Search PubMed⌕ Search

Biomedical subjects

S Paul

Publications and source records attributed to S Paul.

At least 235 records · Page 13Linked to original sources
PubMed1991

Maturation and function of cystic fibrosis transmembrane conductance regulator variants bearing mutations in putative nucleotide-binding domains 1 and 2.

One feature of the mutations thus far found to be associated with the disease cystic fibrosis (CF) is that many of them are clustered within the first nucleotide-binding domain (NBD) of the CF transmembrane conductance regulator (CFTR). We sought to discover the molecular basis for this clustering by introducing into the two NBDs of CFTR mutations either mimicking amino acid changes associated with CF or altering residues within highly conserved motifs. Synthesis and maturation of the mutant CFTR were studied by transient expression in COS cells. The ability of the altered proteins to generate cyclic AMP-stimulated anion efflux was assessed by using 6-methoxy-N-(sulfopropyl) quinolinium (SPQ) fluorescence measurements in HeLa cells expressing mutated plasmids. The results show that (i) all CF-associated mutants, with one exception, lack functional activity as measured in the SPQ assay, (ii) mutations in NBD1 are more sensitive to the effects of the same amino acid change than are the corresponding mutations in NBD2, (iii) cells transfected with plasmids bearing CF-associated mutations commonly but not exclusively lack mature CFTR, (iv) NBD mutants lacking mature CFTR fail to activate Cl- channels, and (v) the glycosylation of CFTR, per se, is not required for CFTR function. We reason that the structure of NBD1 itself or of the surrounding domains renders it particularly sensitive to mutational changes. As a result, most NBD1 mutants, but only a few NBD2 mutants, fail to mature or lack functional activity. These findings are consistent with the observed uneven distribution of CFTR missense mutations between NBD1 and NBD2 of CF patients.

Amino Acid Sequence↗
PubMed1991

Caffeine and theophylline as adenosine receptor antagonists in humans.

Substantial in vitro and animal data suggest that methylxanthines, such as caffeine and theophylline, act as adenosine receptor antagonists. To test this hypothesis in humans, we first determined if theophylline would antagonize the effects of adenosine. Intravenous administration of adenosine, 80 micrograms/kg/min, increased heart rate 28 +/- 6 bpm, systolic blood pressure 19 +/- 5 mm Hg and minute ventilation 6.1 +/- 2.2 liters/min. All these changes were significantly attenuated during theophylline administration (17 +/- 3 bpm and 1 +/- 2 mm Hg and 1.6 +/- 0.6 liters/min, respectively, P less than .05), at a dose (10 mg/kg over 1 hr, followed by 1.8 micrograms/kg/min i.v.) that produced plasma theophylline levels of 17 +/- 2 micrograms/ml (94 microM). We then determined if chronic caffeine consumption resulted in upregulation of platelet adenosine receptors in eight normal volunteers. After 7 days of caffeine abstinence, the adenosine analog 5'-N-ethylcarboxamidoadenosine produced a dose-dependent inhibition of thrombin-induced aggregation (EC50 = 69 nM). Subjects then were given caffeine, 250 mg p.o. 3 times a day for 7 days. Actual caffeine withdrawal, that is, virtual disappearance of caffeine in plasma, was apparent 60 hr after the last dose of caffeine. Caffeine withdrawal produced a significant shift to the left of 5'-N-ethylcarboxamidoadenosine inhibition of aggregation (EC50 = 49 nM, P less than .01), implying sensitization and/or upregulation of adenosine receptors as seen after chronic exposure to an antagonist. These results suggest that methylxanthines act as adenosine receptor antagonists in humans.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1990

Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.

The gene associated with cystic fibrosis (CF) encodes a membrane-associated, N-linked glycoprotein called CFTR. Mutations were introduced into CFTR at residues known to be altered in CF chromosomes and in residues believed to play a role in its function. Examination of the various mutant proteins in COS-7 cells indicated that mature, fully glycosylated CFTR was absent from cells containing delta F508, delta 1507, K464M, F508R, and S5491 cDNA plasmids. Instead, an incompletely glycosylated version of the protein was detected. We propose that the mutant versions of CFTR are recognized as abnormal and remain incompletely processed in the endoplasmic reticulum where they are subsequently degraded. Since mutations with this phenotype represent at least 70% of known CF chromosomes, we argue that the molecular basis of most cystic fibrosis is the absence of mature CFTR at the correct cellular location.

Animals↗
PubMed1990

Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in cultured cystic fibrosis airway epithelial cells and Cl- channel activation assessed in single cells using a fluorescence microscopic assay and the patch-clamp technique. Expression of CFTR, but not of a mutant form of CFTR (delta F508), corrected the Cl- channel defect. Correction of the phenotypic defect demonstrates a causal relationship between mutations in the CFTR gene and defective Cl- transport which is the hallmark of the disease.

Cell Membrane Permeability↗
PubMed1990

Expression and characterization of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is a common lethal genetic disease that manifests itself in airway and other epithelial cells as defective chloride ion absorption and secretion, resulting at least in part from a defect in a cyclic AMP-regulated, outwardly-rectifying Cl- channel in the apical surface. The gene responsible for CF has been identified and predicted to encode a membrane protein termed the CF transmembrane conductance regulator (CFTR). Identification of a cryptic bacterial promoter within the CFTR coding sequence led us to construct a complementary DNA in a low-copy-number plasmid, thereby avoiding the deleterious effects of CFTR expression on Escherischia coli. We have used this cDNA to express CFTR in vitro and in vivo. Here we demonstrate that CFTR is a membrane-associated glycoprotein that can be phosporylated in vitro by cAMP-dependent protein kinase. Polyclonal and monoclonal antibodies directed against distinct domains of the protein immunoprecipitated recombinant CFTR as well as the endogenous CFTR in nonrecombinant T84 cells. Partial proteolysis fingerprinting showed that the recombinant and non-recombinant proteins are indistinguishable. These data, which establish several characteristics of the protein responsible for CF, will now enable CFTR function to be studied and will provide a basis for diagnosis and therapy.

Chloramphenicol O-Acetyltransferase↗
PubMed1990

Affinity chromatography of catalytic autoantibody to vasoactive intestinal peptide.

An autoantibody in human IgG that hydrolyzes vasoactive intestinal peptide (VIP) was identified. The IgG did not hydrolyze VIP unless an unidentified inhibitor was removed by dialysis. The VIP antibody was fractionated by affinity chromatography on immobilized VIP by using IgG without VIP-hydrolytic activity as the starting material. The affinity-purified antibody catalyzed the hydrolysis of VIP (nominal kcat/Km: 1.1 X 10(6) M-1 min-1). The values of Km for the affinity-purified antibody preparation and unfractionated IgG (110 nM and 112 nM) suggested relatively tight antibody-VIP binding. A comparison of the reverse phase HPLC profiles of antibody-treated [Tyr10-125I]VIP with that of synthetic [125I]VIP(1-16) suggested that unfractionated IgG and the affinity-purified antibody cleaved the same peptide bond in VIP (Gln16-Met17).

Autoantibodies↗
PubMed1990

Site specificity of a catalytic vasoactive intestinal peptide antibody. An inhibitory vasoactive intestinal peptide subsequence distant from the scissile peptide bond.

Ten fragments of vasoactive intestinal peptide (VIP) were tested for reactivity with a human catalytic autoantibody that cleaves full-length VIP(1-28) at the Gln16-Met17 peptide bond. A large COOH-terminal subsequence, VIP(15-28), was bound by the autoantibody with high affinity (Ki 1.25 nM), suggesting that it is the antibody binding epitope. VIP(22-28), a short subsequence distant from the scissile bond, inhibited the binding (Ki 242 microM) and hydrolysis (Ki 260 microM) of full-length VIP by the catalytic autoantibody in a competitive fashion. The autoantibody did not show detectable binding of short VIP subsequences that encompass the scissile bond (VIP(15-21), VIP(11-17), and VIP(13-20]. These data show that residues 22-28, located four amino acids distant from the scissile bond, contribute in recognition of VIP by the catalytic autoantibody.

Amino Acid Sequence↗
PubMed1990

Lipid A mutants of Vibrio cholerae: isolation and partial characterization.

Vibrio cholerae mutants resistant to common antibiotics and neutral and anionic detergents were isolated. Analysis of isolated outer membranes revealed a significant deficiency in the acylation of lipid A in the resistant strains. The content of amide-linked and ester-bound fatty acids in the lipid A of the mutant strains compared to that of the wild type was about 50-56% and 29-37% respectively. This defect was specific for lipid A as there was no change in the acylation of phospholipids. The reduction in fatty acid content of lipid A was reflected in the altered endotoxic properties in the mutant strains.

Drug Resistance, Microbial↗
PubMed1990

Protein phosphorylation in isolated human adipocytes-adrenergic control of the phosphorylation of hormone-sensitive lipase.

The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with 32PO4 in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the beta-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (Mr 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the alpha-2 agonist clonidine. Epinephrine, a combined alpha and beta agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the alpha-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes.

Adipose Tissue↗
PubMed1990

Adenosine inhibits the rise in intracellular calcium and platelet aggregation produced by thrombin: evidence that both effects are coupled to adenylate cyclase.

Platelet aggregation and secretion are associated with a rise in intracellular calcium concentration ([Ca2+]i). Adenosine has been postulated as an endogenous inhibitor of platelet aggregation. The antiaggregatory effects of adenosine are related to activation of adenylate cyclase. We studied the effect of adenosine on the rise in [Ca2+]i and platelet aggregation produced by thrombin. Human platelets were obtained from dextrose/citrate-treated plasma. [Ca2+]i was determined by fluorescence-dye techniques (fura-2). Adenosine inhibited the slope of the first phase of aggregation and the rise in [Ca2+]i produced by thrombin, in a dose-dependent manner. The dose that produced 50% inhibition of both aggregation and the rise in [Ca2+]i was approximately 500 nM. The effects of adenosine on [Ca2+]i were shared by its stable analogs, 5'-N-ethylcarboxamidoadenosine being approximately 10-fold more potent than (-)N6-phenylisopropyladenosine, suggesting that these effects were mediated through adenosine A2 receptors. Furthermore, caffeine antagonized the inhibitory effects of adenosine on platelet aggregation and [Ca2+]i. The effects of adenosine on [Ca2+]i appear to be mediated through a rise in intracellular cAMP, because they were prevented by the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (1 mM) and were potentiated by phosphodiesterase inhibition with papaverine (1 microM). Adenosine also inhibits the rise in [Ca2+]i produced by thrombin in a calcium-free medium, suggesting that adenosine inhibits both calcium influx and the release of calcium from intracellular stores.

Adenosine↗
PubMed1989

Catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody.

Vasoactive intestinal peptide (VIP) labeled with 125I, [Tyr10-125I]VIP, can be hydrolyzed by immunoglobulin G (IgG) purified from a human subject, as judged by trichloroacetic acid precipitation and reversed-phase high-performance liquid chromatography (HPLC). The hydrolytic activity was precipitated by antibody to human IgG, it was bound by immobilized protein G and showed a molecular mass close to 150 kilodaltons by gel filtration chromatography, properties similar to those of authentic IgG. The Fab fragment, prepared from IgG by papain treatment, retained the VIP hydrolytic activity of the IgG. Peptide fragments produced by treatment of VIP with the antibody fraction were purified by reversed-phase HPLC and identified by fast atom bombardment-mass spectrometry and peptide sequencing. The scissile bond in VIP deduced from these experiments was Gln16-Met17. The antibody concentration (73.4 fmol per milligram of IgG) and the Kd (0.4 nM) were computed from analysis of VIP binding under conditions that did not result in peptide hydrolysis. Analysis of the antibody-mediated VIP hydrolysis at varying concentrations of substrate suggested conformity with Michaelis-Menton kinetics (Km). The values for Km (37.9 X 10(-9) M) and the turnover number kcat (15.6 min-1) suggested relatively tight VIP binding and a moderate catalytic efficiency of the antibody.

Amino Acid Sequence↗
PubMed1989

Characterization of autoantibodies to vasoactive intestinal peptide in asthma.

Vasoactive intestinal peptide (VIP) is a potent relaxant of the airway smooth muscle. In this study, VIP-binding autoantibodies were observed in the plasma of 18% asthma patients and 16% healthy subjects. Immunoprecipitation studies and chromatography on DEAE-cellulose and immobilized protein G indicated that the plasma VIP-binding activity was largely due to IgG antibodies. Saturation analysis of VIP binding by the plasmas suggested the presence of one or two classes of autoantibodies, distinguished by their apparent equilibrium affinity constants (Ka). The autoantibodies from asthma patients exhibited a larger VIP-binding affinity compared to those from healthy subjects (Ka 7.8 x 10(9) M-1 and 0.13 x 10(9) M-1, respectively; P less than 0.005). The antibodies were specific for VIP, judged by their poor reaction with peptides bearing partial sequence homology with VIP (peptide histidine isoleucine, growth hormone releasing factor and secretin). IgG prepared from the plasma of an antibody-positive asthma patient inhibited the saturable binding of 125I-VIP by receptors in guinea pig lung membranes (by 39-59%; P less than 0.001). These observations are consistent with a role for the VIP autoantibodies in the airway hyperresponsiveness of asthma.

Adult↗