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R J Ryan

Publications and source records attributed to R J Ryan.

At least 19 recordsLinked to original sources
PubMed1992

Inhibition of TSH bio-activity by synthetic beta TSH peptides.

The in vitro bioactivity of the human beta TSH subunit was investigated utilizing eleven overlapping synthetic peptides representing the entire 112 residue sequence. The peptides were tested for both stimulatory and inhibitory activity in two sensitive bioassay systems: the first based on cAMP production in FRTL-5 rat thyroid cells, and the second based on stimulation of iodine trapping by the same continuous cell line. Peptides from three distinct regions of the beta-subunit showed concentration dependent inhibition of TSH bio-activity, including beta 1-15, beta 11-25, beta 31-45, beta 81-95, and beta 91-105 with IC50 values ranging from 150 to 304 microM. An additional peptide representing the entire sequence of the "intercysteine loop" region of beta TSH, beta 31-52, also inhibited TSH activity with somewhat higher potency than its fragment peptide beta 31-45 (IC50 of 87.5 +/- 14.7 microM for beta 31-52 versus 207 +/- 92.4 microM for beta 31-45). Three of these, beta 1-15, beta 31-45, and beta 31-52, also inhibited binding of TSH to the receptor in a radio-receptor assay, as previously reported (1), supporting their importance in receptor interaction. None of the synthetic peptides stimulated either cAMP production or iodine trapping. Two other overlapping peptides, beta 81-95 and beta 91-105, possessed bio-inhibitory activity but did not inhibit binding of labeled TSH. Computer analysis of this sequence predicted an extended turn structure for this region. This region has been referred to as the "determinant loop" as it is bounded by cysteine residues at positions 88 and 95 that many believe form a disulfide bond in the native subunit. The current data suggests the beta 88-95 region may play a role in receptor activation after initial binding of hormone to receptor.

Amino Acid Sequence↗
PubMed1992

The identification of spinal pathology in chronic low back pain using single photon emission computed tomography.

Bone scintigraphy with single photon emission computed tomography (SPECT) offers improved lesion detection and localization when compared to conventional planar imaging. The SPECT findings were investigated in 80 consecutive patients (aged 18-70 years, median 44) referred to a rheumatology outpatient clinic with low back pain persisting for more than 3 months. Lesions of the lumbar spine were demonstrated in 60% of patients using SPECT but in only 35% with planar imaging. Fifty-one per cent of all lesions were only detected by SPECT, and lesions visualized on SPECT could be precisely localized to the vertebral body, or different parts of the posterior elements. Fifty per cent of lesions involved the facetal joints of which almost 60% were identified on SPECT alone. X-rays of the lumbar spine, with posterior oblique views, failed to demonstrate abnormalities corresponding to almost all SPECT posterior element lesions although it identified abnormalities corresponding to over 60% of anterior SPECT lesions. Computed tomography (CT) was performed in 30 patients with a SPECT lesion and sites of facetal joint activity corresponded to facetal osteoarthritis in 82%. It is concluded that bone scintigraphy with SPECT in patients with chronic low back pain demonstrates many lesions not seen with either X-ray or conventional planar imaging. In addition anatomical localization is greatly enhanced with bone SPECT. The technique offers improved diagnosis in a group of patients often difficult to evaluate, and in particular a means of detecting apophyseal joint pathology which may be responsive to treatment.

Adult↗
PubMed1979

Premature menopause. I. Etiology.

The premature menopause syndrome has been regarded as one of the organ-specific autoimmune disorders because circulating antibodies to ovarian tissue have been demonstrated. Fifteen women with spontaneous cessation of menses after initial menarche before they were 35 years old were seen between 1975 and 1977. Increased serum levels of gonadotropin and failure of estrogen secretion were documented. Serum from each woman was examined for antibodies to normal ovary. Proteins from ovaries of normal premenopausal women were extracted and iodinated with 125I. The labeled proteins were incubated with sera for 48 hours, after which goat antihuman gamma-globulin was added and allowed to incubate for 72 hours. The precipitate was washed and evaluated for radioactive label. The binding of antibodies increased in the sera of patients with the premature menopause syndrome, compared with the control sera. This suggests that the etiology of premature menopause may be mediated by circulating antibodies to ovarian tissue.

Adult↗
PubMed1979

Interactions of gonadotropins with corpus luteum surface membranes. V. Differential effects of digitonin on the buoyant densities of light and heavy rat ovarian membrane fractions.

Previous studies have indicated that rat luteal cells at certain stages of development can be fractionated so as to obtain two plasma membrane fractions with different densities and different profiles of marker enzymes. The light membrane fractions (density 1.13) contain the majority of hCG-binding sites and little or no cyclase enzyme, while the heavy membranes (density 1.17) contain the majority of cyclase enzyme and lesser quantities of hormone-binding sites. These membrane fractions were further compared with respect to their susceptibility to perturbation by digitonin. The buoyant density of luteal cell light membrane fractions, as marked by [125I]iodo-hCG binding, Mg2+-dependent ATPase, and 5'-nucleotidase, were highly perturbable by digotonin (delta density, greater than 0.05), while adenylate cyclase activity and phosphodiesterase activity associated with this fraction were only slightly perturbed (delta density, less than 0.02). The buoyant density of luteal cell heavy membrane fractions, as marked by adenylate cyclase, ATPase, and nucleotidase, was not significantly perturbed by digotonin. The hCG binding associated with the heavy membrane fraction was not perturbed by digitonin. From these studies, we conclude that the adenylate cyclase activity associated with light membrane fractions is due to contamination by heavy membranes, while the hCG-binding activity in heavy membrane fractions is intrinsic to that membrane. Except for the lysosomal marker (glucuronidase), which was solubilized by digitonin, the detergent had no significant effect on the density of mitochondrial, Golgi, GERL (Golgi, endoplasmic reticulum, and lysomal), or endoplasmic reticulum membranes. Plasma membranes from isolated granulosa cells and ovaries obtained 24 h after priming with PMS gonadotropin-hCG behaved as heavy membranes (density, 1.17) which contained hCG-binding sites, adenylate cyclase, nucleotidase, and Mg2+-dependent ATPase. These were not significantly perturbed by digitonin. The appearance of light membranes and the segregation of adenylate cyclase from the majority of hCG-binding sites is a development feature of the luteal cell.

Adenylyl Cyclases↗
PubMed1979

FSH stimulation of 3H-glucosamine-incorporation into proteoglycans by porcine granulosa cells in vitro.

Porcine granulosa cells from small follicles were incubated in the presence of 1 muCi/ml 3H-glucosamine and various hormones. Proteoglycans were digested with protease, and the mucopolysaccharides (MPS) were recovered. Most of the labeled MPS was found in secretory products. Maximum incorporation of 3H-glucosamine into MPS occurred after incubation with FSH for 8--16 h. Cell concentrations of 0.5--4.0 x 10(6)/ml proved to be ample. Highly purified porcine or human FSH gave consistent, significant stimulation by doses in the order of 100 pg/ml. Linear log-dose response curves occurred from 0.1 to 10.0 ng/ml, and maximal stimulation at 10 ng/ml was 22-fold above control. Preparations of hCG, hTSH and NIH-LH-S18 were not effective. Incubations with 1--10 mM dibutyryl cyclic AMP significantly increased incorporation into MPS, suggesting that effects of FSH may be mediated by cyclic AMP.

Animals↗
PubMed1979

A search for antibodies to luteinizing hormone receptors in premature ovarian failure.

We recently published evidence of the presence of circulating antibodies in the sera of patients who had the diagnosis of premature menopause syndrome. This led to the question: Is there a circulating antibody in the sera of patients with premature menopause syndrome which interferes with gonadotropin-luteinizing hormone (LH) receptor interaction on the luteal cell surface? Serum samples from 14 women with the diagnosis of Premature menopause syndrome were examined to see whether they would block the binding of 125I-labeled human chorionic gonadotropin (hCG) to human corpora lutea. All patients had spontaneous cessation of menses before the age of 35 years and documentation of increased gonadotropin secretion and failure of estrogen secretion. When the sera of the study group were incubated with 125I-labeled hCG and LH receptor from human corpora lutea, no difference in binding was observed when compared with controls. This suggests that there is no circulating antibody that interferes with hCG-LH receptor interaction and that would thereby lead to loss of ovarian function in premature menopause syndrome.

Adult↗
PubMed1978

Interations of gonadotropins with corpus luteum membranes. VII. Association of hCG-binding and adenylate cyclase activities with rabbit corpus luteum plasma-membranes.

Previous studies with rat luteal cell plasma membranes indicated a dissociation of hCG binding sites and adenylate cyclase activity. The hormone binding sites were associated with a light membrane (d 1.13) fraction derived from the microvillous surface while the cyclase enzyme was associated with a heavy membrane (d 1.17) fraction derived from the basolateral surface of the cell. In this study of plasma membranes from 8-day pseudopregnant rabbit corpora lutea both hCG binding and adenylate cyclase activity were associated with a light membrane (d 1.13) fraction. No heavy membrane material was detected.

Adenylyl Cyclases↗
PubMed1978

Structure of human luteinizing hormone alpha subunit.

Structural studies have substantiated the concept that the glycoprotein hormones consist of a "common" alpha subunit and "hormone-specific" beta subunit. Despite this, consensus is still lacking concerning certain portions of the amino acid sequences, including alignment of residues 81--82 in the alpha subunit. We have carried out sequence analysis of the alpha subunit of human luteinizing hormone (hLH) to clarify the assignment of these residues and to examine further the nature of the amino-terminal heterogeneity found among the different alpha subunits. Our structure showed residues 80--84 to be -His-Cys-Ser-Thy-Cys-, consistent with findings of others for human follicle stimulating hormone (hFSH) and human chorionic gonadotropin (hCG). The extensive degree of heterogeneity found in the amino terminal region of hFSH and hCG is present to only a minor extent in hLH. The differences in the pattern of amino-terminal heterogeneity among the various hormones may result from differences in the nature of cleavage of subunit from a larger intracellular precursor peptide.

Amino Acid Sequence↗
PubMed1978

Studies on the isolation and chemical-physical properties of porcine follicle-stimulating hormone.

Methods are described for isolating highly purified FSH from porcine pituitary glands. The biological activity of pure material was 22 NIH-FSH-P2 U/mg, as judged by the ovarian weight augmentation bioassay. Virtually no immunoreactive LH was indicated by RIA, and only one component was detected by both gel electrophoresis and immunoprecipitation. Amino acid and carbohydrate analyses of the highly purified FSH indicated the presence of 1-tryptophan, a high content of aspartic and glutamic acids, and 6% sialic acid. Molecular weights of untreated and guanidine-treated porcine FSH were estimated from hydrodynamic measurements of Stokes radii and sedimentation coefficients. A highly specific and sensitive RIA was also developed for this hormone.

Amino Acids↗
PubMed1978

Interactions of gonadotropins with corpus luteum membranes. I. Properties and distributions of some marker enzyme activities after subcellular fractionation of the superovulated rat ovary.

The properties of a number of enzyme activities of the superovulated rat ovary have been studied to establish optimal assay conditions and specific assay procedures for each activity. The activities were chosen on the basis of their extensive use in other tissues of the rat as marker enzymes for the major cell organelles. Homogenates of superovulated rat ovaries were subjected to fractionation by differential rate centrifugation, and sedimentation profiles were constructed for each marker enzyme activity. The various subcellular fractions were also monitored by electron microscopy. The enrichment of fractions with particular organelles by electron microscopy, and enrichment of the appropriate organelle marker enzyme activities correlated well. Sedimentation profiles of a number of plasma membrane marker enzymes demonstrated a marked discrepancy between hCG-binding activity, and 5'-nucleotidase-, alkaline phosphatase-, and Mg2+-dependent ATP-ase on the one hand, and basal, hCG-stimulated, and fluoride-stimulated adenylate cyclase activities on the other hand. Fractions enriched in hCG-binding and adenylate cyclase activities were subjected to further fractionation on discontinuous sucrose density gradients. The distributions of the various plasma membrane markers again indicated a partial dissociations between hCG-binding and adenylate cyclase activities of luteinized rat ovaries, suggesting the existence of two distinct major plasma membrane populations, with different buoyant densities, marker enzyme profiles and adenylate cyclase and hormone-binding levels.

Adenylyl Cyclases↗