PubMed Health⌕ Search

Biomedical subjects

R J Ryan

Publications and source records attributed to R J Ryan.

At least 37 records · Page 2Linked to original sources
PubMed1988

Follicle-stimulating hormone (FSH) immunoactivity in porcine follicular fluid is not pituitary FSH.

Two inhibitors of FSH binding to receptor have been isolated from porcine follicular fluid and shown to have in vitro biological activity. These inhibitors were distinct separable entities with opposite biological effects (agonist and antagonist) on cultured FSH-responsive Sertoli cells. In light of the fact that the agonist-containing fraction (P4) inhibited [125I]human (h) FSH binding to anti-hFSH antiserum as well as to receptor, characterization of this factor was undertaken to determine its relationship to pituitary FSH. The P4 fraction was further purified by affinity chromatography, which removed a major protein from immunoreactive components. Western blotting of sodium dodecyl sulfate-polyacrylamide gels using polyclonal (anti-hFSH) and monoclonal (anti-hFSH beta) antibodies revealed a major immunoreactive band at 55,000 mol wt (Mr). When electrophoresed under reducing conditions, major immunoreactive proteins at 58,000 and 45,000 Mr were identified. These bands were also observed in extracts from bovine testes and raw porcine follicular fluid after electrophoresis and Western blotting. Whereas the monoclonal antibody used to characterize this inhibitor does not recognize porcine pituitary FSH, the Mr of the immunoreactive proteins are greater than that of pituitary FSH, and the immunoreactive bands do not reduce to subunits, as observed for pituitary FSH under reducing conditions, we conclude that gonadal extracts contain FSH-immunoreactive proteins that are immunologically and biochemically distinguishable from pituitary FSH. While the physiological role of these proteins remains to be determined, their presence in gonadal extracts or fluids vitiates assessment of FSH within the gonad by RIA using antiserum against hFSH.

Animals↗
PubMed1988

The effects of synthetic alpha-subunit peptides on thyroid-stimulating immunoglobulin activity.

Synthetic peptides, representing specific portions of the alpha-subunit of the human glycoprotein hormones, can inhibit both the binding of labeled TSH to thyroid membranes and adenylate cyclase stimulation by TSH in vitro. The same synthetic peptides (alpha 26-46 and alpha 31-45) significantly (P less than 0.05) inhibited the adenylate cyclase-stimulating activity of thyroid-stimulating immunoglobulins (TSI) from 10 patients with hyperthyroid Graves' disease. Peptide alpha 26-46 was the most potent, resulting in 79.1 +/- 8.8% (+/- SE) inhibition at 133 micrograms/mL, while peptide alpha 31-45 inhibited TSI activity by 36.3 +/- 5.2%. Peptides alpha 61-75 and alpha 81-92, that had only minimal ability to inhibit TSH-mediated cAMP generation, did not significantly inhibit TSI activity. The inhibitory action of alpha 26-46 was dose dependent, and a significant negative correlation was found between the maximum TSI activity of the serum sample and the inhibition achieved by the synthetic peptide, suggesting that differences in TSI affinity and/or titer may account for the variable inhibitory activity of the peptides. These results suggest that TSI interact with the TSH receptor at the site that recognizes the portion of the TSH alpha-subunit represented by the synthetic peptide alpha 26-46 and, thus, support the concept that the TSH-binding site of the TSH receptor is the site of antigen binding between TSI and the thyroid cell.

Cyclic AMP↗
PubMed1988

Replication of mitochondrial DNA in the gonadotropin-primed corpus luteum of the rat.

The effect on mitochondria isolated from corpus luteal tissues following priming of immature female rats with pregnant mares serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) was studied. Ultrastructural studies showed intramitochondrial filamentous nucleic acid networks in situ following intense uranyl acetate staining. The intramitochondrial complexes were sensitive to nuclease treatment. Primed corpora lutea contained a 3.7-fold increase of mitochondrial DNA (mtDNA) per mg of mitochondrial protein when compared to unprimed ovaries. In subsequent experiments female rats were injected with 3H-thymidine 12 h before harvesting gonadotropin-primed corpora lutea from which mitochondria were isolated, purified and lysed. MtDNA was isolated and purified from the lysate by CsCl-ethidium bromide equilibrium buoyant density gradient centrifugation. Both the upper and lower bands of mtDNA as well as the intermediate region of the gradient contained radioactive label. When mtDNA from a fractionated gradient was mounted for electron microscopy and examined, replicative forms of mtDNA were observed. The mechanism of replication appears to be by the displacement-loop model of mtDNA replication. Ultrastructural as well as biochemical evidence indicate that a consequence of corpora lutea formation is the replication of mtDNA.

Animals↗
PubMed1987

Inhibition of human choriotropin binding to receptor by human choriotropin alpha peptides. A comprehensive synthetic approach.

Synthetic overlapping peptides of the alpha-subunit of human chorionic gonadotropin (hCG) were made by solid-phase peptide synthesis employing a comprehensive synthetic approach. The entire primary structure of the alpha-subunit was synthesized as a series of nine consecutive peptides, each 15 residues in length, and overlapping with its two adjacent neighbors by 5 residues on each side. Receptor binding activity of each synthetic peptide was measured by the inhibition of binding of 125I-labeled hCG to rat ovarian receptor. Peptides alpha 21-35, alpha 31-45, alpha 71-85, and alpha 81-92 were shown to compete for binding with native hCG, thus demonstrating that at least two regions on the alpha-subunit may be part of the binding site(s) of the hormone. The low affinity of the peptides (10(-5)-10(-6) M) compared to native hormone (10(-10) M) for receptor is not unexpected due to the probability of discontinuous and multiple sites involved in receptor binding. An ultrapure preparation of hCG alpha-subunit also had low affinity (10(-5), suggesting that conformational changes upon combination with beta-subunit to form dimer or changes in conformation after binding are necessary for high affinity interaction. These results correlate with previous predictions of binding sites based on studies employing chemical and enzymatic modifications of intact hormone and show that synthetic peptide strategies are helpful in the elucidation of protein structure and function.

Amino Acid Sequence↗
PubMed1987

Tryptophan fluorescence studies of subunit interaction and rotational dynamics of human luteinizing hormone.

Human luteinizing hormone (hLH) has a single tryptophan residue occurring in the beta-subunit (beta hLH). This provides an intrinsic fluorescent probe, in native hLH and beta hLH, that is unambiguously assigned. The fluorescence intensities of hLH and beta hLH are, however, significantly different. This difference has been utilized in studying the interaction of fluorescent beta hLH with the nonfluorescent alpha-subunit. The accessibility of the tryptophan residue in native hLH and beta hLH has been assessed by measuring the rate of collisional fluorescence quenching and by solvent perturbation (D2O/H2O) of fluorescence. Fluorescence anisotropy measurements have been used in studying the intramolecular dynamics and segmental tryptophan mobility in hLH and beta hLH. Lifetime-resolved anisotropy, measured by the technique of oxygen quenching of fluorescence, has revealed the presence of segmental tryptophan motion. These data can be satisfactorily explained in terms of fast segmental tryptophan motion and rotational diffusion of the whole protein and do not require that intersubunit motion be invoked for intact hLH as it was suggested earlier on the basis of fluorescence depolarization of fluorescein-labeled hLH [Bishop, W. H., & Ryan, R. J. (1975) Biochem. Biophys. Res. Commun. 65, 1184-1190].

Fluorescence Polarization↗
PubMed1987

A receptor-binding region in human choriogonadotropin/lutropin beta subunit.

Synthetic fragments have not been widely used thus far to evaluate structure-activity relations in the glycoprotein hormones. We prepared a series of peptides representing the intercysteine "loop" sequence (residues 38-57) in human choriogonadotropin (hCG) and lutropin (hLH) beta subunits, anticipating that it might be oriented toward the surface and accessible to receptors. The peptides were characterized chemically and tested for bioactivity by binding to rat ovarian membrane receptor and stimulation of Leydig cell testosterone production. The hCG beta-(38-57) and hLH beta-(38-57) peptides inhibited binding of 125I-labeled hCG half-maximally at 1.51 X 10(-4) and 2.03 X 10(-5) M, respectively, while other peptide hormones and fragments from elsewhere in the beta subunit were inactive. Both peptides stimulated testosterone production, with half-maximal responses at 3.55 X 10(-5) M (hCG) and 2.18 X 10(-5) M (hLH). By radioimmunoassay with an antibody to thyroglobulin-conjugated hCG beta-(38-57) peptide, native hCG and beta subunit were highly reactive, as were the reduced and carboxymethylated subunit and peptide. Helical-wheel projection predicted an amphipathic region in the N-terminal portion of the 38-57 sequence, and circular dichroic measurements showed an increase in ordered structure, especially alpha-helix, when the 38-57 peptides were transferred from an aqueous to a more lipophilic (90% trifluoroethanol) environment. These results indicate that the 38-57 region of beta subunit is exposed on the surface and constitutes a component in the receptor-binding domain for hCG and hLH. A region of amphipathic-helical structure in the 38-57 sequence may promote hormone-receptor interactions in a manner proposed for several other peptide hormones.

Amino Acid Sequence↗
PubMed1986

Deglycosylated human follitropin: characterization and effects on adenosine cyclic 3',5'-phosphate production in porcine granulosa cells.

Chemical deglycosylation of gonadotropins with hydrogen fluoride (HF) has facilitated the investigation of the structure-function relationship of the individual peptide and oligosaccharide moieties in the mechanism of hormone action. These studies have dealt almost exclusively with lutropin or human choriogonadotropin. We report here the chemical characterization and biological properties of deglycosylated human follitropin (degly hFSH). Results indicate that deglycosylation of hFSH by HF removes 89% of the total carbohydrate without disruption of the peptide chain or significant loss of amino acid residues. However, a change in the conformation of the molecule was observed by measurement of the far-ultraviolet circular dichroic spectrum. The degly hFSH showed a 44% reduction in binding when tested in a FSH radioimmunoassay utilizing a polyclonal antibody. Binding of the degly hFSH to FSH-responsive tissues showed that the altered hormone bound with equal or better avidity than the intact hormone while the association constants were approximately the same for both preparations. The degly hFSH alone did not stimulate the FSH-stimulatable adenylyl cyclase (AC) activity of cellular homogenates of small follicle porcine granulosa cells. Furthermore, degly hFSH was a potent antagonist of hFSH-stimulatable AC activity when coincubated with intact hFSH. In intact granulosa cells, both the hFSH and the degly hFSH stimulated cAMP production and release by these cells. However, the degly hFSH was one-tenth as effective as the intact hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1986

Electrophoretic analysis of membrane proteases in the luteinized rat ovary.

Membrane fractions from fully luteinized rat ovaries contained proteolytic enzymes that were solubilized and reversibly inhibited by the anionic detergent sodium dodecyl sulfate. Electrophoretic analysis in substrate-containing sodium dodecyl sulfate-polyacrylamide slab gels revealed the presence of numerous protease bands in the mol wt range of 26,000 to more than 200,000. When casein served as protease substrate in the gels, enzyme bands of Mr 26,000, 28,000, 30,000, and 90,000 were produced by crude (2,000 X g pellet) ovarian membranes. Gels containing casein and plasminogen also revealed the presence of two plasminogen activators of Mr 63,000-65,000 and 42,000. Subcellular fractionation of luteinized ovaries by centrifugation in sucrose density gradients indicated that the Mr 90,000 protease and the two plasminogen activators were uniformly distributed among microvillous membranes, basolateral membranes (BLM), and the mitochondrial-lysosomal fraction (MLF). The Mr 26,000, 28,000, and 30,000 proteases were enriched in BLM and MLF. Analysis of crude membranes in slab gels which contained gelatin as the protease substrate revealed the presence of two additional enzymes of Mr 52,000 and more than 200,000. The Mr greater than 200,000 protease was present in microvillous membranes, BLM, and MLF. The Mr 52,000 protease was found exclusively in BLM. This enzyme was not consistently demonstrable in crude membranes but could be generated upon incubation of membranes at 30 C. This finding indicated that Mr 52,000 protease can exist in an inactive and/or zymogen form. The Mr 90,000 protease was inhibited by tosyl-lysine chloromethyl ketone and dansyl-glutamyl-glycylarginine chloromethyl ketone. The gelatinase activity of the Mr 52,000 protease was blocked by tosyl lysine chloromethyl ketone, dansyl-glutamyl-glycylarginine chloromethyl ketone and tosylamide-2-phenyl chloromethyl ketone. The activity of the Mr 26,000, 28,000, and 30,000 proteases was not affected by any of the above mentioned inhibitors. These findings demonstrate that the proteolytic potential of ovarian membranes is not limited to plasminogen activators. Numerous plasminogen-independent proteases are also present, and these may play a role in ovulation, luteolysis, and mediation of hormonal stimulation.

Animals↗
PubMed1985

Evidence for a conformational change in deglycosylated glycoprotein hormones.

Removal of long-chain asparagine-linked carbohydrates leads to loss of receptor-effector coupling in gonadotropin target tissue. This implies a direct interaction between carbohydrate and cell membrane components. To examine other mechanisms by which carbohydrate could activate post-receptor events, we have used sequence-specific and conformation-specific alpha-subunit radioimmunoassays as probes for conformational changes in deglycosylated choriogonadotropin and follitropin. Immunoreactivity of either hormone was enhanced 8-13-fold after removal of carbohydrate by anhydrous hydrogen fluoride. Removal of sialic acid alone had little effect on reactivity. Based on the specificity of the antisera, the effect could be localized to a region in the amino-terminus remote in the linear sequence from actual sites of carbohydrate attachment. The results suggest that a conformational change in the alpha-subunit could account, at least in part, for the observed effects of deglycosylation on glycoprotein hormone action.

Amino Acid Sequence↗
PubMed1985

Inhibition of adenylyl cyclase activity in rat corpora luteal tissue by glycopeptides of human chorionic gonadotropin and the alpha-subunit of human chorionic gonadotropin.

Indirect evidence has indicated that the carbohydrate moieties of the glycoprotein hormones are involved in the activation of the receptor-adenylyl cyclase system of reproductive tissues. In the present study, we have isolated the glycopeptides (GP) from human chorionic gonadotropin (hCG), the alpha-subunit of hCG, fetuin, and bovine gamma-globulin (b gamma G). These along with a number of synthetic oligosaccharides were tested for their ability to inhibit adenylyl cyclase (AC). There was less than 0.001% cross-reactivity of the GP from hCG, hCG alpha, fetuin, and b gamma G when tested in a double-antibody hCG radioimmunoassay or rat corpora lutea radioreceptor assay. The GP of fetuin, b gamma G, and the synthetic oligosaccharides did not inhibit AC activity of 2000 g corpora lutea membranes when coincubated with 100 ng of hCG/mL (ED50). However, when the GP of hCG and hCG alpha were included with intact hCG, there was a dose-related inhibition. Inhibition of cyclase activity was enhanced when the hCG GP were desialylated. This occurred without a change in the lag time of hCG activation which was calculated to be 1-1.5 min. Changing the concentration of ATP and Mg2+ did not affect the inhibitory effects of the hCG alpha GP on hCG-stimulated AC activity. Inhibition by hCG GP followed uncompetitive kinetics. The inhibition by the GP of hCG seems to be restricted to the LH/hCG-stimulatable AC system because the same dosage of hCG GP which inhibited the rat luteal AC system did not have any effect on the rat hepatocyte AC system when coincubated with glucagon or on NaF-stimulated activity in luteal membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclase Inhibitors↗
PubMed1985

Prevalence of circulating antibodies directed toward ovaries among women with premature ovarian failure.

The presence of circulating antibodies to the ovary in serum samples from women with premature ovarian failure suggests that an autoimmune mechanism is a factor in the pathogenesis of this disorder in some women. Sera from 110 women with elevated serum levels of gonadotropins, low serum levels of estrogens, normal karyotypes, and amenorrhea before the age of 35 years were investigated for the presence of antibodies to the ovary. Of the total sera from these 110 patients with premature ovarian failure, 30 (27%) had results that were positive for the presence of circulating antibodies to ovary.

Antibodies↗
PubMed1985

A simple and rapid method using polyethylenimine-treated filters for assay of solubilized LH/hCG receptors.

Filtration through polyethylenimine (PEI)-treated membrane filters efficiently separates free radiolabeled human (h) LH or hCG from solubilized receptor-hormone complexes. Solubilized LH/hCG receptors of bovine, porcine, and rat origin are suitable for measurement by this method. Specific binding assayed by the PEI-filter technique was 106-138% of specific binding assayed by double polyethylene glycol precipitation, demonstrating that the method gave quantitative recovery of bound hormone. Nonspecific binding was consistently lower with PEI-treated filters. Intra- and interassay variability for the two different methods was similar. These results indicate that filtration through PEI-treated membrane filters is a simple and rapid method for assay of solubilized LH/hCG receptors.

Animals↗
PubMed1985

Monoclonal antibodies against human follicle-stimulating hormone.

Five monoclonal antibodies to human (h) FSH have been prepared, isolated, and characterized. They were produced by hybridomas derived from FO myeloma cells and spleen cells from mice immunized with hFSH or its beta-subunit (hFSH beta). Two of the antibodies (A101 and A102) which recognized the alpha-subunit of hFSH bound much better when alpha was associated with the beta-subunit forming the intact hFSH molecule than when alpha was in free form. These antibodies showed 9-10%, 2-3%, and 1-3% cross-reactions with alpha-subunits in hTSH, hCG, and hLH, respectively. Two antibodies (B201 and B202) recognized only free beta-subunit. One antibody (B305) recognized free beta-subunit and native hFSH. There was no significant cross-reaction of these antibodies to FSH beta with hTSH, hLH, and hCG. Using solid phase competitive binding and sandwich assays, we compared the epitopes for these antibodies. Antibodies A101 and A102 recognize the same epitope on hFSH alpha. Antibodies B201 and B202 recognize different epitopes, but they seemed to be adjacent. Antibody B305 bound a different epitope than B201 and B202. Such characteristics of these antibodies can be useful for sensitive and specific assay of hFSH or hFSH beta and also may be helpful in studying FSH interaction with its receptor.

Animals↗
PubMed1985

The accuracy of clinical parameters in detecting periodontal disease activity.

A review of the literature shows that, currently, no reliable clinical parameter exists either to indicate existing disease activity or to predict future periodontal deterioration. The common clinical parameters used in practice merely assess gingival status or indicate that tissue destruction has occurred earlier (Fig 9). Although many clinicians mistakenly use them as indicators of ongoing destruction, these clinical findings have more value as educational and motivational tools. More sophisticated parameters (that is, antibody tests, and subtraction radiography) are being developed, and their accuracy in identifying disease activity is being tested. These parameters differ in their degrees of accuracy and reliability and, currently, are best suited for research purposes. Quantitative plaque assessments, once shown to have a correlation with incidence of periodontal disease in study populations, have been proved recently to be inaccurate in assessing periodontal disease activity when evaluating specific individuals and sites. Qualitative assessments are more reliable in determining areas that have suffered from specific periodontal disease processes, but their accuracy in identifying ongoing deterioration is questionable. Although it generally is agreed that bacterial plaque is the cause of periodontitis, no plaque assessment to date has been reliable consistently as an indicator of existing or impending periodontal disease activity. Plaque indexes can be used to educate and motivate patients in the removal of this most important etiologic agent. Clinical gingival assessments do not indicate existing or impending periodontal disease activity with loss of attachment and, therefore, are of little value in diagnosing periodontitis. Because gingival inflammation is an undesirable deviation from health, gingival indexes are valuable as a signal to the clinician to manage these patients more closely.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar Process↗