Biomedical subjects
Publications and source records attributed to P Grasso.
Organic solvents have been widely used in industry for decades, and there has been constant concern about their toxic effects on the central and peripheral nervous systems. In this chapter the effects are reviewed and particular attention is paid to the reported clinical effects of long-term exposure.
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We previously reported purification of a protein (approximately equal to 57 kDa) from human follicular fluid having FSH binding inhibitory (FSH-BI) activity. Purified hFSH-BI was cleaved with cyanogen bromide and trypsin. The resulting peptide fragments were separated by HPLC and sequence information for individual fragments was obtained. A ten amino acid sequence of hFSH-BI derived from this procedure was identified, and a corresponding peptide amide (BI-10) was synthesized and utilized for further study. A protein database search revealed no significant identity between this decapeptide and other known proteins. We examined the ability of BI-10 to inhibit binding of 125I-hFSH to FSH-receptor enriched bovine testes membranes utilizing a radioligand receptor assay (RRA). BI-10 inhibited binding of 125I-hFSH to its receptor in a concentration-related manner, with an ED50 of 300 microM. BI-10 had no effect on 125I-hCG binding to receptor even at concentrations up to 1000 microM, suggesting that the effect of BI-10 was specific for the interaction between FSH and its receptor. To assess bioactivity of BI-10, we investigated its effect on FSH-stimulated conversion of androstenedione to estradiol by rat Sertoli cells in primary culture in vitro. Inhibition of FSH-stimulated estradiol synthesis (FSH antagonist activity) was significant at a BI-10 concentration of 1000 microM. BI-10 also significantly inhibited FSH-stimulated cAMP accumulation in primary cultures of Sertoli cells when examined at the same concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
A synthetic peptide strategy was used to study structure-function relationships between residues 32 to 46 of the glycoprotein hormone alpha subunit (GPH alpha) and the testicular follicle-stimulating hormone (FSH) and luteinizing hormone (LH/hCG) receptors. A peptide amide corresponding to this region [GPH-alpha-(32-46)] inhibited both 125I-hFSH and 125I-hCG binding to their respective calf testis membrane receptors. The concentration at which GPH-alpha-(32-46) peptide amide inhibited FSH binding by 50% (IC50) was 36 microM, and for hCG it was 54 microM. GPH-alpha-(32-46) peptide amide also inhibited FSH-stimulated estradiol biosynthesis in cultured rat Sertoli cells. In order to determine the involvement of individual residues within this region of the glycoprotein hormone alpha subunit in receptor binding inhibitory activity, truncated and alanine-substituted peptide analogs were synthesized and tested in both FSH and hCG radioligand receptor competition assays. Based on the relative potency of each peptide, we conclude that Phe-33, Arg-35, Arg-42, Ser-43 and Lys-44 may be important, and Cys-32 is required, for inhibition of FSH and hCG binding to their respective receptor. Our results demonstrate involvement of the glycoprotein hormone alpha-subunit in receptor binding, identify residues 32 to 46 as a receptor binding domain, and define the relative importance of specific residues within this region of the alpha subunit for hormone-receptor interaction.
Two cytosolic regions of the rat testicular FSH receptor (FSHR), residues 533-555 and 645-653, have been identified as G protein-coupling domains. We localized the activity in these domains to their C-terminal sequences, residues 551-555 (KIAKR, net charge +3) and 650-653 (RKSH, net charge +3), and examined the effects of charge on G protein activation by the C-terminal peptides, using synthetic analogs containing additions, through alanine (A) linkages, of arginine (R, +), histidine (H, +) or both. RA-KIAKR (net charge +4) mimicked the effect of FSHR-(551-555) on guanine nucleotide exchange in rat testis membranes, but reduced its ability to inhibit FSH-stimulated estradiol biosynthesis in cultured rat Sertoli cells. Further increasing net charge by the addition of H (HARA-KIAKR, net charge +5) increased guanosine 5'-triphosphate (GTP) binding, but eliminated FSHR-(551-555) effects on FSH-stimulated steroidogenesis. HA-RKSH (net charge +4) significantly inhibited guanine nucleotide exchange in rat testis membranes, but stimulated basal and potentiated FSH-induced estradiol biosynthesis in cultured rat Sertoli cells. Addition of two H residues (HAHA-RKSH, net charge +5) restored GTP binding and further potentiated basal and FSH-stimulated steroidogenesis. These results suggest that positive charges in G protein-coupling domains of the FSHR play a role in modulating G protein activation and postbinding effects of FSH, such as steroidogenesis.
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We have used single- or double-point D-amino acid substitutions to study the structure-function relationships involving residues 32 to 46 of the glycoprotein hormone common alpha-subunit (GPHa) and the testicular follicle-stimulating hormone (FSH) and luteinizing hormone (LH/hCG) receptors. D-Amino acid substitution analogs of GPHa(32-46) were synthesized and tested in both FSH and hCG radioligand receptor assays using bovine calf testis membranes as receptor source. Correct orientation of the amino acid side chains was generally of paramount importance for peptide interaction with receptor and bioactivity. Most substitutions with corresponding D-amino acids did not enhance the potency of native GPHa(32-46). A significant increment in peptide potency, however, was observed by inversion of configuration at positions Ser-34 and Thr-39 with D-amino acid isoforms. Based on the relative potency of each peptide analog. [D-Ser-34, D-Thr-39]GPHa(32-46) was approximately twofold more potent than native peptide GPHa(32-46) in both FSH and hCG radioligand receptor assays. [D-Ser-34, D-Thr-39]GPHa(32-46) also markedly inhibited FSH-stimulated estradiol biosynthesis in cultured rat Sertoli cells. The present study is unique in that it represents the first report of utilizing D-amino acid substitution to develop more potent peptide analogs related to the glycoprotein hormone common alpha-subunit region 32-46. Our results offer hope for the development of more potent and stabile peptide antagonists of possible usefulness in fertility regulation and control.