Conformational changes in the GTPase modules of the signal reception particle and its receptor drive initiation of protein translocation

During cotranslational protein targeting, two guanosine triphosphatase (GTPase) in the signal recognition particle (SRP) and its receptor (SR) form a unique complex in which hydrolyses of both guanosine triphosphates (GTP) are activated in a shared active site. It was thought that GTP hydrolysis drives the recycling of SRP and SR, but is not crucial for protein targeting. Here, we examined the translocation efficiency of mutant GTPases that block the interaction between SRP and SR at specific stages. Surprisingly, mutants that allow SRP-SR complex assembly but block GTPase activation severely compromise protein translocation. These mutations map to the highly conserved insertion box domain loops that rearrange upon complex formation to form multiple catalytic interactions with the two GTPs. Thus, although GTP hydrolysis is not required, the molecular rearrangements that lead to GTPase activation are essential for protein targeting. Most importantly, our results show that an elaborate rearrangement within the SRP-SR GTPase complex is required to drive the unloading and initiate translocation of cargo proteins.     

Redox-dependent coronary metabolic dilation

We have observed that hydrogen peroxide (H2O2), the dismutated product of superoxide, is a coronary metabolic dilator and couples myocardial oxygen consumption to coronary blood flow. Because the chemical activity of H2O2 favors its role as an oxidant, and thiol groups are susceptible to oxidation, we hypothesized that coronary metabolic dilation occurs via a redox mechanism involving thiol oxidation. To test this hypothesis, we studied the mechanisms of dilation of isolated coronary arterioles to metabolites released by metabolically active (paced at 400 min) isolated cardiac myocytes and directly compared these responses with authentic H2O2. Studies were performed under control conditions and using interventions designed to reduce oxidized thiols [0.1 microM dithiothreitol (DTT) and 10 mM N-acetyl-L-cysteine (NAC)]. Aliquots of the conditioned buffer from paced myocytes produced vasodilation of isolated arterioles (peak response, 71% +/- 6% of maximal dilation), whereas H2O2 produced complete dilation (92% +/- 7%). Dilation to either the conditioned buffer or to H2O2 was significantly reduced by the administration of either NAC or DTT. The location of the thiols oxidized by the conditioned buffer or of H2O2 was determined by the administration of the fluorochromes monochlorobimane (20 microM) or monobromotrimethylammoniobimane (20 microM), which covalently label the reduced total or extracellular-reduced thiols, respectively. H2O2 or the conditioned buffer predominantly oxidized intracellular thiols since the fluorescent signal from monochlorobimane was reduced more than that of monobromotrimethylammoniobimane. To determine whether one of the intracellular targets of thiol oxidation that leads to dilation is the redox-sensitive kinase p38 mitogen-activated protein (MAP) kinase, we evaluated dilation following the administration of the p38 inhibitor SB-203580 (10 microM). The inhibition of p38 attenuated dilation to either H2O2 or to the conditioned buffer from stimulated myocytes by a similar degree, but SB-203580 did not attenuate dilation to nitroprusside. Western blot analysis for the activated form of p38 (phospho-p38) in the isolated aortae revealed robust activation of this enzyme by H2O2. Taken together, our results show that an active component of cardiac metabolic dilation, like that of H2O2, produces dilation by the oxidation of thiols, which are predominantly intracellular and dependent activation on the p38 MAP kinase. Thus coronary metabolic dilation appears to be mediated by redox-dependent signals.     

Genome editing of indica rice ASD16 for imparting resistance against rice tungro disease

Journal of Plant Biochemistry and Biotechnology - Rice tungro disease (RTD) is caused by the joint infection of rice tungro bacilliform virus and rice tungro spherical virus (RTSV) and is the most...     

Preventive effect of naringin on cardiac mitochondrial enzymes during isoproterenol-induced myocardial infarction in rats: a transmission electron microscopic study

Dietary flavonoids intake has been reported inversely related to the incidence of cardiovascular diseases (CVD). The present study is undertaken to evaluate the preventive role of naringin on mitochondrial enzymes in isoproterenol (ISO)-induced myocardial infarction in male albino Wistar rats. Rats subcutaneously injected with ISO (85 mg/kg) at an interval of 24 h for 2 days, resulting in significant (p < 0.05) increase in the levels of mitochondrial lipid peroxides. ISO-induction also showed significant (p < 0.05) decrease in the activities of mitochondrial tricarboxylic acid cycle enzymes (isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, and alpha-ketoglutarate dehydrogenase) and respiratory chain enzymes (NADH dehydrogenase and cytochrome c oxidase). Oral pretreatment with naringin (10, 20, and 40 mg/kg) to ISO-induced rats daily for a period of 56 days significantly (p < 0.05) minimized the alterations in all the biochemical parameters and restored the normal mitochondrial function. Transmission electron microscopic (TEM) observations also correlated with these biochemical findings. Thus, our findings demonstrate that naringin prevents the mitochondrial dysfunction during ISO-induced myocardial infarction in rats.     

Plant species diversity and tree population structure of a humid tropical forest in Tamil Nadu, India

Vegetation structure and species composition of tropical ecosystems were studied through nine transects at Veerapuli and Kalamalai reserve forests in the Western Ghats of Tamil Nadu, India. Species diversity, dominance, species richness and evenness indices of plant communities and also population structure of woody plants were enumerated. A total of 244 species (183 genera and 76 families) were recorded. Species richness (number of species) were 82,142 and 96 species per 0.3 ha respectively for the study areas of low-elevation forest (LEF), mid-elevation forest (MEF) and high elevation forest (HEF). Species diversity indices were greater in MEF compared to the other two forests except juveniles. In contrast, greater dominance value indices were recorded in LEF than other forests. Density and basal area of the MEF were twice greater than the LEF, while HEF showed greater tree density and low basal area when compared to LEF. The stem density and species richness (number of species) decreased with increased size classes of trees observed in the present study indicated good regeneration status. Population structure of juveniles and seedlings also reflects good regeneration status. Terminalia paniculata (IVI of 99.9) and Hopea parviflora (IVI of 103.8) were dominant tree species respectively in LEF and MEF whereas in HEF Agrostistachys meeboldii (63.65), Cullenia excelsa (63.67) and Drypetes oblongifolia (39.67) share the dominance. Past damage (anthropogenic perturbation) may be one of the reasons for single species dominance in LEF and MEF. Occurrence of alien species such as Eupatorium odoratum and Ageratum conyzoides also indicated the past disturbance in LEF. The variations in plant diversity and population structure are largely due to anthropogenic perturbation and other abiotic factors.     

Interferon-Gamma and Nitric Oxide Synthase 2 Mediate the Aggregation of Resident Adherent Peritoneal Exudate Cells: Implications for the Host Response to Pathogens

Interferon-gamma (Ifnγ), a key macrophage activating cytokine, plays pleiotropic roles in host immunity. In this study, the ability of Ifnγ to induce the aggregation of resident mouse adherent peritoneal exudate cells (APECs), consisting primarily of macrophages, was investigated. Cell-cell interactions involve adhesion molecules and, upon addition of Ifnγ, CD11b re-localizes preferentially to the sites of interaction on APECs. A functional role of CD11b in enhancing aggregation is demonstrated using Reopro, a blocking reagent, and siRNA to Cd11b. Studies with NG-methyl-L-arginine (LNMA), an inhibitor of Nitric oxide synthase (Nos), NO donors, e.g., S-nitroso-N-acetyl-DL-penicillamine (SNAP) or Diethylenetriamine/nitric oxide adduct (DETA/NO), and Nos2 ^-/- mice identified Nitric oxide (NO) induced by Ifnγ as a key regulator of aggregation of APECs. Further studies with Nos2 ^-/- APECs revealed that some Ifnγ responses are independent of NO: induction of MHC class II and CD80. On the other hand, Nos2 derived NO is important for other functions: motility, phagocytosis, morphology and aggregation. Studies with cytoskeleton depolymerizing agents revealed that Ifnγ and NO mediate the cortical stabilization of Actin and Tubulin which contribute to aggregation of APECs. The biological relevance of aggregation of APECs was delineated using infection experiments with Salmonella Typhimurium (S. Typhimurium). APECs from orally infected, but not uninfected, mice produce high amounts of NO and aggregate upon ex vivo culture in a Nos2-dependent manner. Importantly, aggregated APECs induced by Ifnγ contain fewer intracellular S. Typhimurium compared to their single counterparts post infection. Further experiments with LNMA or Reopro revealed that both NO and CD11b are important for aggregation; in addition, NO is bactericidal. Overall, this study elucidates novel roles for Ifnγ and Nos2 in regulating Actin, Tubulin, CD11b, motility and morphology during the aggregation response of APECs. The implications of aggregation or “group behavior” of APECs are discussed in the context of host resistance to infectious organisms.     

Mechanistic studies on the mononuclear ZnII-containing metallo-beta-lactamase ImiS from Aeromonas sobria

In an effort to understand the reaction mechanism of a B2 metallo-beta-lactamase, steady-state and pre-steady-state kinetic and rapid freeze quench electron paramagnetic resonance (EPR) studies were conducted on ImiS and its reaction with imipenem and meropenem. pH dependence studies revealed no inflection points in the pH range of 5.0-8.5, while proton inventories demonstrated at least 1 rate-limiting proton transfer. Site-directed mutagenesis studies revealed that Lys224 plays a catalytic role in ImiS, while the side chain of Asn233 does not play a role in binding or catalysis. Stopped-flow fluorescence studies on ImiS, which monitor changes in tryptophan fluorescence on the enzyme, and its reaction with imipenem and meropenem revealed biphasic fluorescence time courses with a rate of fluorescence loss of 160 s(-)(1) and a slower rate of fluorescence regain of 98 s(-)(1). Stopped-flow UV-vis studies, which monitor the concentration of substrate, revealed a rapid loss in absorbance during catalysis with a rate of 97 s(-)(1). These results suggest that the rate-limiting step in the reaction catalyzed by ImiS is C-N bond cleavage. Rapid freeze quench EPR studies on Co(II)-substituted ImiS demonstrated the appearance of a rhombic signal after 10 ms that is assigned to a reaction intermediate that has a five-coordinate metal center. A distinct product (EP) complex was also observed and began to appear in 18-19 ms. When these results are taken together, they allow for a reaction mechanism to be offered for the B2 metallo-beta-lactamases and demonstrate that the mono- and dinuclear Zn(II)-containing enzymes share a common rate-limiting step, which is C-N bond cleavage.