Glutathione peroxidase3 of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> protects phospholipids during cadmium-induced oxidative stress

The present study was undertaken to determine the role of glutathione peroxidase3 (gpx3) in phospholipid protection in cells. Wild-type (WT) cells showed an overall increase in phospholipids upon 50 μM cadmium (Cd)-treatment, whereas an untreated gpx3Δ strain showed a drastic reduction in overall phospholipids which was further reduced with 50 μM Cd. In WT cells, Cd-exposure increased the short chain fatty acids and decreased the unsaturated fatty acids and the magnitude was high in Cd-treated gpx3Δ cells. Purified recombinant gpx3p showed higher activity with phospholipid hydroperoxides than shorter hydroperoxides. An increase in gpx activity was observed in Cd-treated WT cells and no such alteration was observed in gpx3Δ. WT cells treated with Cd showed an increase in MDA over untreated, while untreated gpx3Δ cells themselves showed a higher level of MDA which was further enhanced with Cd-treatment. Iron, zinc and calcium levels were significantly altered in WT and gpx3Δ cells during Cd-treatment.     

Mesolimbic and striatal dopamine receptor supersensitivity: prophylactic and reversal effects of L-prolyl-L-leucyl-glycinamide (PLG)

Functional supersensitivity of mesolimbic and striatal dopamine receptors has been suggested to contribute to the pathogenesis of schizophrenia and tardive dyskinesia. Using the rodent model of chronic administration of the neuroleptic haloperidol, we investigated the possible desensitizing effects of a tripeptide structurally unrelated to dopamine agonists, L-prolyl-L-leucyl-glycinamide (PLG) on mesolimbic and striatal dopaminergic receptor supersensitivity. Administration of PLG either prior to or after chronic haloperidol, inhibited the supersensitivity of dopamine receptors. The results have implications for pharmacological intervention in preventing tardive dyskinesia and relapse psychosis of schizophrenia.     

Investigation of Optical Spectroscopic and Computational Binding Mode of Bovine Serum Albumin with 1, 4‐Bis ((4‐((4‐Heptylpiperazin‐1‐yl) Methyl)‐1H‐1, 2, 3‐Triazol‐1‐yl) Methyl) Benzene

A newly synthesized 1, 4‐bis ((4‐((4‐heptylpiperazin‐1‐yl) methyl)‐1H‐1, 2, 3‐triazol‐1‐yl) methyl) benzene from the family of piperazine derivative has good anticancer activity, antibacterial and low toxic nature; its binding characteristics are therefore of huge interest for understanding pharmacokinetic mechanism of the drug. The binding of piperazine derivative to bovine serum albumin (BSA) was investigated using fluorescence spectroscopy. The molecular distance r between the donor (BSA) and acceptor (piperazine derivative) was estimated according to Forster's theory of nonradiative energy transfer. The physicochemical properties of piperazine derivative, which induced structural changes in BSA, have been studied by circular dichroism and those chemical environmental changes were probed using Raman spectroscopic analysis. Further, the binding dynamics was expounded by synchronous fluorescence spectroscopy and molecular modeling studies explored the hydrophobic interaction and hydrogen bonding results, which stabilize the interaction.     

A novel strategy for the identification of genomic islands by comparative analysis of the contents and contexts of tRNA sites in closely related bacteria

We devised software tools to systematically investigate the contents and contexts of bacterial tRNA and tmRNA genes, which are known insertion hotspots for genomic islands (GIs). The strategy, based on MAUVE-facilitated multigenome comparisons, was used to examine 87 Escherichia coli MG1655 tRNA and tmRNA genes and their orthologues in E.coli EDL933, E.coli CFT073 and Shigella flexneri Sf301. Our approach identified 49 GIs occupying ~1.7 Mb that mapped to 18 tRNA genes, missing 2 but identifying a further 30 GIs as compared with Islander [Y. Mantri and K. P. Williams (2004), Nucleic Acids Res., 32, D55–D58]. All these GIs had many strain-specific CDS, anomalous GC contents and/or significant dinucleotide biases, consistent with foreign origins. Our analysis demonstrated marked conservation of sequences flanking both empty tRNA sites and tRNA-associated GIs across all four genomes. Remarkably, there were only 2 upstream and 5 downstream deletions adjacent to the 328 loci investigated. In silico PCR analysis based on conserved flanking regions was also used to interrogate hotspots in another eight completely or partially sequenced E.coli and Shigella genomes. The tools developed are ideal for the analysis of other bacterial species and will lead to in silico and experimental discovery of new genomic islands.     

Synthesis, characterization, and anti-bacterial efficacy of some novel cyclophane amide

Synthesis of macrocyclic di, tetra- and hexaamides with aza and oxy linkages has been achieved and the inhibitory activity of cyclophane amides against human pathogenic bacteria well documented.     

Cardiac expression of kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase inhibits glycolysis, promotes hypertrophy, impairs myocyte function, and reduces insulin sensitivity

Glycolysis is important to cardiac metabolism and reduced glycolysis may contribute to diabetic cardiomyopathy. To understand its role independent of diabetes or hypoxic injury, we modulated glycolysis by cardiac-specific overexpression of kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (kd-PFK-2). PFK-2 controls the level of fructose 2,6-bisphosphate (Fru-2,6-P(2)), an important regulator of glycolysis. Transgenic mice had over 2-fold reduced levels of Fru-2,6-P(2). Heart weight/body weight ratio indicated mild hypertrophy. Sirius red staining for collagen was significantly increased. We observed a 2-fold elevation in glucose 6-phosphate and fructose 6-phosphate levels, whereas fructose 1,6-bisphosphate was reduced 2-fold. Pathways branching off of glycolysis above phosphofructokinase were activated as indicated by over 2-fold elevated UDP-N-acetylglucosamine and glycogen. The kd-PFK-2 transgene significantly inhibited glycolysis in perfused hearts. Insulin stimulation of metabolism and Akt phosphorylation were sharply reduced. In addition, contractility of isolated cardiomyocytes was impaired during basal and hypoxic incubations. The present study shows that cardiac overexpression of kinase-deficient PFK-2 reduces cardiac glycolysis that produced negative consequences to the heart including hypertrophy, fibrosis, and reduced cardiomyocyte function. In addition, metabolic and signaling responses to insulin were significantly decreased.     

Nucleotide sequence of the rhamnose biosynthetic operon of Shigella flexneri 2a and role of lipopolysaccharide in virulence.

N1308, a chromosomal Tn5 mutant of Shigella flexneri 2a, was described previously as a lipopolysaccharide (LPS) mutant with a short O side chain. N1308 formed foci, but not plaques, in LLC-MK2 cell monolayers and was negative in the Serény test. In this study, the wild-type locus inactivated in N1308 was cloned and further defined by means of complementation analysis. A 4.3-kb BstEII-XhoI fragment of S. flexneri 2a YSH6200 DNA was sufficient to restore both normal LPS and virulence phenotype to the mutant. DNA sequencing of this region revealed four genes, rfbA, rfbB, rfbC, and rfbD, encoding the enzymes required for the biosynthesis of activated rhamnose. The four genes were expressed in Escherichia coli, and the expected protein products were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N1308 was shown to have normal levels of surface IpaC and IpaD, while a Western blot (immunoblot) of whole-cell lysates or outer membrane fractions indicated an elevated level of appropriately localized VirG. An in vitro invasion assay revealed that N1308 had normal primary invasive capacity and was able to multiply and move normally within the initial infected cell. However, it exhibited a significant reduction in its ability to spread from cell to cell in the monolayer. A double immunofluorescence assay revealed differences between LLC-MK2 cells infected with the wild-type YSH6000 and those infected with N1308. The wild-type bacteria elicited the formation of the characteristic F-actin tails, whereas N1308 failed to do so. However, N1308 was capable of inducing deposition of F-actin, which accumulated in a peribacterial fashion with only slight, if any, unipolar accumulation of the cytoskeletal protein.     

Changes in calcineurin expression induced in the rat brain by the administration of antipsychotics

Calcineurin (CN) was recently identified as a susceptibility gene for schizophrenia as well as showing altered RNA expression levels in the post-mortem brains of individuals with schizophrenia. CN knockout mice show a number of behaviours associated with schizophrenia, including deficits in sensorimotor gating, suggesting a link between CN and psychosis. Concurrently, we found, using genome screening techniques, that antipsychotics alter CN expression levels. Therefore, western blotting, in situ hybridization, immunocytochemistry and phosphatase assays were employed to determine what effect antipsychotics have on CN. The results indicate that clozapine, risperidone and haloperidol cause substantial reductions in the A subunit of CN but not CN B at both the RNA and protein levels in the striatum and prefrontal cortex. The changes could only be observed after repeated treatment with antipsychotics but not after acute administration. The alterations in CN protein levels were specific to antipsychotics and mediated by D2 dopamine receptor antagonism. However, despite reductions in CN protein levels, the phosphatase activity of CN was significantly elevated after treatment with antipsychotics. Collectively the results suggest that CN may be a common target for antipsychotics and that antipsychotic-induced alterations in CN may represent one of the mechanisms by which antipsychotics alleviate psychosis.     

ArrayOme: a program for estimating the sizes of microarray-visualized bacterial genomes

ArrayOme is a new program that calculates the size of genomes represented by microarray-based probes and facilitates recognition of key bacterial strains carrying large numbers of novel genes. Protein-coding sequences (CDS) that are contiguous on annotated reference templates and classified as 'Present' in the test strain by hybridization to microarrays are merged into ICs (ICs). These ICs are then extended to account for flanking intergenic sequences. Finally, the lengths of all extended ICs are summated to yield the 'microarray-visualized genome (MVG)' size. We tested and validated ArrayOme using both experimental and in silico-generated genomic hybridization data. MVG sizing of five sequenced Escherichia coli and Shigella strains resulted in an accuracy of 97-99%, as compared to true genome sizes, when the comprehensive ShE.coli meta-array gene sequences (6239 CDS) were used for in silico hybridization analysis. However, the E.coli CFT073 genome size was underestimated by 14% as this meta-array lacked probes for many CFT073 CDS. ArrayOme permits rapid recognition of discordances between PFGE-measured genome and MVG sizes, thereby enabling high-throughput identification of strains rich in novel genes. Gene discovery studies focused on these strains will greatly facilitate characterization of the global gene pool accessible to individual bacterial species.