Binding of human chorionic gonadotropin by rat ovarian slices: dependence on the functional state of the ovary (38571).

Ovarian receptors specific for hCG-LH can be induced by PMSG-ovine LH priming. The marked increase in binding activity was accompanied by a rise in ovarian progesterone concentration and preceded by ovarian weight increase. The high binding activity of heavily luteinized ovaries was followed by a decline to prepriming levels during luteolysis. The ovarian progesterone content was decreased simultaneously and no change in ovarian weight was evident. The data demonstrate that ovarian hCG binding activity undergoes variation which depends on the functional state of ovaries.     

Reduction of testicular human chorionic gonadotropin receptors by human chorionic gonadotropin in vivo and in vitro

Changes in rat and human testicular human chorionic gonadotropin (hCG) binding sites induced by hCG were estimated in vivo and in vitro. After a single administration of hCG, the specific 125I-hCG bindings were significantly reduced for 7 and 5 days in rat and human testes, respectively. Thereafter, 125I-hCG bindings had recovered to pretreatment values by the 14th day after the administration. Occupied hCG bindings accounted for about half of the reduced bindings on the day after administration of hCG. After this time, however, the occupancy did not contribute so much to the reduction of the bindings. In experiments in vitro using the organ culture technique, an exposure to hCG for 24 h induced a dose-related significant loss of the specific 125I-hCG bindings for 7 and 5 days in rat and human testes, respectively. Thereafter, the loss was gradually recovered. These patterns of changes in 125I-hCG bindings in vitro were similar to those in vivo. These findings suggest that the reduction in hCG binding sites by hCG is due to not only occupancy but also downregulation of the binding sites and that the testicular organ culture method used in the present study is useful to study hormonal regulation of testicular function, especially in human testes.     

Hormonal modulation of structural alteration of rat ovarian luteinizing/human chorionic gonadotropin receptors

The structure-stabilizing effect of homologous and heterogeneous desensitization and albumin on rat ovarian LH/hCG receptors was analyzed by thermal perturbation technique. HCG-induced down-regulation shifted the heat inactivation profile of hCG-binding sites to a temperature lower by about 7 degrees C (T50 values). In heterogeneous desensitization, which also involves uncoupling of receptors from adenylyl cyclase system, only follicle stimulating hormone (FSH) changed the stability of ovarian LH/hCG receptors. Stimulation of other hormonal receptors, which belong to the family of membrane spanning G protein-linked receptors, i.e. beta-adrenergic, glucagon, serotonin and prostaglandin E (PGE) had no effect on the stability of the LH/hCG receptor. Reduction of the stability of the LH/hCG receptor by about 3 degrees C after PGF2alpha injection to luteinized rats may be connected with specific process of luteolysis. On the other hand, albumin had a stabilizing effect on the receptor. The receptor destabilizing action of oleic acid incorporated into ovarian membranes along with calcium stimulation of endogenous phospholipase A (PLA) activity and reversal of these effects when BSA was used as fatty acid scavenger, may indicate that free fatty acids are responsible for the thermal instability of hCG-binding sites. Fluorescence quenching studies indicated that extraction of free fatty acids by albumin elevated the accessibility of fluorophores for acrylamide, and suggest that modificated lipid-protein interactions may affect the stability of the LH/hCG receptor structure.     

Regulation by human chorionic gonadotropin of protein kinases in rabbit endometrium

The effect of human chorionic gonadotropin (CG) on protein kinase levels has been studied in rabbit endometrium. A good correlation was observed between alterations of total protein kinase activity and DNA content in the tissue. The level of type I cyclic AMP-dependent protein kinase had slightly increased by 6 h after human CG treatment, and then decreased later. In contrast, the type II enzyme gradually increased after 3 days. The increase of the activity at 7 days was mainly due to that of type II enzyme, and the ratio of type II to type I enzyme increased. The activity pattern at the later stage closely resembled that of treatment with progesterone.     

Binding of human recombinant 125I-interferon gamma to receptors on human cells

Recombinant human interferon gamma (rIFN-gamma) produced in Escherichia coli was labeled with 125I to study its binding to receptors of HeLa and lymphoblastoid cells. All the cell lines examined had receptors for rIFN-gamma, although the binding varied considerably among different cell lines. The binding of 125I-rIFN-gamma was competed up to 90% by the addition of unlabeled rIFN-gamma, although not by the addition of IFN-alpha or -beta. By adding increasing concentrations of unlabeled rIFN-gamma to binding assays containing a constant amount of 125I-rIFN-gamma, we determined a KD of 3.7 and 6.3 X 10(-10) M for its binding to Daudi and HeLa cells, respectively. About 13,000 receptors per cell were present in Daudi and 5,000 in HeLa cells. The Mr of the IFN-gamma/receptor complex was determined by cross-linking experiments to be about 125,000. This complex is smaller than the IFN-alpha/receptor complex that has a Mr of about 140,000. The rIFN-gamma receptor was down-regulated when HeLa cells were treated with this interferon, but not when these cells were treated with IFN-beta. These findings suggest that the receptors for IFN-alpha and -gamma differ in several characteristics. The turnover of the rIFN-gamma receptor was measured by inhibiting protein synthesis with cycloheximide and the half-life of this receptor was found to be 2 h. The unglycosylated rIFN-gamma was bound to cellular receptors with an affinity similar to that previously reported for natural IFN-gamma. The lymphoblastoid cell lines examined had high affinity receptors for rIFN-gamma, but did not respond to treatment with this IFN with an induction of the synthesis of the enzyme (2'-5')oligo(A) synthetase, whereas HeLa cells responded to rIFN-gamma. The reason for the lack of response of lymphoblastoid cells is presently unknown.     

Binding and crosslinking of 125I-labeled recombinant human tumor necrosis factor to cell surface receptors

Highly purified recombinant human tumor necrosis factor (TNF) (molecular mass determined as 17 kilodaltons (kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as 36 kDa by Sephadex G-100 gel chromatography) was labeled with 125I to a specific activity of 5 microCi/micrograms without appreciable loss of activity. The binding of 125I-TNF to eighteen human and twelve animal cell lines was examined. The binding varied considerably among different cell lines. In most cell lines, the binding was inhibited up to greater than 90% by the addition of a 100-fold excess of unlabeled TNF. Some human and mouse cell lines showed no significant binding above background levels, suggesting that these cell lines had no receptors for TNF. Among the TNF receptor-positive cell lines, there was no direct correlation between the level of specific TNF binding and the level of sensitivity to the cytotoxic or cytostatic effect of TNF. Some cell lines were sensitive to TNF, whereas others were not affected at all by TNF. The TNF receptor-negative cell lines were also resistant to TNF. Therefore, although the existence of TNF receptor seems to be necessary, it does not alone determine cellular sensitivity to TNF. Scatchard analysis of the binding data revealed that human HeLa S3 and THP-1 had about 50,000 and 10,000 receptors/cell with a dissociation constant (KD) of 0.3-0.5 nM, respectively. Similarly, mouse L-929 and L-M cells had about 5,000 receptors/cell with KD of 3-5 nM. 125I-TNF bound to HeLa S3 cells was rapidly internalized at 37 degrees C, presumably by receptor-mediated endocytosis, and degraded to acid-soluble products. The turnover of TNF receptors on HeLA S3 cells seemed to be rapid, since the level of specific binding quickly decreased after treatment with 100 micrograms/ml of cycloheximide at 37 degrees C with a half-life of about 1.5 h. The crosslinking of the cell-bound 125I-TNF with the use of disuccinimidyl suberate yielded a complex of 105 kDa for HeLa S3 and THP-1 cells, and a complex of 100 kDa for U937 cells. The crosslinking was completely inhibited by the addition of a 100-fold excess of unlabeled TNF. Assuming that the complex was due to a one-to-one association of the dimeric form of TNF (34 kDa) with the receptor, we estimated the molecular size of the human TNF receptor to be 71 kDa for HeLa S3 and THP-1, and 66 kDa for U937.     

Detection of conformational changes in human chorionic gonadotropin upon binding to rat gonadal receptors

After binding to rat testicular or ovarian luteinizing hormone (LH) receptors, human chorionic gonadotropin (hCG) and mammalian LH can be detected with monoclonal antibodies directed against a conserved epitope on the beta subunit of the hormones. Two such anti-hCG/anti-LH monoclonal antibodies, known as B105 and B110, compete with one another for binding to this epitope region on free and receptor-bound hormone. By comparing the affinities of B105 and B110 for these two forms of hCG, we have detected apparent changes in the structure of the hormone which develop subsequent to receptor binding. Whereas the affinity of B105 for receptor-bound hCG is approximately 10-fold lower than that for free hCG, the affinity of B110 for receptor-bound hCG is nearly 20-fold greater than that for free hCG. Both B105.hCG and B110.hCG complexes bind to the receptor; however, they have approximately 25 and 50% lower affinity than hCG. Thus, although B110 binds better to the form of hCG which is bound to receptors, binding of B110 to hCG does not appear to induce a conformational change in the hormone which facilitates hormone-receptor binding. Consequently, both B105 and B110 partially inhibit binding of hCG to its receptors. Fab fragments of B105 and B110 are as effective as intact B105 and B110 in inhibiting the binding of labeled B105 and B110 to hCG-receptor complexes, suggesting that circular complexes which might be formed by the interaction of divalent antibody, two molecules of hCG, and two membrane-bound receptors or one divalent receptor are not contributing to the affinity of the antibodies for receptor-bound hCG. Alternatively, formation of circular complexes can explain an increase in apparent affinity of B105 for ovine or bovine LH-receptor complexes. Data obtained with B105 suggest either that the structure of the epitope is altered following binding or that a portion of the epitope is partially obscured when hCG binds to the receptor. In contrast, the data obtained using B110 are not explained by models in which steric factors reduce the affinity of the antibody for the hormone-receptor complex. Therefore, as a minimal explanation for these observations, we postulate that the conformation of the B105/B110 epitope region is altered following binding of the hormone to receptors. The nature of the conformational change and its relationship to LH/hCG action is unknown.     

Divergent metabolism of human chorionic gonadotropin subunits within rat ovarian lysosomes

Pseudopregnant rats were injected with either native human chorionic gonadotropin or with (125I)-human chorionic gonadotropin and their ovarian homogenates fractionated on Percoll density gradients. The levels of alpha and beta subunits within subcellular fractions were measured using radioimmunoassays specific for each subunit. Radioactivity measurements of fractions obtained from rats injected with (125I)-human chorionic gonadotropin were used as a separate index of alpha subunit distribution. The alpha subunit was primarily restricted to a combined plasma membrane/prelysosomal vesicle fraction. Immunoreactive beta subunit was present at high concentrations within both this plasma membrane/prelysosomal vesicle fraction and within lysosomes. The striking difference in alpha and beta subcellular distribution may arise from differential sensitivities to lysosomal enzymes.     

Localization of human chorionic gonadotropin beta subunit transcripts in ovarian cancer tissue

Recent studies demonstrated that besides placenta and malignant trophoblastic tumors, hCG and especially its beta-subunit is secreted by a varieties of tumors of different origin. The aim of the present investigation was to determine the expression pattern of human chorionic gonadotropin gene in ovarian cancer tissue. The study included 8 patients with epithelial ovarian carcinoma. The expression and distribution of hCGbeta mRNA was assessed by in situ RT-PCR method. The semi-quantitative assessment was performed using computer image analysis. Transformation of the images into the pseudocolour scale showed a clear difference in fluorescence intensity among individual cancer cells. The intensity of ISRT-PCR products corresponding with expression level of hCGbeta demonstrated that its production by individual cancer cells is different. In all studied specimens of the ovarian carcinoma tissue, cancer cells characterized by the presence of active hCGbeta gene were found, whereas noncancerous tissue demonstrated lack of the gene expression. Thus, the study clearly shows that the expression of hCGbeta is the feature of ovarian cancer tissue.     

Binding of 125I-labelled tumor necrosis factor to Pneumocystis carinii and an insoluble cell wall fraction.

We have previously shown that tumor necrosis factor-alpha (TNF-alpha) causes loss of viability of Pneumocystis carinii. In this study, we demonstrate that TNF-alpha irreversibly binds to P. carinii in vitro. The avidity of binding is orders of magnitude greater than that by which it binds to a known sensitive target, L929 cells. A detergent-insoluble fraction of P. carinii, which consists primarily of cyst walls, bound ten times more TNF-alpha per microgram protein as did whole P. carinii.     

Immunobiology of human chorionic gonadotropin

A comprehensive review of the immunobiology of human chorionic gonadotropin (hCG), including the structure of both alpha and beta chains, immunogenicity of various segments and epitopes of each, secretion and function of the hormone, determinants of receptor recognition, and finally, clinical studies of possible contraceptive beta-hCG-based vaccines, is presented. hCG is composed of 2 glycosylated peptides. The alpha subunit is identical to that found in hLH, hFSH and hTSH. The beta subunit, which is limiting in the sense that it is secreted in smaller amounts, defines the biological activity of hCG. hCG is secreted throughout pregnancy from 170 hours after fertilization to a peak at 8-10 weeks of and is essential for maintenance of early pregnancy by progesterone secreted by the corpus luteum. Although native hCG evokes antibodies, they cross react with LH, so such a vaccine would not be useful for contraception. Beta-hCG has been purified and also produced by monoclonal antibodies, and shown to produce antibodies and infertility in baboons. Phase I clinical trials of immunologically purified beta-hCG complexed to tetanus toxoid were conducted on 63 women in an international study in the mid-1970s, but results were mixed in terms of antibody titer and duration. New vaccines have been designed based on more sophisticated adjuvants, beta- hCG-terminal peptides, and polyvalent vaccines and are being tested in 4 Phase I trials currently, sponsored by the Population Council, the Indian government-sponsored program, and the WHO.     

Photo-induced inactivation and uncoupling of gonadotropin receptors in rat ovarian plasma membrane

Extraction of membranes of Lactobacillus plantarum with Triton X-100/glycerol solubilized up to 80% of the undecaprenol kinase activity. Fractionation of the extract by gel chromatography separated endogenous phospholipid from the enzyme but simultaneously inactivated the enzyme. The kinase was reactivated by reconstitution with various synthetic phosphatidylcholines and purified L. plantarum phospholipids. Ditetradecanoylphosphatidylcholine and lysylphosphatidylglycerol were the best activators. Furthermore, the optimal environment for enzyme stimulation was provided by different defined molar ratios of Triton X-100/phospholipid. The ratios for the phospholipids tested ranged from 1.25 to 6.3. Similar substrate specificity and kinetic constants were observed for both the solubilized and reconstituted enzymes suggesting that no fundamental changes in the enzyme activity occurred during the delipidation-reconstitution process.