The effects of isoniazid on the carbohydrates of Mycobacterium tuberculosis BCG

1. Mycobacterium tuberculosis BCG was usually grown in glycerol-asparagine-casein hydrolysate medium. A soluble fraction was obtained from the cells with aq. 50% ethanol; unbound lipids were then removed and the cells were treated with dilute alkali to give, after acidification, an alkali-extractable fraction and an insoluble fraction. On occasion, lipopolysaccharides were obtained by extracting with phenol or dimethyl sulphoxide instead of alkali. The soluble fraction contained, particularly after long extraction, polysaccharide containing mainly glucose, in addition to trehalose and monosaccharides and their derivatives. The alkali-extractable fraction contained polysaccharides containing mannose, glucose, arabinose, galactose and 6-O-methylglucose. These could be resolved into three fractions of markedly different molecular size. It is argued that the high-molecular-weight materials originated from the outside of the cell envelope and the medium-molecular-weight materials from a middle layer of the envelope. 2. Exposure of the growing cells to isoniazid, usually at 1 or 10mug/ml for 6-12h, increased the total cell carbohydrate, mainly due to an increase in trehalose and in insoluble glucan. It also facilitated the extraction of polysaccharide into the medium and the soluble fraction. This produced about a 25% decrease in the amount of carbohydrate in the alkaline-extractable fraction, mainly due to a fall in glucose, arabinose and 6-O-methylglucose. The decrease was confined to polysaccharides of large and medium molecular weight. When intact lipopolysaccharides were extracted, their amount was also decreased by isoniazid. 3. Substitution of ammonium sulphate for asparagine and casein hydrolysate in the medium, so that glycerol was the sole carbon source, decreased the carbohydrate accumulation brought about by isoniazid but did not alter its effect on polysaccharide extraction. 4. Growth with (14)C-labelled substrates showed that glycerol provided two to four times as much of the cell carbon as did asparagine, when both were present. Under these conditions isoniazid inhibited the incorporation of carbon atoms from asparagine into the cells, but had little effect on the total incorporation from glycerol. These experiments also showed that the effect of isoniazid on alkali-extractable polysaccharides was due to their loss to the soluble fraction and external medium. 5. It is suggested that isoniazid inhibits a pathway (probably the synthesis of mycolic acid) involved in the formation of the cell envelope, and that this inhibition results in some re-channelling of intermediates into carbohydrate synthesis and in some loss of polysaccharides through damage to the envelope.     

A proteomics approach to cloning fenestrin from the nuclear exchange junction of tetrahymena

ABSTRACT. We set out to find the " fenestrin " gene, a gene whose protein is associated with numerous cellular apertures, including the nuclear exchange junction in mating Tetrahymena thermophila . First we developed protocols for imaging and isolating intact nuclear exchange junctions from conjugating cells. Proteins from these junctions were purified using SDS-PAGE, subjected to limited proteolysis, and precise molecular weights were determined by mass spectrometry. Using Protein Prospector^® software and the published Tetrahymena Genome Database, genes for 15 of the most abundant proteins found in our extracts were identified. The most promising candidate was cloned by PCR, fused to yellow fluorescent protein (YFP), and placed under the control of an inducible metallothionein promoter. YFP-localization within live Tetrahymena transformants strongly suggested that one of these genes encoded the fenestrin protein, a result that was subsequently confirmed by Western blotting.     

Identification, biochemical characterization, and evolution of the Rhizopus oryzae 99-880 polygalacturonase gene family

A search of the recently sequenced Rhizopus oryzae strain 99-880 genome database uncovered 18 putative polygalacturonase genes with two genes being identical and only one with similarity to a previously reported R. oryzae polygalacturonase gene. The 17 different genes share 50% to greater than 90% identity at the nucleotide level as well as the deduced protein sequence level. The cDNA of the different genes was isolated directly or recombinantly and used to express the encoded proteins in Pichia pastoris. Recombinant protein expression demonstrated that 15 of the 17 genes encode active enzymes with twelve genes encoding for endo-polygalacturonase enzymes and three genes encoding for exo-polygalacturonase enzymes. Phylogenetic analysis indicates that the genes form a distinct monophyletic group among fungal polygalacturonase enzymes. Finally, our results also suggest that the ancestral form of polygalacturonase in fungi is endolytic and exolytic function evolved later, at least two independent times.     

Lake morphometry predicts the degree of habitat coupling by a mobile predator

Habitat coupling is an ecosystem process whereby semi-discontinuous habitats are connected through the movement of energy and nutrients by chemical, physical or biological processes. One oft-cited example is that of littoral–pelagic coupling in lakes. Theory has argued that such habitat coupling may be critical to food web dynamics, yet there have been few empirical studies that have quantified ecological factors that affect the degree of habitat coupling in ecosystems. Specifically, the degree to which habitat coupling occurs across important physical gradients has largely been ignored. To address this, we investigate the degree of littoral habitat coupling (i.e. the degree to which a top predator lake trout, Salvelinus namaycush, derives energy from the littoral zone) along a gradient of lake shape, where lake shape modifies the relative quantity of coupled epilimnetic benthic and pelagic habitats within each lake. Herein we demonstrate that littoral habitat coupling is intensified in simple circular lakes compared to their reticulate counterparts in seven Canadian Shield lakes. Although the more reticulate lakes had larger areas of epilimnetic benthic habitat, littoral food sources comprised 11% compared to 24% of lake trout diet in reticulate and circular lakes, respectively. This heightened interaction in circular lakes also appears to translate into increased omnivory in more circular lakes compared to reticulate lakes such that lake trout of circular lakes have a significantly lower trophic position than lake trout of reticulate lakes (F_1,5=6.71 p=0.05). These results suggest that it is the accessibility of littoral production via thermal refugia, and not the amount of littoral production, that determines the degree to which lake trout couple littoral and pelagic habitats in lakes.     

A genome-wide screen in Saccharomyces cerevisiae reveals pathways affected by arsenic toxicity

We have used Saccharomyces cerevisiae to identify toxicologically important proteins and pathways involved in arsenic-induced toxicity and carcinogenicity in humans. We performed a systemic screen of the complete set of 4733 haploid S. cerevisiae single-gene-deletion mutants to identify those that have decreased or increased growth, relative to wild type, after exposure to sodium arsenite (NaAsO₂). IC₅₀ values for all mutants were determined to further validate our results. Ultimately we identified 248 mutants sensitive to arsenite and 5 mutants resistant to arsenite exposure. We analyzed the proteins corresponding to arsenite-sensitive mutants and determined that they belonged to functional categories that include protein binding, phosphate metabolism, vacuolar/lysosomal transport, protein targeting, sorting, and translocation, cell growth/morphogenesis, cell polarity and filament formation. Furthermore, these data were mapped onto a protein interactome to identify arsenite-toxicity-modulating networks. These networks are associated with the cytoskeleton, ubiquitination, histone acetylation and the MAPK signaling pathway. Our studies have potential implications for understanding toxicity and carcinogenesis in arsenic-induced human conditions, such as cancer and aging.     

The more food webs change,the more they stay the same

Here, we synthesize a number of recent empirical and theoretical papers to argue that food-web dynamics are characterized by high amounts of spatial and temporal variability and that organisms respond predictably, via behaviour, to these changing conditions. Such behavioural responses on the landscape drive a highly adaptive food-web structure in space and time. Empirical evidence suggests that underlying attributes of food webs are potentially scale-invariant such that food webs are characterized by hump-shaped trophic structures with fast and slow pathways that repeat at different resolutions within the food web. We place these empirical patterns within the context of recent food-web theory to show that adaptable food-web structure confers stability to an assemblage of interacting organisms in a variable world. Finally, we show that recent food-web analyses agree with two of the major predictions of this theory. We argue that the next major frontier in food-web theory and applied food-web ecology must consider the influence of variability on food-web structure.     

Synthetic sheep urine alters fungal community structure in an upland grassland soil

Agricultural improvement (fertilisation, liming, intensification of grazing) of acidic upland pastures results in loss of indigenous flora and notable changes in microbial community structure. Such practices have recently raised concerns regarding the possible impacts on natural ecosystem biodiversity and functioning. The effects of synthetic sheep urine (SSU) and plant species on fungal community structure in upland grassland microcosms were investigated. Plant species typical of agriculturally unimproved (Agrostis capillaris) and improved (Lolium perenne) pastures were treated with low, medium or high concentrations of SSU, with harvests carried out 10 d and 50 d after SSU application. Root biomass was negatively affected by SSU addition whereas shoot biomass did not display any significant change. Fungal richness (number of operational taxonomic units) was negatively correlated with SSU concentration (p < 0.001), and also with time (p < 0.001).Multi-dimensional scaling plots revealed significant changes in fungal community composition, depending on concentration of SSU and plant species type, while canonical correspondence analysis also emphasised the importance of interacting environmental variables. In addition, SIMPER analyses supported the finding that shifts in fungal community composition under different SSU and plant treatments had occurred. Overall, while SSU appeared to be influential in determining fungal community structure, community changes were largely driven by interacting environmental factors. This study contributes to our understanding of the potential implications of intensified farming, in particular increased pressure from grazing animals, on fungal community structure in semi-natural grassland systems.     

Quantifying a stress gradient: An objective approach to variable selection,standardization and weighting in ecosystem assessment

Quantifying relative habitat quality is an important means of ecosystem assessment, and an essential step in the development and validation of indices of biotic integrity (IBI). Variables included in multi-metric IBIs are selected on the basis of their correlation with a human disturbance gradient, and the IBI is tested by examining correlation between IBI scores and rankings on the human disturbance gradient for an independent suite of sites. We present an objective approach to develop a disturbance gradient that ranks sites based on local-level measurements of physical and chemical stress; however, it could equally be applied to GIS-derived data. We measured 52 variables at three types of wetland in Alberta: reference wetlands, oil sands reclamation wetlands exposed to mine tailings, and reclamation wetlands free from tailings contamination. We used the data's correlation structure to select a sub-set of variables that minimized redundancy while retaining sensitivity and interpretability. The optimal sub-set included eight variables: chloride, cation and nitrogen content of water; water and oil content of sediment; water depth and amplitude and Secchi depth/total depth. We combined these eight environmental variables using different combinations of standardization (conversion to a common unit) and weighting schemes to produce six multi-metric stress indices. We evaluated the stress indices on their ability to discriminate among our three wetland types. The indices differed in their sensitivity to stress. Standardization had greater influence on index score than weighting. While all stress indices detected a difference among the three wetland types, only two were able to discriminate between the two classes of reclamation wetlands, both of which used percentile binning to standardize variables. The optimal stress index was standardized by percentile binning and weighted such that water quality, sediment chemistry, physical structure, and the level of tailings contamination were weighted equally. The approach we developed is repeatable and produced a sensitive index of wetland condition that is easily interpreted and relies minimally on best professional judgment. It may be suitable for measuring restoration success or the impact of any anthropogenic disturbance in a variety of ecosystem types.     

<Emphasis Type="Italic">mab-31</Emphasis> and the TGF-β pathway act in the ray lineage to pattern <Emphasis Type="Italic">C. elegans</Emphasis>male sensory rays

Background  

C. elegans TGF-β-like Sma/Mab signaling pathway regulates both body size and sensory ray patterning. Most of the components in this pathway were initially identified by genetic screens based on the small body phenotype, and many of these mutants display sensory ray patterning defect. At the cellular level, little is known about how and where these components work although ray structural cell has been implicated as one of the targets. Based on the specific ray patterning abnormality, we aim to identify by RNAi approach additional components that function specifically in the ray lineage to elucidate the regulatory role of TGF-β signaling in ray differentiation.     

Evolution of <Emphasis Type="Italic">trappin</Emphasis> genes in mammals

Background  

Trappin is a multifunctional host-defense peptide that has antiproteolytic, antiinflammatory, and antimicrobial activities. The numbers and compositions of trappin paralogs vary among mammalian species: human and sheep have a single trappin-2 gene; mouse and rat have no trappin gene; pig and cow have multiple trappin genes; and guinea pig has a trappin gene and two other derivativegenes. Independent duplications of trappin genes in pig and cow were observed recently after the species were separated. To determine whether these trappin gene duplications are restricted only to certain mammalian lineages, we analyzed recently-developed genome databases for the presence of duplicate trappin genes.