Population analysis of <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> by using endogenous repetitive DNA sequences and mating-type alleles in different districts of Karnataka,India

Rice is the staple food crop of more than 60% of the population of the world. This crop suffers from blast disease caused by Magnaporthe oryzae. Information on the mating-type allele distribution and diversity of the pathogen population for the state of Karnataka, India is scanty. With this background, a total of 72 isolates of M. oryzae from rice in different districts of Karnataka were examined for identifying sexual mating alleles MAT1, MAT2 and understanding the genetic diversity based on DNA fingerprint of pot2, an inverted repeat transposon. Among 72 isolates, 44 isolates belonged to MAT1 type (male fertile) and 28 isolates were of MAT2 (female fertile) and there were no hermaphrodite isolates. In a given geographical location, only one mating type was identified. Results revealed that the isolates obtained from these regions are not sexually fertile showing predominant asexual reproduction. Hence, genetic variation observed in the pathogen may be mainly because of high copy number of transposons. A high copy number transposon, namely Pot2, was selected in our study to detect genetic diversity of the pathogen. Pot2 rep-PCR DNA fingerprinting profile showed 27 polymorphic bands with bands ranging in size from 0.65 to 4.0 kb and an average of 10 to 14 bands per isolate. Five distinct clusters were formed with two major, two minor, and one outlier. Clusters 4 and 5 are further subdivided into three sub-clusters. Some of the isolates belonging to clusters 3, 4, and 5 are interlinked as these locations are close to one another sharing common geographical parameters and boundaries. This knowledge on the sexual behavior and genetic diversity of M. oryzae is important with respect to breeding for disease resistance.     

Protective Effects of 7‐Hydroxycoumarin on Dyslipidemia and Cardiac Hypertrophy in Isoproterenol‐Induced Myocardial Infarction in Rats

This study evaluates the protective effects of 7‐hydroxycoumarin (7‐HC) on dyslipidemia and cardiac hypertrophy in isoproterenol (ISO) induced myocardial infarction (MI) in rats. Rats were pre‐ and co treated with 7‐HC (16 mg/kg) daily for 8 days. ISO (100 mg/kg) was subcutaneously injected into rats on seventh and eighth days to induce MI. Increased activity/levels of serum creatine kinase‐MB (CK‐MB), troponin‐T, plasma lipid peroxidation products, and altered levels of lipids in the serum and heart and serum lipoproteins were noted in ISO‐induced rats. ISO‐induced myocardial infarcted rats revealed increased hypertrophy (cardiac and left ventricular) and hepatic 3‐hydroxyl 3‐methylglutaryl‐coenzyme‐A reductase (HMG‐CoA reductase) activity. Pre and cotreatment with 7‐HC revealed significant protective effects on all the biochemical parameters evaluated. The in vitro study demonstrated its free radical scavenging property. Thus, 7‐HC protects ISO‐induced MI in rats by its free radical scavenging and antihyperlipidaemic and antihypertrophic properties.     

A novel piperazine linked β-amino alcohols bearing a benzosuberone scaffolds as anti-proliferative agents

A new series of 1-((9-chloro-2,3-dimethyl-6,7-dihydro-5H-benzo[7]annulen-8-yl)methoxy)-3-(4-phenylpiperzin-1-yl) propan-2-ols (6a-k) have been designed, synthesized and their structures were established by spectroscopic data (FT-IR, ¹H NMR, ¹³C NMR, HRMS) and further confirmed by X-ray analysis. The newly synthesized compounds 6a-k were evaluated for their in vitro anti-proliferative activity against four cancer cell lines such as HeLa (cervical), MDA-MB-231 (breast), A549 (lung) and MIAPACA (pancreatic). Among the compounds tested, the compound 6e displayed most potent activity against four cancer cell lines with GI₅₀ values ranging from 0.010 to 0.097 μM. The structure and anti-proliferative activity relationship was further supported by in silico molecular docking study of the active compounds against Colchicine binding site of β-tubulin.     

Role of electrophilic and general base catalysis in the mechanism of Escherichia coli uracil DNA glycosylase.

Escherichia coli uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil bases in DNA by flipping the deoxyuridine from the DNA helix [Stivers, J. T., et al. (1999) Biochemistry 38, 952]. A general acid-base mechanism has been proposed whereby His187 facilitates leaving group departure by protonating the O2 of uracil and Asp64 activates a water molecule for nucleophilic attack at C1' of the deoxyribose. Detailed kinetic studies on the H187Q, H187A, and D64N mutant enzymes indicate that Asp64 and His187 stabilize the chemical transition state by 5.3 and 4.8 kcal/mol, respectively, with little effect on substrate or product binding. The pH dependence of k(cat) for wild-type and H187Q UDG indicates that an unprotonated group in the enzyme-substrate complex (pK(a) = 6.2 +/- 0.2) is required for catalysis. This unprotonated group has a small DeltaH of ionization (-0.4 +/- 1.7 kcal/mol) and is absent in the pH profile for D64N UDG, suggesting that it corresponds to the general base Asp64. The pH dependence of k(cat) for wild-type, H187Q, and D64N UDG shows no evidence for an essential protonated group over the pH range of 5.5-10. Hence, the pK(a) of His187 must be outside this pH range if it serves as an electrophilic catalyst. These results support a mechanism in which Asp64 serves as the general base and His187 acts as a neutral electrophile, stabilizing a developing negative charge on uracil O2 in the transition state. In the following paper of this issue we establish by crystallography and heteronuclear NMR spectroscopy that the imidazole of His187 is neutral during the catalytic cycle of UDG.     

Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria

Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have several positive implications with respect to future development of a transformation system for cellulolytic thermophiles. Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280. Received 12 September 2000/ Accepted in revised form 20 November 2000     

Elucidation of Molecular Identity of the W3 Locus and Its Implication in Determination of Flower Colors in Soybean

The wide range of flower colors in soybean is controlled by six independent loci (W1, W2, W3, W4, Wm, and Wp). Among these loci, mutations in the W3 locus under the w4 allelic background (i.e., w3w4) produce near-white flowers, while the W3w4 genotype produces purple throat flowers. Although a gene encoding dihydroflavonol 4-reductase, DFR1, has been known to be closely associated with the W3 locus, its molecular identity has not yet been characterized. In the present study, we aimed to determine whether DFR1 is responsible for allelic variations in the W3 locus. On the basis of the sequence of a DFR probe, Glyma.14G072700 was identified as a candidate gene for DFR1, and nucleotide sequences of Glyma.14G072700 from cultivars with previously validated genotypes for the W3 locus were determined. As a result, a number of nucleotide polymorphisms, mainly single-base substitutions, between both coding and 5′-upstream region sequences of the W3 and w3 alleles were identified. Among them, an indel of 311-bp in the 5′-upstream region was noteworthy, since the Glyma.14G072700 in all the w3 alleles examined contained the indel, whereas that in all the W3 alleles did not; the former was barely expressed, but the latter was well expressed. These results suggest that Glyma.14G072700 is likely to correspond to DFR1 for the W3 locus and that its expression patterns may lead to allelic color phenotypes of W3 and w3 alleles under the w4 allelic background.     

Susceptibility of Oriental armyworm,Mythimna separata (Lepidoptera: Noctuidae) larvae and pupae to native entomopathogenic nematodes

The Oriental armyworm, Mythimna separata Walker (Lepidoptera: Noctuidae) is a major pest of cereal crops, and its outbreaks result in complete crop loss. In this study, we tested the pathogenicity and reproductive potential of indigenous entomopathogenic nematodes (EPNs), Heterorhabditis indica Poinar, Karunakar, & David NBAIIH38, Steinernema abbasi Elawad NBAIISa01, S. carpocapsae Weiser NBAIISc05 and S. siamkayai Stock, Somsook and Reid NBAIRS92 against larval and pupal stages of M. separata under laboratory conditions. Steinernema carpocapsae caused significantly greater mortality in second- and fourth-instar larval stages than other EPNs species. Steinernema carpocapsae caused greatest mortality 100% in larval stages and 75% in pupal stages. Significant differences were observed in the lethal concentration values (LC₅₀ and LC₉₀) of EPNs species against different stages of M. separata. Differences in penetration and multiplication in the fourth-instar larval stages of M. separata were observed amongst the EPNs species. However, further studies are needed to reveal the field performance of EPN isolates tested to be included in the IPM programme of M. separata.     

Cutting edge: intravenous Ig inhibits invariant NKT cell-mediated allergic airway inflammation through FcγRIIIA-dependent mechanisms

Despite their increasing use in autoimmune, inflammatory, and allergic conditions, the mechanism of action of i.v. Igs (IVIg) is poorly understood. On the basis of the critical role of invariant NKT (iNKT) cells in allergic airway inflammation (AAI) and their constitutive expression of the low-affinity IgG receptor FcγRIIIA, we surmised that IVIg targets iNKT cells to exert their anti-inflammatory effect. We found that IVIg treatment significantly inhibited AAI in OVA-sensitized C57BL/6 mice and downregulated α-galactosylceramide-induced iNKT cell activation and cytokine production. Allergic responses were restored in iNKT cell-deficient mice by transferring iNKT cells from PBS- but not from IVIg-treated mice, suggesting that IVIg acts directly on activated iNKT cells that have a critical role in AAI. The inhibitory effects of IVIg on both iNKT cell activation/function and OVA-driven AAI were lost in FcγRIIIA(-/-) mice. Our data unravel an FcγRIIIA-dependent inhibitory effect of IVIg on activated iNKT cells that confers protection in AAI.     

Bacterial transformation using micro-shock waves

Shock waves are one of the most competent mechanisms of energy dissipation observed in nature. We have developed a novel device to generate controlled micro-shock waves using an explosive-coated polymer tube. In this study, we harnessed these controlled micro-shock waves to develop a unique bacterial transformation method. The conditions were optimized for the maximum transformation efficiency in Escherichia coli. The maximum transformation efficiency was obtained when we used a 30 cm length polymer tube, 100 μm thick metal foil, 200 mM CaCl₂, 1 ng/μl plasmid DNA concentration, and 1 × 10⁹ cell density. The highest transformation efficiency achieved (1 × 10⁻⁵ transformants/cell) was at least 10 times greater than the previously reported ultrasound-mediated transformation (1 × 10⁻⁶ transformants/cell). This method was also successfully employed for the efficient and reproducible transformation of Pseudomonas aeruginosa and Salmonellatyphimurium. This novel method of transformation was shown to be as efficient as electroporation with the added advantage of better recovery of cells, reduced cost (40 times cheaper than a commercial electroporator), and growth phase independent transformation.