Cells transformed by PLC-gamma 1 overexpression are highly sensitive to clostridium difficile toxin A-induced apoptosis and mitotic inhibition

Phospholipase C-γl (PLC-γl) expression is associated with cellular transformation. Notably, PLC-gamma is up-regulated in colorectal cancer tissue and breast carcinoma. Because exotoxins released by Clostridium botulinum have been shown to induce apoptosis and promote growth arrest in various cancer cell lines, we examined here the potential of Clostridium difficile toxin A to selectively induce apoptosis in cells transformed by PLC-γl overexpression. We found that PLC-γl-transformed cells, but not vectortransformed (control) cells, were highly sensitive to C. difficile toxin A-induced apoptosis and mitotic inhibition. Moreover, expression of the proapoptotic Bcl2 family member, Bim, and activation of caspase-3 were significantly up-regulated by toxin A in PLC-γl-transformed cells. Toxin A-induced cell rounding and paxillin dephosphorylation were also significantly higher in PLC-γl-transformed cells than in control cells. These findings suggest that C. difficile toxin A may have potential as an anticancer agent against colorectal cancers and breast carcinomas in which PLC-γl is highly up-regulated.     

Over-expression of a RhoA-specific guanine nucleotide exchange factor, p190RhoGEF, in mouse dendritic cells negatively regulates cellular responses to bacterial lipopolysaccharide

We studied the role of a RhoA-specific guanine nucleotide exchange factor (p190RhoGEF) in dendritic cells (DCs), using transgenic (TG) mice that over-express a full gene of p190RhoGEF under the control of an invariant chain promoter. TG mice lacked localization of activated DCs to the T cell zone in the spleen and had reduced serum levels of IL-6 in response to lipopolysaccharide (LPS) injection. DCs from these mice also showed reduced surface expression of CD86, CD40, and CD205, but not MHCII, as well as a reduced capability to uptake antigen. Moreover, chemokine-driven migration and secretion of IL-6, but not of IL-12, were impaired after LPS-stimulation of TG DCs. Collectively, these results suggest that over-expressing p190RhoGEF negatively regulates conventional DC function in response to bacterial LPS infection.     

S‐ to N‐Acyl transfer in S‐acylcysteine isopeptides via 9‐, 10‐, 12‐, and 13‐membered cyclic transition states

S‐Acyl cysteine peptides containing α‐, β‐ or γ‐amino acid residues undergo long‐range S‐ to N‐acyl transfer to give analogs of native tripeptides and tetrapeptides containing additional carbon atoms in the chain. The ease of intramolecular S → N‐acyl transfer relative to intermolecular transacylation is favored increasingly for 9 < 12 < 13 ~ 10‐membered cyclic transition states; the observed order is explained on conformational and intermolecular interaction considerations. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

A quantitative and direct PCR assay for the subspecies-specific detection of Clavibacter michiganensis subsp. michiganensis based on a ferredoxin reductase gene

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis is the causal agent of canker disease in tomato. Because it is very important to control newly introduced inoculum sources from commercial materials, the specific detection of this pathogen in seeds and seedlings is essential for effective disease control. In this study, a novel and efficient assay for the detection and quantitation of C. michiganensis subsp. michiganensis in symptomless tomato and red pepper seeds was developed. A pair of polymerase chain reaction (PCR) primers (Cmm141F/R) was designed to amplify a specific 141 bp fragment on the basis of a ferredoxin reductase gene of C. michiganensis subsp. michiganensis NCPPB 382. The specificity of the primer set was evaluated using purified DNA from 16 isolates of five C. michiganensis subspecies, one other Clavibacter species, and 17 other reference bacteria. The primer set amplified a single band of expected size from the genomic DNA obtained from the C. michiganensis subsp. michiganensis strains but not from the other C. michiganensis subspecies or from other Clavibacter species. The detection limit was a single cloned copy of the ferredoxin reductase gene of C. michiganensis subsp. michiganensis. In conclusion, this quantitative direct PCR assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of seeds and seedlings with a low level or latent infection of C. michiganensis subsp. michiganensis.     

Hypolithic microbial communities: between a rock and a hard place

Drylands are the largest terrestrial biome on Earth and a ubiquitous feature is desert pavement terrain, comprising rocks embedded in the mineral soil surface. Quartz and other translucent rocks are common and microbial communities termed hypoliths develop as biofilms on their ventral surfaces. In extreme deserts these represent major concentrations of biomass, and are emerging as key to geobiological processes and soil stabilization. These highly specialized communities are dominated by cyanobacteria that support diverse heterotrophic assemblages. Here we identify global-scale trends in the ecology of hypoliths that are strongly related to climate, particularly with regard to shifts in cyanobacterial assemblages. A synthesis of available data revealed a linear trend for colonization with regard to climate, and we suggest potential application for hypoliths as 'biomarkers' of aridity on a landscape scale. The potential to exploit the soil-stabilizing properties of hypolithic colonization in environmental engineering on dryland soils is also discussed.     

Transcriptional network analysis of the tryptophan-accumulating rice mutant during grain filling

In a previous study, we selected a high tryptophan (Trp)-accumulating rice (Oryza sativa L.) mutant line by in vitro mutagenesis using gamma rays. To obtain detailed information about the Trp biosynthetic pathway during the grain-filling in rice, we investigated the gene expression profiles in the wild-type (cv. Dongan) and the high-level Trp-accumulating mutant line (MRVII-33) at five different grain-filling stages using microarray analysis. The mutant line showed approximately 6.3-fold higher Trp content and 2.3-fold higher amino acids compared with the original cultivar at the final stage (stage V). The intensity of gene expression was analyzed and compared between the wild-type and mutant line at each of the five grain-filling stages using the Rice 4?×?44K oligo DNA microarray. Among the five stages, stage III showed the highest gene expression changes for both up- and down-regulated genes. Among the Trp biosynthesis-related genes, trpG showed high expression in the mutant line during stages I to IV and trpE showed higher at stage III. Gene clustering was performed based on the genes of KEGG's amino acid metabolism, and a total of 276 genes related to amino acid metabolism were placed into three clusters. The functional annotation enrichment analysis of the genes classified into the three clusters was also conducted using ClueGO. It was found that cluster 3 uniquely included biological processes related to aromatic amino acid metabolism. These results suggest that gene analysis based on microarray data is useful for elucidating the biological mechanisms of Trp accumulation in high Trp-accumulating mutants at each of the grain-filling stages.     

Single-molecule pull-down for studying protein interactions

This protocol describes a single-molecule pull-down (SiMPull) assay for analyzing physiological protein complexes. The assay combines the conventional pull-down assay with single-molecule total internal reflection fluorescence (TIRF) microscopy and allows the probing of single macromolecular complexes directly from cell or tissue extracts. In this method, antibodies against the protein of interest are immobilized on a passivated microscope slide. When cell extracts are applied, the surface-tethered antibody captures the protein together with its physiological interaction partners. After washing away the unbound components, single-molecule fluorescence microscopy is used to probe the pulled-down proteins. Captured proteins are visualized through genetically encoded fluorescent protein tags or through antibody labeling. Compared with western blot analysis, this ultrasensitive assay requires considerably less time and reagents and provides quantitative data. Furthermore, SiMPull can distinguish between multiple association states of the same protein. SiMPull is generally applicable to proteins from a variety of cellular contexts and to endogenous proteins. Starting with the cell extracts and passivated slides, the assay requires 1.5-2.5 h for data acquisition and analysis.     

New and already known acanthocephalans mostly from mammals in Vietnam, with descriptions of two new genera and species in Archiacanthocephala

Adults of 2 new species and 2 new genera of acanthocephalans in class Archiacanthocephala, collected between 1998 and 2004 in Vietnam from the intestines of mammals, are described, i.e., Cucullanorhynchus constrictruncatus n. gen., n. sp. (Oligacanthorhynchidae) from a leopard Panthera pardus (Linnaeus) (Mammalia: Felidae) and Paraprosthenorchis ornatus n. gen. n. sp. (Oligacanthorhynchidae) from the Chinese pangolin Manis pentadactyla (Linnaeus) (Mammalia: Manidae). Adult Sphaerechinorhynchus macropisthospinus Amin, Wongsawad, Marayong, Saehoong, Suwattanacoupt, and Sey, 1998 (Plagiorhynchidae) are described for the first time from 2 females collected from a tiger Panthera tigris (Linnaeus) (Mammalia: Felidae) and from 1 male from a water monitor Varanus salvator Laurenti (Reptilia: Varanidae). Characteristic features distinguishing the new species or genera from related taxa are as follows. The trunk of C. constrictruncatus has an anterior hood in both sexes and a posterior constriction in females. The anterior trunk of P. ornatus has many small festoons and proboscis hooks are inserted in elevated papillae separated by beady, near hexagonal, ornate grids.