Investigations on the RNA binding and phosphorylation of groundnut bud necrosis virus nucleocapsid protein

Groundnut bud necrosis virus belongs to the genus Tospovirus, infects a wide range of crop plants and causes severe losses. To understand the role of the nucleocapsid protein in the viral life cycle, the protein was overexpressed in E. coli and purified by Ni-NTA chromatography. The purified N protein was well folded and was predominantly alpha-helical. Deletion analysis revealed that the C-terminal unfolded region of the N protein was involved in RNA binding. Furthermore, the N protein could be phosphorylated in vitro by Nicotiana benthamiana plant sap and by purified recombinant kinases such as protein kinase CK2 and calcium-dependent protein kinase. This is the first report of phoshphorylation of a nucleocapsid protein in the family Bunyaviridae. The possible implications of the present findings for the viral life cycle are discussed.

Ribosome-RNA interaction: a potential target for developing antiviral against hepatitis C virus

Hepatitis C virus (HCV), a member of Flaviviridae, encoding a positive-sense single-stranded RNA translates by cap-independent mechanism using the internal ribosome entry site (IRES) present in the 5' UTR of the virus. The IRES has complex stem loop structures and is capable of recruiting the 40S ribosomal subunit in a factor-independent fashion. As the IRES sequence is highly conserved throughout the HCV genotypes and the translation is the first obligatory step of the HCV life cycle, the IRE'S-mediated translation, or more specifically, the ribosome HCV RNA interaction is an attractive target to design effective antivirals. This article will focus on the mechanism of the HCV IRES translation and the various ways in which the interaction of ribosome and IRES has been targeted.

Rural electrification: Optimising the choice between decentralised renewable energy sources and grid extension

The study presents an analysis aimed at choosing between off-grid solar photovoltaic, biomass gasifier based power generation and conventional grid extension for remote village electrification. The model provides a relation between renewable energy systems and the economical distance limit (EDL) from the existing grid point, based on life cycle cost (LCC) analysis, where the LCC of energy for renewable energy systems and grid extension will match. The LCC of energy feed to the village is arrived at by considering grid availability and operating hours of the renewable energy systems. The EDL for the biomass gasifier system of 25 kW capacities is 10.5 km with 6 h of daily operation and grid availability. However, the EDL for a similar 25 kW capacity photovoltaic system is 35 km for the same number of hours of operation and grid availability. The analysis shows that for villages having low load demand situated far away from the existing grid line, biomass gasification based systems are more cost competitive than photovoltaic systems or even compared to grid extension. (C) 2012 Elsevier Inc. All rights reserved.

Is land really a constraint for the utilization of solar energy in India?

This article compares the land use in solar energy technologies with conventional energy sources. This has been done by introducing two parameters called land transformation and land occupation. It has been shown that the land area transformed by solar energy power generation is small compared to hydroelectric power generation, and is comparable with coal and nuclear energy power generation when life-cycle transformations are considered. We estimate that 0.97% of total land area or 3.1% of the total uncultivable land area of India would be required to generate 3400 TWh/yr from solar energy power systems in conjunction with other renewable energy sources.

Targeting ribosome assembly on the HCV RNA using a small RNA molecule

Translation initiation of hepatitis C virus (HCV) RNA is the initial obligatory step of the viral life cycle, mediated through the internal ribosome entry site (IRES) present in the 5'-untranslated region (UTR). Initiation on the HCV IRES is mediated by multiple structure-specific interactions between IRES RNA and host 40S ribosomal subunit. In the present study we demonstrate that the SLIIIef domain, in isolation from other structural elements of HCV IRES, retain the ability to interact with 40S ribosome subunit. A small RNA SLRef, mimicking the SLIIIef domain was found to interact specifically with human La protein and the ribosomal protein S5 and selectively inhibit HCV RNA translation. More importantly, SLRef RNA showed significant suppression of replication in HCV monocistronic replicon and decrease of negative strand synthesis in HCV cell culture system. Finally, using Sendai virus based virosome, the targeted delivery of SLRef RNA into mice liver succeeded in selectively inhibiting HCV IRES mediated translation in vivo.

Evidence for the role of Mycobacteriumtuberculosis RecG helicase in DNA repair and recombination

In order to survive and replicate in a variety of stressful conditions during its life cycle, Mycobacteriumtuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. In this study, we show that M.tuberculosis RecG (MtRecG) expression was induced in response to different genotoxic agents. Strikingly, expression of MtRecG in Escherichiacoli recG mutant strain provided protection against mitomycin C, methyl methane sulfonate and UV induced cell death. Purified MtRecG exhibited higher binding affinity for the Holliday junction (HJ) compared with a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile HJ, branch migration was evident only in the case of the mobile HJ. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently than mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structure, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Together, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as stress conditions.

Identification of an exported heat shock protein 70 in Plasmodium falciparum

Host cell remodelling is a hallmark of malaria pathogenesis. It involves protein folding, unfolding and trafficking events and thus participation of chaperones such as Hsp70s and Hsp40s is well speculated. Until recently, only Hsp40s were thought to be the sole representative of the parasite chaperones in the exportome. However, based on the re-annotated Plasmodium falciparum genome sequence, a putative candidate for exported Hsp70 has been reported, which otherwise was known to be a pseudogene. We raised a specific antiserum against a C-terminal peptide uniquely present in PfHsp70-x. Immunoblotting and immunofluorescence-based approaches in combination with sub-cellular fractionation by saponin and streptolysin-O have been taken to determine the expression and localization of PfHsp70-x in infected erythrocyte. The re-annotated sequence of PfHsp70-x reveals it to be a functional protein with an endoplasmic reticulum signal peptide. It gets maximally expressed at the schizont stage of intra-erythrocytic life cycle. Majority of the protein localizes to the parasitophorous vacuole and some of it gets exported to the erythrocyte compartment where it associates with Maurer's clefts. The identification of an exported parasite Hsp70 chaperone presents us with the fact that the parasite has evolved customized chaperones which might be playing crucial roles in aspects of trafficking and host cell remodelling.

Shape-function-relationship (SFR) framework for semantic interoperability of product model

The problem of semantic interoperability arises while integrating applications in different task domains across the product life cycle. A new shape-function-relationship (SFR) framework is proposed as a taxonomy based on which an ontology is developed. Ontology based on the SFR framework, that captures explicit definition of terminology and knowledge relationships in terms of shape, function and relationship descriptors, offers an attractive approach for solving semantic interoperability issue. Since all instances of terms are based on single taxonomy with a formal classification, mapping of terms requires a simple check on the attributes used in the classification. As a preliminary study, the framework is used to develop ontology of terms used in the aero-engine domain and the ontology is used to resolve the semantic interoperability problem in the integration of design and maintenance. Since the framework allows a single term to have multiple classifications, handling context dependent usage of terms becomes possible. Automating the classification of terms and establishing the completeness of the classification scheme are being addressed presently.

Trans-spliced Heat Shock Protein 90 Modulates Encystation in Giardia lamblia

Background: Hsp90 from Giardia lamblia is expressed by splicing of two independently transcribed RNA molecules, coded by genes named HspN and HspC located 777 kb apart. The reasons underlying such unique trans-splicing based generation of GlHsp90 remain unclear. Principle Finding: In this study using mass-spectrometry we identify the sequence of the unique, junctional peptide contributed by the 5' UTR of HspC ORF. This peptide is critical for the catalytic function of Hsp90 as it harbours an essential ``Arg'' in its sequence. We also show that full length GlHsp90 possesses all the functional hall marks of a canonical Hsp90 including its ability to bind and hydrolyze ATP. Using qRT-PCR as well as western blotting approach we find the reconstructed Hsp90 to be induced in response to heat shock. On the contrary we find GlHsp90 to be down regulated during transition from proliferative trophozoites to environmentally resistant cysts. This down regulation of GlHsp90 appears to be mechanistically linked to the encystation process as we find pharmacological inhibition of GlHsp90 function to specifically induce encystation. Significance: Our results implicate the trans-spliced GlHsp90 from Giardia lamblia to regulate an essential stage transition in the life cycle of this important human parasite.

Endoplasmic Reticulum Stress Triggers Gametocytogenesis in the Malaria Parasite

The malaria parasite experiences a significant amount of redox stress during its growth in human erythrocytes and heavily relies on secretory functions for pathogenesis. Most certainly, the parasite is equipped with machinery to tackle perturbations in the secretory pathway, like the unfolded protein response pathway in higher eukaryotes. Our bioinformatics analysis revealed the complete absence of genes involved in the canonical unfolded protein response pathway in Plasmodium falciparum. Accordingly, the parasite was unable to up-regulate endoplasmic reticulum (ER) chaperones or ER-associated degradation in response to DTT-mediated ER stress. Global profiling of gene expression upon DTT treatment revealed a network of AP2 transcription factors and their targets being activated. The overall outcome was up-regulation of genes involved in protein export and the sexual stage of the parasite life cycle culminating in gametocytogenesis. Our results suggest that the malaria parasite uses ER stress as a cue to switch to the transmissible sexual stages.

Heat shock protein 90 as a potential drug target against surra

Trypanosomiasis is caused by Trypanosoma species which affect both human and animal populations and pose a major threat to developing countries. The incidence of animal trypanosomiasis is on the rise. Surra is a type of animal trypanosomiasis, caused by Trypanosoma evansi, and has been included in priority list B of significant diseases by the World Organization of Animal Health (OIE). Control of surra has been a challenge due to the lack of effective drugs and vaccines and emergence of resistance towards existing drugs. Our laboratory has previously implicated Heat shock protein 90 (Hsp90) from protozoan parasites as a potential drug target and successfully demonstrated efficacy of an Hsp90 inhibitor in cell culture as well as a pre-clinical mouse model of trypanosomiasis. This article explores the role of Hsp90 in the Trypanosoma life cycle and its potential as a drug target. It appears plausible that the repertoire of Hsp90 inhibitors available in academia and industry may have value for treatment of surra and other animal trypanosomiasis.

Plasmodium berghei glycine cleavage system T-protein is non-essential for parasite survival in vertebrate and invertebrate hosts

T-protein, an aminomethyltransferase, represents one of the four components of glycine cleavage system (GCS) and catalyzes the transfer of methylene group from H-protein intermediate to tetrahydrofolate (THF) forming N-5, N-10-methylene THF (CH2-THF) with the release of ammonia. The malaria parasite genome encodes T-, H- and L-proteins, but not P-protein which is a glycine decarboxylase generating the aminomethylene group. A putative GCS has been considered to be functional in the parasite mitochondrion despite the absence of a detectable P-protein homologue. In the present study, the mitochondrial localization of T-protein in the malaria parasite was confirmed by immunofluorescence and its essentiality in the entire parasite life cycle was studied by targeting the T-protein locus in Plasmodium berghei (Pb). PbT knock out parasites did not show any growth defect in asexual, sexual and liver stages indicating that the T-protein is dispensable for parasite survival in vertebrate and invertebrate hosts. The absence of P-protein homologue and the non-essentiality of T protein suggest the possible redundancy of GCS activity in the malaria parasite. Nevertheless, the H- and L-proteins of GCS could be essential for malaria parasite because of their involvement in alpha-lcetoacid dehydrogenase reactions. (C) 2014 Elsevier B.V. All rights reserved.

A Kunitz-type serine protease inhibitor from Butea monosperma seed and its influence on developmental physiology of Helicoverpa armigera

A protease inhibitor from the seeds of Butea monosperma (BmPI) was purified, characterized and studied for its influence on developmental physiology of Helicover-pa armigera. BmPI on two-dimensional separations indicated the presence of a 14 kDa protein with an isoelectric point in the acidic region (pl 5.6). Multiple Sequence Analysis data suggested that the BmPI contains a sequence motif which is conserved in various trypsin and chymotrypsin inhibitors of Kunitz-type. The inhibitor exhibited trypsin inhibitory activity in a broad range of pH (4-10) and temperature (10-80 degrees C). The enzyme kinetic studies revealed BmPI as a competitive inhibitor with a K-i value of 1.2 x 10(-9) M. In vitro studies with BmPI indicated measurable inhibitory activity on total gut proteolytic enzymes of H. armigera (IC(50)2.0 mu g/ml) and bovine trypsin. BmPI supplemented artificial diet caused dose dependent mortality and reduction in growth and weight. The fertility and fecundity of H. armigera, declined whereas the larval-pupal duration of the insect life cycle extended. These detrimental effects on H. armigera suggest the usefulness of BmPl in insect pest management of food crops. (C) 2014 Elsevier Ltd. All rights reserved.

Different Modes of Transactivation of Bacteriophage Mu Late Promoters by Transcription Factor C

Transactivator protein C is required for the expression of bacteriophage Mu late genes from lys, I, P and mom promoters during lytic life cycle of the phage. The mechanism of transcription activation of mom gene by C protein is well understood. C activates transcription at Pmom by initial unwinding of the promoter DNA, thereby facilitating RNA polymerase (RNAP) recruitment. Subsequently, C interacts with the (sic) subunit of RNAP to enhance promoter clearance. The mechanism by which C activates other late genes of the phage is not known. We carried out promoter-polymerase interaction studies with all the late gene promoters to determine the individual step of C mediated activation. Unlike at P-mom, at the other three promoters, RNAP recruitment and closed complex formation are not C dependent. Instead, the action of C at P-lys, P-I, and P-P is during the isomerization from closed complex to open complex with no apparent effect at other steps of initiation pathway. The mechanism of transcription activation of mom and other late promoters by their common activator is different. This distinction in the mode of activation (promoter recruitment and escape versus isomerization) by the same activator at different promoters appears to be important for optimized expression of each of the late genes.