Characterization of the 3' --> 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues

Nucleoside diphosphate kinases (NDKs), an evolutionarily conserved family of proteins, synthesize nucleoside triphosphates from nucleoside diphosphates and ATP. Here, we have characterized the kinase activity and DNA processing functions of eight human proteins that contain at least one domain homologous to Escherichia coli NDK. Not all human proteins with NDK-like domains exhibited NDK activity when expressed as recombinant proteins in E. coli. Human NDK1 (NM23-H1) has been reported to have 3' --> 5' exonuclease activity. In addition to human NDK1, we also find that human NDK5, NDK7, and NDK8 contain 3' --> 5' exonuclease activity. Site-directed mutagenesis, competition assays between wild-type and mutant NDK proteins, and NMR studies confirmed that the DNA-binding and 3' --> 5' exonuclease activity of human NDK1 is an intrinsic activity of the protein. Using double-stranded DNA substrates containing modified bases, human NDK1 efficiently excised nucleotides from the single-strand break produced by APE1 or Nth1. When human cells were treated with various DNA-damaging agents, human NDK1 translocated from the cytoplasm to the nucleus. These results suggest that, in addition to maintenance of nucleotide pool balance, the human NDK-like proteins may have previously unrecognized roles in DNA nucleolytic processing.     

A proposed role for Leishmania major carboxypeptidase in peptide catabolism

Leishmaniasis is a tropical disease caused by Leishmania, eukaryotic parasites transmitted to humans by sand flies. Towards the development of new chemotherapeutic targets for this disease, biochemical and in vivo expression studies were performed on one of two M32 carboxypeptidases present within the Leishmania major (LmaCP1) genome. Enzymatic studies reveal that like previously studied M32 carboxypeptidases, LmaCP1 cleaves substrates with a variety of C-terminal amino acids—the primary exception being those having C-terminal acidic residues. Cleavage assays with a series of FRET-based peptides suggest that LmaCP1 exhibits a substrate length restriction, preferring peptides shorter than 9-12 amino acids. The in vivo expression of LmaCP1 was analyzed for each major stage of the L. major life cycle. These studies reveal that LmaCP1 expression occurs only in procyclic promastigotes—the stage of life where the organism resides in the abdominal midgut of the insect. The implications of these results are discussed.     

Induction of Th2 responses and IgE is largely due to carbohydrates functioning as adjuvants on Schistosoma mansoni egg antigens

Infection with the helminth parasite Schistosoma mansoni induces a pronounced Th2-type response that is associated with significant IgE production. To better understand how the parasite drives these responses, we investigated the relative roles of proteins and carbohydrates in driving Th2-type and/or IgE responses using a murine model of intranasal sensitization with soluble egg Ags (SEA) of Schistosoma mansoni. We found that repeated intranasal sensitization with soluble egg Ags led to the induction of both total and specific IgE production and nasal eosinophilia. By comparing the responses of mice sensitized with SEA or metaperiodate-treated SEA we were able to demonstrate that carbohydrates on SEA are the major inducers of IgE production and nasal recruitment of eosinophils. Mice sensitized with periodate-treated SEA displayed a significant decrease in both total and specific IgE levels in comparison to mice sensitized with native SEA. Furthermore, sensitization of mice with periodate-treated SEA significantly reduced levels of Ag-specific IgG1, but had no effect on IgG2a production. Nasal lymphocytes from mice sensitized with native SEA, but not with periodate-treated SEA, produced IL-4, IL-5, and IL-10 when restimulated with native SEA in vitro. On the other hand, lymphocytes from mice sensitized with periodate-treated SEA did not produce any of these same cytokines following in vitro restimulation, suggesting that carbohydrates were required for in vivo induction of Th2 response and for that of associated cytokine responses in this model. Lastly, competitive inhibition ELISA showed that although carbohydrates are required for SEA-specific IgE induction, they are not targets of the induced IgE response.     

Conserved motifs in voltage-sensing and pore-forming modules of voltage-gated ion channel proteins

Voltage-gated ion channels (VGCs) mediate selective diffusion of ions across cell membranes to enable many vital cellular processes. Three-dimensional structure data are lacking for VGC proteins; hence, to better understand their function, there is a need to identify the conserved motifs using sequence analysis methods. In this study, we have used a profile-to-profile alignment method to identify several new conserved motifs specific to each transmembrane segment (TMS) of the voltage-sensing and the pore-forming modules of Ca2+, Na+, and K+ channel subfamilies. For Ca2+ and Na+, the functional theme of motif conservation is similar in all segments while they differ with those of the K+ channel proteins. Nevertheless, the conservation is strikingly similar in the S4 segment of the voltage-sensing module across all subfamilies. In each subfamily and for each TMS, we have identified conserved motifs/residues and correlated their functional significance and disease associations in human, using mutational data from the literature.     

Toll-like receptor (TLR)-2 and TLR-4 regulate inflammation, gliosis, and myelin sparing after spinal cord injury

Activation of macrophages via toll-like receptors (TLRs) is important for inflammation and host defense against pathogens. Recent data suggest that non-pathogenic molecules released by trauma also can trigger inflammation via TLR2 and TLR4. Here, we tested whether TLRs are regulated after sterile spinal cord injury (SCI) and examined their effects on functional and anatomical recovery. We show that mRNA for TLR1, 2, 4, 5, and 7 are increased after SCI as are molecules associated with TLR signaling (e.g. MyD88, NFkappaB). The significance of in vivo TLR2 and TLR4 signaling was evident in SCI TLR4 mutant (C3H/HeJ) and TLR2 knockout (TLR2-/-) mice. In C3H/HeJ mice, sustained locomotor deficits were observed relative to SCI wild-type control mice and were associated with increased demyelination, astrogliosis, and macrophage activation. These changes were preceded by reduced intraspinal expression of interleukin-1beta mRNA. In TLR2-/- mice, locomotor recovery also was impaired relative to SCI wild-type controls and novel patterns of myelin pathology existed within ventromedial white matter--an area important for overground locomotion. Together, these data suggest that in the absence of pathogens, TLR2 and TLR4 are important for coordinating post-injury sequelae and perhaps in regulating inflammation and gliosis after SCI.     

Structure based virtual screening of ligands to identify cysteinyl leukotriene receptor 1 antagonist

Montelukast and Zafirlukast are known leukotriene receptor antagonists prescribed in asthma treatment. However, these fall short as mono therapy and are frequently used in combination with inhaled glucocorticosteroids with or without long acting beta 2 agonists. Therefore, it is of interest to apply ligand and structure based virtual screening strategies to identify compounds akin to lead compounds Montelukast and Zafirlukast. Hence, compounds with structures having 95% similarity to these compounds were retrieved from NCBI׳s PubChem database. Compounds similar to lead were grouped and docked at the antagonist binding site of cysteinyl leukotriene receptor 1. This exercise identified compounds UNII 70RV86E50Q (Pub Cid 71587778) and Sure CN 9587085 (Pub Cid 19793614) with higher predicted binding compared to Montelukast and Zafirlukast. It is shown that the compound Sure CN 9587085 showed appreciable ligand receptor interaction compared to UNII 70RV86E50Q. Thus, the compound Sure CN 9587085 is selected as a potent antagonist to cysteinyl leukotriene receptor 1 for further consideration in vitro and in vivo validation.     

Overexpression of human copper/zinc-superoxide dismutase in transgenic animals attenuates the reduction of apurinic/apyrimidinic endonuclease expression in neurons after in vitro ischemia and after transient global cerebral ischemia

Oxidative stress after ischemia/reperfusion has been shown to induce DNA damage and subsequent DNA repair activity. Apurinic/apyrimidinic endonuclease (APE) is a multifunctional protein in the DNA base excision repair pathway which repairs apurinic/apyrimidinic sites in DNA. We investigated the involvement of oxidative stress and expression of APE in neurons after oxygen-glucose deprivation and after global cerebral ischemia. Our results suggest that overexpression of human copper/zinc-superoxide dismutase reduced oxidative stress with a subsequent decrease in APE expression. Production of oxygen free radicals and inhibition of the base excision repair pathway may play pivotal roles in the cell death pathway after ischemia.     

Interactive effects of silicon and arbuscular mycorrhiza in modulating ascorbate-glutathione cycle and antioxidant scavenging capacity in differentially salt-tolerant <Emphasis Type="Italic">Cicer arietinum</Emphasis> L. genotypes subjected to long-term salinity

Salinity is the major environmental constraint that affects legume productivity by inducing oxidative stress. Individually, both silicon (Si) nutrition and mycorrhization have been reported to alleviate salt stress. However, the mechanisms adopted by both in mediating stress responses are poorly understood. Thus, pot trials were undertaken to evaluate comparative as well as interactive effects of Si and/or arbuscular mycorrhiza (AM) in alleviating NaCl toxicity in modulating oxidative stress and antioxidant defence mechanisms in two Cicer arietinum L. (chickpea) genotypes—HC 3 (salt-tolerant) and CSG 9505 (salt-sensitive). Plants subjected to different NaCl concentrations (0–100 mM) recorded a substantial increase in the rate of superoxide radical (O₂ ^·?), H₂O₂, lipoxygenase (LOX) activity and malondialdehyde (MDA) content, which induced leakage of ions and disturbed Ca²⁺/Na⁺ ratio in roots and leaves. Individually, Si and AM reduced oxidative burst by strengthening antioxidant enzymatic activities (superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPOX)). Si was relatively more efficient in reducing accumulation of stress metabolites, while mycorrhization significantly up-regulated antioxidant machinery and modulated ascorbate-glutathione (ASA-GSH) cycle. Combined applications of Si and AM complemented each other in reducing reactive oxygen species (ROS) build-up by further enhancing the antioxidant defence responses. Magnitude of ROS-mediated oxidative burden was lower in HC 3 which correlated strongly with more effective AM symbiosis, better capacity to accumulate Si and stronger defence response when compared with CSG 9505. Study indicated that Si and/or AM fungal amendments upgraded salt tolerance through a dynamic shift from oxidative destruction towards favourable antioxidant defence system in stressed chickpea plants.     

Reconstruction of pathways associated with amino acid metabolism in human mitochondria

We have used a bioinformatics approach for the identification and reconstruction of metabolic pathways associated with amino acid metabolism in human mitochondria. Human mitochondrial proteins determined by experimental and computational methods have been superposed on the reference pathways from the KEGG database to identify mitochondrial pathways. Enzymes at the entry and exit points for each reconstructed pathway were identified, and mitochondrial solute carrier proteins were determined where applicable. Intermediate enzymes in the mitochondrial pathways were identified based on the annotations available from public databases, evidence in current literature, or our MITOPRED program, which predicts the mitochondrial localization of proteins. Through integration of the data derived from experimental, bibliographical, and computational sources, we reconstructed the amino acid metabolic pathways in human mitochondria, which could help better understand the mitochondrial metabolism and its role in human health.     

Purification of α-amylase from Bacillus licheniformis by chromatofocusing and gel filtration chromatography

A combination of chromatofocusing and gel filtration chromatography resulted in a simple purification of -amylase from Bacillus licheniformis. The purification was approximately 77-fold. Identification of the purity was established by SDS–PAGE. Molecular weight and isoelectric point of the purified enzyme were 58 kDa and 7.18 respectively. Western blot analysis confirms the specificity of antibody raised against purified -amylase.