hormone

Using hydrophilicity and recognition values of amino acids, the antigenic sites of theΒ-subunits of human choriogonadotropin and luteinizing hormone were computed from their amino acid sequences. Six antigenic sites were calculated for human choriogonadotropinΒ-subunits: residues 3–8, 17–22, 59–65,100–106,110–116 and 134–139. For luteinizing hormoneΒ-chain three antigenic sites were calculated: residues 17–22,59–65, and 100–106; all these three sites of luteinizing hormoneΒ being identical to the corresponding sites in human choriogonadotropinΒ. There was no antigenic site in luteinizing hormone that was also not found in human choriogonadotropin. On the other hand, there were unique determinants in human choriogonadotropin that were not found in luteinizing hormone; these determinants were residues 3–8, 110–116 and 134–139     

Increased β<Subscript>2</Subscript>-Adrenergic Receptor Activity by Thyroid Hormone Possibly Leads to Differentiation and Maturation of Astrocytes in Culture

Summary (1) Our earlier studies indicate a downsteam regulatory role of the β-adrenergic receptor (β-AR) system in thyroid hormone induced differentiation and maturation of astrocytes. In the present study we have investigated the contributions of the subtypes of β-AR in the above phenomenon. (2) Primary astrocyte cultures were grown under thyroid hormone deficient as well as under euthyroid conditions. [¹²⁵I]Pindolol ([¹²⁵I]PIN) binding studies showed a gradual increase in the specific binding to β₂-AR when observed at 5, 10, 15, and 20 days under both cultural conditions. Thyroid hormone caused an increase in binding of [¹²⁵I]PIN to β₂-AR compared to thyroid hormone deficient controls at all ages of astrocyte culture. (3) Saturation studies using [¹²⁵I]PIN in astrocyte membranes prepared from 20-day-old cultures showed a significant increase in the affinity of the receptors (K _D) in the thyroid hormone treated cells without any change in receptor number (B _max). (4) β₂-AR mRNA levels were measured by real-time PCR during ontogenic development as well as during exposure of 10-day-old hypothyroid cultures to normal levels of thyroid hormone for 2, 6, 12, and 24 h. None of the conditions caused any significant change in the β₂-adrenergic receptor mRNA levels when compared with corresponding hypothyroid controls. (5) Over expression of β₂-AR cDNA in hypothyroid astrocytes caused morphological transformation in spite of the absence of thyroid hormone in the medium. (6) Taken together, results suggest thyroid hormone causes a selective increase in [¹²⁵I]PIN binding to β₂-AR due to increase in receptor affinity, which may lead to maturation of astrocytes.     

The effect of luteinizing hormone releasing hormone and anti-luteinizing hormone releasing hormone antibodies on chorionic gonadotropin and progesterone secretion by human placental villiin vitro

Gonadotropin releasing hormone has been located and found to be secreted by the human placenta in culture. Addition of the releasing hormone upto 1μg concentration in the placental cultures brings about stimulation of chorionic gonadotropin and progesterone secretion. Higher amounts of the decapeptide has an inhibitory influence on both the gonadotropin and the steroid production. The action of the releasing hormone on the placenta could be blocked by the anti-luteinizing hormone releasing hormone monoclonal antibodies indicating a possible site of action of the antibodies for control of fertility     

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum

The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with ¹²⁵I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 × g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with a apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similary, the free receptor also showed higher sedimentation profile with a apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of ¹²⁵I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI.U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the performed ¹²⁵I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex.     

Modification of sialyl residues of gonadotropic hormones by reductive amination. Influence on biological activity and circulating half-life

The sialic acid residues of human chorionic gonadotropin, human lutropin and human follitropin were quantitatively modified by introduction of an amino compound. In radioreceptor assays, the modified chorionic gonadotropin, lutropin and follitropin saturated the receptors. However, in the low nanogram range, the gonadotropic binding was higher for the control compared to the modified sample.The hormonal activity of the chorionic gonadotropin was testedin vitro. The modified preparations were four- to thirteen-fold less stimulatory compared to the control but elicited the same maximal response. The biological activity of follitropin was determinedin vivo. In this case, the modified preparations were four- to five-fold less stimulatory than the control. Both the modified chorionic gonadotropin and follitropin preparations were found to act as agonists. Modification of the gonadotropin hormones did not significantly alter the immune recognition of these glycoproteins.The apparent circulating half-life in rats of the modified chorionic gonadotropin and follitropin was increased six- to nine-fold compared to that of native hormones; this might be a consequence of resistance of the modified sialyl residues to sialidases and the resultant slower exposure of terminal galactosyl residues; the plasma half-life of modified lutropin remained the same as that of the native hormone.Abbreviations hCG human chorionic gonadotropin - hLH human lutropin or luteinizing hormone - hFSF human follitropin or follicle stimulating hormone - mala methyl ester of alanine - hCG(ala, mala, etc.) human chorionic gonadotropin modified on sialicacid by reductive amination with alanine, methyl ester of alanine, etc. - IRP-HMG intact rat prostrate-human menopausal gonadotropin     

SERS quantitative detection of trace human chorionic gonadotropin using a label‐free Victoria blue B as probe in the aggregated immunonanogold sol substrate

Nanogold particles (NG) were modified by anti‐rabbit antibody (RAb) against human chorionic gonadotropin to obtain an immunonanogold probe (ING). In pH 7.0 Na₂HPO₄‐citrate buffer solution containing KCl, ING probes formed large aggregates in which Victoria blue B (VBB) molecules were adsorbed on the surface and which exhibited strong surface‐enhanced Raman scattering (SERS) at a peak of 1612 cm^–1. After addition of human chorionic gonadotropin (hCG) an immune reaction with the ING probe occurred to form dispersive ING–hCG complexes with non‐SERS activity that led to a decreased SERS peak at 1612 cm^–1. The decreased SERS intensity was linear to the concentration of hCG over 2.4–73.2 ng/mL. The ING reaction was studied in detail by SERS, scanning electron microscope (SEM), resonance Rayleigh scattering (RRS), surface plasmon resonance (SPR) absorption and laser scattering techniques. SERS quenching was observed and discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

β-Adrenoreceptor agonists and antagonists modulate the activity of α<Subscript>2</Subscript>-adrenoreceptors in rat cortex cerebral membranes

The influence of isoprenaline- and propranolole-induced activation and inhibition of β-adrenoreceptors on the specific nonselective α₂-antagonist [³H]RX821002 binding was studied on rat cerebral cortex subcellular membrane fractions. It was shown that the ligand-receptor interaction for α₂-adrenoreceptors corresponded to the model that assumed the presence of one receptor pool and binding of two ligand molecules to a receptor dimer. The following parameters were determined for [³H]RX821002 binding to α₂-adrenoreceptors: K _d1 = 1.57 ± 0.27 nM, B _max = 7.24 ± 1.63 fmol/mg of protein, n = 2. In the case of isoprenaline-induced activation of β-adrenoreceptors the binding of radiolabeled ligand to α₂-adrenoreceptors was described by the same model. The affinity of α₂-adrenoreceptors for [³H]RX821002 decreased more than twofold (K _d = 3.55 ± 0.02 nM) and the quantity of active receptors increased by 69% (B _max = 12.24 ± 0.06 fmol/mg of protein). Propranolole changed the model of ligand binding, and two pools of receptors were detected with the following parameters: K _d1 = 0.61 ± 0.02 nM, K _d2 = 3.41 ± 0.13 nM, B _ml = 1.88 ± 0.028 fmol/mg of protein, B _m2 = 9.27 ± 0.08 fmol/mg of protein, n = 2. The data suggest that α₂-adrenoreceptors in subcellular membrane fractions from rat cerebral cortex exist in dimeric form. Isoprenaline and propranolole exhibit modulating effect on the specific antagonist binding to α₂-adrenoreceptors, which results in the inhibition and alteration of [³H]RX821002 binding parameters.     

Inhibition of gonadotropin sensitive adenylate cyclase by ovarian follicular fluid.

Follicular fluid obtained from medium or large bovine ovarian follicles inhibited ovarian luteinizing hormone/human chorionic gonadotropin sensitive adenylate cyclase in a dose-dependent manner (I₅₀ = 3 mg follicular fluid protein/ml). The inhibitory activity was excluded by Sephadex G-10 and was fully retained following treatment with charcoal. Fluoride-stimulated enzyme activity was not inhibited. Binding of ¹²⁵I human chorionic gonadotropin to ovarian plasma membranes was only slightly reduced by the follicular fluid. The post-microsomal supernatant of homogenates from ovaries of immature (27-day-old) rats collected 24–36 h after treatment with 15 i.u. of pregnant mare serum gonadotropin also inhibited luteinizing hormone-sensitive adenylate cyclase. The extent of this inhibition seemed to decline with follicular maturation. The possibility is raised that ovarian sulfated glycosaminoglycans are responsible for the observed inhibition of adenylate cyclase.     

A Point-of-Care Immunosensor for Human Chorionic Gonadotropin in Clinical Urine Samples Using a Cuneated Polysilicon Nanogap Lab-on-Chip

Human chorionic gonadotropin (hCG), a glycoprotein hormone secreted from the placenta, is a key molecule that indicates pregnancy. Here, we have designed a cost-effective, label-free, in situ point-of-care (POC) immunosensor to estimate hCG using a cuneated 25 nm polysilicon nanogap electrode. A tiny chip with the dimensions of 20.5 × 12.5 mm was fabricated using conventional lithography and size expansion techniques. Furthermore, the sensing surface was functionalized by (3-aminopropyl)triethoxysilane and quantitatively measured the variations in hCG levels from clinically obtained human urine samples. The dielectric properties of the present sensor are shown with a capacitance above 40 nF for samples from pregnant women; it was lower with samples from non-pregnant women. Furthermore, it has been proven that our sensor has a wide linear range of detection, as a sensitivity of 835.88 μA mIU^-1 ml^-2 cm^-2 was attained, and the detection limit was 0.28 mIU/ml (27.78 pg/ml). The dissociation constant K_d of the specific antigen binding to the anti-hCG was calculated as 2.23 ± 0.66 mIU, and the maximum number of binding sites per antigen was B_max = 22.54 ± 1.46 mIU. The sensing system shown here, with a narrow nanogap, is suitable for high-throughput POC diagnosis, and a single injection can obtain triplicate data or parallel analyses of different targets.     

Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma

We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4°C with high affinity (K _a = 1.49 ± 0.09 × 10⁹ M⁻¹; 0.17 ± 0.02 × 10⁷ M⁻¹) and low binding capacity (B _max = 3.24 ± 0.84 × 10⁻⁵ M; 0.33 ± 0.06 × 10⁻⁴ M). The binding affinity and capacity of testosterone at 23 and 36°C, respectively were similar to those determined at 4°C. However, oestradiol showed no binding activity at 36°C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36°C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.