Evaluation of a water concentration method for the detection of infectious pancreatic necrosis virus in a fish hatchery

A virus-adsorption-elution (viradel) method using electropositive microporous filters (Virosorb 1-MDS) was used for the concentration and détéction of infectious pancreatic necrosis virus (IPNV) at a hatchery with a past history of IPNV infection. Samples of fish tissue and unconcentrated water were also examined for the presence of IPNV. Virus was isolated from three of the nine concentrated water samples taken from various locations at the hatchery. Virus was also isolated from pooled fish tissues, but not from unconcentrated water samples. Representative numbers of viruses isolated from water and fish tissues were examined for virus neutralization in the presence of anti-IPNV amiserum; resistance to acid, ether, and heat; and replication in the presence of 5-bromo-2'-deoxyuridine (BUDR). When tested by dot-immunobinding assay using a panel of monoclonal antibodies (MABs), the viral isolates were found to belong to the West Buxton serotype of IPNV.     

Functional characterization and mutation analysis of family 11, Carbohydrate-Binding Module (<Emphasis Type="Italic">Ct</Emphasis>CBM11) of cellulosomal bifunctional cellulase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

The non-catalytic, family 11 carbohydrate binding module (CtCBM11) belonging to a bifunctional cellulosomal cellulase from Clostridium thermocellum was hyper-expressed in E. coli and functionally characterized. Affinity electrophoresis of CtCBM11 on nondenaturing PAGE containing cellulosic polysaccharides showed binding with β-glucan, lichenan, hydroxyethyl cellulose and carboxymethyl cellulose. In order to elucidate the involvement of conserved aromatic residues Tyr 22, Trp 65 and Tyr 129 in the polysaccharide binding, site-directed mutagenesis was carried out and the residues were changed to alanine. The results of affinity electrophoresis and binding adsorption isotherms showed that of the three mutants Y22A, W65A and Y129A of CtCBM11, two mutants Y22A and Y129A showed no or reduced binding affinity with polysaccharides. These results showed that tyrosine residue 22 and 129 are involved in the polysaccharide binding. These residues are present in the putative binding cleft and play a critical role in the recognition of all the ligands recognized by the protein.     

Microbial and plant derived biomass for removal of heavy metals from wastewater

Discharge of heavy metals from metal processing industries is known to have adverse effects on the environment. Conventional treatment technologies for removal of heavy metals from aqueous solution are not economical and generate huge quantity of toxic chemical sludge. Biosorption of heavy metals by metabolically inactive non-living biomass of microbial or plant origin is an innovative and alternative technology for removal of these pollutants from aqueous solution. Due to unique chemical composition biomass sequesters metal ions by forming metal complexes from solution and obviates the necessity to maintain special growth-supporting conditions. Biomass of Aspergillus niger, Penicillium chrysogenum, Rhizopus nigricans, Ascophyllum nodosum, Sargassum natans, Chlorella fusca, Oscillatoria anguistissima, Bacillus firmus and Streptomyces sp. have highest metal adsorption capacities ranging from 5 to 641 mg g(-1) mainly for Pb, Zn, Cd, Cr, Cu and Ni. Biomass generated as a by-product of fermentative processes offers great potential for adopting an economical metal-recovery system. The purpose of this paper is to review the available information on various attributes of utilization of microbial and plant derived biomass and explores the possibility of exploiting them for heavy metal remediation.     

Apoptosis triggered by Rv1818c, a PE family gene from Mycobacterium tuberculosis is regulated by mitochondrial intermediates in T cells

Ectopic expression of the Mycobacterium tuberculosis PE-family gene Rv1818c, triggers apoptosis in the mammalian Jurkat T cells, which is blocked by anti-apoptotic protein Bcl-2. Although complete overlap is not observed, a considerable proportion of cellular pools of ectopically expressed Rv1818c localizes to mitochondria. However, recombinant Rv1818c does not trigger release of cytochrome c from isolated mitochondria even though Rv1818c protein induced apoptosis of Jurkat T cells. Apoptosis induced by Rv1818c is blocked by the broad-spectrum caspase inhibitory peptide zVAD-FMK. Unexpectedly, Rv1818c-induced apoptosis is not blocked in a Jurkat sub-clone deficient for caspase-8 (JI 9.2) or in cells where caspase-9 function is inhibited or expression of caspase-9 reduced by siRNA, arguing against a central role for these caspases in Rv1818c-induced apoptotic signaling. Depleting cellular pools of the mitochondrial protein Smac/DIABLO substantially reduces apoptosis consistent with mitochondrial involvement in this death pathway. We present evidence that Rv1818c-induced apoptosis is blocked by the co-transfection of an endogenous inhibitor of caspase activation, XIAP in T cells. Additionally, Rv1818c is released into extracellular environment via exosomes secreted by M. tuberculosis infected BM-DC's and macrophages. Furthermore, the extracellular Rv1818c protein can be detected in T cells co-cultured with infected BM-DC's. Taken together, these data suggest that Rv1818c-induced apoptotic signaling is likely regulated in part by the Smac-dependent activation of caspases in T cells.     

Directional transition from initiation to elongation in bacterial translation

The transition of the 30S initiation complex (IC) to the translating 70S ribosome after 50S subunit joining provides an important checkpoint for mRNA selection during translation in bacteria. Here, we study the timing and control of reactions that occur during 70S IC formation by rapid kinetic techniques, using a toolbox of fluorescence-labeled translation components. We present a kinetic model based on global fitting of time courses obtained with eight different reporters at increasing concentrations of 50S subunits. IF1 and IF3 together affect the kinetics of subunit joining, but do not alter the elemental rates of subsequent steps of 70S IC maturation. After 50S subunit joining, IF2-dependent reactions take place independent of the presence of IF1 or IF3. GTP hydrolysis triggers the efficient dissociation of fMet-tRNA^fMet from IF2 and promotes the dissociation of IF2 and IF1 from the 70S IC, but does not affect IF3. The presence of non-hydrolyzable GTP analogs shifts the equilibrium towards a stable 70S–mRNA–IF1–IF2–fMet-tRNA^fMet complex. Our kinetic analysis reveals the molecular choreography of the late stages in translation initiation.     

Spliceosomal intron insertions in genome compacted ray-finned fishes as evident from phylogeny of MC receptors, also supported by a few other GPCRs

Background

Insertions of spliceosomal introns are very rare events during evolution of vertebrates and the mechanisms governing creation of novel intron(s) remain obscure. Largely, gene structures of melanocortin (MC) receptors are characterized by intron-less architecture. However, recently a few exceptions have been reported in some fishes. This warrants a systematic survey of MC receptors for understanding intron insertion events during vertebrate evolution.

Methodology/Principal Findings

We have compiled an extended list of MC receptors from different vertebrate genomes with variations in fishes. Notably, the closely linked MC2Rs and MC5Rs from a group of ray-finned fishes have three and one intron insertion(s), respectively, with conserved positions and intron phase. In both genes, one novel insertion was in the highly conserved DRY motif at the end of helix TM3. Further, the proto-splice site MAG↑R is maintained at intron insertion sites in these two genes. However, the orthologs of these receptors from zebrafish and tetrapods are intron-less, suggesting these introns are simultaneously created in selected fishes. Surprisingly, these novel introns are traceable only in four fish genomes. We found that these fish genomes are severely compacted after the separation from zebrafish. Furthermore, we also report novel intron insertions in P2Y receptors and in CHRM3. Finally, we report ultrasmall introns in MC2R genes from selected fishes.

Conclusions/Significance

The current repository of MC receptors illustrates that fishes have no MC3R ortholog. MC2R, MC5R, P2Y receptors and CHRM3 have novel intron insertions only in ray-finned fishes that underwent genome compaction. These receptors share one intron at an identical position suggestive of being inserted contemporaneously. In addition to repetitive elements, genome compaction is now believed to be a new hallmark that promotes intron insertions, as it requires rapid DNA breakage and subsequent repair processes to gain back normal functionality.     

Molecular characterization of durum and common wheat recombinant lines carrying leaf rust resistance (Lr19) and yellow pigment (Y) genes from Lophopyrum ponticum

Chromosome 7E from Lophopyrum ponticum carries a valuable leaf rust resistant gene designated Lr19. This gene has not been widely used in common wheat breeding because of linkage with the yellow pigment gene Y. This gene tints flour yellow, reducing its appeal in bread making. However, a high level of yellow pigment is desirable in durum wheat breeding. We produced 97 recombinant chromosomes between L. ponticum transfer 7D.7E#1 and its wheat homoeologues, using the ph1b mutation that promotes homoeologous pairing. We characterized a subset of 37 of these lines with 11 molecular markers and evaluated their resistance to leaf rust and the abundance of yellow pigment. The Lr19 gene was mapped between loci Xwg420 and Xmwg2062, whereas Y was mapped distal to Xpsr687, the most distal marker on the long arm of chromosome 7. A short terminal 7EL segment translocated to 7A, including Lr19 and Y (line 1-23), has been transferred to durum wheat by backcrossing. The presence of this alien segment significantly increased the abundance of yellow pigment. The Lr19 also conferred resistance to a new durum leaf rust race from California and Mexico that is virulent on most durum wheat cultivars. The new durum lines with the recombinant 7E segment will be useful parents to increase yellow pigment and leaf rust resistance in durum wheat breeding programs. For the common wheat breeding programs, we selected the recombinant line 1-96, which has an interstitial 7E segment carrying Lr19 but not Y. This recombinant line can be used to improve leaf rust resistance without affecting flour color. The 7EL/7DL 1-96 recombinant chromosome did not show the meiotic self-elimination previously reported for a 7EL/7BL translocation.     

Synthesis and biological evaluation of 2-thiopyrimidine derivatives

Various 2-thiopyrimidine derivatives have been synthesized by an efficient, one-pot reaction of functionalized amines with either 4-isothiocyanato-4-methyl-2-pentanone or 3-isothiocyanatobutanal. All the synthesized compounds were fully characterized by elemental analysis (CHN), FT-IR, (1)H NMR, and mass spectral data. One of the compounds, 7,7,8a-trimethyl-hexahydro-thiazolo[3,2-c]pyrimidine-5-thione (17) showed good anti-inflammatory (37.4% at 100 mg/kg p.o.) and analgesic activity (75% at 100 mg/kg p.o.). 7-(1-Mercapto-3,3,4a-trimethyl-4,4a,5,9b-tetrahydro-3H-pyrido[4,3-b]indol-7-yl)-3,3,4a-trimethyl-3,4,4a,5-tetrahydro-benzo[4,5]imidazo[1,2-c]pyrimidine-1-thiol (3) showed moderate activity against CDK-1 (IC(50)=5 microM). The other compounds showed moderate anti-inflammatory (5-20%), analgesic (25-75%) and protein kinase (CDK-5, GSK-3) inhibitory activities (IC(50)> 10 microM).     

Inhibition of nuclear import of LIMK2 in endothelial cells by protein kinase C-dependent phosphorylation at Ser-283

LIM kinases (LIMKs) are mainly in the cytoplasm and regulate actin dynamics through cofilin phosphorylation. Recently, it has been reported that nuclear localization of LIMKs can mediate suppression of cyclin D1 expression. Using immunofluorescence monitoring of enhanced green fluorescent protein-tagged LIMK2 in combination with photobleaching techniques and leptomycin B treatment, we demonstrate that LIMK2 shuttles between the cytoplasm and the nucleus in endothelial cells. Sequence analysis predicted two PKC phosphorylation sites in LIMK2 but not in LIMK1. One site at Ser-283 is present between the PDZ and the kinase domain, and the other site at Thr-494 is within the kinase domain. Activation of PKC by phorbol ester treatment of endothelial cells stimulated LIMK2 phosphorylation at Ser-283 and inhibited nuclear import of LIMK2 and the PDZ kinase construct of LIMK2 (amino acids 142-638) but not of LIMK1. The PKC-delta isoform phosphorylated LIMK2 at Ser-283 in vitro. Mutational analysis indicated that LIMK2 phosphorylation at Ser-283 but not Thr-494 was functional. Serum stimulation of endothelial cells also inhibited nuclear import of PDZK-LIMK2 by protein kinase C-dependent phosphorylation of Ser-283. Our study shows that phorbol ester and serum stimulation of endothelial cells inhibit nuclear import of LIMK2 but not LIMK1. This effect was dependent on PKC-delta-mediated phosphorylation of Ser-283. Since phorbol ester enhanced cyclin D1 expression and subsequent G1-to-S-phase transition of endothelial cells, we suggest that the PKC-mediated exclusion of LIMK2 from the nucleus might be a mechanism to relieve suppression of cyclin D1 expression by LIMK2.