Real time changes in the expression of eicosanoid synthesizing enzymes during inflammation.

Immune responses during inflammation involve complex, well-coordinated lipid signaling pathways. Eicosanoids are a class of lipid signaling molecules derived from polyunsaturated fatty acids such as arachidonic acid and constitute a major network that controls inflammation and its subsequent resolution. Arachidonic acid is metabolized by enzymes in three different pathways to form a variety of lipid metabolites that can be either pro- or anti-inflammatory. Therefore, an understanding of the time-dependent gene expression, lipid metabolite profiles and cytokine profiles during the initial inflammatory response is necessary, as it will allow for the design of time-dependent therapeutics. Herein, we investigate the multi-level regulation of this process. After stimulating RAW 264.7 cells, a mouse-derived macrophage cell line commonly used to examine inflammatory responses, we examine the gene expression of 44 relevant lipid metabolizing enzymes from the different eicosanoid synthesizing classes. We also measure the formation of lipid metabolites and production of cytokines at selected time points. Results reveal a dynamic relationship between the time-course of inflammation dependent gene expression of the three eicosanoid synthesizing enzymes.

Multiple enzymatic activities of ParB/Srx superfamily mediate sexual conflict among conjugative plasmids.

Conjugative plasmids are typically locked in intergenomic and sexual conflicts with co-resident rivals, whose translocation they block using fertility inhibition factors (FINs). We describe here the first crystal structure of an enigmatic FIN Osa deployed by the proteobacterial plasmid pSa. Osa contains a catalytically active version of the ParB/Sulfiredoxin fold with both ATPase and DNase activity, the latter being regulated by an ATP-dependent switch. Using the Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS), a relative of the conjugative T4SS, we demonstrate that catalytically active Osa blocks T-DNA transfer into plants. With a partially reconstituted T4SS in vitro, we show that Osa degrades T-DNA in the T-DNA-VirD2 complex before its translocation. Further, we present evidence for conservation and interplay between ATPase and DNase activities throughout the ParB/Sulfiredoxin fold, using other members of the family, namely P1 ParB and RK2 KorB, which have general functional implications across diverse biological contexts.

Host plant induced variation in gut bacteria of Helicoverpa armigera.

Helicoverpa are important polyphagous agricultural insect pests and they have a worldwide distribution. In this study, we report the bacterial community structure in the midgut of fifth instar larvae of Helicoverpa armigera, a species prevalent in the India, China, South Asia, South East Asia, Southern & Eastern Africa and Australia. Using culturable techniques, we isolated and identified members of Bacillus firmus, Bacillus niabense, Paenibacillus jamilae, Cellulomonas variformis, Acinetobacter schindleri, Micrococcus yunnanesis, Enterobacter sp., and Enterococcus cassiliflavus in insect samples collected from host plants grown in different parts of India. Besides these the presence of Sphingomonas, Ralstonia, Delftia, Paracoccus and Bacteriodetes was determined by culture independent molecular analysis. We found that Enterobacter and Enterococcus were universally present in all our Helicoverpa samples collected from different crops and in different parts of India. The bacterial diversity varied greatly among insects that were from different host plants than those from the same host plant of different locations. This result suggested that the type of host plant greatly influences the midgut bacterial diversity of H. armigera, more than the location of the host plant. On further analyzing the leaf from which the larva was collected, it was found that the H. armigera midgut bacterial community was similar to that of the leaf phyllosphere. This finding indicates that the bacterial flora of the larval midgut is influenced by the leaf surface bacterial community of the crop on which it feeds. Additionally, we found that laboratory made media or the artificial diet is a poor bacterial source for these insects compared to a natural diet of crop plant.

Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs--standard single, multiplex and construct-specific PCR assays.

Vegetative insecticidal protein (Vip), a unique class of insecticidal protein, is now part of transgenic plants for conferring resistance against lepidopteron pests. In order to address the imminent regulatory need for detection and labeling of vip3A carrying genetically modified (GM) products, we have developed a standard single PCR and a multiplex PCR assay. As far as we are aware, this is the first report on PCR-based detection of a vip3A-type gene (vip-s) in transgenic cotton and tobacco. Our assay involves amplification of a 284-bp region of the vip-s gene. This assay can possibly detect as many as 20 natural wild-type isolates bearing a vip3A-like gene and two synthetic genes of vip3A in transgenic plants. The limit of detection as established by our assay for GM trait (vip-s) is 0.1%. Spiking with nontarget DNA originating from diverse plant sources had no inhibitory effect on vip-s detection. Since autoclaving of vip-s bearing GM leaf samples showed no deterioration/interference in detection efficacy, the assay seems to be suitable for processed food products as well. The vip-s amplicon identity was reconfirmed by restriction endonuclease assay. The primer set for vip-s was equally effective in a multiplex PCR assay format (duplex, triplex and quadruplex), used in conjunction with the primer sets for the npt-II selectable marker gene, Cauliflower mosaic virus 35S promoter and nopaline synthetase terminator, enabling concurrent detection of the transgene, regulatory sequences and marker gene. Further, the entire transgene construct was amplified using the forward primer of the promoter and the reverse primer of the terminator. The resultant amplicon served as a template for nested PCR to confirm the construct integrity. The method is suitable for screening any vip3A-carrying GM plant and food. The availability of a reliable PCR assay method prior to commercial release of vip3A-based transgenic crops and food would facilitate rapid and efficient regulatory compliance.