Biomedical subjects
Publications and source records attributed to S Formisano.
An epidemiological study into the prevalence and incidence of anti-H.I.V. antibody seropositivity in closed communities produced preliminary data on 746 subjects, the majority of whom admitted potentially dangerous behaviour. These results demonstrated that residence in a closed community in which one can limit the use of drugs and sexual contacts reduces the risk of infection.
Follicular 19 S thyroglobulin (molecular weight 660,000) from rat, human, and bovine thyroid tissues contains approximately 10-12 mol of phosphate/mol of protein. These phosphate residues can be radiolabeled when rat thyroid hemilobes, FRTL-5 rat thyroid cells, or bovine thyroid slices are incubated in vitro with [32P]phosphate. Thus labeled, the [32P]phosphate residues comigrate with unlabeled 19 S follicular thyroglobulin on sucrose gradients and gel filtration columns; are specifically immunoprecipitated by an antibody preparation to rat or bovine thyroglobulin as appropriate; and co-migrate with authentic 19 S thyroglobulin when subjected to analytic or preparative gel electrophoresis. Tunicamycin prevents approximately 50% of the phosphate from being incorporated into FRTL-5 cell thyroglobulin. Approximately one-half of the phosphate in FRTL-5 cell or bovine thyroglobulin can also be released by enzymatic deglycosylation and can be located in Pronase-digested peptides which contain mannose, are endo-beta-N-acetylglucosaminidase H but not neuraminidase-sensitive, and release a dually labeled oligosaccharide containing mannose and phosphate after endo-beta-N-acetylglucosaminidase H digestion. The remainder of the phosphate is in alkali-sensitive phosphoserine residues (3-4/mol of protein) and phosphotyrosine residues (approximately 2/mol of protein). This is evidenced by electrophoresis of acid hydrolysates of 32P-labeled thyroglobulin and by reactivity with antibodies directed against phosphotyrosine residues. The phosphoserine and phosphotyrosine residues do not appear to be randomly located through the thyroglobulin molecule since approximately 75-85% of the phosphotyrosine and phosphoserine residues were recovered in a approximately 15-kDa tryptic peptide or a approximately 24-kDa cyanogen bromide peptide, each almost devoid of carbohydrate. 31P nuclear magnetic resonance studies of bovine thyroglobulin confirm the presence and heterogeneity of the phosphate residues on thyroglobulin preparations.
The mitogenic effect of thyrotropin on functional rat thyroid cells of the line FRTL-5 is correlated with membrane lipid fluidity as evaluated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Continued exposure of FRTL-5 cells to a medium lacking thyrotropin causes cessation of cell proliferation and a decrease in membrane lipid fluidity which reaches its minimum in approximately 8 days. The change in lipid fluidity is due to an absolute increase (greater than 2-fold) of membrane cholesterol, with an increased cholesterol/phospholipid ratio and an increased ratio of saturated to unsaturated fatty acids of the membrane phospholipids, contributed primarily by a nearly 4-fold increase in the ratio of saturated to unsaturated C16 fatty acids. It is also associated with a variation of the relative proportions of the major membrane phospholipids; thus, phosphatidylinositol and phosphatidylethanolamine decrease while phosphatidylcholine increases. Both membrane fluidity and lipid composition can be restored by thyrotropin to their original levels, i.e. levels measured under continuous exposure to the hormone. Complete reversal requires at least 48 h, i.e. approximately the same time required for resumption of growth when FRTL-5 cells, starved in thyrotropin, are re-exposed to the hormone. Changes in lipid composition and fluidity can be prevented or can be reversed if FRTL-5 cells are exposed to dibutyryl cAMP while being deprived of thyrotropin. Dibutyryl cAMP has only a modest direct effect on growth; however, this pretreatment eliminates the 48-h lag phase with respect to thyrotropin stimulation. It is proposed that the effects of thyrotropin on growth of FRTL-5 cells requires a modification of the molecular structure and the physical state of cell membranes, which can be mediated by cAMP, although cAMP is not sufficient by itself to promote growth.
Forskolin, a diterpene compound isolated from the roots of Coleus forskohlii, activates adenylate cyclase in membranes from a variety of mammalian tissues. We found that forskolin (10(-7) to 3 X 10(-5) M) caused a concentration-related inhibition of IgE-mediated release of histamine and peptide leukotriene C4 (LTC4) from human basophils and lung mast cells. There was a significant linear correlation between the per cent inhibition of histamine and LTC4 release from both cell types. However, in both systems forskolin exerted a significantly greater inhibitory effect on LTC4 release than on histamine release. The concentration-response inhibition curve was paralleled by a forskolin-induced rise in cAMP levels in human leukocyte and mast cell preparations. The relationship between the effect of forskolin and the cAMP concentration was supported by the finding that forskolin inhibited the "first stage" of antigen-induced histamine release, but not the release caused by the Ca2+ ionophore, A23187. Propranolol, a competitive beta-receptor antagonist, did not block the inhibition of mediator release or the cAMP accumulation caused by forskolin. These data suggest that forskolin modulates the release of mediators of immediate hypersensitivity reactions via the activation of adenylate cyclase in human basophils and mast cells.
A detailed comparative analysis of the Escherichia coli and Salmonella typhimurium hisIE and hisD gene products and the functionally equivalent, single, HIS4 gene product of Saccharomyces cerevisiae permitted several insights concerning the relationship between these genes. Our analysis supports the idea that HIS4 results from the fusion of his IE and hisD. The comparison permitted a more precise definition of the functional domains of hisI/HIS4A and hisE/HIS4B as well as the two functional domains of hisD/HIS4C. The homologies between the bacterial and yeast sequences suggest a region of the hisD/HIS4C protein that may constitute one of the active centres. A large fragment at the amino terminal region of the yeast protein is missing from the bacterial hisIE gene product and is probably not needed for catalytic activity. Another region of non-homology in the yeast protein is probably a peptide bridge connecting the HIS4AB domain to HIS4C. Although the overall homology at the level of amino acid sequence is modest (about 38%) there is a striking similarity when the hydropathic patterns and predicted secondary structural configurations of these proteins are compared.
We found that forskolin (10(-7) to 3 X 10(-5) M) caused dose-related inhibition of antigen-induced histamine release from human basophil leukocytes. The dose-response inhibition curve was paralleled by a forskolin-induced increase in cyclic AMP (cAMP) levels in human leukocyte preparations. The kinetics of inhibition of histamine release and of the increase in leukocyte cAMP were the same. In a second series of experiments we evaluated the effect of forskolin on antigen-induced histamine release from chopped human lung passively sensitized with serum from an allergic patient. Forskolin (10(-7) to 3 X 10(-5) M) dose-dependently inhibited the release of histamine from human lung mast cells. Thus forskolin appears to modulate the release of mediators of the immediate hypersensitivity reaction, presumably through activation of adenylate cyclase in human basophils and mast cells.
A secondary structure prediction has been made using the available primary sequence data of the proposed carboxy-terminal of rat thyroglobulin. The model predicts 22% alfa-helix, 28% beta-structure and 17% beta turns. Out of the 8 possible carbohydrate acceptor-sites (Asn-x-Ser/Thr), 3 (residues 136, 368, 782) are associated with peptide sequences which favour the formation of beta-turn or loop-structures and are located in high hydrophilic regions. The entire sequence is predicted to be made up of two domains: one of them is highly structured, contains the hormonogenic sites, a cluster of tyrosines and at least one carbohydrate acceptor site.
The present investigation demonstrates that the cross-linking agent, dimethylsuberimidate, is an usefull tool to study thyroglobulin structure. In fact, while reproducible and discrete polymerization products are obtained in strictly controlled conditions, valuable information on the native assemblage of thyroglobulin subunits and the effects of its major post-translational modification (iodination) on its structure, are reported.
The lipophilic probe 1,6-diphenyl-1,3,5-hexatriene was incorporated into erythrocyte ghosts of either normal or obese humans, and the polarization of fluorescence was measured between 0 and 40 C. The membrane lipid fluidity, evaluated by fluorescence polarization, was consistently higher in the ghosts from obese subjects. A strong correlation was found between increased 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization and excess body weight. Measurements of cholesterol and phospholipids indicated increased cholesterol and decreased phospholipids in erythrocyte ghosts from obese subjects. These data suggest that alterations in lipid composition in erythrocytes of obese subjects are responsible for abnormal physical properties of plasma membranes, which, in turn, may cause altered enzymatic activities.
In this paper we report on the peptide maps of the three polypeptides produced by reduction of denatured 19-S thyroglobulin from guinea pig. These maps were obtained by both chemical cleavage (CNBr) and by limited enzymatic proteolysis (Staphylococcus aureus SV 8 protease). Analysis of these peptides showed a strict correspondence among the electrophoretic bands produced by cleavage of the guinea pig thyroglobulin components. These results support the idea that thyroglobulin, even though it has an apparently complex molecular structure, contains polypeptide sequences which are repeated in the elementary chain. Since similar structures should correspond to similar functions, each of these regions may work as a functional domain for the biosynthesis of the thyroid hormones.
Thyrotropin causes a rapid and significant increase in the fluorescence polarization of DPH when this hydrophobic probe is incorporated into a strain of functioning rat thyroid cells (FRTL5). This increase is ligand-specific and is not related to cAMP production. The phenomenon seems to reflect the interaction of thyrotropin with the glycoprotein component of its membrane receptor, as suggested by experiments in which thyrotropin causes increases in DPH fluorescence polarization in liposomes embedded with this receptor component but not with gangliosides. A strain of nonfunctioning rat thyroid cells (FRT), exhibiting no reactivity with monoclonal antibodies to the glycoprotein component of the thyrotropin receptor, requires two orders of magnitude higher concentrations of thyrotropin to exhibit a comparable phenomenon.
Polyethylene glycol solutions (10% w/v) were used to detect the effect of mono- and divalent cations on some properties of thyroglobulin. It is shown that in presence of 10% w/v polyethylene glycol in 0.01 M Tris-HCl, pH 7.5, calcium (less than 0.05 M) modifies the solubility, the sedimentation rate, and the Stokes' radius of thyroglobulin, while monovalent cations up to 0.6 M do not effect any of these properties. These findings can be explained by an increase in molecular compactness of thyroglobulin. Furthermore, it was shown that a synthetic polymer, polyethylene glycol, could be used to detect conformational changes.
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A strongly fluorescent 5-dimethylamino-1-naphthalene sulfonate (dansyl) derivative of bovine thyrotropin has been prepared. The dye-conjugated hormone is bioactive and shares, essentially unchanged, the membrane binding and adenylate cyclase stimulatory activities of the native hormone. Binding of 125I-labeled dansyl-thyrotropin to thyroid plasma membranes is sensitive to inhibition by gangliosides and, as is the case for the binding of 125I-thyrotropin, galactosyl-N-acetylgalactosaminyl[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide (GDIb) is the most potent binding inhibitor. Gangliosides interact with dansyl-thyrotropin, causing a large increase of the quantum yield and a 5- to 10-nm blue shift of the emission maximum of the hormone-bound naphthalene chromophore; gangliosides cause no change in the fluorescent properties of the free dye. The fluorescence enhancement caused by gangliosides can be specifically reversed by unlabeled thyrotropin. The effect of gangliosides on dansyl-thyrotropin fluorescence is strongly salt-dependent; salts cannot, however, reverse the formation of the dansyl-thyrotropin.ganglioside complex once it has formed. The salt data suggest that the association of the ganglioside with dansyl-thyrotropin is dominated by electrostatic interactions, but that salt-independent, short range interactions, most likely hydrophobic, dominate the dissociation of the dansyl-thyrotropin-ganglioside adduct. Sucrose gradient centrifugation, ultracentrifugation, and fluorescence polarization data indicate that the gangliosides are micellar in nature under the conditions of these experiments. Acid titration of dansyl-thyrotropin causes a marked quenching of dansyl fluorescence which in part reflects dissociation of the hormone into its constituent alpha and beta subunits. In the presence of GDIb, but not N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GDIa), pH-dependent quenching and subunit dissociation are essentially eliminated. Circular dichroism results and fluorescence polarization studies support the interpretation that the ganglioside interaction causes a conformational change in the thyrotropin molecule. The acid titration data together with differences in the ability of gangliosides to influence the tyrosine fluorescence of the thyrotropin molecule indicate that different gangliosides induce different conformational perturbations in the thyrotropin molecule.