Cloning and characterization of DGAT1 gene of Riverine buffalo.

The present study was carried out to characterize the DGAT1 gene of Riverine buffalo. Total RNA was extracted from the mammary tissue of buffalo and DGAT1cDNA were synthesized by RT-PCR, then cloned using pDRIVE cloning vector and sequenced. The sequencing revealed that the size of DGAT1 gene was 1470 bp with GC content of 62.30%. The gene encoded for 489 amino acid precursors and that it possessed 32 amino acids signal peptide. The similarity of buffalo DGAT1 mRNA sequence with that of cattle, pig, monkey, human, mice and rat were determined as 98.4, 90.7, 85.4, 85.0, 77.4 and 77.1%, respectively. Phylogenetic tree constructed from the derived DGAT1 protein sequences of 15 different species illustrated a unique branches for mammals, fly, nematode and plants. Among mammals, cattle and buffalo grouped together, whereas swine formed another group in the same branch. Four motifs were predicted in buffalo DGAT1 peptide sequence, one N-linked glycosylation site (246th position), two putative tyrosine phosphorylation site (316 and 261), one putative diacylglycerol binding site (382-392 amino acid position) and a conserved domain MBOAT (membrane bound acyl transferase from 150 to 474 amino acids) with a histidine as an active residue.     

Leishmania donovani: a chemically defined medium suitable for cultivation and cloning of promastigotes and transformation of amastigotes to to promastigotes

A chemically defined medium using commercially available alpha-MEM supplemented with hemin, HEPES, L-glutamine, D-glucose, folic acid, D-biotin and adenine supports the luxuriant growth and propagation of Leishmania donovani promastigotes. A peak parasite population of about 7.0 x 10(7)/ml at stationary phase and a population doubling time of 11.4 h for high-subpassage promastigotes were obtained. The medium was suitable for transformation of isolated amastigotes from infected hamster spleen. Promastigotes could be detected by culturing kala-azar patients' bone-marrow aspirate or spleen puncture material in this medium. Four out of six freshly transformed isolates gradually adapted and grew well in this medium. Macroscopic colonies appeared on agar plates prepared with the medium within 16-20 days after inoculation. The cloning efficiency was increased about five-fold by glycerol supplementation.     

IQGAP1 is necessary for pulmonary vascular barrier protection in murine acute lung injury and pneumonia

We recently reported that integrin α(v)β(3) is necessary for vascular barrier protection in mouse models of acute lung injury and peritonitis. Here, we used mass spectrometric sequencing of integrin complexes to isolate the novel β(3)-integrin binding partner IQGAP1. Like integrin β(3), IQGAP1 localized to the endothelial cell-cell junction after sphingosine-1-phosphate (S1P) treatment, and IQGAP1 knockdown prevented cortical actin formation and barrier enhancement in response to S1P. Furthermore, knockdown of IQGAP1 prevented localization of integrin α(v)β(3) to the cell-cell junction. Similar to β(3)-null animals, IQGAP1-null mice had increased pulmonary vascular leak compared with wild-type controls 3 days after intratracheal LPS. In an Escherichia coli pneumonia model, IQGAP1 knockout mice had increased lung weights, lung water, and lung extravascular plasma equivalents of (125)I-labeled albumin compared with wild-type controls. Taken together, these experiments indicate that IQGAP1 is necessary for S1P-mediated vascular barrier protection during acute lung injury and is required for junctional localization of the barrier-protective integrin α(v)β(3).     

Transforming growth factor-β protein inversely regulates in vivo differentiation of interleukin-17 (IL-17)-producing CD4+ and CD8+ T cells

TGF-β is a pleiotropic cytokine that predominantly exerts inhibitory functions in the immune system. Unexpectedly, the in vitro differentiation of both Th17 and Tc17 cells requires TGF-β. However, animals that are impaired in TGF-β signaling (TGF-βRIIDN mice) display multiorgan autoimmune disorders. Here we show that CD4(+) T cells from TGF-βRIIDN mice are resistant to Th17 cell differentiation and, paradoxically, that CD8(+) T cells from these animals spontaneously acquire an IL-17-producing phenotype. Neutralization of IL-17 or depletion of CD8(+) T cells dramatically inhibited inflammation in TGF-βRIIDN mice. Therefore, the absence of TGF-β triggers spontaneous differentiation of IL-17-producing CD8(+) T cells, suggesting that the in vivo and in vitro conditions that promote the differentiation of IL-17-producing CD8(+) T cells are distinct.     

Synthesis of O-prenylated and O-geranylated derivatives of 5-benzylidene2,4-thiazolidinediones and evaluation of their free radical scavenging activity as well as effect on some phase II antioxidant/detoxifying enzymes

A series of 5-arylidene-2,4-thiazolidinediones and its geranyloxy or prenyloxy derivative were synthesized and studied for their radical scavenging activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Their comparable scavenging activities were expressed as IC50 value. Compounds 2c, 2d, 4d, and 6a showed appreciable radical scavenging activities. The vanillin based thiazolidinedione compound 2c displayed highest activity comparable to that of alpha-tocopherol. But in vivo, compound 6a showed better results in inducing phase II detoxifying/antioxidative enzyme.     

Acute effects of heat on neuropsychological changes and physiological responses under noise condition

To examine the effects of heat and noise individually and jointly on certain physiological responses and cognitive and neuromotor based functions, 12 male participants were tested under 6 experimental conditions which resulted by combining 3 levels of heat (25 degrees, 30 degrees and 35 degrees C) and 2 levels of white noise (70 and 100 dB). The experiment was carried out in a controlled climatic chamber following two 6 x 6 latin square designs. The results indicated elevations in heart rate, oxygen uptake and body temperature due to the independent effect of heat or the combined effects of heat and noise. The independent action of noise was found to be depressive on the first two responses. On the neuropsychological effects, the heat adversely affected the speed in card sorting (by design configuration) and digit symbol tests, and also the accuracy and error rate in the reasoning ability test. The noise caused performance improvements in critical flicker frequency (simultaneous) and in error rates in card sorting (by design configuration). The combined effects of heat and noise indicated higher error rates in card sorting (by face value), decreased accuracy in reasoning ability and improvements in performance in accuracy scores and error rates in digit symbol test.     

Nucleosomal Positioning and Genetic Divergence Study Based on DNA Flexibility Map

Abstract

Based on worm like chain model, DNA structural parameters—tilt, roll and rise, derived from crystallographic database have been used to determine the flexibility of DNA that regulates the nucleosomal translational positioning. Theoretically derived data has been compared to the experimental values available in Ioshikhes and Trifonov's database. The methodology has been extended to determine the flexibility of 18S rRNA genome in eukarya, where yeast shows a distinct difference when compared with mammals like human, mouse and rabbit.     

Identification and characterization of autotransporter proteins of Yersinia pestis KIM

Yersinia pestis is a Gram-negative bacterium that causes plague. Currently, plague is considered a re-emerging infectious disease and Y. pestis a potential bioterrorism agent. Autotransporters (ATs) are virulence proteins translocated by a variety of pathogenic Gram-negative bacteria across the cell envelope to the cell surface or extracellular environment. In this study, we screened the genome of Yersinia pestis KIM for AT genes whose expression might be relevant for the pathogenicity of this plague-causing organism. By in silico analyses, we identified ten putative AT genes in the genomic sequence of Y. pestis KIM; two of these genes are located within known pathogenicity islands. The expression of all ten putative AT genes in Y. pestis KIM was confirmed by RT-PCR. Five genes, designated yapA, yapC, yapG, yapK and yapN, were subsequently cloned and expressed in Escherichia coli K12 for protein secretion studies. Two forms of the YapA protein (130 kDa and 115 kDa) were found secreted into the culture medium. Protease cleavage at the C terminus of YapA released the protein from the cell surface. Outer membrane localization of YapC (65 kDa), YapG (100 kDa), YapK (130 kDa), and YapN (60 kDa) was established by cell fractionation, and cell surface localization of YapC and YapN was demonstrated by protease accessibility experiments. In functional studies, YapN and YapK showed hemagglutination activity and YapC exhibited autoagglutination activity. Data reported here represent the first study on Y. pestis ATs.