Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Injection of D-galactosamine sensitizes mice many thousand-fold to the lethal action of endotoxin (lipopolysaccharide [LPS]). Comparable sensitization was practically absent in LPS-resistant C3H/HeJ mice, which after D-galactosamine treatment were about 500,000 times less sensitive to LPS lethality than histocompatible LPS-sensitive C3H/HeN mice. D-Galactosamine induces changes in the hepatocytes of treated animals, such as depletion of UTP and alterations in the pattern of UDP sugars. These early biochemical changes, which are necessary for development of sensitization, were similar in both mouse strains which we examined. High sensitivity to the lethal effects of LPS was achieved in C3H/HeJ mice after D-galactosamine treatment by transfer of C3H/HeN macrophages obtained in culture from bone marrow precursor cells.
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PMID:Requirement for lipopolysaccharide-responsive macrophages in galactosamine-induced sensitization to endotoxin. 394 85

Inducible nitric oxide synthase (iNOS) activity in the murine macrophage cell line RAW 264.7 was increased from two- to four-fold after co-exposure of the cells to low doses of bacterial lipopolysaccharide (LPS) and micromolar ATP, compared to LPS alone. Extracellular ATP and its analogs "per se", i.e. without LPS, were not able to induce iNOS activity. The stimulating effect of UTP too, the concentration range of activity (1-100 mM nucleotides) and the rank of potency (ATP-gamma-S = AMP-PNP > ATP = ADP >> AMP-CPP = UTP) seem to indicate an involvement of P2y-type purinergic receptors. GTP, CTP and adenosine were virtually ineffective. These data suggest that binding of extracellular nucleotides to purinergic receptors may increase nitric oxide production by macrophages. This effect might occur in pathological conditions (i.e. inflammation/infection or trauma) where significant amounts of intracellular ATP can be released due to cellular damage.
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PMID:Extracellular ATP potentiates nitric oxide synthase expression induced by lipopolysaccharide in RAW 264.7 murine macrophages. 752 Nov 63

1. Purinoceptor agonist-induced currents in untreated (proliferating) and lipopolysaccharide (LPS; 100 ng ml-1)-treated (non-proliferating) rat microglial cells in culture were recorded by the whole-cell patch-clamp technique. These cells have two preferred resting membrane potentials, one at -35 mV and another one at -70 mV. 2. Most experiments were carried out in non-proliferating cells. ATP, ATP-gamma-S and alpha,beta-MeATP (1-1000 microM in all cases) evoked an inward current at a holding potential of -70 mV, followed, in some experiments, by an outward current. At -70 mV 2-methylthio ATP (1-1000 microM) evoked an inward current, whereas at -35 mV it produced an outward current only. 3. When K+ was replaced in the pipette solution by an equimolar concentration of Cs+ (150 mM), the main outward component of the ATP-gamma-S (10 microM) induced response disappeared. Instead, an inward current was obtained. Replacement of K+ by Cs+ did not affect the inward current evoked by 2-methylthio ATP (300 microM). 4-Aminopyridine (1-10 mM), however, almost abolished this current and unmasked a smaller outward current. 4. The rank order of agonist potency was 2-methylthio ATP > ATP > alpha,beta-MeATP. Adenosine and UTP were inactive. Suramin (300 microM) and reactive blue 2 (50 microM) antagonized the effect of 2-methylthio ATP (300 microM). 5. I-V relations were determined by delivering fast voltage ramps before and during the application of 2-methylthio ATP (300 microM). In the presence of extra- (1 mM) and intracellular (150 mM) Cs+, the 2-methylthio ATP-evoked current crossed the zero current level near 0 mV. When both Cs+ (1 mm) and 4-aminopyridine (1 mM) were present in the bath medium, the intersection of the 2-methylthio ATP current with the zero current level was near - 75 mV.6. 2-Methylthio ATP (1-1I000 MicroM) induced the same inward current both in proliferating and nonproliferating microglia. However, the depolarizing response to 2-methylthio ATP (300 MicroM) was larger and longer-lasting in the proliferating cells. When the free Ca2+ concentration in the pipettes was increased from the standard 0.01 to 1 MicroM, the amplitude and duration of this depolarization was increased in non-proliferating cells. 4-Aminopyridine (1 mM) enhanced the duration, but not the amplitude of responses.7. ATP and its structural analogues stimulate microglial purinoceptors of the P2Y-type. This leads to the opening of non-selective cationic channels and potassium channels. Depending on the resting membrane potential, depolarization or hyperpolarization prevails. Although the inward current produced by 2-methylthio ATP is of similar amplitude in proliferating and non-proliferating microglia, the resulting depolarization is smaller in the latter cell type because of the presence of voltage-sensitive, outwardly rectifying potassium channels.
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PMID:Characterization and possible function of adenosine 5'-triphosphate receptors in activated rat microglia. 801 72

The physiological properties of the EcoURF-1 open reading frame, which precedes the glmS gene at 84 min on the Escherichia coli chromosome (J. E. Walker, N. J. Gay, M. Saraste, and A. N. Eberle, Biochem. J. 224:799-815, 1984), were investigated. A thermosensitive conditional mutant in which the synthesis of the gene product was impaired at 43 degrees C was constructed. The inactivation of the gene in exponentially growing cells rapidly inhibited peptidoglycan synthesis. As a result, various alterations of cell shape were observed, and cell lysis finally occurred when the peptidoglycan content was 37% lower than that of normally growing cells. Analysis of the pools of peptidoglycan precursors revealed a large accumulation of N-acetylglucosamine-1-phosphate and the concomitant depletion of the pools of the seven peptidoglycan nucleotide precursors located downstream in the pathway, a result indicating that the mutational block was in the step leading from N-acetylglucosamine-1-phosphate and UTP to the formation of UDP-N-acetylglucosamine. In vitro assays showed that the overexpression of this gene in E. coli cells, directed by appropriate plasmids, led to a high overproduction (from 25- to 410-fold) of N-acetylglucosamine-1-phosphate uridyltransferase activity. This allowed us to purify this enzyme to homogeneity in only two chromatographic steps. The gene for this enzyme, which is essential for peptidoglycan and lipopolysaccharide biosyntheses, was designated glmU.
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PMID:Identification of the glmU gene encoding N-acetylglucosamine-1-phosphate uridyltransferase in Escherichia coli. 840 87

The GlmU protein is a bifunctional enzyme with both acetyltransferase and uridylyltransferase (pyrophosphorylase) activities which catalyzes the transformation of glucosamine-1-P, UTP, and acetyl-CoA to UDP-N-acetylglucosamine [Mengin-Lecreulx, D., & van Heijenoort, J. (1994) J. Bacteriol. 176, 5788-5795], a fundamental precursor in bacterial peptidoglycan biosynthesis and the source of activated N-acetylglucosamine in lipopolysaccharide biosynthesis in Gram-negative bacteria. In the work described here, the GlmU protein and truncation variants of GlmU (N- and C-terminal) were purified and kinetically characterized for substrate specificity and reaction order. It was determined that the GlmU protein first catalyzed acetyltransfer followed by uridylyltransfer. The N-terminal portion of the enzyme was capable of only uridylyltransfer, and the C-terminus catalyzed only acetyltransfer. GlmU demonstrated a 12-fold kinetic preference (kcat/Km, 3.1 x 10(5) versus 2.5 x 10(4) L.mol-1.s-1) for acetyltransfer from acetyl-CoA to glucosamine-1-P as compared to UDP-glucosamine. No detectable uridylyltransfer from UTP to glucosamine-1-P was observed in the presence of GlmU; however, the enzyme was competent in catalyzing the formation of UDP-N-acetylglucosamine from UTP and N-acetylglucosamine-1-P (kcat/Km 1.2 x 10(6) L.mol-1.s-1). A two active site model for the GlmU protein was indicated both by domain dissection experiments and by assay of the bifunctional reaction. Kinetic studies demonstrated that a pre-steady-state lag in the production of UDP-N-acetylglucosamine from acetyl-CoA, UTP, and glucosamine-1-P was due to the release and accumulation of steady-state levels of the intermediate N-acetylglucosamine-1-P.
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PMID:Acetyltransfer precedes uridylyltransfer in the formation of UDP-N-acetylglucosamine in separable active sites of the bifunctional GlmU protein of Escherichia coli. 855 30

Chlamydia trachomatis is a nucleotide parasite, being entirely dependent on its host eukaryotic cell for a supply of ATP, GTP, and UTP. Chlamydiae are not, however, auxotrophic for CTP, as they are able both to transport CTP from the host and synthesize CTP de novo via a chlamydial CTP synthetase. This study addresses the developmental regulation of CTP synthetase over the course of the C. trachomatis life cycle. Given the distinct life stages of C. trachomatis, analysis of temporal changes in gene expression and regulation of protein activity is the key to unravelling the mechanism of pathogenesis of this bacterium. The results of immunodetection analysis indicate that CTP synthetase is present in C. trachomatis elementary bodies and reticulate bodies and that it is widespread in other chlamydial strains. Reverse transcriptase-polymerase chain reaction (RT-PCR) and metabolic labelling experiments show that CTP synthetase is transcribed and translated primarily during the mid- and late stages of the chlamydial growth cycle. In addition, C. trachomatis CTP synthetase was transcribed with the CTP utilizing enzyme CMP-2-keto-3-deoxy-octanoic acid synthetase (CMP-KDO synthetase) as part of a polycistronic mRNA. The co-expression of these two enzymes suggests a role for CTP synthetase in lipopolysaccharide biosynthesis, potentially channelling CTP directly to CMP-KDO synthetase. The ability of the intact operon to complement CTP synthetase and CMP-KDO deficiencies in mutant Escherichia coli strains indicates that both enzymes are efficiently translated from a single messenger RNA. Kinetic analysis revealed that the C. trachomatis CTP synthetase possessed co-operativity patterns typical of both prokaryotic and eukaryotic CTP synthetases. However, the K(m) of the enzyme for UTP was lower than that of E. coli CTP synthetase, presumably in response to the low intracellular concentration of this nucleotide in C. trachomatis.
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PMID:Chlamydia trachomatis CTP synthetase: molecular characterization and developmental regulation of expression. 895 11

Stimulation of mouse RAW 264.7 macrophages with UTP activates both the inositol phosphate signal transduction pathway and the phospholipase A2 pathway. In the present study, we investigated the interactions between bacterial lipopolysaccharide and UTP in these two systems and the underlying mechanisms involved. While the UTP-induced release of arachidonic acid was only 2.9-fold that in controls, priming the cells with 1 microgram/ml lipopolysaccharide for 1 h before UTP treatment resulted in 9.2-fold arachidonic acid release upon stimulation with UTP. Lipopolysaccharide priming was both concentration- and time-dependent with a peak effect after 1 h treatment at a concentration of 1 microgram/ml. Lipopolysaccharide treatment affect neither the basal nor the UTP-stimulated inositol phosphate formation and [Ca2+]i rise. Pretreatment of the cells with staurosporine, calphostin, N-(2-aminoethyl)-5-isoquinolinesulfonamide H-7), genistein or K-252a led marked inhibition of the priming effect, suggesting that both protein kinase C and tyrosine kinase are involved in the lipopolysaccharide effect. Buffering intracellular Ca2+ levels using [1,2-bis-(o-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester] (BAPTA/AM) or pretreatment with either N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89), 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD098059) or {1-N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl] -4-phenyl-piperazine (KN-62) did not affect the lipopolysaccharide-induced priming effect. Primed UTP stimulation was inhibited by actinomycin D and cycloheximide, indicating a requirement for both gene expression and protein translation. To further examine whether the stimulatory effects of lipopolysaccharide on phospholipase A2 activity were independent of [Ca2+]i levels but dependent on protein phosphorylation, a fixed Ca2+ concentration and inhibitors of protein phosphatases were used in primed permeabilized cells. Arachidonic acid release from permeabilized cells containing 100 nM Ca2+ was high in lipopolysaccharide-primed cells and potentiated by addition of microcystin, orthovanadate or FK 506. These results that the Ser/Thr and tyrosine phosphorylation cascades induced by protein kinase C and tyrosine kinase, respectively, are required for the arachidonic acid potentiation effect of lipopolysaccharide, which was independent of modulation of the upper stream signaling pathways of UTP.
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PMID:Priming effects of lipopolysaccharide on UTP-induced arachidonic acid release in RAW 264.7 macrophages. 908 94

1. Nucleotide-induced currents in untreated (proliferating) and lipopolysaccharide (LPS; 100 ng ml(-1)) treated (non-proliferating) rat microglial cells were recorded by the whole-cell patch-clamp technique. Most experiments were carried out on non-proliferating microglial cells. ATP (100 nM-1 mM), ADP (10 nM-10 mM) and UTP (1 microM-100 mM), but not uridine (100 microM-10 mM) produced a slow outward current at a holding potential of 0 mV. The effect of UTP (1 mM) did not depend on the presence of extracellular Mg2+ (1 mM). The outward current response to UTP (1 mM) was similar in non-proliferating and proliferating microglia. 2. In non-proliferating microglial cells, the ATP (10 microM)-induced outward current was antagonized by suramin (300 microM) or reactive blue 2 (50 microM), whereas 8-(p-sulphophenyl)-theophylline (8-SPT; 100 microM) was inactive. By contrast, the current induced by UTP (1 mM) was increased by suramin (300 microM) and was not altered by reactive blue 2 (50 microM) or 8-SPT (100 microM). 3. The current response to UTP (1 mM) disappeared when K+ was replaced in the pipette solution by an equimolar concentration of Cs+ (150 mM). However, the effect of UTP (1 mM) did not change when most Cl- was replaced with an equimolar concentration of gluconate (145 mM). The application of 4-aminopyridine (1 mM) or Cs+ (1 mM) to the bath solution failed to alter the UTP (1 mM)-induced current. UTP (1 mM) had almost no effect in a nominally Ca2+-free bath medium, or in the presence of charybdotoxin (0.1 microM); the inclusion of U-73122 (5 microM) or heparin (5 mg ml(-1)) into the pipette solution also blocked the responses to UTP (1 mM). By contrast, the effect of ATP (10 microM) persisted under these conditions. 4. I-V relations were determined by delivering fast voltage ramps before and during the application of UTP (1 mM). In the presence of extracellular Cs+ (1 mM) and 4-aminopyridine (1 mM) the UTP-evoked current crossed the zero current level near -75 mV. Omission of Ca2+ from the Cs+ (1 mM)- and 4-aminopyridine (1 mM)-containing bath medium or replacement of K+ by Cs+ (150 mM) in the pipette solution abolished the UTP current. 5. Replacement of GTP (200 microM) by GDP-beta-S (200 microM) in the pipette solution abolished the current evoked by UTP (1 mM). 6. When the pipette solution contained Cs+ (150 mM) instead of K+ and in addition inositol 1,4,5,-trisphosphate (InsP3; 10 microM), an inward current absolutely dependent on extracellular Ca2+ was activated after the establishment of whole-cell recording conditions. This current had a typical delay, a rather slow time course and did not reverse its amplitude up to 100 mV, as measured by fast voltage ramps. 7. A rise of the internal free Ca2+ concentration from 0.01 to 0.5 microM on excised inside-out membrane patches produced single channel activity with a reversal potential of 0 mV in a symmetrical K+ solution. The reversal potential was shifted to negative values, when the extracellular K+ concentration was decreased from 144 to 32 mM. By contrast, a decrease of the extracellular Cl- concentration from 164 to 38 mM did not change the reversal potential. 8. Purine and pyrimidine nucleotides act at separate receptors in rat microglial cells. Pyrimidinoceptors activate via a G protein the enzyme phospholipase C with the subsequent release of InsP3. The depletion of the intracellular Ca2+ pool appears to initiate a capacitative entry of Ca+ from the extracellular space. This Ca2+ then activates a Ca2+-dependent K+ current.
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PMID:Coexistence of purino- and pyrimidinoceptors on activated rat microglial cells. 924 43

Cytokine-induced nitric oxide (NO) is produced on glomerular inflammation. Glomerular injury and thrombocyte aggregation result in the release of nucleotides, which may regulate induced NO synthesis in cultured rat mesangial cells (MCs). ATP (10(-3) M) inhibited 24-h nitrite production induced by lipopolysaccharide (LPS, 10 microg/ml)/interferon-gamma (IFN-gamma, 100 U/ml) by 48.2 +/- 6. 3%, as well as induction of inducible NOS (iNOS) protein and mRNA. Also, coincubation with either 10(-4) M of UTP, ATP, or ATPgammaS inhibited LPS/IFN-gamma-induced nitrite production by 29.9 +/- 5.8, 36.4 +/- 4.3, and 50.3 +/- 6.5%, respectively, indicating involvement of purinergic P2Y2 receptors. Correspondingly, cultured MCs expressed P2Y2 receptor mRNA. Agonists for other purinergic receptors [alpha,beta-methylene-ATP, 3'-O-(4-benzoyl)-benzoyl-ATP, 2-methylthio-ATP, ADP, UDP, adenosine] were ineffective. Treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 10(-8) M) reproduced the inhibitory effect of ATP on iNOS protein expression and nitrite inhibition (by 46.6 +/- 10. 4%). The effect of ATP or PMA was reversed by the PKC inhibitors Ro-31-8220 (10(-8) M) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (10(-5) M), indicating that suppression of iNOS is mediated via activation of PKC through stimulated P2Y2 receptors. In conclusion, the release of purine mediators may play a critical role for iNOS expression and synthesis of NO during glomerular inflammatory disorders.
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PMID:Activation of purinergic P2Y2 receptors inhibits inducible NO synthase in cultured rat mesangial cells. 968 11

1. D-Galactosamine (GalN) depletes UTP primarily in the liver, resulting in decreased RNA synthesis in hepatocytes. Co-injection of GalN and lipopolysaccharide (LPS) into mice produces fulminant hepatitis with severe hepatic congestion, resulting in rapid death. Although the underlying mechanism is uncertain, GalN enhances the sensitivity to tumour necrosis factor (TNF). Administration of uridine (a precursor of UTP) prior injection of either LPS itself or interleukin-1 (IL-1) reduces the lethality of GalN+LPS. The present study focused on the effects of these agents on TNF production. 2. Intraperitoneal injection of GalN+LPS into mice greatly elevated serum TNF. Although large doses of LPS alone also greatly elevated serum TNF, LPS itself induced neither hepatic congestion nor rapid death. Administration of a macrophage depletor, liposomes encapsulated with dichloromethylene bisphosphonate, reduced both the TNF production and mortality induced by GalN+LPS. 3. Uridine, when injected 0.5 h after the injection of GalN+LPS, reduced the production of TNF. Prior injection of LPS, but not of IL-1, also reduced this TNF production. 4. Serum from LPS-injected mice reduced the TNF production induced by GalN+LPS, but it was less effective at reducing the lethality. Its ability to reduce TNF production was abolished by heat-treatment. 5. We hypothesize that a factor inhibiting TNF production by macrophages is produced by hepatocytes in response to LPS. Possibly, production of this hepatocyte-derived TNF-down-regulator (TNF-DRh) may be: (i) inhibited by GalN, causing over-production of TNF by macrophages and (ii) stimulated by LPS-pretreatment (and restored by uridine), causing reduced TNF production.
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PMID:Enhancement by galactosamine of lipopolysaccharide(LPS)-induced tumour necrosis factor production and lethality: its suppression by LPS pretreatment. 1049 28


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