Nif-hybrids of Enterobacter: selection for nif gene integration with chlorate

The nif gene group from Klebsiella can be transferred into Enterobacter cloacae by conjugation using Escherichia coli donor cells carrying the composite self-transmissible nif-plasmid pRD1. A small fraction of the hybrids obtained is stable upon prolonged passaging without selection. Their stability is due to integration of pRD1 into the chromosome. Such integration hybrids were chlorate resistant, and nitrate reductase negative, which indicated that integration preferentially occurred within one of the genes for the production or functioning of this enzyme. Chlorate resistance could, therefore, be used to select for additional nitrate reductase-negative sublines with pRD1 in their chromosome. Such sublines have been analyzed further for the presence of nif genes, other pRD1 markers, and for stability. In all except one the complete plasmid seems to have been integrated. Some tend to revert to nitrate utilisation (chlorate sensitivity).     

Transcriptional regulation of the antioxidant protein 2 gene, a thiol-specific antioxidant, by lens epithelium-derived growth factor to protect cells from oxidative stress.

Antioxidant protein 2 (AOP2), a member of the newly defined family of thiol-specific antioxidant proteins, has been shown to remove H(2)O(2) and protect proteins and DNA from oxidative stress. Here we report that LEDGF is one of the regulatory factors for the AOP2 gene. We found that LEDGF bound to the heat shock element and to stress-related elements in the AOP2 promoter. It trans-activated expression of AOP2-CAT in COS-7 cells and lens epithelial cells overexpressing LEDGF. Mutations in the heat shock element and stress-related elements of the AOP2 promoter reduced LEDGF-dependent trans-activation. Lens epithelial cells showed a higher level of AOP2 mRNA in the presence of LEDGF. Cells overexpressing LEDGF exhibited a higher level of AOP2 protein, the level of which was directly related to the increase in cellular protection. Thus, LEDGF, by activating the AOP2 gene, protected and enhanced the survival of cells under oxidative stress.     

Laboratory Estimation of Field Resistance of Potato to Late Blight

Based on quantitative measurement of components of field resistance of potatoes to late blight, a multiple linear regression equation is derived for laboratory estimation of Area Under Disease Progress Curve. The equation accounts for 82% of variation in Area Under Disease Progress Curve. Transformation of data or inclusion of more terms do not improve the equation significantly. The method is suggested as a replacement or supplement to field testing.     

Handling class imbalance problem in miRNA dataset associated with cancer

MiRNAs are small (~22nt long) non-coding RNA sequences; binds to the complementarity target sites in 3'' Untranslated Region (UTR) of mRNA sequences but not restricted to other mRNA regions viz., 5'' UTR and Coding sequences (CDS). Complementarity binding of miRNA to mRNA target sites either results in complete degradation of the mRNA itself or it may regulate the mRNA as an oncogene or as a tumor suppressor gene. However, the exact mechanism involved in identifying a miRNA to be associated with cancer is still unclear. Further, with the outburst in the number of miRNAs sequences recorded every year in miRBase, the gap is still widening mainly due to the laborious and economically unfavorable experimental procedures associated with the functional annotation. Motivated by the fact, we constructed a two-step support vector machine-based predictive model - miRSEQ and miRINT. However, the major pitfall during the construction of the model is the class imbalance problem. Hence, in order to overcome class imbalance problem, in the present study we empirically compare the effectiveness of two different methods viz., Synthetic Minority Oversampling Technique (SMOTE) and cost-senstive learning method. Performance measures were evaluated in terms of Precision and Recall. Based on our result, it was observed that for miRNA dataset with high class imbalance utilized for predicting association of cancer, cost-sensitive method outperformed the oversampling method.     

Identification of UDP-linked murein precursors as contaminants in recombinant proteins of low molecular weight.

The A(280)/A(260) ratio of a purified protein is frequently used as an indication of the purity of the preparation with respect to nucleic acids. We show here that for low-molecular-weight recombinant proteins purified from Escherichia coli, a low A(280)/A(260) ratio can also result from contamination with UDP-linked murein precursors derived from bacterial cell wall metabolism. Although these precursors are small molecules of molecular weight 1000-1200, they comigrate in gel filtration with recombinant human FKBP (MW 11,820). This gel filtration behavior, which is distinct from that of unmodified mononucleotides, does not reflect binding interactions with FKBP, but is an intrinsic property of these precursors. Therefore, these molecules would be expected to copurify with other low-molecular-weight proteins, especially in the abbreviated purification protocols made possible by freeze-thaw release of recombinant proteins from E. coli (Johnson, B. H., and Hecht, M. H. (1994) BioTechnology 12, 1357-1360). Several alternative strategies are discussed for integrating these findings into the design of improved purification procedures for low-molecular-weight recombinant proteins.     

Design,synthesis, and evaluation of curcumin analogues as potential inhibitors of bacterial sialidase

Sialidases are key virulence factors that remove sialic acid from the host cell surface glycan, unmasking receptors that facilitate bacterial adherence and colonisation. In this study, we developed potential agents for treating bacterial infections caused by Streptococcus pneumoniae Nan A that inhibit bacterial sialidase using Turmeric and curcumin analogues. Design, synthesis, and structure analysis relationship (SAR) studies have been also described. Evaluation of the synthesised derivatives demonstrated that compound 5e was the most potent inhibitor of S. pneumoniae sialidase (IC₅₀?=?0.2?±?0.1?µM). This compound exhibited a 3.0-fold improvement in inhibitory activity over that of curcumin and displayed competitive inhibition. These results warrant further studies confirming the antipneumococcal activity 5e and indicated that curcumin derivatives could be potentially used to treat sepsis by bacterial infections.     

Preliminary X-ray investigation of a bifunctional inhibitor from Indian finger millet (ragi).

A bifunctional alpha-amylase/trypsin inhibitor that has two binding sites has been purified from ragi. The inhibitor has been crystallized from its ammonium sulphate solution by the vapour diffusion method. The crystals belong to the orthogonal space group P2(1)2(1)2(1) with unit cell dimensions a = 30.49 A, b = 56.30 A, c = 73.65 A and Z = 4.