An insecticidal GroEL protein with chitin binding activity from Xenorhabdus nematophila

Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an approximately 58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of approximately 3.6 microg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed alpha-chitin binding activity with Kd approximately 0.64 microm and Bmax approximately 4.68 micromol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an approximately 8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity.     

Effect on lycopene, β-carotene,ascorbic acid and phenolic content in tomato fruits infected by Alternaria alternata and its toxins (TeA,AOH and AME)

Tomato is considered as one of the most important sources of nutrients such as lycopene, β-carotene, flavonoids, ascorbic acid (vitamin C) and hydroxyl-cinnamic acid derivatives. The quality and quantity of nutrients in tomato fruits were decreased during the severe infection of Alternaria alternata. The present study deals with the estimation of lycopene, β-carotene, phenolic and ascorbic acid content in tomato fruits which were infected with A. alternata and its toxins such as tenuazonic acid (TeA), alternariol (AOH) and alternariol monomethyl ether (AME). The lycopene, β-carotene, ascorbic acid and phenolic content were found lowest in pathogen-infected fruits i.e. (0.66 ± 0.03 mg/g), (0.14 ± 0.01 mg/g), (1.89 ± 0.2 mg/g) and (0.58 ± 0.05 mg/g), respectively, followed by toxins-treated samples as compared to the control. The results concluded that A. alternata mostly affects the nutritional values of tomato fruits due to the combined effect of the toxins.     

Opposing roles for actin in Cdc42p polarization

In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399-416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site.     

Towards compendia of negative genetic association studies: an example for Alzheimer disease

Most genetic sequence variants that contribute to variability in complex human traits will have small effects that are not readily detectable with population samples typically used in genetic association studies. A potentially valuable tool in the gene discovery process is meta-analysis of the accumulated published data, but in order to be valid these require a sample of studies representative of the true genetic effect and thus hypothetically should include some positive and an abundance of negative reports. A survey of the literature on association studies for Alzheimer disease (AD) from January 2004–April 2005, identified 138 studies, 86 of which reported positive findings other than for apolipoprotein E (APOE), strongly indicative of publication bias. We report here an analysis of 62 genetic markers, tested for association with AD risk as well as for possible effects upon quantitative indices of AD severity (mini-mental state examination scores, age-at-onset, and cerebrospinal fluid (CSF) β-amyloid (Aβ) and CSF tau proteins). Within this set, only modest signals were present that, with the exception of APOE are easily lost when corrections for multiple hypotheses are applied. In isolation, results are thus broadly negative. Genes studied encompass both novel candidates as well as several recently claimed to be associated with AD (e.g. urokinase plasminogen activator (PLAU) and acetyl-coenzyme A acetyltransferase 1 (ACAT1)). By reporting these data we hope to encourage the publication of gene compendia to guide further studies and aid future meta-analyses aimed at resolving the involvement of genes in complex human traits.     

Decolorization and purification of crude protease from Rhizopus oryzae by activated charcoal and its electrophoretic analysis

Activated charcoal decolorized and partially purified the protease from a crude extract of solid state fermentation of wheat bran by Rhizopus oryzae. Treatment for 5 min was sufficient. Depending on the initial colour intensity of crude, the charcoal to crude extract ratio could be optimized to achieve 90% decolorization, 85% enzyme recovery, and over a 3-fold purification, even up to 20-fold variation in batch size (from 1 ml to 20 ml crude extract). Decolorization followed the Freundlich and the Langmuir models, the Freundlich constant, n, being 2.74. Partial purification was confirmed by native PAGE and the protease band identified by gelatin-PAGE. SDS-PAGE showed the protease consisted of two sub-units (about 22 and 24 kDa). List of symbols: c _o, initial solute concentration in liquid before adsorption; c ^*, equilibrium solute concentration in liquid after adsorption; k, empirical constant for Freundlich adsorption isotherm; U, unit of protease activity; v, volume of solution per unit weight of adsorbent.     

Prophylactic and therapeutic use of antibodies for protection against respiratory infection with Francisella tularensis

The role of Abs in protection against respiratory infection with the intracellular bacterium Francisella tularensis is not clear. To investigate the ability of Abs to clear bacteria from the lungs and prevent systemic spread, immune serum was passively administered i.p. to naive mice before intranasal F. tularensis live vaccine strain infection. It was found that immune serum treatment provided 100% protection against lethal challenge while normal serum or Ig-depleted immune serum provided no protection. Protective efficacy was correlated with increased clearance of bacteria from the lung and required expression of FcgammaR on phagocytes, including macrophages and neutrophils. However, complement was not required for protection. In vitro experiments demonstrated that macrophages were more readily infected by Ab-opsonized bacteria but became highly efficient in killing upon activation by IFN-gamma. Consistent with this finding, in vivo Ab-mediated protection was found to be dependent upon IFN-gamma. SCID mice were not protected by passive Ab transfer, suggesting that T cells but not NK cells serve as the primary source for IFN-gamma. These data suggest that a critical interaction of humoral and cellular immune responses is necessary to provide sterilizing immunity against F. tularensis. Of considerable interest was the finding that serum Abs were capable of conferring protection against lethal respiratory tularemia when given 24-48 h postexposure. Thus, this study provides the first evidence for the therapeutic use of Abs in Francisella-infected individuals.     

Sodium butyrate suppresses NOD1-mediated inflammatory molecules expressed in bovine hepatocytes during iE-DAP and LPS treatment

Nucleotide oligomerization domain protein-1 (NOD1), a cytosolic pattern recognition receptor for the γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) is associated with the inflammatory diseases. Very little is known how bovine hepatocytes respond to specific ligands of NOD1 and sodium butyrate (SB). Therefore, the aim of our study was to investigate the role of bovine hepatocytes in NOD1-mediated inflammation during iE-DAP or LPS treatment or SB pretreatment. To achieve this aim, hepatocytes separated from cows at ∼160 days in milk (DIM) were divided into six groups: The nontreated control group (CON), the iE-DAP-treated group (DAP), the lipopolysaccharide-treated group (LPS), iE-DAP with SB group (DSB), LPS with SB group (LSB), and the SB group. Both iE-DAP and LPS highly increased the expression of both NOD1 and RIPK2, the two key factors for the immune response in hepatocytes. IκBα, NF-κB/p65, and MAP kinases (ERK, JNK, and p38) were activated through phosphorylation. The activation of NF-κB and MAPK pathway consequently increased the proinflammatory cytokines, IL-6, TNF-α, IL-8, and IFN-γ and the chemokines CCL5, CCL20, and CXCL-10. Both treatments improved iNOS/NOS2 expression. However, iE-DAP was failed to express acute phase protein SAA3, but HP and LPS HP but SAA3. These ligands also increased LRRK2, TAK1, TAB1, and β-defensins expression. The SB pretreatment at lower dose restored the function of hepatocytes by suppressing these increased molecules, as HDAC3 was inhibited. The activated NOD1 negatively regulated the expression of FOXA2. Altogether these data suggest an important role of bovine hepatocytes to promote immune responses via NOD1 expression during infection in the liver and a key role of SB to attenuate inflammation.     

HHV-5 epitope: A potential vaccine candidate with high antigenicity and large coverage

Outbreak of Human Herpes virus-5 (HHV-5) infection in emerging countries has raised worldwide health concern owing to prevalence of congenital impairments and life threatening consequences in immunocompromised individuals. Thus, there lies an impending need to develop vaccine against HHV-5. HHV-5 enters into host cells with the help of necessary components glycoprotein B (gB) and H/L. In this study, the conformational linear B-cell and T-cell epitopes for gB of HHV-5 have been predicted using conformational approaches, for their possible collective use as vaccine candidates. We examined epitope’s interactions with major histocompatibility complexes using molecular docking and also investigated their stable binding with specific toll like receptor-2 (TLR2), present on host cells during HHV-5 infection. Predicted MHC-I epitope ‘LVAIAVVII’ with high antigenicity and large coverage of HLA alleles was found to superimpose on MHC-II epitope (Rank 1) and was also identified to be the core sequence of putative B cell epitope ‘ILVAIAVVIITYLI’. Resulting epitope was found to have consistent interaction with TLR2 during long term (100?ns) MD run. We also validated this nonamer epitope for its dissimilarity with human genome and high population coverage, suggesting it to be a potential vaccine candidate with higher coverage for both the MHC alleles of Indian population.

Communicated by Ramaswamy H. Sarma