Synthesis of two different molecular forms of ribulose-bisphosphate carboxylase/oxygenase in Rhodopseudomonas blastica grown in batch culture

The expression of the two different molecular forms (form I and form II) of ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) in Rhodopseudomonas blastica during growth in batch on pyruvate–malate medium was investigated. During the early exponential phase of growth, only form I enzyme was synthesized. At the mid-exponential phase of growth, both forms were expressed, although form I enzyme was predominant. At the late exponential phase, form I and form II enzymes were synthesized but form II enzyme predominated. It is concluded that form I and form II RuBisCO enzymes in R. blastica are differentially expressed and this may be mediated by the level of CO2 in the growth medium.     

5'-Haloacetamido-5'-deoxythymidines: novel inhibitors of thymidylate synthase

5'-Bromoacetamido-5'-deoxythymidine (BAT), 5'-iodoacetamido-5'-deoxythymidine (IAT), 5'-chloroacetamido-5'-deoxythymidine (CAT) and [14C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were investigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT greater than IAT greater than CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4-8-fold higher than it was in the absence of dUMP. The I50 values for BAT were 1 X 10(-5) M and 1.2 X 10(-6) M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thymidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The Km for the enzyme was 9.2 microM, and the Ki determined for competitive inhibition by BAT was 5.4 microM. Formation of a tight, irreversible complex is inferred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min-1, and the steady-state constant of inactivation, Ki, was estimated to be 6.6 microM. The 5'-haloacetamido-5'-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.     

Protective effect of resveratrol on acute endotoxemia-induced nephrotoxicity in rat through nitric oxide independent mechanism

Lipopolysaccharide (LPS) is a glycolipid component of the cell wall of gram negative bacteria inducing deleterious effects on the kidney. Endotoxemia-induced nephrotoxicity is characterized by disturbed intracellular redox balance and reactive oxygen species (ROS) accumulation leading to DNA, proteins and membrane lipid damages. Resveratrol (trans-3,5,4′-trihydroxystilbene) is a polyphenol displaying antioxidant and anti-inflammatory properties. This study investigated its effects on LPS-induced nephrotoxicity in rats. Resveratrol counteracted all LPS-induced changes in renal haemodynamic parameters. In the kidney resveratrol abrogated LPS-induced lipoperoxidation and antioxidant enzyme activities depletion as superoxide dismutase (SOD) and catalase (CAT) but not peroxidase (POD) activity. LPS increased plasma and urine nitric oxide (NO) level and resveratrol reversed them. More importantly, LPS-induced iron mobilization from plasma to kidney, which was also abolished by resveratrol treatment. All these results suggest that resveratrol exerted strong antioxidant properties against LPS-induced nephrotoxicity and that its mode of action seemed to involve iron shuttling proteins.     

Integrin expression and H2O2 production in circulating and splenic leukocytes of obese rats

Objective: Obesity is associated with oxidative stress and inflammation. We hypothesized that the pro‐inflammatory state in obesity may result in spontaneous activation and, hence, increased generation of reactive oxygen species (ROS) and integrin expression in the circulating leukocytes. Methods: Flow cytometry was used to determine integrin expression (immunostaining) as well as superoxide and hydrogen peroxide productions (fluorescent probes) in the peripheral blood and splenic leukocyte of 24‐week‐old male obese normotensive and not‐as‐yet diabetic Zucker rats (n = 6) and their lean counterparts (n = 6). Results: Obese rats had hyperlipidemia and normal arterial pressure, plasma glucose, and creatinine concentrations. Nevertheless, obese rats exhibited increased hydrogen peroxide production by circulating and splenic CD4+ and CD8+ T lymphocytes and by splenic macrophages. This was accompanied by up‐regulations of CD11a expression in the peripheral blood and splenic CD4+ T cells, CD11b in circulating macrophages, and CD11a and CD18 in circulating granulocytes. Conclusion: The study revealed direct evidence of spontaneous leukocyte activation and increased ROS generation by T lymphocytes and monocytes in the peripheral blood of obese Zucker rats before the development of diabetes or hypertension. These findings illustrate the link between obesity, oxidative stress, and inflammation.     

Niacin improves renal lipid metabolism and slows progression in chronic kidney disease

Background

Mounting evidence points to lipid accumulation in the diseased kidney and its contribution to progression of nephropathy. We recently found heavy lipid accumulation and marked dysregulation of lipid metabolism in the remnant kidneys of rats with chronic renal failure (CRF). Present study sought to determine efficacy of niacin supplementation on renal tissue lipid metabolism in CRF.

Methods

Kidney function, lipid content, and expression of molecules involved in cholesterol and fatty acid metabolism were determined in untreated CRF (5/6 nephrectomized), niacin-treated CRF (50 mg/kg/day in drinking water for 12 weeks) and control rats.

Results

CRF resulted in hypertension, proteinuria, renal tissue lipid accumulation, up-regulation of scavenger receptor A1 (SR-A1), acyl-CoA cholesterol acyltransferase-1 (ACAT1), carbohydrate-responsive element binding protein (ChREBP), fatty acid synthase (FAS), acyl-CoA carboxylase (ACC), liver X receptor (LXR), ATP binding cassette (ABC) A-1, ABCG-1, and SR-B1 and down-regulation of sterol responsive element binding protein-1 (SREBP-1), SREBP-2, HMG-CoA reductase, PPAR-α, fatty acid binding protein (L-FABP), and CPT1A. Niacin therapy attenuated hypertension, proteinuria, and tubulo-interstitial injury, reduced renal tissue lipids, CD36, ChREBP, LXR, ABCA-1, ABCG-1, and SR-B1 abundance and raised PPAR-α and L-FABP.

Conclusions and general significance

Niacin administration improves renal tissue lipid metabolism and renal function and structure in experimental CRF.     

Microbial Diversity in Uranium Mining-Impacted Soils as Revealed by High-Density 16S Microarray and Clone Library

Microbial diversity was characterized in mining-impacted soils collected from two abandoned uranium mine sites, the Edgemont and the North Cave Hills, South Dakota, using a high-density 16S microarray (PhyloChip) and clone libraries. Characterization of the elemental compositions of soils by X-ray fluorescence spectroscopy revealed higher metal contamination including uranium at the Edgemont than at the North Cave Hills mine site. Microarray data demonstrated extensive phylogenetic diversity in soils and confirmed nearly all clone-detected taxonomic levels. Additionally, the microarray exhibited greater diversity than clone libraries at each taxonomic level at both the mine sites. Interestingly, the PhyloChip detected the largest number of taxa in Proteobacteria phylum for both the mine sites. However, clone libraries detected Acidobacteria and Bacteroidetes as the most numerically abundant phyla in the Edgemont and North Cave Hills mine sites, respectively. Several 16S rDNA signatures found in both the microarrays and clone libraries displayed sequence similarities with yet-uncultured bacteria representing a hitherto unidentified diversity. Results from this study demonstrated that highly diverse microbial populations were present in these uranium mine sites. Diversity indices indicated that microbial communities at the North Cave Hills mine site were much more diverse than those at the Edgemont mine site.     

Spatial heterogeneity in the perception of face and form attributes

The identity of an object is a fixed property, independent of where it appears, and an effective visual system should capture this invariance [1-3]. However, we now report that the perceived gender of a face is strongly biased toward male or female at different locations in the visual field. The spatial pattern of these biases was distinctive and stable for each individual. Identical neutral faces looked different when they were presented simultaneously at locations maximally biased to opposite genders. A similar effect was observed for perceived age of faces. We measured the magnitude of this perceptual heterogeneity for four other visual judgments: perceived aspect ratio, orientation discrimination, spatial-frequency discrimination, and color discrimination. The effect was sizeable for the aspect ratio task but substantially smaller for the other three tasks. We also evaluated perceptual heterogeneity for facial gender and orientation tasks at different spatial scales. Strong heterogeneity was observed even for the orientation task when tested at small scales. We suggest that perceptual heterogeneity is a general property of visual perception and results from undersampling of the visual signal at spatial scales that are small relative to the size of the receptive fields associated with each visual attribute.     

Circadian rhythms in toxic effects of the serotonin antagonist ondansetron in mice

The aim of the study was to learn whether the lethal and the motor incoordination (ataxia) side effect of ondansetron (Zophren) administration is dosing-time dependent. Ondansetron is a serotonin 5-HT3 receptor antagonist used primarily to control nausea and vomiting arising from cytotoxic chemo- and radiotherapy. A total of 210 male Swiss mice 10 to 12 weeks of age were synchronized for 3 weeks by 12 h light (rest span)/12 h dark (activity span). Different doses of ondansetron were injected intraperitoneally (i.p.) at fixed times during the day to determine both the sublethal (TD50) and lethal (LD50) doses, which were, respectively, 3.7 +/- 0.6 mg/kg and 4.6 +/- 0.5 mg/kg. In the chronotoxicologic study a single dose of ondansetron (3.5 mg/kg, i.p.) was administered to different and comparable groups of animals at four different circadian stages [1, 7, 13, and 19 h after light onset (HALO)]. The lethal toxicity was statistically significantly dosing time-dependent (chi2 = 21.51, p < 0.0001). Drug dosing at 1 HALO resulted in 100% survival rate whereas drug dosing at 19 HALO was only one-half that (52%). Similarly, lowest and highest ataxia occurred when ondansetron was injected at 1 and 19 HALO, respectively (chi2 = 22.24, p < 0.0001). Effects on rectal temperature were also dosing-time related (Cosinor analysis, p < 0.0001). The characteristics of the waveform describing the temporal patterns differed between the studied variables, e.g., lethal toxicity and survival rate showing two peaks and rectal temperature showing one peak in the 24 h time series waveform pattern. Cosinor analysis also revealed a statistically significant ultradian (tau = 8 h) rhythmic component in the considered variables. Differences in curve patterns in toxicity elicited by ondansetron on a per end point basis are hypothesized to represent the phase relations between the identified 24 h and 8 h periodicities.