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

The lipopolysaccharide structure of the nitrogen-fixing bacterium Rhizobium leguminosarum differs from that of Escherichia coli in several ways, one of which is the sugar composition of the core. The E. coli inner core consists of 3-deoxy-D-manno-octulosonic acid (Kdo) and L-glycero-D-manno-heptose (heptose), while the inner core of R. leguminosarum contains 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo), mannose, galactose, and galacturonic acid. The two Kdo residues and their linkages appear to be identical in both species. The linkages of heptose in E. coli and of mannose in R. leguminosarum to Kdo are both alpha1-5. We now characterize a membrane-associated glycosyl transferase in R. leguminosarum extracts that incorporates mannose into nascent lipopolysaccharide, using Kdo2-lipid IVA as the acceptor and GDP-mannose (or synthetic ADP-mannose) as the donor. The mannosyl transferase is associated with the inner membrane. The apparent Km values for GDP-mannose and Kdo2-lipid IVA are 4.3 microM and 7.1 microM, respectively, in the presence of excess co-substrate. Extracts of E. coli do not catalyze GDP-mannose-dependent glycosylation of Kdo2-lipid IVA, but they are active when ADP-mannose is substituted for GDP-mannose. Given the structural similarity of ADP-mannose to ADP-heptose, we examined the possibility that heptosyl transferase I of E. coli (the product of the rfaC gene) catalyzes mannose transfer from ADP-mannose to Kdo2-lipid IVA. Extracts of E. coli mutants defective in the rfaC gene are unable carry out ADP-mannose-dependent glycosylation of Kdo2-lipid IVA. Plasmids bearing rfaC+ not only restore the missing activity but also direct its overexpression. Our assay using ADP-mannose as a substitute for ADP-heptose (which is not readily available) should facilitate the purification and characterization of heptosyl transferase I of E. coli. The GDP-mannose-dependent enzyme of R. leguminosarum may represent a functional equivalent of E. coli RfaC.
 ...
PMID:Lipopolysaccharide core glycosylation in Rhizobium leguminosarum. An unusual mannosyl transferase resembling the heptosyl transferase I of Escherichia coli. 894 65

Haemophilus influenzae is an important human pathogen. The lipooligosaccharide (LOS) of H. influenzae has been implicated as a virulence determinant. To better understand the assembly of LOS in nontypeable H. influenzae (NtHi), we have cloned and characterized the rfaD and rfaF genes of NtHi 2019, which encode the ADP-L-glycero-D-manno-heptose-6-epimerase and heptosyltransferase II enzymes, respectively. This cloning was accomplished by the complementation of Salmonella typhimurium lipopolysaccharide (LPS) biosynthesis gene mutants. These deep rough mutants are novobiocin susceptible until complemented with the appropriate gene. In this manner, we are able to use novobiocin resistance to select for specific NtHi LOS inner core biosynthesis genes. Such a screening system yielded a plasmid with a 4.8-kb insert. This plasmid was able to complement both rfaD and rfaF mutants of S. typhimurium. The LPS of these complemented strains appeared identical to the wild-type Salmonella LPS. The genes encoding the rfaD and rfaF genes from NtHi 2019 were sequenced and found to be similar to the analogous genes from S. typhimurium and Escherichia coli. The rfaD gene encodes a polypeptide of 35 kDa and the rfaF encodes a protein of 39 kDa, as demonstrated by in vitro transcription-translation studies. Isogenic mutants which demonstrated truncated LOS consistent with inner core biosynthesis mutants were constructed in the NtHi strain 2019. Primer extension analysis demonstrated the presence of a strong promoter upstream of rfaD but suggested only a very weak promoter upstream of rfaF. Complementation studies, however, suggest that the rfaF gene does have an independent promoter. Mass spectrometric analysis shows that the LOS molecules expressed by H. influenzae rfaD and rfaF mutant strains have identical molecular masses. Additional studies verified that in the rfaD mutant strain, D-glycero-D-manno-heptose is added to the LOS molecule in place of the usual L-glycero-D-manno-heptose. Finally, the genetic organizations of the inner core biosynthesis genes of S. typhimurium, E. coli, and several strains of H. influenzae were examined, and substantial differences were uncovered.
 ...
PMID:Identification of the ADP-L-glycero-D-manno-heptose-6-epimerase (rfaD) and heptosyltransferase II (rfaF) biosynthesis genes from nontypeable Haemophilus influenzae 2019. 911 77

The proinflammatory cytokine, interleukin-1 (IL-1) is elevated in the Alzheimer's disease (AD) brain. Studies from our laboratory have demonstrated that beta-amyloid (A beta) 1-42, fibrillar A beta 1-40 and A beta 25-35 induce the release of IL-1 beta from activated THP-1 cells, a human monocyte cell line. A beta also is chemotactic for primary rodent microglia and peritoneal macrophages. We hypothesize that A beta is a chemokine and induces these responses by interaction with chemotactic receptors. If this is true, then these A beta-induced responses should be calcium-dependent and require activation of pertussis toxin-sensitive G-proteins. To test this hypothesis, THP-1 cells were grown in culture with lipopolysaccharide (LPS) and incubated with A beta 1-42 (5 muM) in the presence and absence of a calcium chelator, an inhibitor of intracellular calcium mobilization, a calcium channel blocker, or pertussis toxin, a bacterial endotoxin which uncouples G proteins from receptors by catalyzing the ADP ribosylation of cysteine near the carboxy-terminus of the alpha subunit. The media was collected and IL-1 beta present in the media was measured using an ELISA. Treatment of LPS-activated THP-1 cells with A beta 1-42 significantly elevated IL-1 beta released into the media as previously shown. Addition or ethylene glycol-bis (beta-aminothyl ether) N,N,N'N'-tetraacetic acid (EGTA) (0.5 mM), a calcium chelator, to the media blocked A beta-induced IL-1 beta release, but had no effect on LPS-activated THP-1 cell release of IL-1 beta. The presence of 3,4,5-trimethoxybenzoic acid 8-(diethyl amino)-octyl ester (TMB-8), an inhibitor of intracellular calcium mobilization, as well as nickel chloride, a non-specific calcium channel blocker, in the media also inhibited A beta-induced IL-1 release from LPS-activated THP-1 cells. IL- 1 beta release from activated THP-1 monocytes incubated with TMB-8 and nickel chloride without A beta remained at baseline values. Pretreatment of THP-1 monocytes with pertussis toxin for 4 h, followed by LPS activation and incubation with A beta, antagonized the release of IL-1 beta from these cells, but did not alter IL-1 beta release from activated THP-1 monocytes. These data suggest that A beta-induced IL-1 beta release from these cells is calcium-dependent and requires the activation of specific G-proteins. These findings are consistent with known second messengers that are activated following stimulation of chemotactic receptors.
 ...
PMID:beta-Amyloid-induced IL-1 beta release from an activated human monocyte cell line is calcium- and G-protein-dependent. 914 72

In rats injected with bacterial lipopolysaccharide (LPS; 5 gamma mg/g body weight [BWT]), the toxin provokes death within 24 h in 23% of the animals and, in surviving rats, causes a decrease in BWT, hyperlactacidemia, hyperlipacidemia, and hyperketonemia, as well as depletion of both liver and muscle glycogen content. In the liver, LPS severely lowers the ATP and total adenine nucleotide content, ATP/ADP ratio, and adenylate charge. In hepatocytes from LPS-injected rats, the oxidation of D-glucose is first increased 2 h after administration of the toxin, despite close-to-normal phosphorylation of the hexose. In hepatocytes prepared from rats killed 24 h after injection of LPS, the phosphorylation of D-glucose, its incorporation into glycogen, and its oxidation are all severely impaired. This sequence of changes, which coincides with a decreased ratio between pyruvate and lactate production from exogenous D-glucose, is comparable to that found with agents that uncouple oxidative phosphorylation. The injection of LPS also alters the metabolic response of hepatocytes to the dimethyl ester of succinic acid (SAD), in terms, for instance, of the sparing action of the ester upon both the production of 14CO2 by hepatocytes prelabeled with L-[U-14C] glutamine and the output of NH4+, and its inhibitory action on glycogenolysis and futile cycling in the reactions catalyzed by glucokinase and glucose-6-phosphatase. Nevertheless, the infusion of SAD protects the rats against the deleterious effect of LPS upon such variables as the plasma concentration of free fatty acids and beta-hydroxybutyrate, the liver ATP content, and the oxidation of D-glucose, as well as the pyruvate/lactate ratio, in hepatocytes prepared from the LPS-injected rats. The infusion of SAD also virtually suppresses lethality in the LPS-injected animals. It is proposed, therefore, that the infusion of succinic acid esters may represent a novel therapeutic approach in endotoxemia and multiple-organ failure.
 ...
PMID:Protective effects of succinic acid dimethyl ester infusion in experimental endotoxemia. 917 84

Stimulating monocytes/macrophages with bacterial lipopolysaccharide (LPS) results in TNF-alpha, IL-1, IL-6 and nitrite (NO2-) formation. Inhibitors of poly(ADP-ribose)polymerase inhibit release of these mediators by preventing mRNA expression indicating that ADP-ribosylation plays a crucial role in the synthesis of these mediators. Furthermore we present evidence that ADP-ribosylation is involved in modifying cellular proteins. In murine macrophages a 33 kDa cytosolic protein could be identified that in response to LPS changed its state of ADP-ribosylation, and in human monocytes we showed that the inhibitor nicotinamide prevents LPS induced phosphorylation of two cytosolic proteins of 36 kDa and 38 kDa (p36/38) LPS. Taken together these data indicate that protein modification by ADP-ribosylation may control cellular processes involved in distinct steps of monocyte/macrophage activation.
 ...
PMID:Role of ADP-ribosylation in activated monocytes/macrophages. 919 61

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.
 ...
PMID:Coexistence of purino- and pyrimidinoceptors on activated rat microglial cells. 924 43

A moderate elevation of plasma homocysteine is a risk factor for atherosclerosis and arterial and veinous thrombosis. However, the mechanisms leading to vascular disorders are poorly understood because studies that have investigated the potential atherothrombogenicity of hyperhomocysteinemia in vivo are scarce. Using a rat model, we were the first to show that dietary folic acid deficiency, a major cause of basal hyperhomocysteinemia, is associated with enhanced macrophage-derived tissue factor and platelet activities. We proposed that an homocysteine-induced oxidative stress may account for this hypercoagulable state. To determine the true thrombogenicity of moderate hyperhomocysteinemia and better understand its etiology, we have carried out an acute methionine load in control and folate-deficient animals. When rats were fed the control diet, a transient fourfold increase in plasma homocysteine levels was observed 2 h after the methionine administration. As with prolonged dietary folic acid deficiency, this methionine load potentiated the platelet aggregation in response to thrombin and ADP as well as the thrombin-induced thromboxane synthesis. It also stimulated the basal and lipopolysaccharide-induced tissue factor activity of peritoneal macrophages. These prothrombotic effects were associated with an increased lipid peroxidation characterized by an elevation of plasma conjugated dienes, lipid hydroperoxides, and thiobarbituric acid-reactive substances. When rats were fed a folic acid-deficient diet, the methionine load did not cause any further increase in plasma homocysteine concentration, platelet activation, macrophage tissue factor-dependent coagulation, or lipoperoxidation. Altogether, our data showed that the prethrombotic state due to both the altered remethylation and transsulfuration pathways resulted from the moderate elevation of circulating homocysteine. We conclude that moderate hyperhomocysteinemia plays a role in the development of a thrombogenic state that might be mediated by the occurrence of oxidative stress.
 ...
PMID:Acute methionine load-induced hyperhomocysteinemia enhances platelet aggregation, thromboxane biosynthesis, and macrophage-derived tissue factor activity in rats. 936 51

Heptosyltransferase I, encoded by the rfaC(waaC) gene of Escherichia coli, is thought to add L-glycero-D-manno-heptose to the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide core. Lipopolysaccharide isolated from mutants defective in rfaC lack heptose and all other sugars distal to heptose. The putative donor, ADP-L-glycero-D-manno-heptose, has never been fully characterized and is not readily available. In cell extracts, the analog ADP-mannose can serve as an alternative donor for RfaC-catalyzed glycosylation of the acceptor, Kdo2-lipid IVA. Using a T7 promoter construct that overexpresses RfaC approximately 15,000-fold, the enzyme has been purified to near homogeneity. NH2-terminal sequencing confirms that the purified enzyme is the rfaC gene product. The subunit molecular mass is 36 kDa. Enzymatic activity is dependent upon the presence of Triton X-100 and is maximal at pH 7.5. The apparent Km (determined at near saturating concentrations of the second substrate) is 1.5 mM for ADP-mannose and 4.5 microM for Kdo2-lipid IVA. Chemical hydrolysis of the RfaC reaction product at 100 degrees C in the presence of sodium acetate and 1% sodium dodecyl sulfate generates fragments consistent with the inner Kdo residue of Kdo2-lipid IVA as the site of mannosylation. The analog, Kdo-lipid IVA, functions as an acceptor, but is mannosylated at less than 1% the rate of Kdo2-lipid IVA. The purified enzyme displays no activity with ADP-glucose, GDP-mannose, UDP-glucose, or UDP-galactose. Mannosylation of Kdo2-lipid IVA catalyzed by RfaC proceeds in high yield and may be useful for the synthesis of lipopolysaccharide analogs. Pure RfaC can also be used together with Kdo2-[4'-32P]lipid IVA to assay for the physiological donor (presumably ADP-L-glycero-D-manno-heptose) in a crude, low molecular weight fraction isolated from wild type cells.
 ...
PMID:Enzymatic synthesis of lipopolysaccharide in Escherichia coli. Purification and properties of heptosyltransferase i. 944 88

Elucidation of a signal transduction pathway essential to lipopolysaccharide (LPS)-induced macrophage activation has the capacity to provide new targets for the treatment of septic shock. In this regard, activation of the transcription factor NF-kappaB is commonly thought to be critical to LPS-stimulated macrophage inflammatory mediator production, although certain immunological, genetic, and molecular evidence suggests that other factors are involved. To address this issue, we hypothesized that the degree of LPS-induced NF-kappaB mobilization should correlate with the murine endotoxicity of the species of LPS used for in vitro study. Therefore, using D-galactosamine-sensitized mice, we assessed the lethal potencies of eight LPS preparations from Escherichia, Salmonella, Klebsiella, Bacteroides, Pseudomonas, Neisseria, and Rhodobacter species as well as that of the endotoxin substructure lipid X. The lethal potencies of these LPS preparations varied by > 160-fold. Treatment of RAW 264.7 cells with the same LPS preparations induced levels of tumor necrosis factor alpha (TNF-alpha) and NO production that correlated with the LPS 50% lethal dose. The combined analysis of the levels of these two mediators produced in response to LPS in RAW cells was found to be a strong predictor of murine endotoxic lethality. Interestingly, while relatively nontoxic in mice, Rhodobacter capsulatus LPS stimulated RAW cell NF-kappaB-like DNA binding protein mobilization and TNF-alpha production to levels comparable to those of more toxic species of LPS but was unable to induce NO generation in RAW cells. These data indicate that neither NF-kappaB activation nor TNF-alpha production alone is a dependable predictor of LPS lethality. Additionally, cotreatment of RAW cells with the potent inflammatory mediator ADP had no effect on the ability of R. capsulatus LPS to stimulate NO production but significantly enhanced induction of NO production by the toxic species of LPS. In contrast, cotreatment of RAW cells or peritoneal macrophages with gamma interferon (IFN-gamma) normalized the abilities of both toxic and nontoxic LPS preparations to induce NO production, suggesting that selected preparations of LPS may preferentially generate an IFN-gamma-like signal that accounts for enhanced toxicity. In sum, the activation of NF-kappaB does not correspond to LPS lethality, thereby complicating models of macrophage activation that highlight NF-kappaB alone as a signal transduction factor necessary for LPS-mediated toxicity.
 ...
PMID:Nuclear translocation of NF-kappaB in lipopolysaccharide-treated macrophages fails to correspond to endotoxicity: evidence suggesting a requirement for a gamma interferon-like signal. 952 92

We show that the coumeromycin antibiotic novobiocin, a potent inhibitor of ADP ribosylation, prevents lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, and IL-10 secretion in human peripheral blood mononuclear cells. It shares these cytokine-suppressing properties with other inhibitors of ADP ribosylation. We found that novobiocin prevents TNF-alpha production by inhibiting translation of the TNF-alpha mRNA. Elevated TNF-alpha levels in mice treated with D-galactosamine (GalN)-LPS or GalN-TNF were not reduced by novobiocin; however, the drug exhibited hepatoprotective properties. Novobiocin causes downregulation of the surface molecules on monocytes, among which CD14 was the most affected. The diminished expression of surface molecules was not observed on T and B lymphocytes. Similar to other inhibitors of ADP ribosylation, novobiocin prevents LPS-induced phosphate labelling of gamma-actins.
 ...
PMID:Immunomodulating properties of the antibiotic novobiocin in human monocytes. 968 83


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>