Cysteine auxotrophs of Salmonella typhimurium which grow without cysteine in a hydrogen/carbon dioxide atmosphere.

Cysteine auxotrophs of Salmonella typhimurium mutated in cysB, cysI or cysJ grew with sulphate as a sulphur source when incubated under a hydrogen/carbon dioxide atmosphere. Yields obtained under these conditions were equivalent to those characteristic of wild-type S. typhimurium. The same mutants failed to grow with sulphate as a sulphur source when incubated aerobically. Auxotrophs mutated in cysA, cysC, cysD, cysE, cysG and cysH required cysteine for growth under both incubation conditions. The results suggest that mutations in cysB (regulation of the several cys operons) and also cysI and cysJ (sulphite reductase activity) can be circumvented during anaerobic growth under hydrogen.     

Resistance of Cysts of Amoebae to Microbial Decomposition

Cyst walls of Acanthamoeba rhysodes, A. palestinensis, A. castellanii, and one other strain of Acanthamoeba contain 36 to 45% protein and 20 to 34% carbohydrate. More than half of the protein in the walls of A. palestinensis, A. castellanii and Acanthamoeba sp. is accessible to and hydrolyzed by protease, and 67 to 69% of the carbohydrate of A. palestinensis and A. rhysodes walls is hydrolyzed by cellulase. The extent of hydrolysis of walls of the other amoebae by these enzymes is appreciably less, and chitinase and β-1,3-glucanase have no detectable effect. Protease solubilizes 10% or less of the weight of intact cysts, and no solubilization is observed with cellulase. Walls of A. palestinensis are extensively degraded in soil, the activity is less with A. rhysodes, and little attack on the other amoebae occurs. When added to soil, the protozoa excyst and grow for short periods, the trophozoites then die, and chiefly cysts persist thereafter.     

Explant culture of rat colon: A model system for studying metabolism of chemical carcinogens

Summary An explant culture system has been developed for the long-term maintenance of colonic tissue from the rat. Explants of 1 cm² in size were placed in tissue-culture dishes to which was added 2 ml of CMRL-1066 medium supplemented with glucose, hydrocortisone, β-retinyl acetate, and either 2.5% bovine albumin or 5% fetal bovine serum. The dishes were placed in a controlied-atmosphere chamber which was gassed with 95% O₂ and 5% CO₂. The chamber then was placed on a rocker platform which rocked at 10 cycles per min causing the medium to flow intermittently over the epithelial surface. The explants were incubated at 30°C. The viability of the tissue was measured both by incorporation of specific precursors into cellular macromolecules and by monitoring of tissue morphology with light and electron microscopy. Cultured rat colon was able to metabolize benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, aflatoxin B₁, dimethylnitrosamine, 1,2-dimethylhydrazine, and methylazoxymethanol acetate into chemical species that bind to cellular DNA and protein.     

Superinfection exclusion by bacteriophage T7.

Only two of the early genes of bacteriophage T7 were found to play a significant role in exclusion of superinfecting bacteriophage T3 particles; genes 0.3 and 1. Protein synthesis by the preinfecting phage particle was not required for efficient exclusion. These findings are discussed with regard to the known functions of these genes during T7 development.     

Dynamite extended: two new services to simplify protein dynamic analysis

We describe two additional services now available as part of the previously described Dynamite protein dynamics web service. Dynatraj provides principle component analysis and visualization of modes of motion for a user's own ensemble of protein structures, e.g. from Molecular Dynamics, NMR or experimental ensembles. Dynapocket predicts probable configurations of a protein pocket from a single known structure. Both have been provided in response to requests from users for additional functionality from the Dynamite server. Like Dynamite, both are available free of charge to all users.     

The 2 microm plasmid causes cell death in Saccharomyces cerevisiae with a mutation in Ulp1 protease

The 2 microm circle plasmid confers no phenotype in wild-type Saccharomyces cerevisiae but in a nib1 mutant, an elevated plasmid copy number is associated with cell death. Complementation was used to identify nib1 as a mutant allele of the ULP1 gene that encodes a protease required for removal of a ubiquitin-like protein, Smt3/SUMO, from protein substrates. The nib1 mutation replaces conserved tryptophan 490 with leucine in the protease domain of Ulp1. Complete deletion of ULP1 is lethal, even in a strain that lacks the 2 microm circle. Partial deletion of ULP1, like the nib1 mutation, results in clonal variations in plasmid copy number. In addition, a subset of these mutant cells produces lineages in which all cells have reduced proliferative capacity, and this phenotype is dependent upon the presence of the 2 microm circle. Segregation of the 2 microm circle requires two plasmid-encoded proteins, Rep1 and Rep2, which were found to colocalize with Ulp1 protein in the nucleus and interact with Smt3 in a two-hybrid assay. These associations and the observation of missegregation of a fluorescently tagged 2 microm circle reporter plasmid in a subset of ulp1 mutant cells suggest that Smt3 modification plays a role in both plasmid copy number control and segregation.     

Development of a large-scale chemogenomics database to improve drug candidate selection and to understand mechanisms of chemical toxicity and action

Successful drug discovery requires accurate decision making in order to advance the best candidates from initial lead identification to final approval. Chemogenomics, the use of genomic tools in pharmacology and toxicology, offers a promising enhancement to traditional methods of target identification/validation, lead identification, efficacy evaluation, and toxicity assessment. To realize the value of chemogenomics information, a contextual database is needed to relate the physiological outcomes induced by diverse compounds to the gene expression patterns measured in the same animals. Massively parallel gene expression characterization coupled with traditional assessments of drug candidates provides additional, important mechanistic information, and therefore a means to increase the accuracy of critical decisions. A large-scale chemogenomics database developed from in vivo treated rats provides the context and supporting data to enhance and accelerate accurate interpretation of mechanisms of toxicity and pharmacology of chemicals and drugs. To date, approximately 600 different compounds, including more than 400 FDA approved drugs, 60 drugs approved in Europe and Japan, 25 withdrawn drugs, and 100 toxicants, have been profiled in up to 7 different tissues of rats (representing over 3200 different drug-dose-time-tissue combinations). Accomplishing this task required evaluating and improving a number of in vivo and microarray protocols, including over 80 rigorous quality control steps. The utility of pairing clinical pathology assessments with gene expression data is illustrated using three anti-neoplastic drugs: carmustine, methotrexate, and thioguanine, which had similar effects on the blood compartment, but diverse effects on hepatotoxicity. We will demonstrate that gene expression events monitored in the liver can be used to predict pathological events occurring in that tissue as well as in hematopoietic tissues.     

Na(+)/Ca(2+) exchange regulates Ca(2+)-dependent duodenal mucosal ion transport and HCO(3)(-) secretion in mice

Stimulation of muscarinic receptors in duodenal mucosa raises intracellular Ca(2+), which regulates ion transport, including HCO(3)(-) secretion. However, the underlying Ca(2+) handling mechanisms are poorly understood. The aim of the present study was to determine whether Na(+)/Ca(2+) exchanger (NCX) plays a role in the regulation of duodenal mucosal ion transport and HCO(3)(-) secretion by controlling Ca(2+) homeostasis. Mouse duodenal mucosa was mounted in Ussing chambers. Net ion transport was assessed as short-circuit current (I(sc)), and HCO(3)(-) secretion was determined by pH-stat. Expression of NCX in duodenal mucosae was analyzed by Western blot, and cytosolic Ca(2+) in duodenocytes was measured by fura 2. Carbachol (100 muM) increased I(sc) in a biphasic manner: an initial transient peak within 2 min and a later sustained plateau starting at 10 min. Carbachol-induced HCO(3)(-) secretion peaked at 10 min. 2-Aminoethoxydiphenylborate (2-APB, 100 muM) or LiCl (30 mM) significantly reduced the initial peak in I(sc) by 51 or 47%, respectively, and abolished the plateau phase of I(sc) without affecting HCO(3)(-) secretion induced by carbachol. Ryanodine (100 muM), caffeine (10 mM), and nifedipine (10 muM) had no effect on either response to carbachol. In contrast, nickel (5 mM) and KB-R7943 (10-30 muM) significantly inhibited carbachol-induced increases in duodenal mucosal I(sc) and HCO(3)(-) secretion. Western blot analysis showed expression of NCX1 proteins in duodenal mucosae, and functional NCX in duodenocytes was demonstrated in Ca(2+) imaging experiments where Na(+) depletion elicited Ca(2+) entry via the reversed mode of NCX. These results indicate that NCX contributes to the regulation of Ca(2+)-dependent duodenal mucosal ion transport and HCO(3)(-) secretion that results from stimulation of muscarinic receptors.     

Mining parasite data using genetic programming

Genetic programming is a technique that can be used to tackle the hugely demanding data-processing problems encountered in the natural sciences. Application of genetic programming to a problem using parasites as biological tags demonstrates its potential for developing explanatory models using data that are both complex and noisy.