Isolation and characterization of isoperoxidases from Dee-Geo-Woo-Gen rice seedlings

Multiple forms of peroxidase from ten-day-old Dee-Geo-Woo-Gen (DGWG) rice seedlings were isolated by ammonium sulphate precipitation, polyacrylamide gel electrophoresis (PAGE) and DEAE-cellulose chromatography. The pH optima for P1-A and P1-B are 6.5–7.0 and 7.0 respectively. These isoperoxidases have apparently similar MWs and differ only in their electrophoretic and catalytic properties.     

Assessment of Genetic Relatedness of Crossbred Chicken Populations Using Microsatellite Markers

To measure genetic relatedness between populations, for breeding purposes, we analyzed 170 birds from six crossbred populations of three pure lines of White Leghorn chickens, using 14 microsatellite markers. All the microsatellites were polymorphic, with 2–6 alleles. The mean number of alleles per locus was 3.21. The effective number of alleles varied from 1.14 to 3.94. The observed heterozygosity varied from 0.133 to 1.00, with a mean of 0.748. The F _IS values were mostly negative, with an average of ?0.345. The mean F _ST value was 0.056. The Nm values ranged from 1.91 to 42.17. The highest genetic identity was observed between IWI × IWK and IWK × IWI. The relation between any two groups of crosses was more than 85%. The results suggest that the crossbred populations were very closely related.     

Highly sensitive quenched fluorescent substrate of Legionella major secretory protein (msp) based on its structural analysis

Legionella pneumophila has been shown to secrete a protease termed major secretory protein (Msp). This protease belongs to the M4 family of metalloproteases and shares 62.9% sequence similarity with pseudolysin (EC 3.4.24.26). With the aim of developing a specific enzymatic assay for the detection and quantification of Msp, the Fluofast substrate library was screened using both enzymes in parallel. Moreover, based on the crystal structure of pseudolysin, a model of the Msp structure was built. Screening of the peptide library identified a lead substrate specifically cleaved by Msp that was subsequently optimized by rational design. The proposed model for Msp is consistent with the enzymatic characteristics of the studied peptide substrates and provides new structural information useful for the characterization of the protease. This study leads to the identification of the first selective and high affinity substrate for Msp that is able to detect picomolar concentrations of the purified enzyme. The identified substrate could be useful for the development of a novel method for the rapid detection of Legionella.     

Discovery of novel hydroxy pyrazole based factor IXa inhibitor

Synthesis and biological data of a novel selective and efficacious factor IXa inhibitor are described along with its crystal structure in factor VIIa.     

Polymeric black tea polyphenols modulate the localization and activity of 12-O-tetradecanoylphorbol-13-acetate-mediated kinases in mouse skin: Mechanisms of their anti-tumor-promoting action

Polymeric black tea polyphenols (PBPs) have been shown to possess anti-tumor-promoting effects in two-stage skin carcinogenesis. However, their mechanisms of action are not fully elucidated. In this study, mechanisms of PBP-mediated antipromoting effects were investigated in a mouse model employing the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Compared to controls, a single topical application of TPA to mouse skin increased the translocation of protein kinase C (PKC) from cytosol to membrane. Pretreatment with PBPs 1-3 decreased TPA-induced translocation of PKC isozymes (α, β, η, γ, ε) from cytosol to membrane, whereas PBPs 4 and 5 were less effective. The levels of PKCs δ and ζ in cytosol/membrane were similar in all the treatment groups. Complementary confocal microscopic evaluation showed a decrease in TPA-induced PKCα fluorescence in PBP-3-pretreated membranes, whereas pretreatment with PBP-5 did not show a similar decrease. Based on the experiments with specific enzyme inhibitors and phosphospecific antibodies, both PBP-3 and PBP-5 were observed to decrease TPA-induced level and/or activity of phosphatidylinositol 3-kinase (PI3K) and AKT1 (pS473). An additional ability of PBP-3 to inhibit site-specific phosphorylation of PKCα at all three positions responsible for its activation [PKCα (pT497), PKC PAN (βII pS660), PKCα/βII (pT638/641)] and AKT1 at the Thr308 position, along with a decrease in TPA-induced PDK1 protein level, correlated with the inhibition of translocation of PKC, which may impart relatively stronger chemoprotective activity to PBP-3 than to PBP-5. Altogether, PBP-mediated decrease in TPA-induced PKC phosphorylation correlated well with decreased TPA-induced NF-κB phosphorylation and downstream target proteins associated with proliferation, apoptosis, and inflammation in mouse skin. Results suggest that the antipromoting effects of PBPs are due to modulation of TPA-induced PI3K-mediated signal transduction.     

Optimization of anaerobically digested distillery molasses spent wash decolorization using soil as inoculum in the absence of additional carbon and nitrogen source

The aim of this study was to achieve maximum decolorization of molasses spent wash (MSW) in absence of any additional carbon or nitrogen source using soil as inoculum. Soil samples were collected from the MSW disposal site. Colored soil samples exhibited higher pH, sugar and protein as compare to less colored samples. A decolorization of 69% was obtained using 10% (w/v) soil and 12.5% (v/v) MSW after 7 days incubation. Optimized parameters including days--6 days, pH--6, MSW--12.5% and soil concentration--40%, were obtained for maximum decolorization. A decolorization of 81% was achieved using 10% soil and 12.5% MSW after 18 days incubation in absence of any media supplement. Nearly 12% reduction in decolorization activity of the soil sample was observed over a period of 12 months when stored at 6 degrees C. It could be concluded that the decolorization of MSW might be achieved using soil as inoculum without addition of chemical amendments.     

Efforts toward oral bioavailability in factor VIIa inhibitors

Efforts toward developing orally bioavailable factor VIIa inhibitors starting from parenteral lead compound 1 are described. SAR resulted in improved physicochemical properties, leading to enhanced oral absorption in rat.     

In vivo single-particle imaging of nuclear mRNA export in budding yeast demonstrates an essential role for Mex67p

Many messenger RNA export proteins have been identified; yet the spatial and temporal activities of these proteins and how they determine directionality of messenger ribonucleoprotein (mRNP) complex export from the nucleus remain largely undefined. Here, the bacteriophage PP7 RNA-labeling system was used in Saccharomyces cerevisiae to follow single-particle mRNP export events with high spatial precision and temporal resolution. These data reveal that mRNP export, consisting of nuclear docking, transport, and cytoplasmic release from a nuclear pore complex (NPC), is fast (∼200 ms) and that upon arrival in the cytoplasm, mRNPs are frequently confined near the nuclear envelope. Mex67p functions as the principal mRNP export receptor in budding yeast. In a mex67-5 mutant, delayed cytoplasmic release from NPCs and retrograde transport of mRNPs was observed. This proves an essential role for Mex67p in cytoplasmic mRNP release and directionality of transport.     

Proteasomes Associated with the Blm10 Activator Protein Antagonize Mitochondrial Fission through Degradation of the Fission Protein Dnm1

The conserved Blm10/PA200 activators bind to the proteasome core particle gate and facilitate turnover of peptides and unfolded proteins in vitro. We report here that Blm10 is required for the maintenance of functional mitochondria. BLM10 expression is induced 25-fold upon a switch from fermentation to oxidative metabolism. In the absence of BLM10, Saccharomyces cerevisiae cells exhibit a temperature-sensitive growth defect under oxidative growth conditions and produce colonies with dysfunctional mitochondria at high frequency. Loss of BLM10 leads to reduced respiratory capacity, increased mitochondrial oxidative damage, and reduced viability in the presence of oxidative stress or death stimuli. In the absence of BLM10, increased fragmentation of the mitochondrial network under oxidative stress is observed indicative of elevated activity of the mitochondrial fission machinery. The degradation of Dnm1, the main factor mediating mitochondrial fission, is impaired in the absence of BLM10 in vitro and in vivo. These data suggest that the mitochondrial functional and morphological changes observed are related to elevated Dnm1 levels. This hypothesis is supported by the finding that cells that constitutively overexpress DNM1 display the same mitochondrial defects as blm10Δ cells. The data are consistent with a model in which Blm10 proteasome-mediated turnover of Dnm1 is required for the maintenance of mitochondrial function and provides cytoprotection under conditions that induce increased mitochondrial damage and programmed cell death.     

Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study

All molecular traffic between nucleus and cytoplasm occurs via the nuclear pore complex (NPC) within the nuclear envelope. In this study we analyzed the interactions of the nuclear transport receptors kapα2, kapβ1, kapβ1ΔN44, and kapβ2, and the model transport substrate, BSA-NLS, with NPCs to determine binding sites and kinetics using single-molecule microscopy in living cells. Recombinant transport receptors and BSA-NLS were fluorescently labeled by AlexaFluor 488, and microinjected into the cytoplasm of living HeLa cells expressing POM121-GFP as a nuclear pore marker. After bleaching the dominant GFP fluorescence the interactions of the microinjected molecules could be studied using video microscopy with a time resolution of 5 ms, achieving a colocalization precision of 30 nm. These measurements allowed defining the interaction sites with the NPCs with an unprecedented precision, and the comparison of the interaction kinetics with previous in vitro measurements revealed new insights into the translocation mechanism.