Influence of elevated CO(2) on the sensitivity of two soybean cultivars to sulphur dioxide

Two cultivars of soybean (Glycine max cv. Bragg and PK 472) were subjected to elevated concentrations of CO(2) (600 &mgr;l l(-1)) and/or SO(2) (0.06 &mgr;l l(-1)), for 8 h from germination to grain maturity in open top chambers under field conditions to assess the modification in response to SO(2) exposure resulting form CO(2) enrichment. Exposure to SO(2) alone resulted in reductions in plant growth, biomass and yield, as well as declines in foliar starch and protein content in both the cultivars of soybean. Elevated CO(2) stimulated plant growth, yield and enhanced foliar starch content, photosynthesis and WUE in both the cultivars. In plants exposed to the combination of elevated CO(2)+SO(2), the adverse influence of SO(2) was mitigated by CO(2) enrichment. This effect was considered to result from the provision of extra carbon sources required for repair and detoxification processes and a reduction in SO(2) uptake through reduction in stomatal conductance. PK 472 exhibited greater sensitivity to SO(2) than Bragg. PK 472 also showed greater stimulation of yield under CO(2)+SO(2) treatment than Bragg.     

Human DNA polymerase iota incorporates dCTP opposite template G via a G.C + Hoogsteen base pair

Human DNA polymerase iota (hPoliota), a member of the Y family of DNA polymerases, differs in remarkable ways from other DNA polymerases, incorporating correct nucleotides opposite template purines with a much higher efficiency and fidelity than opposite template pyrimidines. We present here the crystal structure of hPoliota bound to template G and incoming dCTP, which reveals a G.C + Hoogsteen base pair in a DNA polymerase active site. We show that the hPoliota active site has evolved to favor Hoogsteen base pairing, wherein the template sugar is fixed in a cavity that reduces the C1'-C1' distance across the nascent base pair from approximately 10.5 A in other DNA polymerases to 8.6 A in hPoliota. The rotation of G from anti to syn is then largely in response to this curtailed C1'-C1' distance. A G.C+ Hoogsteen base pair suggests a specific mechanism for hPoliota's ability to bypass N(2)-adducted guanines that obstruct replication.     

High-throughput microplate phosphorylation assays based on DevR-DevS/Rv2027c 2-component signal transduction pathway to screen for novel antitubercular compounds

DevR-DevS (Rv3133c-Rv3132c) and DevR-Rv2027c have been established through their autophosphorylation and phospho-transfer properties to constitute bonafide regulatory 2-component systems of Mycobacterium tuberculosis. DevR has also been shown by others to play a key regulatory role in the expression of M. tuberculosis genes comprising the dormancy regulon. The authors describe high-throughput phosphorylation assays in a microplate format using DevS and Rv2027c histidine kinases and DevR response regulator proteins from M. tuberculosis. The assays were designed to measure [gamma-(32)P]ATP-dependent autophosphorylation of DevS/Rv2027c and also the phosphotransfer reaction to DevR. First, the optimal reaction conditions were established using the conventional method of radiolabeling the 2-component proteins by [gamma-(32)P]ATP and followed by gel electrophoresis-based analysis. Next, the assays were converted to a high-throughput format in which the radiolabeled protein retained on a filter using mixed cellulose ester-based 96-well filter plates was analyzed for radioactivity retention by scintillation counting. The utility of these assays to screen for inhibitors is illustrated using 2-mercaptobenzimidazole, ethidium bromide, and EDTA. The high quality and flexibility of these assays will enable their use in high-throughput screening for new antitubercular compounds directed against 2-component systems that comprise a novel target in dormant mycobacteria.     

Prediction of glutathionylation sites in proteins using minimal sequence information and their experimental validation

S-glutathionylation of proteins plays an important role in various biological processes and is known to be protective modification during oxidative stress. Since, experimental detection of S-glutathionylation is labor intensive and time consuming, bioinformatics based approach is a viable alternative. Available methods require relatively longer sequence information, which may prevent prediction if sequence information is incomplete. Here, we present a model to predict glutathionylation sites from pentapeptide sequences. It is based upon differential association of amino acids with glutathionylated and non-glutathionylated cysteines from a database of experimentally verified sequences. This data was used to calculate position dependent F-scores, which measure how a particular amino acid at a particular position may affect the likelihood of glutathionylation event. Glutathionylation-score (G-score), indicating propensity of a sequence to undergo glutathionylation, was calculated using position-dependent F-scores for each amino-acid. Cut-off values were used for prediction. Our model returned an accuracy of 58% with Matthew’s correlation-coefficient (MCC) value of 0.165. On an independent dataset, our model outperformed the currently available model, in spite of needing much less sequence information. Pentapeptide motifs having high abundance among glutathionylated proteins were identified. A list of potential glutathionylation hotspot sequences were obtained by assigning G-scores and subsequent Protein-BLAST analysis revealed a total of 254 putative glutathionable proteins, a number of which were already known to be glutathionylated. Our model predicted glutathionylation sites in 93.93% of experimentally verified glutathionylated proteins. Outcome of this study may assist in discovering novel glutathionylation sites and finding candidate proteins for glutathionylation.     

E3 Ubiquitin Ligases in Protein Quality Control Mechanism

In living cells, polypeptide chains emerging from ribosomes and preexisting polypeptide chains face constant threat of misfolding and aggregation. To prevent protein aggregation and to fulfill their biological activity, generally, protein must fold into its proper three-dimensional structure throughout their lifetimes. Eukaryotic cell possesses a quality control (QC) system to contend the problem of protein misfolding and aggregation. Cells achieve this functional QC system with the help of molecular chaperones and ubiquitin-proteasome system (UPS). The well-conserved UPS regulates the stability of various proteins and maintains all essential cellular function through intracellular protein degradation. E3 ubiquitin ligase enzyme determines specificity for degradation of certain substrates via UPS. New emerging evidences have provided considerable information that various E3 ubiquitin ligases play a major role in cellular QC mechanism and principally designated as QC E3 ubiquitin ligases. Nevertheless, very little is known about how E3 ubiquitin ligase maintains QC mechanism against abnormal proteins under various stress conditions. Here in this review, we highlight and discuss the functions of various E3 ubiquitin ligases implicated in protein QC mechanism. Improving our knowledge about such processes may provide opportunities to modulate protein QC mechanism in age-of-onset diseases that are caused by protein aggregation.     

Prevalence of Dyslipidemia in Urban and Rural India: The ICMR–INDIAB Study

Aim

To study the pattern and prevalence of dyslipidemia in a large representative sample of four selected regions in India.

Methods

Phase I of the Indian Council of Medical Research–India Diabetes (ICMR-INDIAB) study was conducted in a representative population of three states of India [Tamil Nadu, Maharashtra and Jharkhand] and one Union Territory [Chandigarh], and covered a population of 213 million people using stratified multistage sampling design to recruit individuals ≥20 years of age. All the study subjects (n = 16,607) underwent anthropometric measurements and oral glucose tolerance tests were done using capillary blood (except in self-reported diabetes). In addition, in every 5th subject (n = 2042), a fasting venous sample was collected and assayed for lipids. Dyslipidemia was diagnosed using National Cholesterol Education Programme (NCEP) guidelines.

Results

Of the subjects studied, 13.9% had hypercholesterolemia, 29.5% had hypertriglyceridemia, 72.3% had low HDL-C, 11.8% had high LDL-C levels and 79% had abnormalities in one of the lipid parameters. Regional disparity exists with the highest rates of hypercholesterolemia observed in Tamilnadu (18.3%), highest rates of hypertriglyceridemia in Chandigarh (38.6%), highest rates of low HDL-C in Jharkhand (76.8%) and highest rates of high LDL-C in Tamilnadu (15.8%). Except for low HDL-C and in the state of Maharashtra, in all other states, urban residents had the highest prevalence of lipid abnormalities compared to rural residents. Low HDL-C was the most common lipid abnormality (72.3%) in all the four regions studied; in 44.9% of subjects, it was present as an isolated abnormality. Common significant risk factors for dyslipidemia included obesity, diabetes, and dysglycemia.

Conclusion

The prevalence of dyslipidemia is very high in India, which calls for urgent lifestyle intervention strategies to prevent and manage this important cardiovascular risk factor.     

Overexpression of α2,3sialyl T-antigen in breast cancer determined by miniaturized glycosyltransferase assays and confirmed using tissue microarray immunohistochemical analysis

Glycan structure alterations during cancer regulate disease progression and represent clinical biomarkers. The study determined the degree to which changes in glycosyltransferase activities during cancer can be related to aberrant cell-surface tumor associated carbohydrate structures (TACA). To this end, changes in sialyltransferase (sialylT), fucosyltransferase (fucT) and galactosyltransferase (galT) activity were measured in normal and tumor tissue using a miniaturized enzyme activity assay and synthetic glycoconjugates bearing terminal LacNAc Type-I (Galβ1-3GlcNAc), LacNAc Type-II (Galβ1-4GlcNAc), and mucin core-1/Type-III (Galβ1-3GalNAc) structures. These data were related to TACA using tissue microarrays containing 115 breast and 26 colon cancer specimen. The results show that primary human breast and colon tumors, but not adjacent normal tissue, express elevated β1,3GalT and α2,3SialylT activity that can form α2,3SialylatedType-IIIglycans (Siaα2-3Galβ1-3GalNAc). Prostate tumors did not exhibit such elevated enzymatic activities. α1,3/4FucT activity was higher in breast, but not in colon tissue. The enzymology based prediction of enhanced α2,3sialylated Type-III structures in breast tumors was verified using histochemical analysis of tissue sections and tissue microarrays. Here, the binding of two markers that recognize Galβ1-3GalNAc (peanut lectin and mAb A78-G/A7) was elevated in breast tumor, but not in normal control, only upon sialidase treatment. These antigens were also upregulated in colon tumors though to a lesser extent. α2,3sialylatedType-III expression correlated inversely with patient HER2 expression and breast metastatic potential. Overall, enzymology measurements of glycoT activity predict truncated O-glycan structures in tumors. High expression of the α2,3sialylated T-antigen O-glycans occur in breast tumors. A transformation from linear core-1 glycan to other epitopes may accompany metastasis.     

Mode of Action of the Toxin from Pseudomonas phaseolicola: I. Toxin Specificity, Chlorosis, and Ornithine Accumulation

The specificity of the Pseudomonas phaseolicola toxin for enzyme inhibition and its relationship to toxin-induced chlorosis in bean leaves (Phaseolus vulgaris L.) was examined. The toxin showed no significant inhibitory activity against glutamine synthetase, glutamine transferase, carbamyl phosphate synthetase, aspartate carbamoyltransferase, or arginase at concentrations 100-fold higher than that needed to inhibit ornithine carbamoyltransferase by 50%.