Possible involvement of queuine in regulation of cell proliferation

An increase in cell number is one of the most prominent characteristics of cancer cells. This may be caused by an increase in cell proliferation or decrease in cell death. Queuine is one of the modified base which is found at first anticodon position of specific tRNAs. It is ubiquitously present throughout the living system except mycoplasma and yeast. The tRNAs of Q-family are completely modified to Q-tRNAs in terminally differentiated somatic cells, however hypomodification of Q-tRNA is closely associated with cell proliferation and malignancy. Queuine participates at various cellular functions such as regulation of cell proliferation, cell signaling and alteration in the expression of growth associated proto-oncogenes. Like other proto-oncogenes bcl2 is known to involve in cell survival by inhibiting apoptosis. Queuine or Q-tRNA is suggested to inhibit cell proliferation but the mechanism of regulation of cell proliferation by queuine or Q-tRNA is not well understood. Therefore, in the present study regulation in cell proliferation by queuine in vivo and in vitro as well as the expression of cell death regulatory protein Bcl2 are investigated. For this DLAT cancerous mouse, U87 cell line and HepG2 cell line are treated with different concentrations of queuine and the effect of queuine on cell proliferation and apoptosis are studied. The results indicate that queuine down regulates cell proliferation and expression of Bcl2 protein, suggesting that queuine promotes cell death and participates in the regulation of cell proliferation.     

Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure

Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, we analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. We show that Mst77F on its own causes severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F binds DNA via electrostatic interaction. This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm. Based on these findings we postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure.     

Investigation on the role of p53 codon 72 polymorphism and interactions with tobacco, betel quid, and alcohol in susceptibility to cancers in a high-risk population from North East India

The association of TP53 codon 72 polymorphism with cancer susceptibility remains uncertain and varies with ethnicity. Northeast India represents a geographically, culturally, and ethnically isolated population. The area reports high rate of tobacco usage in a variety of ways of consumption, compared with the rest of Indian population. A total of 411 cancer patients (161 lung, 134 gastric, and 116 oral) and 282 normal controls from the ethnic population were analyzed for p53 codon 72 polymorphism by polymerase chain reaction-restriction fragment length polymorphism. No significant difference in genotypic distribution of p53 between cases and controls was observed. Results suggested betel quid chewing as a major risk factor for all the three cancers (odds ratio [OR]=3.54, confidence interval [CI]=2.01-6.25, p<0.001; OR=1.74, CI=1.04-2.92, p=0.03; and OR=1.85, CI=1.02-3.33, p=0.04 for lung, gastric, and oral cancers, respectively). Tobacco smoking was associated with risk of lung and oral cancers (OR=1.88, CI=1.11-3.19, p=0.01 and OR=1.68, CI=1.00-2.81, p=0.04). Interactions between p53 genotypes and risk factors were analyzed to look for gene-environment interactions. Interaction of smoking and p53 genotype was significant only for oral cancer. Interactions of betel quid with p53 genotypes in lung cancer showed significant increase for all the three genotypes, indicating a major role of betel quid (OR=5.90, CI=1.67-20.81, p=0.006; OR=5.44, CI=1.67-17.75, p=0.005; and OR=5.84, CI=1.70-19.97, p=0.005 for Arg/Arg, Arg/Pro, and Pro/Pro, respectively). In conclusion, high incidence of these cancers in northeast India might be an outcome of risk habits; further, tissue- and carcinogen-specific risk modification by p53 gene is probable.     

Multiple analytical approaches reveal distinct gene-environment interactions in smokers and non smokers in lung cancer

Complex disease such as cancer results from interactions of multiple genetic and environmental factors. Studying these factors singularly cannot explain the underlying pathogenetic mechanism of the disease. Multi-analytical approach, including logistic regression (LR), classification and regression tree (CART) and multifactor dimensionality reduction (MDR), was applied in 188 lung cancer cases and 290 controls to explore high order interactions among xenobiotic metabolizing genes and environmental risk factors. Smoking was identified as the predominant risk factor by all three analytical approaches. Individually, CYP1A1*2A polymorphism was significantly associated with increased lung cancer risk (OR = 1.69;95%CI = 1.11–2.59,p = 0.01), whereas EPHX1 Tyr113His and SULT1A1 Arg213His conferred reduced risk (OR = 0.40;95%CI = 0.25–0.65,p<0.001 and OR = 0.51;95%CI = 0.33–0.78,p = 0.002 respectively). In smokers, EPHX1 Tyr113His and SULT1A1 Arg213His polymorphisms reduced the risk of lung cancer, whereas CYP1A1*2A, CYP1A1*2C and GSTP1 Ile105Val imparted increased risk in non-smokers only. While exploring non-linear interactions through CART analysis, smokers carrying the combination of EPHX1 113TC (Tyr/His), SULT1A1 213GG (Arg/Arg) or AA (His/His) and GSTM1 null genotypes showed the highest risk for lung cancer (OR = 3.73;95%CI = 1.33–10.55,p = 0.006), whereas combined effect of CYP1A1*2A 6235CC or TC, SULT1A1 213GG (Arg/Arg) and betel quid chewing showed maximum risk in non-smokers (OR = 2.93;95%CI = 1.15–7.51,p = 0.01). MDR analysis identified two distinct predictor models for the risk of lung cancer in smokers (tobacco chewing, EPHX1 Tyr113His, and SULT1A1 Arg213His) and non-smokers (CYP1A1*2A, GSTP1 Ile105Val and SULT1A1 Arg213His) with testing balance accuracy (TBA) of 0.6436 and 0.6677 respectively. Interaction entropy interpretations of MDR results showed non-additive interactions of tobacco chewing with SULT1A1 Arg213His and EPHX1 Tyr113His in smokers and SULT1A1 Arg213His with GSTP1 Ile105Val and CYP1A1*2C in nonsmokers. These results identified distinct gene-gene and gene environment interactions in smokers and non-smokers, which confirms the importance of multifactorial interaction in risk assessment of lung cancer.     

NTPase and 5'-RNA triphosphatase activities of Chikungunya virus nsP2 protein

Chikungunya virus (CHIKV) is an insect borne virus (genus: Alphavirus) which causes acute febrile illness in humans followed by a prolonged arthralgic disease that affects the joints of the extremities. Re-emergence of the virus in the form of outbreaks in last 6-7 years has posed a serious public health problem. CHIKV has a positive sense single stranded RNA genome of about 12,000 nt. Open reading frame 1 of the viral genome encodes a polyprotein precursor, nsP1234, which is processed further into different non structural proteins (nsP1, nsP2, nsP3 and nsP4). Sequence based analyses have shown helicase domain at the N-terminus and protease domain at C-terminus of nsP2. A detailed biochemical analysis of NTPase/RNA helicase and 5'-RNA phosphatase activities of recombinant CHIKV-nsP2T protein (containing conserved NTPase/helicase motifs in the N-terminus and partial papain like protease domain at the C-terminus) was carried out. The protein could hydrolyze all NTPs except dTTP and showed better efficiency for ATP, dATP, GTP and dGTP hydrolysis. ATP was the most preferred substrate by the enzyme. CHIKV-nsP2T also showed 5'-triphosphatase (RTPase) activity that specifically removes the γ-phosphate from the 5' end of RNA. Both NTPase and RTPase activities of the protein were completely dependent on Mg(2+) ions. RTPase activity was inhibited by ATP showing sharing of the binding motif by NTP and RNA. Both enzymatic activities were drastically reduced by mutations in the NTP binding motif (GKT) and co-factor, Mg(2+) ion binding motif (DEXX) suggesting that they have a common catalytic site.     

Key to the management of the Western Himalayan Hillstreams in relation to

A key to the management of hillstreams in relation to Fish Species Richness (FSR) and Shannon and Weaver Diversity Index (H) (Shannon and Weaver, 1949. The Mathematical Theory of Communications. Urbana, University of Illinois Press, IL, USA) using a multitude of factors such as altitude, water source, bed gradient, dominant channel materials and habitat type has been proposed on the basis of 10 study sites, belonging to three north Indian rivers namely; Ghaggar, Yamuna and Ganga. The selected streams are located between the altitudes ranging from 380 to 1524m. It has been observed that high altitude streams having steep gradient, boulder dominated substrate with rapids as the main habitat type had lower FSR (3–4) and H (0.55–0.99) as compared to hillstreams located in the lower altitudes having gentle gradient, cobble dominated substrate with pools, riffles, runs, rapids and cascades as the main habitats and have high FSR (6–14) and H (1.67–2.35). Types – A, B and F hillstreams were encountered when the hillstreams are classified after Rosgen (Applied River Morphology, Wildland Hydrology, Co, USA).     

Acetoxy drug: protein transacetylase of buffalo liver-characterization and mass spectrometry of the acetylated protein product

The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3-3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4-methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K(m) (1667 microM) and V(max) (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3-3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3-3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3-3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3-3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys(51), Lys(82), Lys(123), Lsy(181), Lys(191) and Lys(210)) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions.     

The structure and ligand binding properties of the B. subtilis YkoF gene product, a member of a novel family of thiamin/HMP-binding proteins

The crystal structure of the Bacillus subtilis YkoF gene product, a protein involved in the hydroxymethyl pyrimidine (HMP) salvage pathway, was solved by the multiwavelength anomalous dispersion (MAD) method and refined with data extending to 1.65 A resolution. The atomic model of the protein shows a homodimeric association of two polypeptide chains, each containing an internal repeat of a ferredoxin-like betaalphabetabetaalphabeta fold, as seen in the ACT and RAM-domains. Each repeat shows a remarkable similarity to two members of the COG0011 domain family, the MTH1187 and YBL001c proteins, the crystal structures of which were recently solved by the Northeast Structural Genomics Consortium. Two YkoF monomers form a tightly associated dimer, in which the amino acid residues forming the interface are conserved among family members. A putative small-ligand binding site was located within each repeat in a position analogous to the serine-binding site of the ACT-domain of the Escherichia coli phosphoglycerate dehydrogenase. Genetic data suggested that this could be a thiamin or HMP-binding site. Calorimetric data confirmed that YkoF binds two thiamin molecules with varying affinities and a thiamine-YkoF complex was obtained by co-crystallization. The atomic model of the complex was refined using data to 2.3 A resolution and revealed a unique H-bonding pattern that constitutes the molecular basis of specificity for the HMP moiety of thiamin.     

NTPase and 5′ to 3′ RNA Duplex-Unwinding Activities of the Hepatitis E Virus Helicase Domain

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.