Synthesis, structure and biological evaluation of bis salicylaldehyde-4(N)-ethylthiosemicarbazone ruthenium(iii) triphenylphosphine

Bis salicylaldehyde-4(N)-ethylthiosemicarbazone ruthenium(iii) triphenylphosphine [Ru(Sal-etsc)(H-Sal-etsc)(PPh(3))] was synthesized and structurally characterized by spectral and X-ray crystallographic studies and it showed 100% inhibition on the DPPH radical. It also exhibited a significant lymphocyte activity and inhibitory effect on the lung carcinoma A549 cell.     

Description of a new species of the genus Awaous Valenciennes, 1837 (Gobiiformes: Oxudercidae) from the middle stretch of the Mahanadi River,Odisha, India,with comments on the Awaous species from India

A new species of the genus Awaous (Oxudercidae), Awaous motla sp. nov., is described based on 18 specimens collected from the Mahanadi River near Sonepur, Subarnapur District, and 3 specimens from the same river near Boudh bridge, Boudh District of Odisha, India. This species is distinct from its congeners by having a combination of characteristics: relatively small eyes, diameter of 6.6–8.4 in head length (LH); robust and long snout, 2.0–2.6 in LH; eye diameter 2.7–4.1 in snout length; cephalic sensory pore system interrupted with eight pores; predorsal scales 13–15; longitudinal scale series 55–58; gill rakers 2 + 1 + (6–7) on the first gill arch; teeth small, conical, and in a single row on the upper jaw and multiserial (2–3) on the lower jaw. This species is also differentiated from some of its congeners in the nucleotide composition of the cytochrome c oxidase I gene by 8.3%–13.8% Kimura two-parameter (K2P) distance and belongs to a separate cluster in the maximum likelihood tree analysis. This finding is also supported by the species delimitation analysis based on Assemble Species by Automatic Partitioning. The new species holds high commercial value in its locality and needs special conservation attention for sustainable utilization.     

Analysis of TRPC3-interacting proteins by tandem mass spectrometry

Mammalian transient receptor potential canonical (TRPC) channels are a family of nonspecific cation channels that are activated in response to stimulation of phospholipase C (PLC)-dependent hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate. Despite extensive studies, the mechanism(s) involved in regulation of mammalian TRPC channels remains unknown. Presence of various protein-interacting domains in TRPC channels have led to the suggestion that they associate with proteins that are involved in their function and regulation. This study was directed toward identifying the proteins associated with native TRPC3 using a shotgun proteomic approach. Anti-TRPC3 antibody was used to immunoprecipitate TRPC3 from solubilized rat brain crude membranes under conditions that allow retention of TRPC3 function. Proteins in the TRPC3 (using anti-TRPC3 antibody) and control (using rabbit IgG) immunoprecipitates were separated by SDS-PAGE, the gel was sectioned, and the resolved proteins were digested by trypsin in situ. After extraction of the peptides, the peptides were separated by HPLC and sequences derived by MS/MS. Analysis of the data revealed 64 specific TRPC3-associated proteins which can be grouped in terms of their cellular location and involvement in specific cellular function. Many of the proteins identified have been previously reported as TRPC3-regulatory proteins, such as IP3Rs and vesicle trafficking proteins. In addition, we report novel putative TRPC3-interacting proteins, including those involved in protein endocytosis and neuronal growth. To our knowledge, this is the first comprehensive proteomic analysis of a native TRPC channel. These data reveal potential TRPC3 regulatory proteins and provide novel insights of the mechanism(s) regulating TRPC3 channels as well as the possible cellular functions where the channel might be involved.     

Functions of the ORF9-to-ORF12 gene cluster in varicella-zoster virus replication and in the pathogenesis of skin infection

The gene cluster composed of varicella-zoster virus (VZV) open reading frame 9 (ORF9) to ORF12 encodes four putative tegument proteins and is highly conserved in most alphaherpesviruses. In these experiments, the genes within this cluster were deleted from the VZV parent Oka (POKA) individually or in combination, and the consequences for VZV replication were evaluated with cultured cells in vitro and with human skin xenografts in SCID mice in vivo. As has been reported for ORF10, ORF11 and ORF12 were dispensable for VZV replication in melanoma and human embryonic fibroblast cells. In contrast, deletion of ORF9 was incompatible with the recovery of infectious virus. ORF9 localized to the virion tegument and formed complexes with glycoprotein E, which is an essential protein, in VZV-infected cells. Recombinants lacking ORF10 and ORF11 (POKADelta10/11), ORF11 and ORF12 (POKADelta11/12), or ORF10, ORF11 and ORF12 (POKADelta10/11/12) were viable in cultured cells. Their growth kinetics did not differ from those of POKA, and nucleocapsid formation and virion assembly were not disrupted. In addition, these deletion mutants showed no differences compared to POKA in infectivity levels for primary human tonsil T cells. Deletion of ORF12 had no effect on skin infection, whereas replication of POKADelta11, POKADelta10/11, and POKADelta11/12 was severely reduced, and no virus was recovered from skin xenografts inoculated with POKADelta10/11/12. These results indicate that with the exception of ORF9, the individual genes within the ORF9-to-ORF12 gene cluster are dispensable and can be deleted simultaneously without any apparent effect on VZV replication in vitro but that the ORF10-to-ORF12 cluster is essential for VZV virulence in skin in vivo.     

Synthesis and in vivo evaluation of [11C]SN003 as a PET ligand for CRF1 receptors

Synthesis and evaluation of [O-methyl-11C](4-methoxy-2-methylphenyl)[1-(1-methoxymethylpropyl)-6-methyl-1H-[1,2,3]triazolo[4,5-c]pyridin-4-yl]amine or [11C]SN003 ([11C]6), as a PET imaging agent for CRF1 receptors, in baboons is described. 4-[1-(1-Methoxymethylpropyl)-6-methyl-1H-[1,2,3]triazolo[4,5-c]pyridin-4-ylamino]-3-methylphenol (5), the precursor molecule for the radiolabeling, was synthesized from 2,4-dichloro-6-methyl-3-nitropyridine in seven steps with 20% overall yield. The total time required for the synthesis of [11C]SN003 is 30 min from EOB using [11C]methyl triflate in the presence of NaOH in acetone. The yield of the synthesis is 22% (EOS) with >99% chemical and radiochemical purities and a specific activity of >2000 Ci/mmol. PET studies in baboon show that [11C]6 penetrates the BBB and accumulates in brain. No detectable specific binding was observed, likely due to the rapid metabolism or low density of CRF1 receptors in primate brain.     

17 beta-Estradiol inhibits angiotensin II activation of area postrema neurons

It is well established that the area postrema, as a circumventricular organ, is susceptible to modulation by circulating hormones and peptides. Furthermore, activation of the area postrema has been shown to modulate central neurons involved in the regulation of cardiovascular function and blood pressure. In particular, the vasoactive peptide angiotensin II (ANG II) has been shown to inhibit baroreflex regulation of heart rate and increase sympathetic outflow and blood pressure via activation of area postrema neurons. Estrogen is thought to protect against hypertension in both humans and animal models and has been shown in a number of systems to alter the effects of ANG II. The purpose of the present study was to determine the effects of estrogen on ANG II activation of area postrema neurons. In this study, the effects of ANG II and KCl on fura 2-measured cytosolic Ca2+ concentration ([Ca2+]i) responses in cultured area postrema neurons in the presence and absence of 12-h exposure to 100 nM 17 beta-estradiol (E2) were evaluated. In neurons incubated in control vehicle media, 50 nM ANG II increased [Ca2+]i by 92 +/- 12%. In neurons preincubated with 100 nM E2, ANG II increased [Ca2+]i by only 68 +/- 11%, for a total inhibition of the ANG II-evoked response of 24%. Coapplication of the estrogen receptor antagonist ICI-182,780 did not inhibit the effects of E2. In the same cells in which the effects of E2 on ANG II-evoked responses were tested, the effects of incubation in E on the depolarization-induced increased [Ca2+2]i due to 60 mM KCl were also tested. Incubation of the cells with 100 nM E increased the KCl-evoked [Ca2+2]i response, and this response was blocked by ICI-182,780. These results suggest that in the area postrema, estrogen may utilize multiple pathways to modulate neural activity and responses to ANG II.     

The Effect of Mutation and Selection on Codon Adaptation in Escherichia coli Bacteriophage

Studying phage codon adaptation is important not only for understanding the process of translation elongation, but also for reengineering phages for medical and industrial purposes. To evaluate the effect of mutation and selection on phage codon usage, we developed an index to measure selection imposed by host translation machinery, based on the difference in codon usage between all host genes and highly expressed host genes. We developed linear and nonlinear models to estimate the C→T mutation bias in different phage lineages and to evaluate the relative effect of mutation and host selection on phage codon usage. C→T-biased mutations occur more frequently in single-stranded DNA (ssDNA) phages than in double-stranded DNA (dsDNA) phages and affect not only synonymous codon usage, but also nonsynonymous substitutions at second codon positions, especially in ssDNA phages. The host translation machinery affects codon adaptation in both dsDNA and ssDNA phages, with a stronger effect on dsDNA phages than on ssDNA phages. Strand asymmetry with the associated local variation in mutation bias can significantly interfere with codon adaptation in both dsDNA and ssDNA phages.     

Cloning,expression and characterization of a highly active thermostable alkaline phosphatase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> MTCC 1483 in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 (DE3)

Alkaline phosphatase gene of the bacterium, Bacillus licheniformis MTCC 1483 was cloned and successfully expressed in Escherichia coli BL21 (DE3). Sequence analysis revealed an open reading frame of 1662 bp encoding a 553 amino acid protein with a molecular mass of 62 kDa, as determined by SDS-PAGE. The recombinant enzyme was purified using Ni-NTA affinity column and the purified enzyme showed a specific activity of 24890 U/mg protein, which is the highest value among any other bacterial recombinant alkaline phosphatases reported so far. The enzyme exhibited optimum activity at 50°C and pH 10.0 and showed high thermostability. The recombinant alkaline phosphatase from B. licheniformis MTCC 1483 exhibited a dephosphorylation efficiency of 92.9% to dephosphorylate linear DNA fragments. The recombinant enzyme with high catalytic efficiency and thermostability has the potential for applications in clinical diagnostics which require enzyme stability against thermal deactivation during preparation or labeling procedures.