A mutant of escherichia coli defective in removing 3′ terminal nucleotides from some transfer RNA precursor molecules

The conversion of precursor RNA into bacteriophage T4 proline and serine transfer RNAs includes two steps for the enzymatic removal of nucleotides from the 3′ ends of RNA chains. Neither of these steps occur following infection of a mutant of Escherichia coli that was previously shown to block the suppressor function of T4 serine transfer RNA. Cell-free extracts of this mutant are furthermore deficient in a wild type enzyme activity that removes nucleotides from the 3′ ends of one of the RNA chains described above. The relation of this enzyme to other 3′ ribonucleases is not known.We subsequently examined the mutant for its ability to support the biosynthesis of other bacteriophage transfer RNAs. In one instance that is analogous to the proline-serine precursor RNA, maturation of the precursor RNA was blocked during infection of mutant cells. In another instance, precursor RNA maturation was normal, even though this involved the removal of 3′ nucleotides. These observations point to the possible existence of at least two 3′ ribonucleases for the biosynthesis of transfer RNAs.     

An algorithm for discriminating sequences and its application to yeast transfer RNA

We describe an algorithm for finding nucleotide residues stronglycorrelated with the amino acid acceptor functions of transferRNAs. The algorithm exploits the fact that each tRNA acceptsonly one of 20 amino acids. The algorithm is applied to 37 Saccharomycescerevisiae transfer RNAs. Received on January 28, 1987     

Activation of peroxisome proliferator-activated receptor-γ by rosiglitazone improves lipid homeostasis at the adipose tissue-liver axis in ethanol-fed mice

The development of alcohol-induced fatty liver is associated with a reduction of white adipose tissue (WAT). Peroxisome proliferator-activated receptor (PPAR)-γ prominently distributes in the WAT and plays a crucial role in maintaining adiposity. The present study investigated the effects of PPAR-γ activation by rosiglitazone on lipid homeostasis at the adipose tissue-liver axis. Adult C57BL/6 male mice were pair fed liquid diet containing ethanol or isocaloric maltose dextrin for 8 wk with or without rosiglitazone supplementation to ethanol-fed mice for the last 3 wk. Ethanol exposure downregulated adipose PPAR-γ gene and reduced the WAT mass in association with induction of inflammation, which was attenuated by rosiglitazone. Ethanol exposure stimulated lipolysis but reduced fatty acid uptake capacity in association with dysregulation of lipid metabolism genes. Rosiglitazone normalized adipose gene expression and corrected ethanol-induced lipid dyshomeostasis. Ethanol exposure induced steatosis and upregulated inflammatory genes in the liver, which were attenuated by rosiglitazone. Hepatic peroxisomal fatty acid β-oxidation was suppressed by ethanol in associated with inhibition of acyl-coenzyme A oxidase 1. Rosiglitazone elevated plasma adiponectin level and normalized peroxisomal fatty acid β-oxidation rate. However, rosiglitazone did not affect ethanol-reduced very low-density lipoprotein secretion from the liver. These results demonstrated that activation of PPAR-γ by rosiglitazone reverses ethanol-induced adipose dysfunction and lipid dyshomeostasis at the WAT-liver axis, thereby abrogating alcoholic fatty liver.     

Pancreatic response to endotoxin after chronic alcohol exposure: switch from apoptosis to necrosis?

Chronic alcohol consumption is known to increase the susceptibility to acute and chronic pancreatitis, and it is likely that a cofactor is required to initiate the progression to alcoholic pancreatitis. The severity and complications of alcoholic and nonalcoholic acute pancreatitis may be influenced by a number of cofactors, including endotoxemia. To explore the effect of a possible cofactor, we used endotoxin [lipopolysaccharide (LPS)] as a tool to induce cellular injury in the alcoholic pancreas. Single, increasing doses of endotoxin were injected in rats fed an alcohol or control diet and killed 24 h after the injection. We examined the mechanism by which LPS exacerbates pancreatic injury in alcohol-fed rats and whether the injury is associated with apoptosis or necrosis. We showed that chronic alcohol exposure alone inhibits apoptosis through the intrinsic pathway and the downstream apoptosis executor caspase-3 compared with the controls. Pancreatic necrosis and inflammation increased after LPS injection in control and alcohol-fed rats in a dose-dependent fashion but with a significantly greater response in the alcohol-fed animals. Caspase activities and TdT-mediated dUTP nick-end labeling positivity were lower in the alcoholic pancreas injected with LPS, whereas the histopathology and inflammation were more severe compared with the control-fed animals. Assessment of a putative indicator of necrosis, the ratio of ADP to ATP, indicated that alcohol exposure accelerates pancreatic necrosis in response to endotoxin. These findings suggest that the pancreas exposed to alcohol is more sensitive to LPS-induced damage because of increased sensitivity to necrotic cell death rather than apoptotic cell death. Similar to the liver, the pancreas is capable of responding to LPS with a more severe response in alcohol-fed animals, favoring pancreatic necrosis rather than apoptosis. We speculate that this mechanism may occur in acute alcoholic pancreatitis patients.     

Protein-protein interactions among Helicobacter pylori cag proteins

Many Helicobacter pylori isolates contain a 40-kb region of chromosomal DNA known as the cag pathogenicity island (PAI). The risk for development of gastric cancer or peptic ulcer disease is higher among humans infected with cag PAI-positive H. pylori strains than among those infected with cag PAI-negative strains. The cag PAI encodes a type IV secretion system that translocates CagA into gastric epithelial cells. To identify Cag proteins that are expressed by H. pylori during growth in vitro, we compared the proteomes of a wild-type H. pylori strain and an isogenic cag PAI deletion mutant using two-dimensional difference gel electrophoresis (2D-DIGE) in multiple pH ranges. Seven Cag proteins were identified by this approach. We then used a yeast two-hybrid system to detect potential protein-protein interactions among 14 Cag proteins. One heterotypic interaction (CagY/7 with CagX/8) and two homotypic interactions (involving H. pylori VirB11/ATPase and Cag5) were similar to interactions previously reported to occur among homologous components of the Agrobacterium tumefaciens type IV secretion system. Other interactions involved Cag proteins that do not have known homologues in other bacterial species. Biochemical analysis confirmed selected interactions involving five of the proteins that were identified by 2D-DIGE. Protein-protein interactions among Cag proteins are likely to have an important role in the assembly of the H. pylori type IV secretion apparatus.     

Calls,colours, shape,and genes: a multi‐trait approach to the study of geographic variation in the Amazonian frog Allobates femoralis

Evolutionary divergence in behavioural traits related to mating may represent the initial stage of speciation. Direct selective forces are usually invoked to explain divergence in mate‐recognition traits, often neglecting a role for neutral processes or concomitant differentiation in ecological traits. We adopted a multi‐trait approach to obtain a deeper understanding of the mechanisms behind allopatric divergence in the Amazonian frog, Allobates femoralis. We tested the null hypothesis that geographic distance between populations correlates with genetic and phenotypic divergence, and compared divergence between mate‐recognition (acoustic) and ecological (coloration, body‐shape) traits. We quantified geographic variation in 39 phenotypic traits and a mitochondrial DNA marker among 125 individuals representing eight populations. Geographic variation in acoustic traits was pronounced and tracked the spatial genetic variation, which appeared to be neutral. Thus, the evolution of acoustic traits tracked the shared history of the populations, which is unexpected for pan‐Amazonian taxa or for mate‐recognition traits. Divergence in coloration appeared uncorrelated with genetic distance, and might be partly attributed to local selective pressures, and perhaps to Batesian mimicry. Divergence in body‐shape traits was low. The results obtained depict a complex evolutionary scenario and emphasize the importance of considering multiple traits when disentangling the forces behind allopatric divergence. ©2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 826–838.     

Dominant-negative Inhibitors of the Clostridium perfringens ?-Toxin

The Clostridium perfringens ϵ-toxin is responsible for a severe, often lethal intoxication. In this study, we characterized dominant-negative inhibitors of the ϵ-toxin. Site-specific mutations were introduced into the gene encoding ϵ-toxin, and recombinant proteins were expressed in Escherichia coli. Paired cysteine substitutions were introduced at locations predicted to form a disulfide bond. One cysteine in each mutant was introduced into the membrane insertion domain of the toxin; the second cysteine was introduced into the protein backbone. Mutant proteins with cysteine substitutions at amino acid positions I51/A114 and at V56/F118 lacked detectable cytotoxic activity in a MDCK cell assay. Cytotoxic activity could be reconstituted in both mutant proteins by incubation with dithiothreitol, indicating that the lack of cytotoxic activity was attributable to the formation of a disulfide bond. Fluorescent labeling of the cysteines also indicated that the introduced cysteines participated in a disulfide bond. When equimolar mixtures of wild-type ϵ-toxin and mutant proteins were added to MDCK cells, the I51C/A114C and V56C/F118C mutant proteins each inhibited the activity of wild-type ϵ-toxin. Further analysis of the inhibitory activity of the I51C/A114C and V56C/F118C mutant proteins indicated that these proteins inhibit the ability of the active toxin to form stable oligomeric complexes in the context of MDCK cells. These results provide further insight into the properties of dominant-negative inhibitors of oligomeric pore-forming toxins and provide the basis for developing new therapeutics for treating intoxication by ϵ-toxin.The Clostridium perfringens ϵ-toxin is one of the most potent bacterial toxins (1, 2). The ϵ-toxin can lead to a fatal enterotoxemia characterized by widespread vascular permeability and edema in the heart, lungs, brain, and kidneys (3–6). The disease most frequently affects livestock animals, though the toxin may also affect humans (7–9). Because of its extreme potency and the possibility of intoxicating humans, the C. perfringens ϵ-toxin is considered a select agent by the United States Department of Health and Human Services. A vaccine currently is approved for veterinary use, though multiple immunizations are required to provide long-term immunity (10–13). There also is an antitoxin approved for veterinary use. However, in the event that an animal exhibits symptoms of intoxication by ϵ-toxin, it is typically too late for the current antitoxin to be effective, and use of the antitoxin is typically limited to prophylactic treatment of unvaccinated animals within a herd (14). There is no treatment currently approved for use in humans. Thus, alternative countermeasures are needed that inhibit the activity of the toxin.One alternative method of countering the cytotoxic activity of bacterial toxins is through dominant-negative inhibitors. Dominant-negative inhibitors are non-cytotoxic mutant forms of active toxins that are able to inhibit the activity of wild-type toxin when the two proteins are mixed together. Such dominant-negative inhibitors have been described for a diverse set of toxins, including Helicobacter pylori VacA (15–19), Bacillus anthracis anthrax toxin protective antigen (20–25), Bacillus thuringiensis Cry1Ab (26), and Escherichia coli ClyA cytotoxin (27). Like VacA, protective antigen, Cry1Ab, and ClyA, the ϵ-toxin assembles into oligomeric complexes containing multiple toxin monomers (28–30). In the case of VacA and protective antigen, the most extensively studied examples of toxins inhibited by dominant-negative mutants, the number of mutations that inactivate the toxins is substantially greater than the number of mutations that lead to a dominant-negative phenotype (16, 17, 24, 31, 32). Although many of the mutations leading to dominant-negative toxins are located within regions of the toxins that are believed to form the membrane insertion domain, some mutations that inactivate the toxins (but are not dominant-negative) also map within the predicted membrane insertion domains (24, 32). Thus, a deeper understanding of the nature of the dominant-negative phenotype is needed.In this study, we sought to generate dominant-negative mutants of the ϵ-toxin. We hypothesized that mutations within the membrane insertion domain of ϵ-toxin, particularly mutations that are expected to restrict movement of this domain, would lead to dominant-negative inhibitors. We expressed wild-type and site-specific mutants of the ϵ-toxin as recombinant proteins in E. coli. The recombinant proteins were purified, and cytotoxicity was assessed using an established cell culture assay. Using this approach, we identified mutant proteins that inhibited the activity of wild-type ϵ-toxin in vitro and determined the mechanism of inhibition.