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61.
Hepatitis E virus (HEV) is a causative agent of acute hepatitis, and it is the sole member of the genus Hepevirus in the family Hepeviridae. The open reading frame 1 (ORF1) protein of HEV encodes nonstructural polyprotein with putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase. It is not yet known whether ORF1 functions as a single protein with multiple domains or is processed to form separate functional units. On the basis of amino acid conserved motifs, HEV helicase has been grouped into helicase superfamily 1 (SF-1). In order to examine the RNA helicase activity of the NTPase/helicase domain of HEV, the region (amino acids 960 to 1204) was cloned and expressed as histidine-tagged protein in Escherichia coli (HEV Hel) and purified. HEV Hel exhibited NTPase and RNA unwinding activities. Enzyme hydrolyzed all rNTPs efficiently, dATP and dCTP with moderate efficiency, while it showed less hydrolysis of dGTP and dTTP. Enzyme showed unwinding of only RNA duplexes with 5′ overhangs showing 5′-to-3′ polarity. We also expressed and purified two HEV Hel mutants. Helicase mutant I, with substitution in the nucleotide-binding motif I (GKS to GAS), showed 30% ATPase activity. Helicase mutant II, with substitutions in the Mg2+ binding motif II (DEAP to AAAP), showed 50% ATPase activity. Both mutants completely lost ability to unwind RNA duplexes with 5′ overhangs. These findings represent the first report demonstrating NTPase/RNA helicase activity of the helicase domain of HEV ORF1.Viruses with single-strand positive-sense RNA genomes represent the largest class of viruses, which includes numerous pathogens of humans, plants, and animals. In these viruses, RNA replication occurs through negative-strand RNA intermediate, which may also act as the template for synthesis of subgenomic RNAs in some viruses. During replication, various nonstructural proteins remain associated with the viral polymerase in a small compartmentalized replisome. Most of the other accessory proteins are obtained from the cellular machinery.Helicase seems to be essential for RNA replication by many positive-sense RNA viruses (19). Many positive-strand RNA viruses encode their own RNA helicases and besides RNA-dependent RNA polymerase, helicase is the most conserved viral sequence in these viruses. It has been shown by direct mutagenesis studies in poliovirus (26, 39), alphaviruses (31), brome mosaic virus (2, 41), nidoviruses (40), and flaviviruses (15) that helicase functions are essential for viral replication. In addition, it may be involved in RNA translocation, genome packaging, protection of RNA at the replication center, modulating RNA-protein interactions, etc.Helicases are classified into six superfamilies, SF-1 to SF-6 (11, 35), and can be classified further into subfamilies, A (3′→5′) or B (5′→3′) depending on their unwinding directionality. Classic helicases (exhibiting both NTPase and unwinding activities) are referred to as subtype α, while translocases (with no unwinding activity) are referred to as subtype β (35). SF-1 and SF-2 constitute largest of these superfamilies with seven signature motifs (I, Ia, II, III, IV, V, and VI), which form core of the enzyme. Although these motifs are not comparable between SF-1 and SF-2, universal features of core domains include (i) conserved residues involved in binding and hydrolysis of the NTP and (ii) an arginine finger that plays a key role in energy coupling.Hepatitis E virus (HEV) is a nonenveloped virus in the genus Hepevirus of the family Hepeviridae. Hepatitis E is an important public health disease in many developing countries and is also endemic in some industrialized countries (8). Infection by HEV has a known association with increased mortality during pregnancy (22, 23). HEV has a positive-sense RNA genome of ∼7.2 kb, consisting of a 5′ noncoding region (5′NCR) of 27 to 35 nucleotides (nt), followed by three open reading frames (ORFs)—ORF1, ORF2, and ORF3—and a 3′NCR of 65 to 74 nt, ending with a poly(A) tail of variable length (37). The 5′ end has m7G cap (18). ORF1 is known to encode for the viral nonstructural polyprotein with a proposed molecular mass of ∼186 kDa (3). Based on protein sequence homology, the ORF1 polyprotein is proposed to contain four putative domains indicative of methyltransferase, papain-like cysteine protease, RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp) (24). ORF2 encodes the major structural protein (capsid protein), which has N-terminal signal peptide and three glycosylation sites and is translocated across the endoplasmic reticulum (ER). ORF2 protein associates with the 5′ end of the viral RNA, suggesting its regulatory role in the virus replication (36, 37, 44, 45). ORF3 encodes a protein which gets phosphorylated by the cellular mitogen activated protein kinase and is associated with cellular membranes and cytoskeleton fractions (43).HEV belongs to an “alpha-like” supergroup of positive-sense single-stranded RNA (+ssRNA) viruses with conserved motifs of replication-related proteins in the ORF1, with typical signature sequences homologous with the other members of the family (11, 12, 13). ORF1 of HEV encodes additional domains such as the Y domain, papainlike protease, “proline-rich hinge,” and the X domain. Methyltransferase (25), RdRp (1), and X domain (binding to poly-ADP-ribose) (9) in ORF1 have been characterized, whereas the functions of the other domains are yet to be identified. Intracellularly expressed RdRp localizes itself in the ER membranes (30), suggesting that HEV replicates probably in ER in the cytosolic compartment of the cells. It is still unknown whether ORF1 polyprotein undergoes cleavages to form separate functional units of the replication machinery or functions as a single protein with multiple functional domains.The putative RNA helicase of HEV contains all of the seven conserved segments typical of the SF-1 helicase (12, 13). Putative SF-1 helicases are extremely widespread among +ssRNA viruses. Based on sequence comparisons, such helicases have been identified in a variety of plant virus families, as well as in animal viruses such as alphavirus, rubivirus, hepatitis E virus, and coronavirus (11). When compared to other +ssRNA viral helicases belonging to SF-1, HEV helicase showed the highest overall similarity with the helicase of beet necrotic yellow vein virus, a plant furovirus. HEV helicase was speculated to have N-terminal NTPase and C-terminal RNA-binding domains (24). A major obstacle in studying HEV replication has been lack of cell culture system. We report here experimental verification of the helicase activity of the recombinant helicase domain protein of HEV. 相似文献
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Pandey GN Dwivedi Y Ren X Rizavi HS Faludi G Sarosi A Palkovits M 《Neurochemical research》2006,31(2):167-176
Abnormalities in serotonin receptor subtypes have been observed in the postmortem brain of suicide victims. We examined the regional distribution of serotonin (5HT)(2C) receptor mRNA in several areas of the human brain and also compared its protein and mRNA expression in the prefrontal cortex (PFC), hippocampus, and choroid plexus between suicide victims and normal control subjects. 5HT(2C) receptors were found to be distributed in several areas of the human brain (in order of abundance): highly concentrated and richest in choroid plexus; hypothalamus; nucleus accumbens; with the lowest abundance in PFC and cerebellum. Comparison of 5HT(2C) receptors between suicide victims and control subjects showed higher protein levels in the PFC but not the hippocampus or choroid plexus of suicide victims. However, there were no significant differences in mRNA levels between suicide victims and control subjects in these brain areas. These results suggest that 5HT(2C) receptors are richly distributed throughout the brain with the highest level in the choroid plexus and that abnormalities in protein expression of 5HT(2C) receptors in the PFC may be associated with suicide. 相似文献
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Thakker C Burhanpurwala Z Rastogi G Shouche Y Ranade D 《Indian journal of experimental biology》2006,44(2):142-150
In the present study, isolation of anaerobic bacteria from 24 different eco-niches was carried out. A total number of 300 bacterial isolates, including 230 obligate and 70 facultative anaerobes were obtained using anaerobic techniques. All the isolates were initially screened for succinic acid production by Fluorescein test and TLC method. During screening, 10 isolates found to produce succinic acid were further examined by HPLC and then finally confirmed for succinic acid by LC-MS analysis. Amongst 10 isolates, isolate SAP, a facultative anaerobe isolated from buffalo rumen fluid, showed maximum yield of 2.1 g/l of succinic acid from 10 g of glucose in 24 hr under anaerobic condition. This isolate was identified as Klebsiella pneumoniae strain SAP by 16S rDNA sequence and signature sequence analysis. Mouse lethality test for the strain SAP showed LD50 value of 3.3 x 10(8) CFU/ml, which shows non-virulent nature of the strain. This strain may become a candidate strain for succinic acid production because of its osmotolerant nature and higher succinate:acetate ratio. 相似文献
66.
Singhal SS Wickramarachchi D Singhal J Yadav S Awasthi YC Awasthi S 《FEBS letters》2006,580(9):2258-2264
Doxorubicin (DOX) transport activity of Ral-interacting protein (RLIP76) in non-small cell lung cancer (NSCLC) is approximately twice that of in small cell lung cancer (SCLC). Since protein-kinase-C (PKC)alpha mediated phosphorylation of RLIP76 causes doubling of the specific activity of RLIP76, and NSCLC cells are known to have greater PKCalpha activity, we examined the contribution of PKC mediated phosphorylation of RLIP76 towards intrinsic DOX-resistance in human NSCLC. Expression of a deletion mutant RLIP76(delPKCalpha-sites) followed by depletion of the wild-type RLIP76 using a siRNA targeted at one of the deleted regions resulted in generation of cells expressing only the mutant protein, which could not be phosphorylated by PKCalpha. DOX-transport activity of the mutant RLIP76 purified from NSCLC and SCLC was similar and comparable to that of RLIP76 purified from the wild-type SCLC. However, this activity was significantly lower than that of RLIP76 purified from the wild-type NSCLC. After siRNA mediated depletion of PKCalpha, DOX-transport activities of RLIP76 purified from SCLC and NSCLC were indistinguishable. Depletion of PKCalpha inhibited the growth of NSCLC more than SCLC cells (70+/-3% vs. 43+/-5%, respectively). PKCalpha-depletion lowered the IC(50) of NSCLC cell lines for DOX to the same level as that observed for SCLC. RLIP76(-/-) mouse embryonic fibroblasts (MEFs) were significantly more sensitive to DOX as compared with RLIP76(+/+) MEFs (IC(50) 25 vs. 125nM, respectively). However, PKCalpha-depletion did not affect DOX-cytotoxicity towards RLIP76(-/-) MEFs, as opposed to RLIP76(+/+) MEFs which were sensitized by 2.2-fold. These results demonstrate that RLIP76 is a primary determinant of DOX-resistance, and that PKCalpha mediated accumulation defect and DOX-resistance in NSCLC is primarily due to differential phosphorylation of RLIP76 in SCLC and NSCLC. 相似文献
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Raghav PK Verma YK Gangenahalli GU 《International journal of biological macromolecules》2012,50(3):796-814
Bcl-2 (B cell lymphoma-2) is an anti-apoptotic member of Bcl-2 family and its overexpression causes development of several types of cancer. The BH3 domain of pro-apoptotic and BH3-only proteins is capable of binding to Bcl-2 protein to induce apoptosis. This binding is the basis for the development of novel anticancer drug which would likely antagonize Bcl-2 overexpression. In this study we have identified BH3 domain of Bax (Bax BH3) as potentially the best Bcl-2 antagonist by performing docking of BH3 peptides (peptides representing BH3 domain of pro-apoptotic and BH3-only proteins) into the Bcl-2 hydrophobic groove formed by BH3, BH1 and BH2 domains (also referred as BH3 cleft). To predict the best small antagonist for Bcl-2, three groups of small peptides (pentapeptide, tetrapeptide and tripeptide) were designed and screened against Bcl-2 which revealed the structural importance of a set of residues playing a vital role in interaction with Bcl-2. The docking and scoring function identified KRIG and KRI as specific peptides among the screened small peptides responsible for Bcl-2 neutralization and would induce apoptosis. The applied pharmacokinetic and pharmacological filters to all small peptides signify that only IGD has drug-like properties and displayed good oral bioavailability. However, the obtained binding affinity of IGD to Bcl-2 was diminutive. Hence deprotonation, amidation, acetylation, benzoylation, benzylation, and addition of phenyl, deoxyglucose and glucose fragments were performed to increase the binding affinity and to prevent its rapid degradation. Benzoylated IGD tripeptide (IGD(bzo)) was observed to have increased binding affinity than IGD with acceptable pharmacokinetic filters. In addition, stability of Bcl-2/IGD(bzo) complex was validated by Molecular Dynamics (MD) simulations revealing improved binding energy, salt bridges and strong interaction energies. This study suggests a new molecule that inhibits Bcl-2 associated cancer/tumor regression. 相似文献
69.
P Singh C Raghukumar P Verma Y Shouche 《World journal of microbiology & biotechnology》2012,28(2):659-667
Increasing evidence of the fungal diversity in deep-sea sediments has come from amplification of environmental DNA with fungal
specific or eukaryote primer sets. In order to assess the fungal diversity in deep-sea sediments of the Central Indian Basin
(CIB) at ~5,000 m depth, we amplified sediment DNA with four different primer sets. These were fungal-specific primer pair
ITS1F/ITS4 (internal transcribed spacers), universal 18S rDNA primers NS1/NS2, Euk18S-42F/Euk18S-1492R and Euk18S-555F/Euk18S-1269R.
One environmental library was constructed with each of the primer pairs, and 48 clones were sequenced per library. These sequences
resulted in 8 fungal Operational Taxonomic Units (OTUs) with ITS and 19 OTUs with 18S rDNA primer sets respectively by taking
into account the 2% sequence divergence cut-off for species delineation. These OTUs belonged to 20 distinct fungal genera
of the phyla Ascomycota and Basidiomycota. Seven sequences were found to be divergent by 79–97% from the known sequences of
the existing database and may be novel. A majority of the sequences clustered with known sequences of the existing taxa. The
phylogenetic affiliation of a few fungal sequences with known environmental sequences from marine and hypersaline habitat
suggests their autochthonous nature or adaptation to marine habitat. The amplification of sequences belonging to Exobasidiomycetes
and Cystobasidiomycetes from deep-sea is being reported for the first time in this study. Amplification of fungal sequences
with eukaryotic as well as fungal specific primers indicates that among eukaryotes, fungi appear to be a dominant group in
the sampling site of the CIB. 相似文献
70.