Expression,purification and molecular modeling of the NIa protease of Cardamom mosaic virus

The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8?M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease.     

Molecular modeling and conformational analysis of native and refolded viral genome-linked protein of cardamom mosaic virus

The viral genome-linked protein (VPg) of Potyviruses is covalently attached to the 5' end of the genomic RNA. Towards biophysical characterization, the VPg coding region of Cardamom mosaic virus (CdMV) was amplified from the cDNA and expressed in E. coli. Most of the expressed VPg aggregated as inclusion bodies that were solubilized with urea and refolded with L-arginine hydrochloride. The various forms of CdMV VPg (native, denatured and refolded) were purified and the conformational variations between these forms were observed with fluorescence spectroscopy. Native and refolded CdMV VPg showed unordered secondary structure in the circular dichroism (CD) spectrum. The model of CdMV VPg was built based on the crystal structure of phosphotriesterase (from Pseudomonas diminuta), which had the maximum sequence homology with VPg to identify the arrangement of conserved amino acids in the protein to study the functional diversity of VPg. This is the first report on the VPg of CdMV, which is classified as a new member of the Macluravirus genus of the Potyviridae family.     

Isozymes variability among <Emphasis Type="Italic">Fusarium udum</Emphasis> resistant cultivars of pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) (Millsp)

The present study was carried out to select the different pigeonpea cultivars for resistance against wilt caused by Fusarium udum and to assess the genetic variability among the resistant and susceptible cultivars. These cultivars were screened by root dip inoculation and classified into resistant (ICP 8863 and 9145), moderately resistant (ICP 11681 and Selection-1), susceptible (ICP 7118, TRG-1 and LRG-30) and highly susceptible cultivars (ICP-2376 and LRG-41). The peroxidase activity (PEO) in both leaf and root tissues of four pigeonpea cultivars (ICP 8863, Selection-1, ICP 2376 and LRG-30) were determined at 1^st, 4^th and 7^th day after inoculation (DAI) in healthy and F. udum infected tissues. Higher PEO activity in both leaf and root was observed and at 4^th DAI in susceptible cultivars. In native-PAGE analysis of isozymes, the induction of specific leaf peroxidase band (Em=0.17) and two root peroxidase bands (Em=0.24 and 0.55) were observed in ICP 8863 after inoculation. Significant differences were observed in the leaf phosphatase and esterase banding profiles of all the cultivars. The presence of leaf phosphatase band at Em of 0.04 was observed only in ICP 8863 and 11681. The leaf esterase band (Em=0.3) was well expressed in ICP 8863 when compared to other cultivars. The significance of peroxidase in plant defense mechanism and utility of biochemical markers in breeding programmes are discussed. Part of M.Sc. (Ag) thesis of the first author and approved by the Acharya N.G. Ranga Agricultural University during March 2002.     

Reagent Strips and Conventional Tests for Acid Production from Mannitol and Coagulase Activity of Staphylococcus: a Comparative Study

A total of 244 Staphylococcus strains were tested simultaneously for acid production from mannitol and for coagulase activity with reagent-impregnated paper strips and with their conventional counterparts. Significant correlation was obtained with 97.9% of the strains for mannitol and with 95% for the coagulase test. The paper strip method is a combined test for both mannitol and coagulase tests, thus making it more convenient and simpler than conventional methods. The results are obtained rapidly within 6 hr by the paper strip method. However, as the paper strip method is designed for the aerobic system, the conventional tests were also carried out under aerobic conditions to compare the results.     

Melatonin enhances the sensitivity of circadian pacemakers to light in the nocturnal field mouse Mus booduga

The effect of exogenous melatonin (1 mg/kg) on light pulse (LP) induced phase shifts of the circadian locomotor activity rhythm was studied in the nocturnal field mouse Mus booduga. Three phase response curves (PRCs: LP, control, and experimental) were constructed to study the effect of co-administration of light and melatonin at various circadian times (CTs). The LP PRC was constructed by exposing animals free-running in constant darkness (DD) to LPs of 100-lux intensity and 15-min duration, at various CTs. The control and experimental PRCs were constructed by using a single injection of either 50% DMSO or melatonin (1 mg/kg dissolved in 50% DMSO), respectively, administered 5 min before LPs, to animals free-running in DD. A single dose of melatonin significantly modified the waveform of the LP PRC. The experimental PRC had significantly larger areas under advance and delay regions of the PRC compared to the control PRC. This was also confirmed when the phase shifts obtained at various CTs were compared between the three PRCs. The phase delays at three phases (CT12, CT14, and CT16) of the experimental PRCs were significantly greater than those of the control and the LP PRCs. Based on these results we conclude that phase shifting effects of melatonin and light add up to produce larger responses.     

C-Dimethylated Flavones as Possible Potential Anti-Tubercular and Anticancer Agents.

The present investigation describes an intramolecular Oxa-Michael addition of penta-substituted phenols to the enone of the tether in the presence of iodine as the oxidizing agent. Ten C-Dimethylated flavones with moderate to good yields ( 10a – j , 60–89 %) were isolated by heating the corresponding C-dimethylated chalcones using iodine in DMSO. Using the Microplate Alamar Blue test (MABA) technique, the drugs′ quantitative drug susceptibility against the H37Rv strain of replicating Mycobacterium TB was determined. The sensitivity of two of the developed compounds ( 10e , 10h ) was up to 6.25 g/mL. The human lung adenocarcinoma cell lines (A549) were used in the anticancer study, which was carried out using the MTT cell proliferation assay. In A549 cell lines, four flavones demonstrated anticancer activity with IC₅₀ values between 39 and 48 μM. The C-dimethylated flavones, 10b (3,4-dimethoxy), 10c (2,3,4-trimethoxy), 10e (p-fluoro) and 10 g (N-methyl indole) substitutions on ring ‘B’ showed good anticancer activity with IC₅₀ values 39.17, 39.21, 48.43 and 43.48 μM, respectively. The compounds 10b , 10c , 10d , 10e , and 10i had improved binding and interaction profiles among all the compounds examined during the current In Silico research, as shown by the docking simulations against two targets EGFR and MTB MurI.     

Induction of the Endoplasmic Reticulum Stress and Autophagy in Human Lung Carcinoma A549 Cells by Anacardic Acid

Anacardic acid (AA, 2-hydroxy-6-pentadecylbenzoic acid), a constituent of the cashew-nut shell, has a variety of beneficial effects on the treatment of cancer and tumors. However, the fact that AA induces ER stress and autophagy in cancer cell is not known. We investigated the effect of AA on ER-stress and autophagy-induced cell death in cancer cells. Because of our interest in lung cancer, we used the non-small cell lung adenocarcinoma A549 cells treated with 3.0 μg/ml of AA for this research. In this research we found that AA induces intracellular Ca²⁺ mobilization and ER stress. AA induced the ER stress-inducing factors, especially IRE1α, and the hallmarks of UPR, Grp78/Bip and GADD153/CHOP. AA inhibited the expression of p-PERK and its downstream substrate, p-elF2α. We also demonstrated that AA induces autophagy. Up-regulation of autophagy-related genes and the appearance of autophagosome in transfected cells with green fluorescent protein (GFP)-LC3 and GFP-Beclin1 plasmids showed the induction of autophagy in AA-treated A549 cells. The morphological analysis of intracellular organelles by TEM also showed the evidence that AA induces ER stress and autophagy. For the first time, our research showed that AA induces ER stress and autophagy in cancer cells.