Mutual co-regulation between GPI-N-acetylglucosaminyltransferase and ergosterol biosynthesis in Candida albicans

A novel co-regulation exists between the first step of GPI (glycosylphosphatidylinositol) anchor biosynthesis and the rate-determining step of ergosterol biosynthesis in Candida albicans. Depleting CaGpi19p, an accessory subunit of the enzyme complex that initiates GPI biosynthesis, down-regulates ERG11, altering ergosterol levels and drug response. This effect is specific to CaGpi19p depletion and is not due to cell wall defects or GPI deficiency. Additionally, down-regulation of ERG11 down-regulates CaGPI19 and GPI biosynthesis.     

Steatohepatitis in laboratory opossums exhibiting a high lipemic response to dietary cholesterol and fat

Plasma VLDL and LDL cholesterol were markedly elevated (>40-fold) in high-responding opossums, but moderately elevated (6-fold) in low-responding opossums after they had consumed a high-cholesterol and high-fat diet for 24 wk. In both high- and low-responding opossums, plasma triglycerides were slightly elevated, threefold and twofold, respectively. Dietary challenge also induced fatty livers in high responders, but not in low responders. We studied the lipid composition, histopathological features, and gene expression patterns of the fatty livers. Free cholesterol (2-fold), esterified cholesterol (11-fold), and triglycerides (2-fold) were higher in the livers of high responders than those in low responders, whereas free fatty acid levels were similar. The fatty livers of high responders showed extensive lobular disarray by histology. Inflammatory cells and ballooned hepatocytes were also present, as were perisinusoidal fibrosis and ductular proliferation. In contrast, liver histology was normal in low responders. Hepatic gene expression revealed differences associated with the development of steatohepatitis in high responders. The accumulation of hepatic cholesterol was concomitant with upregulation of the HMGCR gene and downregulation of the CYP27A1, ABCG8, and ABCB4 genes. Genes involved in inflammation (TNF, NFKB1, and COX2) and in oxidative stress (CYBA and NCF1) were upregulated. Upregulation of the growth factor genes (PDGF and TGFB1) and collagen genes (Col1A1, Col3A1, and Col4A1) was consistent with fibrosis. Some of the histological characteristics of the fatty livers of high-responding opossums imitate those in the livers of humans with nonalcoholic steatohepatitis.     

Comparative structural modeling of a monothiol GRX from chickpea: insight in iron-sulfur cluster assembly

Glutaredoxins (GRXs) are small, ubiquitous, multifunctional, heat-stable and glutathione-dependent thiol-disulphide oxidoreductases, classified under thioredoxin-fold superfamily. In the green lineage, GRXs constitute a complex family of proteins. Based on their active site, GRXs are classified into two subfamilies: dithiol and monothiol. Monothiol GRXs contain 'CGFS' as a redox active motif and assist in maintaining redox state and iron homeostasis within the cell. Using RACE strategy, a full length cDNA of chickpea (Cicer arietinum) glutaredoxin 3 (CarGRX3) was cloned and sequenced. The cDNA contains open reading frame of 537 bp encoding 178 amino acids and exhibits features of other known 'CGFS' type GRXs. Based on the multiple sequence alignment among CarGRX3 and monothiol GRXs of other photosynthetic organisms, the characteristic motif (KGX4PXCGFSX([29/30/32])KX4WPTXPQX4GX3GGXDI) with 18 invariant residues was observed. The proposed structure of CarGRX3 was compared with structurally resolved monothiol GRXs of other organisms. The CarGRX3 and nearest Arabidopsis homolog (AtGRXcp) shares 76% sequence identity which was reflected by their 3D-structure conservation. The structure of chickpea monothiol GRX (CarGRX3) coordinates glutathione ligated [2Fe-2S] cluster in a homodimeric form, highlighting the structural basis for iron-sulfur cluster (ISC) assembly and delivery to acceptor proteins. The present study on CarGRX3 model highlighted the utility of the theoretical approaches to understand complex biological phenomena such as glutathione docking and incorporation of GSH-ligated [2Fe-2S] cluster.     

Caenorhabditis elegans battling starvation stress: low levels of ethanol prolong lifespan in L1 larvae

The nematode Caenorhabditis elegans arrests development at the first larval stage if food is not present upon hatching. Larvae in this stage provide an excellent model for studying stress responses during development. We found that supplementing starved larvae with ethanol markedly extends their lifespan within this L1 diapause. The effects of ethanol-induced lifespan extension can be observed when the ethanol is added to the medium at any time between 0 and 10 days after hatching. The lowest ethanol concentration that extended lifespan was 1 mM (0.005%); higher concentrations to 68 mM (0.4%) did not result in increased survival. In spite of their extended survival, larvae did not progress to the L2 stage. Supplementing starved cultures with n-propanol and n-butanol also extended lifespan, but methanol and isopropanol had no measurable effect. Mass spectrometry analysis of nematode fatty acids and amino acids revealed that L1 larvae can incorporate atoms from ethanol into both types of molecules. Based on these data, we suggest that ethanol supplementation may extend the lifespan of L1 larvae by either serving as a carbon and energy source and/or by inducing a stress response.     

Recombinant trehalose-6-phosphate phosphatase of Brugia malayi cross-reacts with human Wuchereria bancrofti immune sera and engenders a robust protective outcome in mice

Trehalose-6-phosphate phosphatase of Brugia malayi (Bm-TPP) represents an attractive vaccine candidate because it is present in all the major life stages of parasite, but is absent in mammals. We have previously cloned, purified and biochemically characterized Bm-TPP. In the present study, we investigated the cross-reactivity of recombinant Bm-TPP (r-Bm-TPP) with the sera of human bancroftian patients belonging to different disease categories. In silico study using bioinformatics tool demonstrated that Bm-TPP is highly immunogenic in nature. BALB/c mice administered with r-Bm-TPP alone or in combination with Freund's complete adjuvant (FCA) generated a strong IgG response. Further investigations on the antibody isotypes showed generation of a mixed T helper cell response which was marginally biased towards Th1 phenotype. r-Bm-TPP with or without adjuvant lead to significantly increased accumulation of CD4+ and CD8+ T cells in the spleen of infected mice and increased the activation of peritoneal macrophages. Additionally, r-Bm-TPP enhanced the production of both proinflammatory (IL-2, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines and mice immunized with r-Bm-TPP alone or in combination with FCA showed 54.5% and 67% protection respectively against B. malayi infective larvae challenge. Taken together, our findings suggest that Bm-TPP is protective in nature and might be a potential candidate for development of vaccine against lymphatic filarial infections.     

Physiological and biochemical effect of 24-epibrassinoslide on cold tolerance in maize seedlings

Germination and early seedling growth are important for establishment of maize because maize is chilling sensitive crop and low temperature during early period of growth can be detrimental to subsequent crop growth and productivity. Therefore, it is important to protect maize seedling from cold stress. A study was conducted on induced cold tolerance by 24-epibrassinoslide (EBR) at the Indian Agricultural Research Institute, New Delhi, India. Maize seedlings were raised in green house condition (25/18 °C day-night temperatures). Ten days old seedlings were treated with EBR (0.0, 0.01, 0.1, 1.0 and 10 μM) and then divided into two sets, one set was kept in greenhouse (25/18 °C day-night temperatures) and another was transferred to net house (cold stress). Data on various morpho-physiological traits was recorded after 7, 14 and 21 days of treatment. Exogenous application of 1.0 μM EBR had significant effect on growth and morpho-physiological traits under both conditions. The maize seedlings treated with EBR were more tolerant to cold stress than the untreated one. Significant increase in plant height, dry matter accumulation, chlorophyll content, total soluble proteins and starch contents was observed under both conditions, however, the results were more pronounced under cold stress. 1.0 μ M concentration being the most effective under both conditions. Maintenance of high tissue water content, reduced membrane injury index, increased total chlorophyll, soluble sugar and protein content were taken as the possible indicators of EBR induced chilling tolerance.     

Native X-DING-CD4 protein secreted by HIV-1 resistant CD4+ T cells blocks activity of IL-8 promoter in human endothelial cells infected with enteric bacteria

Onsets of bacterial infections devastate the compromised immune system in AIDS patients. Damaged gut mucosa permits dissemination of bacterial toxins into deeper layers and hyper-activation of the immune system. We previously reported that the unfractionated supernatants of HIV-resistant CD4(+) T cells impeded the NF-κB/DNA binding in macrophages induced by either HIV-1 or LPS. The active component of this soluble material was identified as X-DING-CD4 (extracellular DING from CD4 T cells). We hypothesized that the anti-inflammatory effect of the X-DING-CD4 protein might extend to non-immune cells, for example endothelial cells, undergoing persistent endotoxin stimulation in the course of advanced HIV disease. To test this proposition, we evaluated the efficiency of NF-κB and Ap-1 binding to the IL-8 promoter in LPS-activated endothelial cells and control human macrophages exposed to native X-DING-CD4 protein. We found a deficiency of NF-κB- but not AP-1-DNA binding in the systems where cells were treated with native soluble X-DING-CD4 protein. The X-DING-CD4-mediated inhibition of the IL-8 promoter also resulted in a reduction of the soluble IL-8 protein in endothelial cells and human macrophages infected with a subset of enteric bacteria frequently causing diarrhea in progressive HIV disease. Bacterial endotoxin did not induce the endogenous X-DING-CD4 mRNA activity in human macrophages and transformed CD4(+)T cells, indicating that the reduction of LPS-mediated IL-8 promoter activation was not related to de novo X-DING-CD4 protein synthesis, but depended on function of the exogenous X-DING-CD4 protein. This study provides evidence that the X-DING-CD4 protein might be developed as a novel biotherapeutic to control LPS-mediated inflammation in advanced HIV disease.     

Ozone treatment of process water from a dry-mill ethanol plant

Fuel ethanol production in corn dry milling plants is a rapidly expanding industrial sector. Whole stillage, the residue from the distillation of the fermented corn, is centrifuged and the concentrate, thin stillage, is found to have a chemical oxygen demand (COD) of approximately 75,000 mg/L. This thin stillage is partly recycled, but much of it needs to be evaporated to concentrate the solubles for addition to the animal feed coproduct from corn dry milling. This research is an exploration into lowering COD from thin stillage using ozonation as a simple single-step unit process to facilitate a larger reusable fraction. The ozonation would usually be a pretreatment before additional flocculation or biological treatment. Also, COD removal by ozonation with and without a catalyst has been studied. Three different application rates of ozone O(3,1)=7 mg/min, O(3,2)=21 mg/min, and O(3,3)=33 mg/min were used for 8h into samples of 2L each of three dilutions 20x, 30x and 40x. COD removal of 85% was observed with an ozone dosage of 4000 mg/L into a 40x-diluted sample. This would correspond to about 0.5 mg COD removed per mg ozone dosed. However, at lower dosages and smaller dilutions, more than 1mg COD removal was achieved per mg ozone dosed. Two different catalysts, Fe(II) and Fe(III), were used and the samples were ozonated for 4 h. Five different dosages of each of the two catalysts were used and better COD removal was observed compared to ozonation alone with all 5 dosages. COD removal rate was increased from 45% to 74% with Fe(III) and was increased to 77% with Fe(II). Up to 10mg/L COD was removed per mg O(3) dosed well below the maximum dosage. Both the catalysts resulted in almost the same improved COD removal rates when compared to ozonation alone.