Reconstitution of R6K DNA replication in vitro using 22 purified proteins

We have reconstituted a multiprotein system consisting of 22 purified proteins that catalyzed the initiation of replication specifically at ori gamma of R6K, elongation of the forks, and their termination at specific replication terminators. The initiation was strictly dependent on the plasmid-encoded initiator protein pi and on the host-encoded initiator DnaA. The wild type pi was almost inert, whereas a mutant form containing 3 amino acid substitutions that tended to monomerize the protein was effective in initiating replication. The replication in vitro was primed by DnaG primase, whereas in a crude extract system that had not been fractionated, it was dependent on RNA polymerase. The DNA-bending protein IHF was needed for optimal replication and its substitution by HU, unlike in the oriC system, was less effective in promoting optimal replication. In contrast, wild type pi-mediated replication in vivo requires IHF. Using a template that contained ori gamma flanked by two asymmetrically placed Ter sites in the blocking orientation, replication proceeded in the Cairns type mode and generated the expected types of termination products. A majority of the molecules progressed counterclockwise from the ori, in the same direction that has been observed in vivo. Many features of replication in the reconstituted system appeared to mimic those of in vivo replication. The system developed here is an important milestone in continuing biochemical analysis of this interesting replicon.     

Changes in Sugar Content and Activities of Sucrose Metabolizing Enzymes in Roots and Nodules of Lentil

Activities of acid and alkaline invertases and sucrose synthase were determined in roots and nodules of lentil at various stages of development. Alkaline invertase and sucrose synthase were both involved in sucrose metabolism in the nodule cytosol, but there was only a small amount of acid invertase present. Activity of sucrose metabolizing enzymes in roots was significantly less than that observed in the nodules. Amongst sugars, sucrose was found to be the main component in the host cytosol. Lentil neutral invertase (LNI) was partially purified from nodules at 50 days after sowing (DAS). Two forms of invertase were identified, i.e., a major form of 71 kDa which was taken for enzyme characterization and a minor form of 270 kDa which was not used for further studies. The purified enzyme exhibited typical hyperbolic saturation kinetics for sucrose hydrolysis. It had a Km of 11.0 to 14.0 mM for sucrose depending upon the temperature, a pH optimum of 6.8 and an optimum temperature of 40 °C. Compared with raffinose and stachyose, sucrose was better substrate for LNI. The enzyme showed no significant hydrolysis of maltose and p-nitrophenyl--D-glucopyranoside, showing its true -fructosidase nature. LNI is completely inhibited by HgCl₂, MnCl₂ and iodoacetamide but not by CaCl₂, MgCl₂ or BaCl₂.     

Comparative study of perturbations of peripheral markers in different stressors in rats

Stress has been implicated in the etiopathogenesis of several diseases. In the present study, the effects of acute (AS), chronic (CS), and chronic unpredictable stress (CUS) were studied on the ulcer index, adrenal gland mass, and biochemical and hormonal changes in rats. The stress was provided in the form of immobilization-immobilization for 150 min, once only, and for 10 consecutive days in CS and CUS. In CUS, animals received variable unpredictable stressors. Immediately after stress, animals were decapitated, blood was collected, and plasma was separated for the estimation of plasma glucose, triglyceride, cholesterol, creatine kinase (CK), corticosterone, and insulin. The adrenal gland and stomach were also dissected for mass and ulcer scoring, respectively. AS significantly increased the ulcer index, plasma glucose, CK, corticosterone, and insulin. CS and CUS significantly increased the ulcer index, adrenal gland mass, and corticosterone. In CS, a significant decrease in plasma triglyceride and cholesterol levels was found, but in CUS only cholesterol was decreased significantly. High CK activity and hyperglycemia maintain the energy demands of metabolism, and elevated corticosterone desensitizes the insulin receptor in AS. In CS and CUS, prolonged elevation of corticosterone shifts metabolism to utilization of lipids as a secondary substrate by gluconeogenesis. From our experiment, it is clear that AS causes maximum activation of energy metabolism, which becomes specific after habituation in prolonged CS. These biochemical manipulations in the body by using different types of stressors are good markers that can be of great use to understand, target, and manage stress-induced etiologies.     

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.     

Dothistroma pini,a forest pathogen,contains homologs of aflatoxin biosynthetic pathway genes

Homologs of aflatoxin biosynthetic genes have been identified in the pine needle pathogen Dothistroma pini. D. pini produces dothistromin, a difuranoanthraquinone toxin with structural similarity to the aflatoxin precursor versicolorin B. Previous studies with purified dothistromin suggest a possible role for this toxin in pathogenicity. By using an aflatoxin gene as a hybridization probe, a genomic D. pini clone was identified that contained four dot genes with similarity to genes in aflatoxin and sterigmatocystin gene clusters with predicted activities of a ketoreductase (dotA), oxidase (dotB), major facilitator superfamily transporter (dotC), and thioesterase (dotD). A D. pini dotA mutant was made by targeted gene replacement and shown to be severely impaired in dothistromin production, confirming that dotA is involved in dothistromin biosynthesis. Accumulation of versicolorin A (a precursor of aflatoxin) by the dotA mutant confirms that the dotA gene product is involved in an aflatoxin-like biosynthetic pathway. Since toxin genes have been found to be clustered in fungi in every case analyzed so far, it is speculated that the four dot genes may comprise part of a dothistromin biosynthetic gene cluster. A fifth gene, ddhA, is not a homolog of aflatoxin genes and could be at one end of the dothistromin cluster. These genes will allow comparative biochemical and genetic studies of the aflatoxin and dothistromin biosynthetic pathways and may also lead to new ways to control Dothistroma needle blight.     

Agrobacterium-mediated transformation of cauliflower: optimization of protocol and development of Bt-transgenic cauliflower

A number of factors that are known to influence genetic transformation were evaluated to optimizeAgrobacterium-mediated transformation of hypocotyl explants of cauliflower variety Pusa Snowball K-1. The binary vector p35SGUSINT mobilized intoAgrobacterium strain GV2260 was used for transformation and transient GUS expression was used as the basis for identifying the most appropriate conditions for transformation. Explant age, preculture period, bacterial strain and density were found to be critical determinants of transformation efficiency. Using the optimized protocol, the syntheticcryIA(b) gene was mobilized into cauliflower. Molecular analyses of transgenics established the integration and expression of the transgene. Insect bioassays indicated the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae.     

Phospholipase A2-activating protein--an important regulatory molecule in modulating cyclooxygenase-2 and tumor necrosis factor production during inflammation

Inflammation is a complex multifactorial process and a hallmark of many inflammatory diseases. Most of the tissue destruction that occurs in these diseases is the result of an aberrant or often uncontrolled immune response. Factors that play an important role in such diseases include pro-inflammatory cytokines, complement, and eicosanoids. This review focuses on eicosanoids and their regulation via phospholipase A2-activating protein, which could be targeted as a new therapeutic tool to control inflammatory diseases.