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Biomedical subjects

R J Ryan

Publications and source records attributed to R J Ryan.

At least 55 records · Page 3Linked to original sources
PubMed1984

Specific binding to cultured cells of 125I-labeled type beta transforming growth factor from human platelets.

Purified type beta transforming growth factor from human platelets (TGF beta) radioiodinated with 125I-labeled Bolton and Hunter reagent was found to bind to a variety of cultured cells of both epithelial and mesenchymal origin, including normal human fibroblasts and keratinocytes. TGF beta binding sites have also been found on three mouse embryo-derived fibroblast-like cell lines with lower levels of TGF beta binding on the chemically transformed derivatives of these cell lines. A variety of human tumor cell lines was shown to have an inverse correlation between their level of TGF beta binding and their ability to form colonies in soft agar. The mouse embryo-derived AKR-2B (clone 84A) cells reached maximal binding of 125I-labeled TGF beta after 2 hr at 22 degrees C. Scatchard analysis of the equilibrium binding of TGF beta to AKR-2B (clone 84A) cells gives a Kd of 33 pM with approximately equal to 10,500 binding sites per cell. This Kd for TGF beta binding to AKR-2B (clone 84A) cells agreed well with the ED50 of 40 pM for stimulation of colony formation of these cells by TGF beta. The TGF beta binding sites on the AKR-2B cells were shown to be specific for TGF beta with no significant competition with epidermal growth factor, fibroblast growth factor, or insulin and only a small level of competition with high concentrations of platelet-derived growth factor. Partially purified preparations with TGF beta-like activity from mouse embryos and medium conditioned by mouse embryo-derived cells competed effectively for binding to the TGF beta receptor.

Animals↗
PubMed1983

Phosphorylation of human choriogonadotropin. Stoichiometry and sites of phosphate incorporation.

Human chorionic gonadotropin (hCG) and its subunits were studied as substrates for cAMP-dependent protein kinase. Phosphorylated residues were identified in peptide fragments by sequence analysis following appropriate hydrolysis and purification. Only the free beta-subunit was phosphorylated. Intact beta subunit incorporated 1 mol of phosphate/mol of peptide. This was localized to Thr 97, within the sequence -Arg-Arg-Ser-Thr-, which resembles one type of acceptor site for cAMP-dependent phosphorylation. Since beta Thr 97 did not phosphorylate in native hCG, this residue may be masked by the alpha subunit. Reduced and carboxymethylated hCG beta phosphorylated to 2.8 mol of phosphate/mol of peptide. Besides Thr 97, phosphate was also found at Ser 66 and Ser 96. Ser 66 occurs within a segment recognized to be favored for phoshorylation with the general sequence -Arg-X-X-Arg-X-X-Ser-. The appearance of this site in the linearized peptide suggests that Ser 66 is "buried" in the native conformation of the beta subunit. Two synthetic peptides representing residues 93-102 of hCG beta were also examined. The disulfide form of the peptide phosphorylated at Thr 97 while the linear peptide phosphorylated at Ser 96. Conformation as well as primary structure can thus influence the site of phosphate incorporation.

Amino Acid Sequence↗
PubMed1983

Properties of the phosphorylated beta subunit of human choriogonadotropin.

The beta subunit of human choriogonadotropin (hCG) was previously shown to be phosphorylated by beef skeletal muscle cAMP-dependent protein kinase (Keutmann, H. T., Ratanabanangkoon, K., Pierce, M. W., Kitzmann, K., and Ryan, R. J. (1983) J. Biol. Chem. 258, 14522-14527). The phosphorylation site was primarily at Thr 97, located within the "determinant loop" region proposed by Ward and Moore (Ward, D. N., and Moore, W. T. (1979) Animal Models for Research in Fertility and Contraception (Alexander, N. J., ed) pp. 151-164, Harper and Row, Baltimore, MD) to be important for hormonal activity and specificity. Biological and immunological studies were carried out to determine the effect of this modification on the activity of hCG. The phosphorylated hCG beta recombined with hCG alpha to form phosphorylated hCG (*hCG). The recombined *hCG retained full immunological activity in a radioimmunoassay using anti-hCG serum and 125I-hCG. The biological activities of the *hCG on appropriate rat gonadal cells, as studied by hCG radioreceptor assay, follitropin radioreceptor assay, and adenylate cyclase assay, were 0.29 +/- 0.04, 0.29 +/- 0.07, and 0.69 +/- 0.13 times as potent as hCG, respectively. The reduced receptor binding activity of *hCG was not due to dissociation of the hormone into its subunits. The far ultraviolet CD spectrum of the *hCG showed no gross conformational change induced by phosphorylation. We conclude the following: 1) Thr 97 is not involved in subunit-subunit interaction and is not part of the hCG antigenic site recognized by this particular antiserum; 2) Thr 97 does appear to participate in the hCG-receptor interactions; 3) modification of Thr 97 does not result in an enhancement of follitropin-like activity.

Adenylyl Cyclases↗
PubMed1983

Synergistic effects of chymotrypsin and human chorionic gonadotrophin on rat luteal adenylate cyclase.

The effects of chymotrypsin on rat luteal adenylate cyclase have been investigated. Maximal stimulation occurred at a concentration of 10 micrograms/ml. This dose of chymotrypsin stimulated basal, GTP-,5'-(beta, gamma-imido)triphosphate-, and NaF-stimulated adenylate cyclase activity 2- to 3-fold. hCG-stimulated activities were stimulated 5- to 6-fold by the proteinase and showed a synergistic effect. The proteinase did not alter the hCG response curve, and the effect could not be mimicked by trypsin. Proteolytic activity was required, since chymotrypsinogen and inactivated chymotrypsin were not effective. The presence of chymotrypsin could not restore the hormonal sensitivity of a proteinase inhibitor-inactivated system. The results suggest that chymotrypsin stimulated rat luteal adenylate cyclase activity by two or more mechanisms: 1) a direct action on the catalytic subunit or a closely associated protein, and 2) an action somewhere in the pathway of hormonal stimulation.

Adenylyl Cyclases↗
PubMed1983

Effects of dansyl-arginyl-(4'-ethyl)piperidine amide on gonadotropin binding to rat tissues.

The effects of dansyl-arginyl-(4'-ethyl)-piperidine amide (DAPA) on the binding of gonadotropins has been investigated. The compound inhibited the binding of hCG, human LH, and porcine LH to rat luteal membranes with an ED50 of 70 microM and the binding of human FSH to rat testicular membranes with an ED50 of 350 microM. Alteration of the substitution at the 4 carbon of the piperidine moiety altered the effect for hCG binding to luteal tissue, the ED50 being lowered with increased chain length (ED50 for H greater than methyl greater than ethyl greater than propyl = isobutyl) and shifted approximately half an order of magnitude for each carbon added. Analysis of equilibrium binding data for hCG to rat luteal membranes showed a decrease in the Ka with increasing DAPA concentrations (2.2 X 10(10), 1.0 X 10(10), and 0.4 X 10(10) M-1 for 0, 50, and 150 microM DAPA, respectively), with little or no effect on the number of sites. At a concentration of 50 microM, the compound did not affect the initial rate of association of the hormone and its receptor, but did change the dissociative behavior. After a short period of association, dissociation was followed in the absence and presence of DAPA. In all cases a biphasic dissociation was observed. The presence of DAPA slowed the rate constant for the fast phase (DAPA-treated, 4.7 X 10(-3) min-1; control, 7.6 X 10(-3) min-1) and reduced the fraction of total hCG undergoing the slow phase of dissociation (DAPA-treated, 60.6%; control, 77.4%). After a long period of association, the dissociation of hCG was monophasic, and the presence of DAPA increased the rate constant from 2.0 X 10(-4) to 4.4 X 10(-4) min-1. The results show that DAPA acted as an inhibitor of gonadotropin binding. The kinetic data suggest a sequential model of hormone binding in which the compound affected a step subsequent to the initial interaction.

Animals↗
PubMed1982

Circular dichroic and immunological properties of human choriogonadotropin- beta carboxyl terminal peptides.

Circular dichroic spectra have been obtained in aqueous solution and in trifluoro-ethanol for several synthetic (non-glycosylated) human choriogonadotropin carboxyl terminal peptides of the beta-subunit ranging in size from 10 residues to 40 residues. These was no evidence for formation of alpha-helicity or beta-structure, but the spectra in 90% (v/v) trifluoroethanol were consistent with the occurrence of beta-turns. The Chou-fasman predictive rules also suggest a high probability of beta-turns in these peptides which could result in the occurrence of repeating kinks. Disulfide-linked dimers were also investigated by circular dichroism, and there was evidence of stabilization of particular skewness of the disulfide dihedral angle depending upon the location of the disulfide bond. The single phenylalanyl residue at position 115 in the beta-subunit also contributed to the circular dichroic spectra above 250 nm. Antibodies raised to a peptide consisting of residues 111-145 have been shown to contain two immunological determinants, but the sum of antibodies raised to separate determinant sequences do not equal those raised to the full length peptide. These data could reflect the existence of a conformation-related determinant on the 111-145 peptide or stearic hindrance of immunoglobulin binding of two antibodies to the same peptide.

Chorionic Gonadotropin↗
PubMed1981

Decreased epidermal growth factor binding in cells growth arrested in G1 by nutrient deficiency.

Previous studies have shown that the nontransformed AKR-2B mouse embryo derived cell line may growth arrest by two separate mechanisms in the G1 phase of the cell cycle--growth factor deficiency arrest (G0) and low molecular weight nutrient deficiency arrest. An examination of epidermal growth factor (EGF) receptors under the different resting or growth conditions has shown that rapidly growing cells or cells arrested due to growth factor deficiency have the expected amount of 125I-EGF binding with approximately 10(5) receptors per cell being present in G0 arrested cells. In contrast, cells arrested due to nutrient deficiency show a reduction in 125I-EGF binding to 10-20% of that observed under the other conditions. This effect appears to be due to decreased receptor number and not to a change in the affinity of the receptor. Stimulation of DNA synthesis by nutrient replenishment causes a tenfold increase in EGF binding 20 hours later, with some increase in binding being detectable as early as six hours. The increase in binding is inhibited by cycloheximide and actinomycin D. This suggests that new mRNA synthesis as well as increased protein synthesis is required for the increase in EGF binding.

Amino Acids↗
PubMed1981

Isolation and characterization of the subunits of porcine follicle-stimulating hormone.

The alpha and beta subunits of porcine FSH were isolated and purified to homogeneity as judged by SDS disc gel electrophoresis. The isolated subunits had less than 1% of the biological activity of the native hormone but were capable of recombining to generate at least 23% of the activity of the native hormone. The molecular weights calculated from hydrodynamic properties were 12,600 for pFSH alpha and 17,200 for pFSH beta. Total carbohydrate (g/100 g) was 18.9 for alpha and 15.1 for beta. The sialic acid content of alpha (3.8 g/100 g) exceeded that of beta (0.2 g/100 g). The alpha subunit contained significantly more lysine, alanine phenylalanine and methionine and significantly less aspartic acid, threonine, valine, isoleucine, leucine and tryptophan than the beta subunit. Evidence was found for N-terminal heterogeneity and an internal cleavage in the isolated alpha subunit.

Amino Acids↗
PubMed1981

Porcine granulosa cells in suspension culture. II. Luteinization and hCG responsiveness.

Granulosa cells from small follicles were cultured as suspensions in spinner flasks for 10 days in the absence or presence of follicle-stimulating hormone (FSH). With or without FSH, the cultured cells ultrastructurally resembled luteinized cells to different degrees. FSH increased progesterone accumulation in the culture medium. Ovine prolactin potentiated the effect of FSH in terms of the quantity of progesterone produced and the duration of accumulation. FSH increased acute human chorionic gonadotropin (hCG)-responsive progesterone secretion in short-term incubations of cultured granulosa cells. Responsiveness of FSH-cultured cells was maximal at day 4; that of control cultured cells was maximal at day 6. Adenylate cyclase activity of homogenates of cells cultured for 4, 6, or 8 days was measured. FSH induced in cultured cells an hCG sensitivity of the adenylate cyclase enzyme. These results indicate that FSH induced hCG-responsive progesterone secretion and hCG-responsive adenylate cyclase activity that correlate with ultrastructural signs of luteinization and with the previously reported FSH induction of hCG receptors.

Adenylyl Cyclases↗
PubMed1981

Tungstate stimulates adenylate cyclase.

Tungstate stimulates adenylate cyclase (E.C. 4.6.1.1) in ovarian homogenates of the rat; maximal stimulation (approximately 3-fold) is achieved at a concentration of 1 mM. This stimulation of cAMP production cannot be explained by the inhibition of phosphodiesterase activity or of ATP hydrolysis. Activation of adenylate cyclase by tungstate is rapid and reversible. The effects of tungstate and hCG on cyclase activity are additive, but tungstate does not augment fluoride-stimulated activity. At higher concentrations (5 and 10 mM), tungstate inhibits basal as well as hCG- and fluoride-stimulated cyclase activity in an irreversible manner. After solubilization by Lubrol-PX, the cyclase enzyme is inhibited by tungstate at concentrations ranging from 0.1-10 mM. Tungstate also activates adenylate cyclase in the brain, heart, lungs, kidneys, and liver of the rat. This suggests that tungstate activation of adenylate cyclase is a general phenomenon and may be mediated by a similar mechanism in different tissues. Tungstate provides an additional tool by which the molecular basis of adenylate cyclase activation can be probed. (Endocrinology 108: 435, 1981)

Adenylyl Cyclases↗
PubMed1981

Porcine granulosa cells in suspension culture. I. follicle-stimulating hormone induction of human chorionic gonadotropin-binding sites on cells from small follicles.

Granulosa cells were aspirated from small follicles from the ovaries of 3- to 6-month-old pigs, washed, and cultured in sealed spinner flasks. Medium 199 was supplemented with 10% fetal bovine serum, insulin, dexamethasone, T4, and antibiotics. FSH, LH, or hCG was added daily where appropriate. Cultures were sampled every other day for 10 days. Cells were examined microscopically, and cellular [125I]hCG binding and DNA content were measured. The cells aggregated and underwent morphological luteinization within 4 days in the absence or presence of FSH, LH, or hCG. Aggregation in culture was independent of clumping at the time of harvest. There was no measurable cell proliferation, based on the lack of mitotic figures and the lack of any increase in DNA. The average yield of luteinized cells from small follicle granulosa cells was 7% at 6 days. Cell concentration remained constant between days 6-10. Between days 0-2, [125I]iodo-hCG binding declined in all cultures. It continued to decline through day 10 in control cultures. In cultures containing FSH, binding increased to a maximum on day 6. The effect was specific for FSH. Increased binding was due to increased numbers of binding sites per cell. Equilibrium saturation binding data indicated that [125I]iodo-hCG binding to freshly harvested and cultured granulosa cells was specific, saturable, and of high affinity. The numbers of binding sites were 938 +/- 49 and 7602 +/- 156 sites/cell for control and FSH on day 6, respectively, compared to 498 +/- 14 sites/cell on day 0. The effect of FSH on the increase in binding sites was specific and dose related. Autoradiographic studies indicated a skewed normal frequency distribution of [125I]iodo-hCG-binding sites on cultured cells

Animals↗
PubMed1981

Photodynamic effect of hematoporphyrin derivative as a function of optical spectrum and incident energy density.

Tumor cell killing effect of hematoporphyrin derivative (HPD) and light was studied in culture to determine the dependence of this effect on treatment variables. Particular attention has been given to the spectral characteristics of the light and the absorption properties of hematoporphyrin. A human tumor cell line was treated using HPD and three broad bands of light ranging from the short- to the long-wavelength end of the visible spectrum. Cell killing was assessed by trypan blue exclusion. A transformed mouse embryo cell line was treated in a similar manner, and its reproductive efficiency was determined following treatment. Results of both studies are consistent with the hypothesis that the photocytotoxic action of HPD plus light is directly proportional to the number of light quanta absorbed by the HPD in each cell. For thin layers of cells, such as in situ carcinoma, it appears that short-wavelength radiation falling in the porphyrin Soret band around 400 nm may have from 12 to 30 times the killing power as does red light.

Animals↗
PubMed1980

Effects of proteinase inhibitors on adenylate cyclase.

The effects of a number of proteinase inhibitors on rat ovarian and rat hepatic adenylate cyclase preparations were examined. N alpha-tosylarginine methyl ester, 7-amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one, 1-chloro-4-methyl-3-L-tosylamidopentan-2-one and other low-molecular-weight proteinase inhibitors blocked hormonally stimulated adenylate cyclase from either source with hepatic preparations requiring higher concentrations. Addition of nucleotides (ATP, GTP, GDP, CTP or ITP) to inhibited ovarian preparations did not reverse inhibition, nor did dithiothreitol reverse phenylmethanesulphonyl fluoride-inhibited ovarian adenylate cyclase. The kinetics of the inhibition of rat ovarian adenylate cyclase were examined by following the production of cyclic AMP after the addition of inhibitors to membrane preparations preincubated under assay conditions with human choriogonadotropin, guanosine 5'-[beta gamma-imido]triphosphate of NaF. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one, 1-chloro-4-phenyl-3-L-tosylamidobutan-2-one and 1-chloro-4-methyl-3-L-tosylamidopentan-2-one had two effects on human-choriogonadotropin-stimulated adenylate cyclase. At low concentrations (less than or equal to 0.2 mM) there was an irreversible inhibition of hormonally-stimulated cyclase with maximum first-order inhibitory rate constants of 0.05--0.08 min-1. At higher concentrations the irreversible effect persisted, but, in addition, there was a marked decrease in the cyclase initial velocity to 25--50% of that of control values. N alpha-tosylarginine methyl ester had similar effects; at low concentrations (less than or equal to 2 mM) it inhibited irreversibly, and at higher concentrations it decreased the initial velocity (50% at 10 mM). At high concentrations (greater than 3 mM) N alpha-tosylarginine methyl ester also inhibited NaF- and guanosine 5'-[beta gamma-imidol]-triphosphate-stimulated cyclase but in a reversible manner. 7-Amino-1-chloro-3-L-tosylamidoheptan-2-one inhibited NaF-stimulated adenylate cyclase in two ways, as for human-choriogonadotropin-stimulated adenylate cyclase, but required 10--20-fold higher concentrations. The low-concentration irreversible effect can be explained by a continual inactive in equilibrium active conversion of adenylate cyclase during hormonal stimulation in which the inactive to active conversion is blocked by the inhibitors. The high-concentration effect is a direct one on the active catalytic moiety of the enzyme.

Adenylyl Cyclase Inhibitors↗