<Emphasis Type="Italic">Streptomyces</Emphasis> sp. strain SK68, isolated from peanut rhizosphere,promotes growth and alleviates salt stress in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> cv. Micro-Tom)

A novel actinobacterium, strain SK68, was isolated from the rhizosphere of peanut plant and its salinity stress alleviation ability was studied using tomato (Solanum lycopersicum cv. Micro-Tom) plants. Based on 16S rDNA based phylogenetic analysis, strain SK68 has been identified as a Streptomyces sp. Strain SK68 had branched substrate mycelium bearing smooth surfaced spores and the spore colour is brownish grey on ISP4 medium. It exhibited enzyme activities such as xylanase, cellulase, amylase, and pectinase and degraded hypoxanthine, casein, and L-tyrosine. The strain SK68 differed in its banding pattern in BOX-PCR and RAPD fingerprinting compared to the closely matching type strains Streptomyces erythrochromogenes NBRC 3304^T (AB184746), S. flavotricini NBRC 12770^T (AB184132), S. racemochromogenes NBRC 12906^T (AB184235), and S. polychromogenes NBRC 13072^T (NR041109). Strain SK68 was evaluated for its salinity stress-alleviating activity in tomato plants with 180 mmol/L NaCl under gnotobiotic condition. A significant increase in plant biomass was observed in strain SK68-inoculated tomato plants under salt stress compared to control and salt-stressed non-inoculated plants.     

The unknown face of IRE1α – Beyond ER stress

IRE1α (Inositol Requiring kinase Enzyme 1 alpha), a transmembrane protein localized to the endoplasmic reticulum (ER) is a master regulator of the unfolded protein response (UPR) pathway. The fate determining steps during ER stress-induced apoptosis are greatly attributed to IRE1α’s endoribonuclease and kinase activities. Apart from its role as a chief executioner in ER stress, recent studies have shown that upon activation in the presence or absence of ER stress, IRE1α executes multiple cellular processes such as differentiation, immune response, progression and repression of the cell cycle. Besides its crucial role in protein misfolding, the versatile contributions of IRE1α in other cellular functions are greatly unknown. In this review, we have discussed the structural conservation of IRE1 among eukaryotes, the mechanisms underlying its activation and the recent understandings of the non-apoptotic functions of IRE1 other than ER stress-induced cell death.     

Stabilization of Membrane Bound Enzyme Profiles and Lipid Peroxidation by Withania Somnifera Along with Paclitaxel on Benzo(a)pyrene Induced Experimental Lung Cancer

The present study was aimed to evaluate the therapeutic effects of Withania somnifera along with paclitaxel on lung tumor induced by benzo(a)pyrene in male Swiss albino mice. The levels of ATPase enzymes and lipid peroxidation were evaluated in lung cancer bearing mice, in erythrocyte membrane and tissues. The extent of peroxidation was estimated by measuring the thiobarbituric acid-reactive substances. Simultaneously the activities of different ATPases (Na⁺/K⁺-ATPases, Mg²⁺-ATPases and Ca²⁺-ATPases) were determined. The alterations of these enzyme activities in membrane and tissues were indicative of the tumor formation caused by benzo(a)pyrene (50 mg/kg body weight, orally) in cancer bearing animals. The activities of these enzymes were reversed to near normal control values in animals treated with Withania somnifera (400 mg/kg b.wt, orally) along with paclitaxel (33 mg/kg b.wt, i.p). Treatment with Withania somnifera along with paclitaxel altered these damage mediated through free radicals, and the treatment displays the protective role of these drugs by inhibiting free radical mediated cellular damages. Over, based on the data providing a correlation Withania somnifera along with paclitaxel provide stabilization of membrane bound enzyme profiles and decreased lipid peroxidation against benzo(a)pyrene induced lung cancer in mice.     

Nonhomologous End Joining during Restriction Enzyme-Mediated DNA Integration in Saccharomyces cerevisiae

The BamHI restriction enzyme mediates integration of nonhomologous DNA into the Saccharomyces cerevisiae genome (R. H. Schiestl and T. D. Petes, Proc. Natl. Acad. Sci. USA 88:7585–7589, 1991). The present study investigates the mechanism of such events: in particular, the mediating activity of various restriction enzymes and the processing of resultant fragment ends. Our results show that in addition to BamHI, BglII and KpnI increase DNA integration efficiencies severalfold, while Asp718, HindIII, EcoRI, SalI, SmaI, HpaI, MscI, and SnaBI do not. Secondly, the three active enzymes stimulated integrations only of fragments containing 5′ or 3′ overhangs but not of blunt-ended fragments. Thirdly, integrations mediated by one enzyme and utilizing a substrate created by another required at least 2 bp of homology. Furthermore, an Asp718 fragment possessing a 5′ overhang integrated into a KpnI (isoschizomer) site possessing a 3′ overhang, most likely by filling of the 5′ overhang followed by 5′ exonuclease digestion to produce a 3′ end. We classified and analyzed the restriction enzyme-mediated integration events in the context of their genomic positions. The majority of events integrated into single sites. In the remaining 6 of 19 cases each end of the plasmid inserted into a different sequence, producing rearrangements such as duplications, deletions, and translocations.DNA double-strand breaks occur either as a result of assaults by external agents or spontaneously during DNA metabolism, repair, or replication. Double-strand breaks may cause genome rearrangements, such as deletions, duplications, and translocations, which have been implicated in carcinogenesis. For any cell, double-strand break repair is essential, since these cytotoxic DNA lesions may cause potentially lethal losses of chromosomes. In the yeast Saccharomyces cerevisiae, DNA repair enzymes encoded by genes belonging to the RAD52 epistasis group repair double-strand breaks by homologous recombination. This process requires homologous DNA sequences, usually present on sister chromatids and on homologous chromosomes in diploids. In mammalian cells, however, the majority of double-strand breaks are repaired by nonhomologous end joining (NHEJ) (32). This event in S. cerevisiae occurs either in rad52 mutants in the presence of homology (18) or in the wild type in the absence of homology (26, 36). Joining reactions of restriction enzyme-produced DNA ends have frequently been used to study NHEJ both in vivo and in vitro. NHEJ of substrates with defined terminal configurations produced by different enzyme digestions were studied in vitro in the presence of Xenopus laevis extracts (2, 30, 43) and in vivo in mammalian cells (32) and fission yeast cells (12). In S. cerevisiae, illegitimate repair of a double-strand break in a plasmid was studied by Mezard and Nicolas (25) and the repair of double-strand breaks produced by an inducible HO endonuclease in the absence of homology was studied by Moore and Haber (26) (for the conclusions of those studies see Discussion).Schiestl and Petes (36) studied illegitimate integration events by transforming a BamHI URA3 fragment into yeast cells lacking homology to the transforming DNA (in a ura3 deletion mutant), so that integration into the genome was by illegitimate recombination. With BamHI in the transformation mixture, the URA3 fragment integrated into genomic BamHI sites and the frequency of integration increased sixfold (36). These experiments suggest that the BamHI restriction enzyme can cut chromosomal DNA in vivo and thus mediate integration of the transforming DNA into that site. Subsequently, restriction enzyme-mediated integration (REMI) has been used in a variety of organisms for insertional mutagenesis. For example, Kuspa and Loomis (20) first adapted this technology to Dictyostelium discoideum, where previously cloning of developmental genes by complementation of mutant phenotypes was not feasible. Application of REMI has led to construction of REMI-restriction fragment length polymorphism maps (19, 23) and the cloning of most developmental genes in Dictyostelium. REMI has also been adapted successfully to the ascomycete Cochliobolus heterostrophus (24) and the maize pathogenic fungus Ustilago maydis (3) to tag genes by insertional mutagenesis.Here we find that enzymes vary in their ability to mediate integration into the yeast genome. Furthermore, we present model mechanisms based on the products created from various blunt 5′ protruding single strand (PSS) and 3′ PSS joining combinations.     

Design,synthesis and evaluation of inhibitor of apoptosis protein (IAP) antagonists that are highly selective for the BIR2 domain of XIAP

We recently reported the systematic ligand-based rational design and synthesis of monovalent Smac mimetics that bind preferentially to the BIR2 domain of the anti-apoptotic protein XIAP. Expanded structure–activity relationship (SAR) studies around these peptidomimetics led to compounds with significantly improved selectivity (>60-fold) for the BIR2 domain versus the BIR3 domain of XIAP. The potent and highly selective IAP antagonist 8q (ML183) sensitized TRAIL-resistant prostate cancer cells to apoptotic cell death, highlighting the merit of this probe compound as a valuable tool to investigate the biology of XIAP.     

Induction of autophagic cell death in human ovarian carcinoma cells by Antrodia salmonea through increased reactive oxygen species generation

We reported in our previously executed studies that the fermented culture broth of Antrodia salmonea (AS), a mushroom used in Taiwanese folk medicine induced reactive oxygen species (ROS)-mediated apoptosis in human ovarian carcinoma cells. In this study, we studied the anticancer efficacies of AS (0–240 μg/ml) by examining the key molecular events implicated in cell death associated with autophagy in SKOV-3 and A2780 human ovarian carcinoma cells and clarified the fundamental molecular mechanisms. Treatment of ovarian carcinoma cells with AS-induced autophagic cell death mediated by increased microtubule-associated protein LC3-II, GFP-LC3 puncta, and acidic vesicular organelle (AVO) formation. These events are linked with the activation of p62/SQSTM1, the inhibition of ATG4B, the expression of ATG7, and the dysregulation of Beclin-1/Bcl-2 (i.e., B-cell lymphoma 2). N-acetylcysteine inhibited AS-induced ROS generation, which in turn constricted AS-induced LC3 conversion, AVO formation, and ATG4B inhibition, indicating ROS-mediated autophagy cell death. In addition, the 3-methyladenine (3-MA) or chloroquine (CQ)-induced autophagy inhibition decreased AS-induced apoptosis. Additionally, apoptosis inhibition by Z-VAD-FMK, a pan-caspase inhibitor, substantially suppressed AS-induced autophagy. Furthermore, AS-inhibited HER-2/ neu and PI3K/AKT signaling pathways which were reversed by autophagy inhibitors 3-MA and CQ. Thus, A. salmonea is a potential chemopreventive agent that is capable of activating ROS-mediated autophagic cell death in ovarian carcinoma cells.     

Co-encapsulation of lactic acid bacteria and prebiotic with alginate-fenugreek gum-locust bean gum matrix: Viability of encapsulated bacteria under simulated gastrointestinal condition and during storage time

The aim of this study was to enhance the viability of probiotic strains Pediococcus pentosaceus KID7, Lactobacillus plantarum KII2, Lactobacillus fermentum KLAB6 and Lactobacillus helveticus KII13 in gastrointestinal transit, freeze-drying condition and during storage time by microencapsulation using a combination of alginate, fenugreek gum and locust bean gum. The microcapsules were prepared using various ratio of alginate to fenugreek gum to locust bean gum and tested for its dissolution in colonic fluid. The combination that efficiently dissolved in colonic fluid was selected for co-encapsulation of the probiotic strains and prebiotics to produce synbiotic microcapsules. Further, we observed that the bacteria encapsulated with alginate-fenugreek gum-locust bean gum (AFL) matrix tolerated gastrointestinal condition efficiently compared to non-encapsulated bacteria. The encapsulated bacterial cells retained higher viability than non-encapsulated cells during freeze-drying condition and subsequent storage for 3 months at 4°C. These results show the utility of AFL matrix in microencapsulation of probiotics for use in food industry.     

Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in mammalian cells

Mammalian cells repair DNA double-strand breaks by illegitimate end-joining or by homologous recombination. We investigated the effects of restriction enzymes on illegitimate and homologous DNA integration in mammalian cells. A plasmid containing the neo^R expression cassette, which confers G418 resistance, was used to select for illegitimate integration events in CHO wild-type and xrcc5 mutant cells. Co-transfection with the restriction enzymes BamHI, BglII, EcoRI and KpnI increased the efficiency of linearized plasmid integration up to 5-fold in CHO cells. In contrast, the restriction enzymes did not increase the integration efficiency in xrcc5 mutant cells. Effects of restriction enzymes on illegitimate and homologous integration were also studied in mouse embryonic stem (ES) cells using a plasmid containing the neo^R gene flanked by exon 3 of Hprt. The enzymes BamHI, BglII and EcoRI increased the illegitimate integration efficiency of transforming DNA several-fold, similar to the results for CHO cells. However, all three enzymes decreased the absolute frequency of homologous integration ~2-fold, and the percentage of homologous integration decreased >10-fold. This suggests that random DNA breaks attract illegitimate recombination (IR) events that compete with homology search.     

Modulation of Macrophage Polarization and HMGB1-TLR2/TLR4 Cascade Plays a Crucial Role for Cardiac Remodeling in Senescence-Accelerated Prone Mice

The aim of this study was to investigate the role of macrophage polarization in aging heart. Macrophage differentiation is pathogenically linked to many inflammatory and immune disorders. It is often preceded by myocardial inflammation, which is characterized by increased cardiac damage and pro-inflammatory cytokine levels. Therefore, we investigated the hypothesis that senescence accelerated-prone (SAMP8) mice cardiac tissue would develop macrophage polarization compared with senescence-resistant control (SAMR1) mice. Both SAMP8 and SAMR1 mice were sacrificed when they became six month old. We evaluated, histo-pathological changes and modifications in protein expression by Western blotting and immuno-histochemical staining for M1 and M2 macrophage markers, high mobility group protein (HMG)B1 and its cascade proteins, pro-inflammatory factors and inflammatory cytokines in cardiac tissue. We observed significant upregulation of HMGB1, toll-like receptor (TLR)2, TLR4, nuclear factor (NF)κB p65, tumor necrosis factor (TNF)α, cyclooxygenase (COX)2, interferon (IFN)γ, interleukin (IL)-1β, IL-6 and M1 like macrophage specific marker cluster of differentiation (CD)68 expressions in SAMP8 heart. In contrast, M2 macrophage specific marker CD36, and IL-10 expressions were down-regulated in SAMP8 mice. The results from the study demonstrated that, HMGB1-TLR2/TLR4 signaling cascade and induction of phenotypic switching to M1 macrophage polarization in SAMP8 mice heart would be one of the possible reasons behind the cardiac dysfunction and thus it could become an important therapeutic target to improve the age related cardiac dysfunction.