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Biomedical subjects

J Moss

Publications and source records attributed to J Moss.

At least 469 records · Page 26Linked to original sources
PubMed1984

NIH conference. Cyclic nucleotides: mediators of bacterial toxin action in disease.

In several bacterial diseases, the clinical, laboratory, and histologic findings result from the elaboration by the organism of a toxic product that binds to and may enter the host cell to alter its metabolism. In some cases, the intracellular mediators of toxin action are the cyclic nucleotides, cyclic adenosine 5'-monophosphate (cAMP) and cyclic guanosine 5'-monophosphate (cGMP), the ubiquitous second messengers through which numerous hormones, neurotransmitters, and drugs exert their effects. Certain toxins act by enhancing the activity of cellular enzymes that synthesize cAMP or cGMP; and others, by themselves catalyzing cAMP synthesis after entering the cell. Studies of the mechanism of action of these toxins have helped in deciphering the enzymatic components within animal cells that are responsible for cyclic nucleotide synthesis, degradation, and function as well as in understanding the pathogenesis of the diseases in which they are involved.

Adenosine Diphosphate Ribose↗
PubMed1983

Selective regulation by pertussis toxin of insulin-induced activation of particulate cAMP phosphodiesterase activity in 3T3-L1 adipocytes.

Incubation of 3T3-L1 adipocytes with insulin or isoproterenol for 10 min increased particulate "low Km" cAMP phosphodiesterase activity by 42% and 50%, respectively. Pertussis toxin catalyzed the [32P]-ADP ribosylation of a 41,000 dalton protein in adipocyte particulate fractions; prior incubation of adipocytes with toxin markedly reduced incorporation of radiolabel. Exposure of adipocytes to pertussis toxin (0.3 microgram, 18 hr) increased glycerol production and inhibited activation of cAMP phosphodiesterase by insulin, but not by isoproterenol. These results suggest that pertussis toxin can interfere with receptor-mediated processes that stimulate cAMP hydrolysis as well as those that inhibit cAMP formation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Activation by thiol of the latent NAD glycohydrolase and ADP-ribosyltransferase activities of Bordetella pertussis toxin (islet-activating protein).

Pertussis toxin (islet-activating protein) activates adenylate cyclase in susceptible cells by ADP-ribosylating an inhibitory component of the cyclase system. This toxin, assayed in a cell-free system in the presence of high concentrations of thiol, catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. This NAD glycohydrolase activity co-chromatographed on Sephacryl G-200 in 6.5 M urea, pH 3.2, 0.1 M glycine with the ADP-ribosyltransferase activity of the toxin, as monitored by the transfer of [32P]ADP-ribose from [32P]NAD to a 41,000-Da protein in NG108-15 neuroblastoma X glioma hybrid cells. In the absence of thiol, the native holotoxin was enzymatically inactive. Following addition of 250 mM dithiothreitol to the assay, maximal enzymatic activity was evident after a delay of approximately 1 h; with 20 mM thiol, the delay was longer. The Km for NAD with the fully activated enzyme was 25 microM; the Km did not appear to vary with the extent of activation. Thiol was necessary in a cell-free system to demonstrate NAD glycohydrolase activity. When extensively washed membranes were used as a source of 41,000-Da substrate, thiol was necessary to observe ADP-ribosylation in some cases (human erythrocytes) and significantly stimulated activity in others (NG108-15 cells). In contrast to the bacterial toxins choleragen and Escherichia coli heat-labile enterotoxin that ADP-ribosylate stimulatory components of the cyclase system, pertussis toxin did not transfer ADP-ribose to low molecular weight guanidino compounds, such as arginine or agmatine.

Adenosine Diphosphate Ribose↗
PubMed1983

Binding of choleragen and anti-ganglioside antibodies to gangliosides incorporated into preformed liposomes.

Exogenously added gangliosides were taken up and incorporated into liposomes just as they are incorporated into cells. Ganglioside GM1 was rapidly taken up by liposomes containing dimyristoyl- or dipalmitoylphosphatidylcholine, cholesterol and dicetyl phosphate. When incubated with a wide range of GM1 concentrations for 18 h, the liposomes incorporated about 10% of the added ganglioside. The rate of GM1 uptake by preformed liposomes was both time- and temperature-dependent. The liposomes also incorporated other gangliosides to a similar extent. The GM1 taken up by preformed liposomes was predominantly located on the outer surface of the liposomes and did not appear to be internalized into the inner half of the lipid bilayer. Liposomes containing GM1 added after liposome formation bound as many anti-GM1 antibodies and as much choleragen as liposomes having GM1 added during the formation of the lipid bilayers. Thus, preformed liposomes sensitized by incubation with GM1 are a good model system for studying the interactions of antibodies and toxins with membrane-associated gangliosides.

Antibodies↗
PubMed1983

Amino acid-specific ADP-ribosylation.

[adenine-U-14C]ADP-ribose-agmatine and [adenine-U-14C ))ADP-ribose-histone were synthesized by an NAD:arginine ADP-ribosyltransferase from [14C]NAD and agmatine and histone, respectively. The pseudo-first order rate constants for breakdown of the two components either in 0.4 N NaOH or in 0.4 M neutral hydroxylamine were identical. Hydroxylamine treatment of [14C]ADP-ribose-agmatine or [32P]ADP-ribose-histone yielded a single radioactive product which was separated by high pressure liquid chromatography and identified as ADP-ribose-hydroxamate by the formation of a ferric chloride complex. Hydrolysis of ADP-ribose-hydroxamate with snake venom phosphodiesterase resulted in the formation of 5'-AMP, consistent with the presence of a pyrophosphate bond. Incubation of ADP-ribose-[14C]agmatine, synthesized by the ADP-ribosyltransferase from NAD and [14C]agmatine, with 0.4 M neutral hydroxylamine resulted in the release of [14C]agmatine rather than phosphoribosyl[14C]agmatine. In addition, neither NAD nor ADP-ribose reacts with hydroxylamine; i.e. there was no evidence of nucleophilic attack by hydroxylamine at the pyrophosphate bond. The ADP-ribosyl-protein linkage formed by the NAD:arginine ADP-ribosyltransferase is considerably more stable to hydroxylamine than is the ADP-ribose-glutamate bond. The presence of ADP-ribose-arginine and ADP-ribose-glutamate synthesized by the ADP-ribosyltransferase and poly(ADP-ribose) synthetase, respectively, may be the chemical basis for the "hydroxylamine-stable" and "hydroxylamine-labile" bonds described by Hilz (Hilz, H. (1981) Hoppe-Seyler's Z. Physiol. Chem. 362, 1415-1425).

ADP Ribose Transferases↗
PubMed1983

Amino acid-specific ADP-ribosylation. Evidence for two distinct NAD:arginine ADP-ribosyltransferases in turkey erythrocytes.

An NAD:arginine mono-ADP-ribosyltransferase has been purified 270,000-fold from turkey erythrocytes through a five-step chromatographic procedure to apparent electrophoretic homogeneity. Molecular weight determinations of the enzyme by sodium dodecyl sulfate-gel electrophoresis, Ultrogel AcA 54, and TSK 3000 SW gel filtration were consistent with Mr = 32,000. The purified enzyme utilized arginine, other low molecular weight guanidino compounds, and various proteins as ADP-ribose acceptors. The Km values for NAD and arginine methyl ester were 36 and 3,000 microM, respectively. Unlike another transferase purified from turkey erythrocytes (Moss, J., and Stanley, S.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4809-4812), this enzyme was not activated by chaotropic salts or micromolar concentrations of histone. Thus, two distinct soluble ADP-ribosyltransferases may be present in turkey erythrocytes.

ADP Ribose Transferases↗
PubMed1983

Identification of thiol:protein disulfide oxidoreductase activity in cultured human fibroblasts: dependence of enzyme activity on growth conditions.

Thiol:protein disulfide oxidoreductase activity was assayed in extracts of cultured normal human skin fibroblasts. Enzyme activity in confluent fibroblasts was dependent on growth conditions. In serum-deprived fibroblasts grown in minimal medium enzyme activity was approximately 40% of that observed in fibroblasts maintained in medium supplemented with 10% fetal calf serum. In fibroblasts cultured in medium supplemented only with insulin, activity was 35% greater than that in fibroblasts cultured in unsupplemented defined medium. Antibodies raised against purified bovine liver thiol:protein disulfide oxidoreductase immunoprecipitated all of the activity present in fibroblast extracts. The thiol:protein disulfide oxidoreductase from human fibroblasts thus appears to share antigenic determinants with the bovine liver enzyme. The human fibroblast may serve as an in vitro model to study the regulation of the oxidoreductase.

Blood↗
PubMed1983

Pertussis toxin inhibits enkephalin stimulation of GTPase of NG108-15 cells.

In neuroblastoma-glioma (NG108-15) hybrid cells, opiates inhibit adenylate cyclase and stimulate a low Km GTPase. It has been postulated that the stimulation of GTPase plays a role in opiate inhibition of adenylate cyclase (Koski, G., and Klee, W. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4185-4189). Treatment of NG108-15 cells with pertussis toxin attenuates receptor-mediated inhibition of adenylate cyclase. The toxin acts by catalyzing the ADP-ribosylation of a 41,000-dalton substrate believed to be a part of the receptor-adenylate cyclase complex. We have found that toxin treatment of NG108-15 results in inhibition of the opiate-stimulated GTPase. The concentration of toxin required for inhibition of this GTPase was similar to that needed for both attenuation of opiate inhibition of adenylate cyclase and ADP ribosylation of the 41,000-dalton substrate. Inhibition of the opiate-induced GTPase by pertussis toxin in isolated membranes required NAD, consistent with the hypothesis that this effect of the toxin resulted from ADP ribosylation of a protein component of the system. Since the opiate-stimulated GTPase is believed to play a role in the receptor-mediated decrease in adenylate cyclase activity, inhibition of this GTPase may be an important part of the mechanism by which the toxin interferes with opiate action on adenylate cyclase.

Adenosine Diphosphate Ribose↗
PubMed1983

Release of guanyl nucleotides from the regulatory subunit of adenylate cyclase.

Choleragen and beta-adrenergic agonists, both of which activate turkey erythrocyte adenylate cyclase, have been reported to accelerate release of bound [3H]guanyl nucleotides from turkey erythrocyte membranes. We have now obtained evidence that choleragen- or isoproterenol-stimulated release reflects a change in the affinity of the regulatory subunit (G/F) of adenylate cyclase for guanyl nucleotides. Solubilized preparations of turkey erythrocytes that had bound radiolabeled GTP were chromatographed on Ultrogel AcA 34. The protein from which guanyl nucleotide was released upon incubation with choleragen or isoproterenol was co-eluted with G/F activity. Furthermore, this protein appears to be the same size as the complex containing the 42,000-dalton peptide, ADP*-ribosylated by choleragen, which is presumably a subunit of G/F. ADP ribosylation of the 42,000-dalton subunit of G/F by choleragen occurred with a half-time of about 5 min, whereas choleragen-stimulated release of guanyl nucleotides was much slower (t1/2 greater than or equal to 60 min). When membranes were treated with choleragen and NAD, the delay in activation of adenylate cyclase by guanylyl imidodiphosphate was decreased but not abolished, a finding consistent with the idea that release of endogenously bound nucleotide (and subsequent binding of the nonhydrolyzable GTP analog) occurs only slowly following ADP ribosylation. In contrast, activation of the adenylate cyclase of either toxin-treated or untreated membranes in the presence of isoproterenol and guanylyl imidodiphosphate was very rapid. These data support the hypothesis that isoproterenol and choleragen may activate adenylate cyclase, at least in part, by increasing the rate of release of guanyl nucleotides from G/F.

Adenylyl Cyclases↗
PubMed1983

Degradation of choleragen bound to cultured human fibroblasts and mouse neuroblastoma cells.

125I-choleragen bound to human fibroblasts was degraded slowly with a t1/2 of 2-3 days; the radiolabel in bound 125I-choleragen was present in both the A and B subunits. During degradation, radiolabel was lost more rapidly from the 125I-A1 (t1/2 approximately 2 days) than from the 125I-B peptides (t1/2 greater than 5 days). 125I-Choleragen bound to neuroblastoma cells showed a considerably shorter t1/2 for both the 125I-A1 and 125I-B peptides; as with the fibroblasts, radiolabel was lost more rapidly from the 125I-A1 than from the 125I-B peptides. The continued presence of choleragen in the fibroblasts and neuroblastoma cells was associated with a prolonged activation of adenylate cyclase. In addition, fibroblasts, previously exposed to toxin and then washed free of unbound choleragen, only slowly recovered their ability to bind 125I-choleragen with a t1/2 of 7 days. Fibroblasts exposed to choleragen also showed evidence of persistent toxin on the surface based on the ability of the cells to bind antitoxin, antisubunit A or antisubunit B antibodies followed by 3H-protein A. It appears that choleragen remains persistently bound to fibroblasts, is degraded at a slow rate, and may prevent the binding of new toxin molecules to the fibroblast. The relatively slow degradation of toxin by fibroblasts may explain the prolonged activation of adenylate cyclase by toxin. The loss of 125I-toxin binding capacity following incubation with toxin may result from continued presence of toxin subunits on the cell surface.

Adenylyl Cyclases↗
PubMed1983

3T3-L1 preadipocyte differentiation and poly(ADP-ribose) synthetase.

Differentiation of 3T3-L1 preadipocytes, induced by methyl-isobutylxanthine (MIX), dexamethasone (DEX), and insulin, results in cells with the morphological and biochemical characteristics of adipocytes. Following incubation of 3T3-L1 cells with MIX, DEX, and insulin, poly(ADP-ribose) synthetase activity decreased abruptly, remained low for several hours and then increased; this rise was delayed by readdition of MIX, DEX, and insulin. The transient reduction in poly(ADP-ribose) synthetase activity in 3T3-L1 cells occurred prior to the appearance of the adipocyte phenotype induced by the above agents. It was not observed when preparations were assayed in the presence of DNase I, indicating that poly(ADP-ribose) synthetase activity was masked following treatment with MIX, DEX, and insulin. The change in synthetase activity represents the earliest alteration of a specific enzyme yet detected during the differentiation of 3T3-L1 cells. It appears to be differentiation specific since nondifferentiating 3T3-C2 control cells did not exhibit changes in poly(ADP-ribose) synthetase activity when treated with MIX, DEX, and insulin. The transient reduction in activity may be an early event in differentiation which reflects changes in chromatin structure.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Exogenous gangliosides enhance the interaction of fibronectin with ganglioside-deficient cells.

The major cell-surface glycoprotein fibronectin mediates a variety of cellular adhesive interactions that have been reported to be competitively inhibited by gangliosides. These effects suggest a possible function of gangliosides as receptors for fibronectin. To test this hypothesis more directly, we examined the interaction of endogenous fibronectin with a ganglioside-deficient cell line, NCTC 2071. These cells, which grow in serum-free medium, synthesized fibronectin. The fibronectin did not bind to these cells, but instead bound diffusely to the culture substratum. When the cells were cultured in medium containing ganglioside, the fibronectin became bound to the cell surface in fibrillar strands. The order of effectiveness of purified gangliosides was GT1b greater than GD1a greater than GM1 greater than GM2 greater than GM3. The effect with mixed gangliosides was accompanied by a restoration of cellular capacity to bind and to respond to cholera toxin. Treatment of the cells with several phospholipids did not alter fibronectin binding. Our results support the hypothesis that gangliosides can help mediate the binding of fibronectin to fibroblasts.

Animals↗