Effects of thiols, sugars, and proteins on nitric oxide activation of guanylate cyclase.

Purification of soluble guanylate cyclase from rat liver resulted in an apparent loss of enzyme activation by nitric oxide that could be restored by dithiothreitol. methemoglobin, bovine serum albumin, or sucrose. Although hemoglobin also permitted some activation with nitric oxide, the effect of other agents to restore enzyme activation was prevented with hemoglobin. As a result of enzyme purification, there is an alteration of the dose-response relationship for nitric oxide activation. After partial enzyme purification, relatively high concentrations of nitric oxide that were stimulatory in crude enzyme preparations had no effect on enzyme activity. However, partially purified or homogeneous enzyme was activated by lower concentrations of nitric oxide. The bell-shaped dose-response curve for nitric oxide was shifted to the left with guanylate cyclase purification. The addition of dithiothreitol, methemoglobin, bovine serum albumin, or sucrose to enzyme markedly broadens the dose-response curve for nitric oxide. Thus, the apparent loss of responsiveness to nitric oxide with purification is a function of increased sensitivity of guanylate cyclase to nitric oxide. Increased sensitivity to nitric oxide with enzyme purification probably results from the removal of heme, proteins, and small molecules that can serve as scavengers or sinks for nitric oxide and prevent excessive oxidation of the enzyme.     

Enhancing Data Reliability in TOMAHAQ for Large‐Scale Protein Quantification

The analytical scale of most mass‐spectrometry‐based targeted proteomics assays is usually limited by assay performance and instrument utilization. A recently introduced method, called triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantitation (TOMAHAQ), combines both peptide and sample multiplexing to simultaneously improve analytical scale and quantitative performance. In the present work, critical technical requirements and data analysis considerations for successful implementation of the TOMAHAQ technique based on the study of a total of 185 target peptides across over 200 clinical plasma samples are discussed. Importantly, it is observed that significant interference originate from the TMTzero reporter ion used for the synthetic trigger peptides. This interference is not expected because only TMT10plex reporter ions from the target peptides should be observed under typical TOMAHAQ conditions. In order to unlock the great promise of the technique for high throughput quantification, here a post‐acquisition data correction strategy to deconvolute the reporter ion superposition and recover reliable data is proposed.     

Association genetics of the parameters related to nitrogen use efficiency in Brassica juncea L.

Key message

Genome wide association studies allowed prediction of 17 candidate genes for association with nitrogen use efficiency. Novel information obtained may provide better understanding of genomic controls underlying germplasm variations for this trait in Indian mustard.

Abstract

Nitrogen use efficiency (NUE) of Indian mustard (Brassica juncea (L.) Czern & Coss.) is low and most breeding efforts to combine NUE with crop performance have not succeeded. Underlying genetics also remain unexplored. We tested 92 SNP-genotyped inbred lines for yield component traits, N uptake efficiency (NUPEFF), nitrogen utilization efficiency (NUTEFF), nitrogen harvest index (NHI) and NUE for two years at two nitrogen doses (N_o without added N and N₁₀₀ added @100 kg/ha). Genotypes IC-2489-88, M-633, MCP-632, HUJM 1080, GR-325 and DJ-65 recorded high NUE at low N. These also showed improved crop performance under high N. One determinate mustard genotype DJ-113 DT-3 revealed maximum NUTEFF. Genome wide association studies (GWAS) facilitated recognition of 17 quantitative trait loci (QTLs). Environment specificity was high. B-genome chromosomes (B02, B03, B05, B07 and B08) harbored many useful loci. We also used regional association mapping (RAM) to supplement results from GWAS. Annotation of the genomic regions around peak SNPs helped to predict several gene candidates for root architecture, N uptake, assimilation and remobilization. CAT9 (At1g05940) was consistently envisaged for both NUE and NUPEFF. Major N transporter genes, NRT1.8 and NRT3.1 were predicted for explaining variation for NUTEFF and NUPEFF, respectively. Most significant amino acid transporter gene, AAP1 appeared associated with NUE under limited N conditions. All these candidates were predicted in the regions of high linkage disequilibrium. Sequence information of the predicted candidate genes will permit development of molecular markers to aid breeding for high NUE.

     

Methylation Status of Arabidopsis DNA Repair Gene Promoters During Agrobacterium Infection Reveals Epigenetic Changes in Three Generations

Agrobacterium tumefaciens is a unique pathogen with the ability to transfer a portion of its DNA, the T-DNA, to other organisms. The role of DNA repair genes in Agrobacterium transformation remains controversial. In order to understand if the host DNA repair response and dynamics was specific to bacterial factors such as Vir proteins, T-DNA, and oncogenes, we profiled the expression and promoter methylation of various DNA repair genes. These genes belonged to nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), homologous recombination (HR), and non-homologous end joining (NHEJ) pathways. We infected Arabidopsis plants with different Agrobacterium strains that lacked one or more of the above components so that the influence of the respective factors could be analysed. Our results revealed that the expression and promoter methylation of most DNA repair genes was affected by Agrobacterium, and it was specific to Vir proteins, T-DNA, oncogenes, or the mere presence of bacteria. In order to determine if Agrobacterium induced any transgenerational epigenetic effect on the DNA repair gene promoters, we studied the promoter methylation in two subsequent generations of the infected plants. Promoters of at least three genes, CEN2, RAD51, and LIG4 exhibited transgenerational memory in response to different bacterial factors. We believe that this is the first report of Agrobacterium-induced transgenerational epigenetic memory of DNA repair genes in plants. In addition, we show that Agrobacterium induces short-lived DNA strand breaks in Arabidopsis cells, irrespective of the presence or absence of virulence genes and T-DNA.

     

EFFECT OF FERMENTATION PARAMETERS ON BIO-ALCOHOLS PRODUCTION FROM GLYCEROL USING IMMOBILIZED Clostridium pasteurianum: AN OPTIMIZATION STUDY

This article addresses the issue of effect of fermentation parameters for conversion of glycerol (in both pure and crude form) into three value-added products, namely, ethanol, butanol, and 1,3-propanediol (1,3-PDO), by immobilized Clostridium pasteurianum and thereby addresses the statistical optimization of this process. The analysis of effect of different process parameters such as agitation rate, fermentation temperature, medium pH, and initial glycerol concentration indicated that medium pH was the most critical factor for total alcohols production in case of pure glycerol as fermentation substrate. On the other hand, initial glycerol concentration was the most significant factor for fermentation with crude glycerol. An interesting observation was that the optimized set of fermentation parameters was found to be independent of the type of glycerol (either pure or crude) used. At optimum conditions of agitation rate (200 rpm), initial glycerol concentration (25 g/L), fermentation temperature (30°C), and medium pH (7.0), the total alcohols production was almost equal in anaerobic shake flasks and 2-L bioreactor. This essentially means that at optimum process parameters, the scale of operation does not affect the output of the process. The immobilized cells could be reused for multiple cycles for both pure and crude glycerol fermentation.     

Genetic polymorphisms in GSTM1, GSTT1, GSTP1, GSTM3 and the susceptibility to gallbladder cancer in North India

Abstract

The glutathione S-transferase (GSTs) are polymorphic supergene family of detoxification enzymes that are involved in the metabolism of numerous potential carcinogens. Several allelic variants of polymorphic GSTs show impaired enzyme activity and are suspected to increase the susceptibility to various cancers. To find out the association of GST variants with risk of gallbladder cancer, the distribution of polymorphisms in the GST family of genes (GSTT1, GSTM1, GSTP1, and GSTM3) were studied in 106 cancer patients and 201 healthy controls. Genotypes were analysed by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP). The frequencies of GSTM1 null and GSTM3*BB genotypes did not differ between patients and controls. The overall frequency of GSTT1 null was lower in cases as compared with controls (p=0.003, Odds ratio (OR)?=?0.2, 95% confidence interval (CI), 0.1–0.6). After sex stratification, the GSTT1 null frequency was reduced only in female patients (p=0.008, OR?=?0.2, 95% CI?=?0.1–0.6). However, the GSTP1, ile/val genotype and the val allele were significantly higher in cases than controls (p=0.013, OR?=?1.9, 95% CI?=?1.1–3.1; p=0.027, OR?=?1.5, 95% CI?=?1.0–2.1), respectively. To study gene–gene interactions, a combined risk of gallbladder cancer due to ile/val or val/val were calculated in combination with null alleles of GSTM1 and GSTT1 or the *B allele of GSTM3, but there was no enhancement of risk. Gallstones were present in 57.5% of patients with gallbladder cancer, but there were no significant differences between allelic/genotype frequencies of the studied GST genes polymorphisms between patients with or without gallstones. To best of our knowledge, this is the first paper showing ile/val genotypes and val allele of GSTP1 to be associated with higher risk of gallbladder cancer.     

Improved Detection of Remote Homologues Using Cascade PSI-BLAST: Influence of Neighbouring Protein Families on Sequence Coverage

Background

Development of sensitive sequence search procedures for the detection of distant relationships between proteins at superfamily/fold level is still a big challenge. The intermediate sequence search approach is the most frequently employed manner of identifying remote homologues effectively. In this study, examination of serine proteases of prolyl oligopeptidase, rhomboid and subtilisin protein families were carried out using plant serine proteases as queries from two genomes including A. thaliana and O. sativa and 13 other families of unrelated folds to identify the distant homologues which could not be obtained using PSI-BLAST.

Methodology/Principal Findings

We have proposed to start with multiple queries of classical serine protease members to identify remote homologues in families, using a rigorous approach like Cascade PSI-BLAST. We found that classical sequence based approaches, like PSI-BLAST, showed very low sequence coverage in identifying plant serine proteases. The algorithm was applied on enriched sequence database of homologous domains and we obtained overall average coverage of 88% at family, 77% at superfamily or fold level along with specificity of ∼100% and Mathew’s correlation coefficient of 0.91. Similar approach was also implemented on 13 other protein families representing every structural class in SCOP database. Further investigation with statistical tests, like jackknifing, helped us to better understand the influence of neighbouring protein families.

Conclusions/Significance

Our study suggests that employment of multiple queries of a family for the Cascade PSI-BLAST searches is useful for predicting distant relationships effectively even at superfamily level. We have proposed a generalized strategy to cover all the distant members of a particular family using multiple query sequences. Our findings reveal that prior selection of sequences as query and the presence of neighbouring families can be important for covering the search space effectively in minimal computational time. This study also provides an understanding of the ‘bridging’ role of related families.