Respiratory chain strongly oxidizes the CXXC motif of DsbB in the Escherichia coli disulfide bond formation pathway.
Escherichia coli DsbB has four essential cysteine residues, among which Cys41 and Cys44 form a CXXC redox active site motif and the Cys104-Cys130 disulfide bond oxidizes the active site cysteines of DsbA, the disulfide bond formation factor in the periplasm. Functional respiratory chain is required for the cell to keep DsbA oxidized. In this study, we characterized the roles of essential cysteines of DsbB in the coupling with the respiratory chain. Cys104 was found to form the inactive complex with DsbA under respiration-defective conditions. While DsbB, under normal aerobic conditions, is in the oxidized state, having two intramolecular disulfide bonds, oxidation of Cys104 and Cys130 requires the presence of Cys41-Cys44. Remarkably, the Cys41-Cys44 disulfide bond is refractory to reduction by a high concentration of dithiothreitol, unless the membrane is solubilized with a detergent. This reductant resistance requires both the respiratory function and oxygen, since Cys41-Cys44 became sensitive to the reducing agent when membrane was prepared from quinone- or heme-depleted cells or when a membrane sample was deaerated. Thus, the Cys41-Val-Leu-Cys44 motif of DsbB is kept both strongly oxidized and strongly oxidizing when DsbB is integrated into the membrane with the normal set of respiratory components. (+info)
ApoB100 secretion from HepG2 cells is decreased by the ACAT inhibitor CI-1011: an effect associated with enhanced intracellular degradation of ApoB.
The concept that hepatic cholesteryl ester (CE) mass and the rate of cholesterol esterification regulate hepatocyte assembly and secretion of apoB-containing lipoproteins remains controversial. The present study was carried out in HepG2 cells to correlate the rate of cholesterol esterification and CE mass with apoB secretion by CI-1011, an acyl CoA:cholesterol acyltransferase (ACAT) inhibitor that is known to decrease apoB secretion, in vivo, in miniature pigs. HepG2 cells were incubated with CI-1011 (10 nmol/L, 1 micromol/L, and 10 micromol/L) for 24 hours. ApoB secretion into media was decreased by 25%, 27%, and 43%, respectively (P<0.0012). CI-1011 (10 micromol/L) inhibited HepG2 cell ACAT activity by 79% (P<0.002) and cellular CE mass by 32% (P<0.05). In contrast, another ACAT inhibitor, DuP 128 (10 micromol/L), decreased cellular ACAT activity and CE mass by 85% (P<0.002) and 42% (P=0.01), respectively, but had no effect on apoB secretion into media. To characterize the reduction in apoB secretion by CI-1011, pulse-chase experiments were performed and analyzed by multicompartmental modelling using SAAM II. CI-1011 did not affect the synthesis of apoB or albumin. However, apoB secretion into the media was decreased by 42% (P=0.019). Intracellular apoB degradation increased proportionately (P=0.019). The secretion of albumin and cellular reuptake of labeled lipoproteins were unchanged. CI-1011 and DuP 128 did not affect apoB mRNA concentrations. These results show that CI-1011 decreases apoB secretion by a mechanism that involves an enhanced intracellular degradation of apoB. This study demonstrates that ACAT inhibitors can exert differential effects on apoB secretion from HepG2 cells that do not reflect their efficacy in inhibiting cholesterol esterification. (+info)
Anaerobic oxidations of cysteate: degradation via L-cysteate:2-oxoglutarate aminotransferase in Paracoccus pantotrophus.
Anoxic, fresh-water enrichment cultures to oxidize different organosulfonates were set up with nitrate, ferric iron or sulfate as electron acceptors. Pure cultures were easily obtained with two naturally occurring sulfonates, cysteate (2-amino-3-sulfopropionate) and taurine (2-aminoethanesulfonate), under nitrate-reducing conditions. These two sulfonates were also oxidized during reduction of iron(III), though isolation of pure cultures was not successful. One nitrate-reducing cysteate-oxidizing bacterium, strain NKNCYSA, was studied in detail. It was identified as Paracoccus pantotrophus. Eighteen sulfonates were tested, and the organism degraded cysteate, taurine, isethionate (2-hydroxyethanesulfonate), sulfoacetate or 3-aminopropanesulfonate with concomitant reduction of nitrate, presumably to molecular nitrogen. The carbon skeleton of these substrates was converted to cell material and, presumably, CO2. The amino group was released as ammonia and the sulfono moiety was recovered as sulfate. Cell-free extracts of P. pantotrophus NKNCYSA contained constitutive L-cysteate:2-oxoglutarate aminotransferase (EC 2.6.1.-) and glutamate dehydrogenase (EC 188.8.131.52). Taurine:pyruvate aminotransferase, in contrast, was inducible. (+info)
Modulation of fibroblast growth factor-2 receptor binding, signaling, and mitogenic activity by heparin-mimicking polysulfonated compounds.
Basic fibroblast growth factor (FGF-2) interacts with high-affinity tyrosine-kinase fibroblast growth factor receptors (FGFRs) and low-affinity heparan sulfate proteoglycans (HSPGs) in target cells. Both interactions are required for FGF-2-mediated biological responses. Here we report the FGF-2 antagonist activity of novel synthetic sulfonic acid polymers with distinct chemical structures and molecular masses (MMs). PAMPS [poly(2-acrylamido-2-methyl-1-propanesulfonic acid)], (MM approximately 7,000-10,000), PAS [poly(anetholesulfonic acid)], (MM approximately 9,000-11,000), PSS [poly(4-styrenesulfonic acid)], (MM = 70,000), and poly(vinylsulfonic acid) (MM = 2,000), inhibited FGF-2 binding to HSPGs and FGFRs in fetal bovine aortic endothelial GM 7373 cells. They also abrogated the formation of the HSPG/FGF-2/FGFR ternary complex, as evidenced by their capacity to prevent FGF-2-mediated cell-cell attachment of FGFR-1-overexpressing, HSPG-deficient Chinese hamster ovary cells to wild-type HSPG-bearing cells. Direct interaction of the polysulfonates with FGF-2 was demonstrated by their ability to protect the growth factor from proteolytic cleavage. Accordingly, molecular modeling, based on the crystal structure of the interaction of FGF-2 with a heparin hexamer, showed the feasibility of docking PAMPS into the heparin-binding domain of FGF-2. In agreement with their FGF-2-binding capacity, PSS, PAS, and PAMPS inhibited FGF-2-induced cell proliferation in GM 7373 cells and murine brain microvascular endothelial cells. The antiproliferative activity of these compounds was associated with the abrogation of FGF-2-induced tyrosine phosphorylation of FGFR-1. Moreover, the polysulfonates PSS and PAS inhibited FGF-2-induced activation of mitogen-activated protein kinase-1/2, involved in FGF-2 signal transduction. In conclusion, sulfonic acid polymers bind FGF-2 by mimicking heparin interaction. These compounds may provide a tool to inhibit FGF-2-induced endothelial cell proliferation in angiogenesis and tumor growth. (+info)
Inhibition of ACAT by avasimibe decreases both VLDL and LDL apolipoprotein B production in miniature pigs.
An orally bioavailable acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, avasimibe (CI-1011), was used to test the hypothesis that inhibition of cholesterol esterification, in vivo, would reduce hepatic very low density (VLDL) apolipoprotein (apo) B secretion into plasma. ApoB kinetic studies were carried out in 10 control miniature pigs, and in 10 animals treated with avasimibe (10 mg/kg/d, n = 6; 25 mg/kg/d, n = 4). Pigs were fed a diet containing fat (34% of calories) and cholesterol (400 mg/d; 0.1%). Avasimibe decreased the plasma concentrations of total triglyceride, VLDL triglyceride, and VLDL cholesterol by 31;-40% 39-48%, and 31;-35%, respectively. Significant reductions in plasma total cholesterol (35%) and low density lipoprotein (LDL) cholesterol (51%) concentrations were observed only with high dose avasimibe. Autologous 131I-labeled VLDL, 125I-labeled LDL, and [3H]leucine were injected simultaneously into each pig and apoB kinetic data were analyzed using multicompartmental analysis (SAAM II). Avasimibe decreased the VLDL apoB pool size by 40;-43% and the hepatic secretion rate of VLDL apoB by 38;-41%, but did not alter its fractional catabolism. Avasimibe decreased the LDL apoB pool size by 13;-57%, largely due to a dose-dependent 25;-63% in the LDL apoB production rate. Hepatic LDL receptor mRNA abundances were unchanged, consistent with a marginal decrease in LDL apoB FCRs. Hepatic ACAT activity was decreased by 51% (P = 0.050) and 68% (P = 0.087) by low and high dose avasimibe, respectively. The decrease in total apoB secretion correlated with the decrease in hepatic ACAT activity (r = 0.495; P = 0.026). We conclude that inhibition of hepatic ACAT by avasimibe reduces both plasma VLDL and LDL apoB concentrations, primarily by decreasing apoB secretion. (+info)
Low-molecular-weight sulfonates, a major substrate for sulfate reducers in marine microbial mats.
Several low-molecular-weight sulfonates were added to microbial mat slurries to investigate their effects on sulfate reduction. Instantaneous production of sulfide occurred after taurine and cysteate were added to all of the microbial mats tested. The rates of production in the presence of taurine and cysteate were 35 and 24 microM HS(-) h(-1) in a stromatolite mat, 38 and 36 microM HS(-) h(-1) in a salt pond mat, and 27 and 18 microM HS(-) h(-1) in a salt marsh mat, respectively. The traditionally used substrates lactate and acetate stimulated the rate of sulfide production 3 to 10 times more than taurine and cysteate stimulated the rate of sulfide production in all mats, but when ethanol, glycolate, and glutamate were added to stromatolite mat slurries, the resulting increases were similar to the increases observed with taurine and cysteate. Isethionate, sulfosuccinate, and sulfobenzoate were tested only with the stromatolite mat slurry, and these compounds had much smaller effects on sulfide production. Addition of molybdate resulted in a greater inhibitory effect on acetate and lactate utilization than on sulfonate use, suggesting that different metabolic pathways were involved. In all of the mats tested taurine and cysteate were present in the pore water at nanomolar to micromolar concentrations. An enrichment culture from the stromatolite mat was obtained on cysteate in a medium lacking sulfate and incubated anaerobically. The rate of cysteate consumption by this enrichment culture was 1.6 pmol cell(-1) h(-1). Compared to the results of slurry studies, this rate suggests that organisms with properties similar to the properties of this enrichment culture are a major constituent of the sulfidogenic population. In addition, taurine was consumed at some of highest dilutions obtained from most-probable-number enrichment cultures obtained from stromatolite samples. Based on our comparison of the sulfide production rates found in various mats, low-molecular-weight sulfonates are important sources of C and S in these ecosystems. (+info)
Antioxidant activity: what do we measure?
Inhibition of oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by free radicals generated by decomposition of 2,2'-azobis(2-amidopropane) (ABAP) by antioxidants and biological material was studied. A correlation was found between the ability of various substances to delay the onset of ABTS oxidation and their rapid reduction of the ABTS+* cation radical, and between the ability to reduce the maximal rate of ABTS oxidation and slow reduction of ABTS+*. The length of the lag period of ABTS oxidation was found to be independent of ABTS concentration. Similar decrease of peroxynitrite-induced ABTS+* formation by antioxidants was observed when the antioxidants were added before and after peroxynitrite. All these findings indicate that the main effect of antioxidants in this system is reduction of ABTS+* and not prevention of its formation. Reduction of oxidation products rather than inhibition of their formation may be the predominant mode of action of antioxidants in various assays of antioxidant activity. (+info)
Malaria vectors in a traditional dry zone village in Sri Lanka.
Malaria transmission by anopheline mosquitoes was studied in a traditional tank-irrigation-based rice-producing village in the malaria-endemic low country dry zone of northcentral Sri Lanka during the period August 1994-February 1997. Adult mosquitoes were collected from human and bovid bait catches, bovid-baited trap huts, indoor catches, and pit traps. Mosquito head-thoraces were tested for the presence of Plasmodium falciparum and P. vivax, and blood-engorged abdomens for the presence of human blood by ELISAs. House surveys were done at two-day intervals to record cases of blood film-confirmed malaria among the villagers. A total of 7,823 female anophelines representing 14 species were collected. Trends in anopheline abundance were significantly correlated with rainfall of the preceding month in An. annularis, An. barbirostris, An. subpictus, An. vagus, and An. varuna, but were not significant in An. culicifacies and An. peditaeniatus. Malaria parasite infections were seen in seven mosquito species, with 75% of the positive mosquitoes containing P. falciparum and 25% P. vivax. Polymorph PV247 was recorded from a vector (i.e., An. varuna) for the first time in Sri Lanka. Computations of mean number of infective vector (MIV) rates using abundance, circumsporozoite (CS) protein rate, and human blood index (HBI) showed the highest rate in An. culicifacies. A malaria outbreak occurred from October 1994 to January 1995 in which 45.5% of village residents experienced at least a single disease episode. Thereafter, malaria incidence remained low. Anopheles culicifacies abundance lagged by one month correlated positively with monthly malaria incidence during the outbreak period, and although this species ranked fifth in terms of abundance, infection was associated with a high MIV rate due to a high CS protein rate and HBI. Abundance trends in other species did not correlate significantly with malaria. It was concluded that An. culicifacies was epidemiologically the most important vector in the study area. (+info)