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C Nathan

Publications and source records attributed to C Nathan.

At least 91 records · Page 5Linked to original sources
PubMed1993

Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon-gamma and bacterial lipopolysaccharide.

We have proved that iNOS is regulated by induction of transcription. The induction of iNOS mRNA requires synthesis of an intermediary protein(s) and probably involves action of a protein kinase or kinases. We have cloned and sequenced a 1.7-kilobase pair fragment from the 5'-flanking region of the iNOS gene and proved that this region contains a promoter which confers inducibility of iNOS by LPS and IFN-gamma.

Amino Acid Oxidoreductases↗
PubMed1992

Contrasting mechanisms for suppression of macrophage cytokine release by transforming growth factor-beta and interleukin-10.

Transforming growth factor (TGF)-beta and interleukin (IL)-10 inhibited lipopolysaccharide (LPS)-induced macrophage production of the inflammatory cytokines tumor necrosis factor-alpha (TNF), IL-1 alpha, and IL-1 beta by contrasting post-transcriptional mechanisms. TGF-beta acted slowly and late, as it required 12-16 h to exert a suppressive effect, and inhibited TNF production even when added 6 h after LPS. TGF-beta affected neither the level of TNF mRNA, the release of preformed TNF nor the degradation of TNF. Thus, TGF-beta appeared to inhibit translation of TNF mRNA. IL-10 not only suppressed TNF release to a 25-fold greater extent than TGF-beta, but also inhibited release of IL-1. In contrast to TGF-beta, IL-10 acted on an early step in cytokine production, its effect being maximal 3 h after addition of LPS. Unlike TGF-beta, IL-10 markedly suppressed TNF, IL-1 alpha, and IL-1 beta mRNA levels. However, this was accomplished without suppressing transcription of the corresponding genes. Moreover, cycloheximide antagonized the IL-10-dependent reduction in cytokine mRNA levels. Thus, IL-10 may induce a ribonuclease active on cytokine transcripts or may induce a protein that enhances the susceptibility of TNF, IL-1 alpha, and IL-1 beta mRNAs to ribonucleolytic action. We conclude that IL-10 and TGF-beta induce different phenotypes of macrophage deactivation, and deactivate macrophages by different mechanisms: IL-10 promotes degradation of cytokine mRNA, while TGF-beta primarily suppresses translation.

Animals↗
PubMed1992

Calmodulin is a subunit of nitric oxide synthase from macrophages.

A central issue in nitric oxide (NO) research is to understand how NO can act in some settings as a servoregulator and in others as a cytotoxin. To answer this, we have sought a molecular basis for the differential regulation of the two known types of NO synthase (NOS). Constitutive NOS's in endothelium and neurons are activated by agonist-induced elevation of Ca2+ and resultant binding of calmodulin (CaM). In contrast, NOS in macrophages does not require added Ca2+ or CaM, but is regulated instead by transcription. We show here that macrophage NOS contains, as a tightly bound subunit, a molecule with the immunologic reactivity, high performance liquid chromatography retention time, tryptic map, partial amino acid sequence, and exact molecular mass of CaM. In contrast to most CaM-dependent enzymes, macrophage NOS binds CaM tightly without a requirement for elevated Ca2+. This may explain why NOS that is independent of Ca2+ and elevated CaM appears to be activated simply by being synthesized.

Amino Acid Oxidoreductases↗
PubMed1992

Interactions of bacterial lipopolysaccharide with microtubule proteins.

Bacterial LPS is a potent stimulator of immune cells, but its mechanisms are unknown. A possible role for microtubules in LPS actions has been indicated by previous findings that the microtubule-active agent, taxol, can mimic some effects of LPS in macrophages from normal strains of mice, but not from genetically LPS-hyporesponsive strains. In this report we demonstrate that isolated microtubules from mouse brain can bind LPS in vitro. LPS and tubulin coeluted through a gel filtration column, and LPS was cross-linked to microtubule proteins with an iodinatable, photoreactive agent, sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl-1,3'-dithiopropionate. beta-Tubulin and microtubule-associated protein-2 (MAP), a predominant MAP in the brain, bound LPS specifically. Cross-linking was inhibited by an excess of unlabeled LPS or partially by unlabeled lipid A, but not by 2 M NaCl. Under the same conditions, neither myosin nor soybean trypsin inhibitor was labeled by the photoaffinity LPS probe, nor did these proteins compete for binding of LPS to beta-tubulin. These findings support the hypothesis that the microtubule network could be an intracellular target for LPS, and suggest further that a beta-tubulin-associated MAP could have an important role in LPS actions.

Affinity Labels↗
PubMed1992

Cloning and characterization of inducible nitric oxide synthase from mouse macrophages.

Nitric oxide (NO) conveys a variety of messages between cells, including signals for vasorelaxation, neurotransmission, and cytotoxicity. In some endothelial cells and neurons, a constitutive NO synthase is activated transiently by agonists that elevate intracellular calcium concentrations and promote the binding of calmodulin. In contrast, in macrophages, NO synthase activity appears slowly after exposure of the cells to cytokines and bacterial products, is sustained, and functions independently of calcium and calmodulin. A monospecific antibody was used to clone complementary DNA that encoded two isoforms of NO synthase from immunologically activated mouse macrophages. Liquid chromatography-mass spectrometry was used to confirm most of the amino acid sequence. Macrophage NO synthase differs extensively from cerebellar NO synthase. The macrophage enzyme is immunologically induced at the transcriptional level and closely resembles the enzyme in cytokine-treated tumor cells and inflammatory neutrophils.

Amino Acid Oxidoreductases↗
PubMed1992

Acute and chronic effects of dexfenfluramine on glucose and insulin response to intravenous glucose in diabetic and non-diabetic obese subjects.

Fenfluramine improves glucose tolerance in obese subjects independently of its anorectic effect. Increased insulin action has been reported, but such an effect may be secondary to reduced hyperglycemia following augmented insulin secretion. For this reason, we investigated whether the dextro-enantiomer of fenfluramine (dexfenfluramine, dF) has a direct effect on insulin secretion using the glucose infusion test, a technique that can also be used to indirectly evaluate insulin action. Ten lean controls (BMI 21 +/- 0.4 kg/m2), 9 non-diabetic obese subjects (BMI 32.3 +/- 1.1) and 10 obese mild non-insulin dependent diabetics (BMI 36 +/- 2.6, fasting plasma glucose (FPG) 7.9 +/- 0.9 mmol/l) were studied with a random, double blind cross-over protocol. Each subject received 15 mg dF (or placebo) twice daily for 3 days prior to glucose infusion; 30 mg dF (or placebo) were given 90 min before the test. Obese control and diabetic subjects continued treatment with either dF or placebo for one month after which they were retested. There was no significant change in weight or fasting plasma glucose or insulin in any group. Biphasic insulin secretion was demonstrated in both non-diabetic groups, whereas a complete lack of first-phase and a delayed and reduced second phase insulin response was demonstrated in the diabetic subjects. There was no acute or chronic (obese subjects) effect of dF on insulin secretion in any group. In lean control subjects, plasma glucose curves during glucose infusion were unchanged by dF, whereas in non-diabetic obese subjects the glucose slope was improved after both acute and chronic dF, implying augmented glucose disposal. In diabetic patients, no significant acute or chronic effect of dF on glucose response was registered. When all obese subjects were re-grouped according to fasting plasma insulin levels (FPI) and not glucose tolerance, those with relative fasting hyperinsulinemia (> 100 pmol/l, mean +/- SE = 144 +/- 12 pmol/l) showed significant improvement of insulin action after dF, whereas those with low FPI (< 100 pmol/l, mean +/- SE = 65 +/- 7 pmol/l) did not. These findings, together with previously published results of improved insulin action in diabetics during hyperinsulinemic clamps, suggest that dF-mediated improvement in glucose clearance may require substantial plasma insulin concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1992

Nitric oxide as a secretory product of mammalian cells.

Evolution has resorted to nitric oxide (NO), a tiny, reactive radical gas, to mediate both servoregulatory and cytotoxic functions. This article reviews how different forms of nitric oxide synthase help confer specificity and diversity on the effects of this remarkable signaling molecule.

Amino Acid Oxidoreductases↗
PubMed1991

Macrophage deactivation by interleukin 10.

Recombinant mouse interleukin 10 (IL-10) was exceedingly potent at suppressing the ability of mouse peritoneal macrophages (m phi) to release tumor necrosis factor alpha (TNF-alpha). The IC50 of IL-10 for the suppression of TNF-alpha release induced by 0.5 microgram/ml lipopolysaccharide was 0.04 +/- 0.03 U/ml, with as little as 1 U/ml suppressing TNF-alpha production by a factor of 21.4 +/- 2.5. At 10 U/ml, IL-10 markedly suppressed m phi release of reactive oxygen intermediates (ROI) (IC50 3.7 +/- 1.8 U/ml), but only weakly inhibited m phi release of reactive nitrogen intermediates (RNI). Since TNF-alpha is a T cell growth and differentiation factor, whereas ROI and RNI are known to inhibit lymphocyte function, it is possible that m phi exposed to low concentrations of IL-10 suppress lymphocytes. m phi deactivated by higher concentrations of IL-10 might be permissive for the growth of microbial pathogens and tumor cells, as TNF-alpha, ROI, and RNI are major antimicrobial and tumoricidal products of m phi. IL-10's effects on m phi overlap with but are distinct from the effects of the two previously described cytokines that suppress the function of mouse m phi, transforming growth factor beta and macrophage deactivation factor. Based on results with neutralizing antibodies, all three m phi suppressor factors appear to act independently.

Animals↗
PubMed1991

Complementary DNA sequence of human neutrophil azurocidin, an antibiotic with extensive homology to serine proteases.

Human neutrophils contain in their azurophil granules four antibiotic proteins with extensive homology to serine proteases, collectively termed serprocidins. Azurocidin is the only member of the group that lacks proteolytic activity. Using a monospecific antibody, we isolated from human bone marrow a cDNA encoding the complete azurocidin protein in its mature form, along with an N-terminal 24 residue hydrophobic peptide. The N-terminal third of the mature protein sequence contains a cluster of positively charged amino acid residues, many of which are predicted to be surface exposed. The primary sequence is highly homologous to elastase, proteinase 3, cathepsin G, T-cell granzymes and other serine proteases. However, azurocidin has Gly for Ser and Ser for His substitutions in the catalytic triad. Southern blot analysis of human genomic DNA suggests the existence of a single azurocidin coding sequence.

Amino Acid Sequence↗
PubMed1991

The mode of action and clinical pharmacology of gliclazide: a review.

Gliclazide is a sulphonylurea drug with an intermediate half-life of around 11 hours. It is extensively metabolised, and renal clearance accounts for only 4% of total drug clearance. The molecule contains an azabicyclo-octyl group which confers special properties on the basic sulphonylurea moiety. Gliclazide stimulates insulin secretion through the beta cell sulphonylurea receptor, and possibly through a direct effect on intracellular calcium transport. It specifically improves the abnormal first phase insulin release in type 2 diabetes, and also has an effect on the second phase. This pattern of insulin release is thought to explain the lower incidence of hypoglycaemic episodes and weight gain compared with some other sulphonylureas. There is also a reduction in hepatic glucose production and improvement in glucose clearance, without changes in insulin receptors. This suggests a possible post-receptor effect on insulin action, perhaps by stimulation of hepatic fructose-2,6-bisphosphatase and muscle glycogen synthase. Gliclazide reduces platelet adhesion, aggregation and hyperactivity and increases fibrinolysis. These actions, thought to be independent of its hypoglycaemic activity, may make gliclazide useful in halting the progression of diabetic microangiopathy.

Adult↗
PubMed1991

Eradication of methicillin-resistant Staphylococcus aureus vaginitis with mupirocin.

We report the case of a 54-year-old, quadriplegic woman with a methicillin-resistant Staphylococcus aureus (MRSA) vaginal infection. After failing a five-day course of intravenous vancomycin therapy, the patient was treated for ten days with mupirocin ointment applied intravaginally twice daily, which resulted in eradication of the infection. This report details a novel approach in treating MRSA vaginal infections.

Administration, Intravaginal↗
PubMed1991

Mechanisms and modulation of macrophage activation.

In the two decades following the discovery that macrophages can be activated by cytokines, there have been four major advances in our understanding of this phenomenon: (i) the identification of two biochemically defined, cytokine-inducible antimicrobial pathways, the enzymatic generation of superoxide and the enzymatic generation of nitric oxide; (ii) the identification of individual cytokines of known amino acid sequence capable of inducing these antimicrobial pathways and enhancing macrophage antimicrobial function; (iii) the demonstration of the utility of macrophage activating factors in the prevention and treatment of infectious diseases in man; and (iv) the discovery of the phenomenon of macrophage deactivation, together with the identification of several macrophage deactivating cytokines. This review briefly surveys each of these points, with emphasis on the regulation of production of the reactive oxygen intermediates and reactive nitrogen intermediates by two distinct but comparably suppressive cytokines, macrophage deactivation factor (MDF) and transforming growth factor-beta (TGF-beta).

Animals↗
PubMed1990

Cloning of cDNA for proteinase 3: a serine protease, antibiotic, and autoantigen from human neutrophils.

Closely similar but nonidentical NH2-terminal amino acid sequences have been reported for a protein or proteins in human neutrophils whose bioactivities is/are diverse (as a serine protease, antibiotic, and Wegener's granulomatosis autoantigen) but that share(s) several features: localization in the azurophil granules, a molecular mass of approximately 29 kD, reactivity with diisopropylfluorophosphate, and the ability to degrade elastin. We previously purified one such entity, termed p29b. Using a monospecific antibody, we have cloned from human bone marrow a cDNA encoding the complete p29b protein in its mature form, along with pre- and pro-sequences. The predicted amino acid sequence agrees closely with the NH2-terminal sequence obtained previously from purified p29b, as well as with sequences newly obtained from CNBr fragments. The primary structure is highly homologous to elastase, cathepsin G, T cell granzymes, and other serine proteases, and shares both the catalytic triad and substrate binding pocket of elastase. Hybridization of the full-length cDNA with restriction enzyme digests of human genomic DNA revealed only one fragment. This suggests that the closely related species described previously are the same, and can be subsumed by the term used for the first-described activity, proteinase 3. Proteinase 3 is more abundant in neutrophils than elastase and has a similar proteolytic profile and specific activity. Thus, proteinase 3 may share the role previously attributed to neutrophil elastase in tissue damage, and has the potential to function as an antimicrobial agent.

Amino Acid Sequence↗
PubMed1990

Widespread intradermal accumulation of mononuclear leukocytes in lepromatous leprosy patients treated systemically with recombinant interferon gamma.

Intradermal administration of recombinant interferon gamma (rIFN-gamma) to lepromatous leprosy patients has converted the local histology toward a tuberculoid pattern. However, such changes have been confined to the site of injection. In contrast, in the present study, marked, intradermal accumulation of CD3+, CD4+, CD8+, and CD1a+ T cells and Leu-M5+ mononuclear phagocytes was induced at a distance from the sites of administration, in a dose-dependent manner, by 10 daily intramuscular injections of 10-30 micrograms rIFN-gamma/m2. Mononuclear cell infiltration began within 3 d of onset of rIFN-gamma therapy and persisted at least 8 wk. Intramuscular administration of rIFN-gamma to lepromatous patients receiving concurrent chemotherapy can safely induce widespread histologic features of an upgrading reaction.

Adult↗
PubMed1990

Shedding of tumor necrosis factor receptors by activated human neutrophils.

The capacity of human neutrophils (PMN) to bind tumor necrosis factor (TNF) was rapidly lost when the cells were incubated in suspension with agents that can stimulate their migratory and secretory responses. Both physiological (poly)peptides (FMLP, C5a, CSF-GM) and pharmacologic agonists (PMN, calcium ionophore A23187) induced the loss of TNF receptors (TNF-R) from the cell surface. Half-maximal loss in TNF-R ensued after only approximately 2 min with 10(-7) M FMLP at 37 degrees C, and required only 10(-9) M FMLP during a 30-min exposure. However, there were no such changes even with prolonged exposure of PMN to FMLP at 4 degrees or 16 degrees C. Scatchard analysis revealed loss of TNF-binding sites without change in their affinity (Kd approximately 0.4 nM) as measured at incompletely modulating concentrations of FMLP, C5a, PMA, or A23187. The binding of anti-TNF-R mAbs to PMN decreased in parallel, providing independent evidence for the loss of TNF-R from the cell surface. At the same time, soluble TNF-R appeared in the medium of stimulated PMN. This inference was based on the PMN- and FMLP-dependent generation of a nonsedimentable activity that could inhibit the binding of TNF to fresh human PMN or to mouse macrophages, and the ability of mAbs specific for human TNF-R to abolish inhibition by PMN-conditioned medium of binding of TNF to mouse macrophages. Soluble TNF-R activity was associated with a protein of Mr approximately 28,000 by ligand blot analysis of cell-free supernatants of FMLP-treated PMN. Thus, some portion of the FMLP-induced loss of TNF-R from human PMN is due to shedding of TNF-R. Shedding was unaffected by inhibitors of serine and thiol proteases and could not be induced with phosphatidylinositol-specific phospholipase C. Loss of TNF-R from PMN first stimulated by other agents may decrease their responsiveness to TNF. TNF-R shed by PMN may be one source of the TNF-binding proteins found in body fluids, and may blunt the actions of the cytokine on other cells.

Biological Factors↗
PubMed1990

Purification of macrophage deactivating factor.

Macrophage deactivation factor (MDF) in P815 tumor cell-conditioned medium was assayed by its suppression of the ability of activated mouse peritoneal macrophages to release hydrogen peroxide. MDF displayed properties of a soluble protein(s) associated with both low (8-25,000) and high (greater than 450,000) Mr fractions. MDF was purified 6,140-fold by a seven-step procedure: extraction with acid-ethanol; precipitation with ether; and fractionation on gel filtration, anion-exchange, diphenyl reversed-phase and C4 reversed-phase HPLC columns, the last column twice. The final preparation contained two species: (a) a approximately 13,000 Mr band on reducing or nonreducing SDS-PAGE and on autoradiograms after radioiodination with chloramine T, and (b) a 66,000 Mr species ranging from approximately 5% to approximately 50% of the protein detectable by silver strain. The 66,000 Mr species was identified as albumin from its NH2-terminal amino acid sequence. However, no amino acid sequence could be obtained for the approximately 13,000 Mr species, either in fluid phase or after electroelution of the corresponding SDS-PAGE band. Thus, approximately 13,000 Mr MDF associates tightly with albumin through a variety of separation techniques, and may have a blocked NH2 terminus. Purified MDF afforded 50% inhibition of activated macrophage H2O2 releasing capacity at a concentration of 1-10 nM. Separation of MDF from most higher Mr moieties was associated with disproportionately small increases in specific activity, suggesting MDF might be partially inactivated by purification. As purified, MDF was approximately 1,000-fold less potent at deactivating macrophages than TGF-beta. Antibodies that neutralized the macrophage-deactivating effect of TGF-beta did not inhibit deactivation by MDF.

Animals↗