In vitro cytotoxic and in vivo anxiolytic study of methanolic crude extract of Streculia villosa seeds

We have aimed to evaluate the in vitro cytotoxic and in vivo anxiolytic and sedative activities of the methanolic extract of Sterculia villosa roxb seeds. The dried powder of the seeds was extracted with methanol which was then tested to ascertain the neuropharmacological and cytotoxic potentials. The methanolic extract of Sterculia villosa roxb were subjected to Brine Shrimp lethality bioassay for possible cytotoxicity having LC 50 of 8.672μg/ml. However, fractions produced concentration dependent increase in percent of mortality of Brine Shrimp nauplii indicates the presence of cytotoxic property. We also have studied for possible sedative and anxiolytic activity of the methanolic seed extract of Sterculia villosa roxb in mice. This study includes hole cross, open field, thiopental- sodium induced sleeping time and elevated-plus maze (EPM) tests at the dose of 200 mg/kg while on the peripheral and central nervous system the extract mild to moderately decreased the locomotor activity of mice in hole cross, open field and EPM test. However, the extract moderately has minimized the onset of sleep and slightly has maximized the duration of sleep while administered with thiopental sodium

Variations in TcdB Activity and the Hypervirulence of Emerging Strains of Clostridium difficile

Hypervirulent strains of Clostridium difficile have emerged over the past decade, increasing the morbidity and mortality of patients infected by this opportunistic pathogen. Recent work suggested the major C. difficile virulence factor, TcdB, from hypervirulent strains (TcdBHV) was more cytotoxic in vitro than TcdB from historical strains (TcdBHIST). The current study investigated the in vivo impact of altered TcdB tropism, and the underlying mechanism responsible for the differences in activity between the two forms of this toxin. A combination of protein sequence analyses, in vivo studies using a Danio rerio model system, and cell entry combined with fluorescence assays were used to define the critical differences between TcdBHV and TcdBHIST. Sequence analysis found that TcdB was the most variable protein expressed from the pathogenicity locus of C. difficile. In line with these sequence differences, the in vivo effects of TcdBHV were found to be substantially broader and more pronounced than those caused by TcdBHIST. The increased toxicity of TcdBHV was related to the toxin's ability to enter cells more rapidly and at an earlier stage in endocytosis than TcdBHIST. The underlying biochemical mechanism for more rapid cell entry was identified in experiments demonstrating that TcdBHV undergoes acid-induced conformational changes at a pH much higher than that of TcdBHIST. Such pH-related conformational changes are known to be the inciting step in membrane insertion and translocation for TcdB. These data provide insight into a critical change in TcdB activity that contributes to the emerging hypervirulence of C. difficile

Heterologous delivery of the TcdB enzymatic domain.

<p>CHO cells were treated with LFnTcdB_HIST(enz) or LFnTcdB_HV(enz) in the presence of PA for 24 h and cell viability was determined by WST-8 staining. The error bars represent the standard deviation from the mean of three samples.</p

Representative photographs of zebrafish after 24 h exposure to TcdB.

<p>(A) Zebrafish after exposure to 10 nM TcdB_HIST. Cardiac damage is evident by pericardial edema (black arrow) and blood accumulation (white arrow). (B) Exposure to 10 nM TcdB_HV causes tissue necrosis and death of the zebrafish. (C) Zebrafish treated with 1 nM TcdB_HIST appear normal, with little to no edema. (D) Zebrafish after exposure to 1 nM TcdB_HV. Arrow indicates damage to the yolk sac, visualized by tissue discoloration and necrosis. (E) Untreated control.</p

Comparison of the timing of cell entry between TcdB_HV and TcdB_HIST.

<p>CHO cells were pretreated with TcdB_HIST or TcdB_HV and the lysosomotropic inhibitor, ammonium chloride, was added at the indicated time points. Cytopathic effects were determined at 2 h (A) and 12 h (B), and black bars represent cells treated with TcdB_HV while gray bars represent TcdB_HIST. The error bars mark the standard deviation from the mean. C, untreated control. I, inhibitor alone. T, TcdB alone.</p

Representation of sequence variation between TcdB_HIST and TcdB_HV.

<p>The illustration depicts TcdB_HIST (top) and TcdB_HV (bottom) divided into functional domains: glucosyltransferase (A), cysteine protease (B), translocation (C), and receptor binding (D). (A) Trp 102 and the DXD motif of the glucosyltransferase domain are conserved between TcdB_HIST and TcdB_HV. The amino acids making up the substrate recognition region (SR) show 99% similarity between the strains, and the overall amino acid identity of the domain is 96%. (B) The catalytic triad of the cysteine protease domain remains unchanged between TcdB_HIST and TcdB_HV, and the overall identity of the domain is 96%. TcdB_HIST contains a cysteine at residue 870, while TcdB_HV contains a tyrosine at residue 870. (C) Amino acid identity of the translocation domain is 91%, with a 97% sequence identity occurring in the hydrophobic region (HR). TcdB_HV contains two cysteines in this domain, which are not found in this region of TcdB_HIST. (D) TcdB_HIST and TcdB_HV share an identity of 88% in the putative receptor binding domain. Gray boxes symbolize the CROP (clostridial repetitive oligopeptide) regions, 4 large repeats and 18 small repeats. White boxes indicate TcdB_HV CROPs that have less than 80% similarity to TcdB_HIST. (E) Coomassie stained SDS-PAGE analysis of 1 µg of each TcdB_HIST and TcdB_HV.</p

TNS analysis of pH-induced hydrophobic transitions in TcdB_HIST and TcdB_HV.

<p>TcdB_HIST or TcdB_HV was incubated with TNS for 20 min at 37°C. Samples were analyzed for changes in TNS fluorescence, and the emission profile of each pH is shown and labeled. Panels (A) and (B) represent pH 4.0 to pH 7.0 and panels (C) and (D) show TNS fluorescence of TcdB between pH 5.0 and 6.0. Each spectrum represents the experimental sample with background (TNS and buffer alone) subtracted.</p

Tryptophan emission of TcdB_HIST and TcdB_HV at acidic and neutral pH.

<p>The fluorescent spectrum of each sample is shown and labeled; each spectrum represents the experimental sample minus background fluorescence of buffer alone. Panels (A) and (B) show tryptophan emission of TcdB_HIST and TcdB_HV from pH 4.0 to pH 7.0 while panels (C) and (D) highlight the changes in tryptophan fluorescence between pH 5.0 and pH 6.0.</p

Flow-based analysis of cell binding.

<p>TcdB_HIST and TcdB_HV were tested for their ability to bind CHO (A) and HL-1 cells (B). 40 nM of fluorescently labeled TcdB was incubated with cells on ice, and binding was determined by flow cytometry. TcdB_HIST and TcdB_HV are indicated, and shaded peaks represent cells incubated with unlabeled TcdB. (C) Mean fluorescence intensity (MFI) vs TcdB concentration on HL-1 cells. Please note the difference in axis for TcdB_HIST and TcdB_HV.</p

The Mechanism of Bacillus anthracis Intracellular Germination Requires Multiple and Highly Diverse Genetic Loci▿

In an effort to better understand the mechanisms by which Bacillus anthracis establishes disease, experiments were undertaken to identify the genes essential for intracellular germination. Eighteen diverse genetic loci were identified via an enrichment protocol using a transposon-mutated library of B. anthracis spores, which was screened for mutants delayed in intracellular germination. Fourteen transposon mutants were identified in genes not previously associated with B. anthracis germination and included disruption of factors involved in membrane transport, transcriptional regulation, and intracellular signaling. Four mutants contained transposon insertions in gerHA, gerHB, gerHC, and pagA, respectively, each of which has been previously associated with germination or survival of B. anthracis within macrophages. Strain MIGD101 (named for macrophage intracellular germination defective 101) was of particular interest, since this mutant contained a transposon insertion in an intergenic region between BAs2807 and BAs2808, and was the most highly represented mutant in the enrichment. Analysis of B. anthracis MIGD101 by confocal microscopy and differential heat sensitivity following macrophage infection revealed ungerminated spores within the cell. Moreover, B. anthracis MIGD101 was attenuated in cell killing relative to the parent strain. Further experimental analysis found that B. anthracis MIGD101 was defective in five known B. anthracis germination pathways, supporting a mechanism wherein the intergenic region between BAs2807 and BAs2808 has a global affect on germination of this pathogen. Collectively, these findings provide insight into the mechanisms supporting B. anthracis germination within host cells