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1.  Characterization of the Proliferating Cell Nuclear Antigen of Leishmania donovani Clinical Isolates and Its Association with Antimony Resistance 
Previously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate of Leishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance in L. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by ≥3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ∼5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate. L. donovani PCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate of L. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (SbV) and trivalent antimonial (SbIII) drugs was assessed in vitro against promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of SbV (from 41.2 ± 0.6 μg/ml to 66.5 ± 3.9 μg/ml) and SbIII (from 24.0 ± 0.3 μg/ml to 43.4 ± 1.8 μg/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon SbIII treatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance in L. donovani clinical isolates, which warrants detailed investigations regarding its mechanism.
PMCID: PMC4068424  PMID: 24614385
2.  An Intermediate Alemtuzumab Schedule Reduces the Incidence of Mixed Chimerism Following Reduced-Intensity Conditioning Hematopoietic Cell Transplantation for Hemophagocytic Lymphohistiocytosis 
Reduced-intensity conditioning (RIC) improves the outcomes of hematopoietic cell transplantation (HCT) in patients with hemophagocytic lymphohistiocytosis (HLH). Proximal (ie, close to graft infusion) dosing of alemtuzumab is associated with a high incidence of mixed chimerism, whereas distal (ie, distant from graft infusion) dosing is associated with less mixed chimerism but more acute graft-versus-host disease (GVHD). The alemtuzumab dose per kilogram of body weight also influences these outcomes. We hypothesized that an intermediate alemtuzumab dosing schedule would reduce mixed chimerism and maintain a low incidence of acute GVHD. In this study, 24 consecutive HCTs were performed in patients with HLH or a related disorder using a novel intermediate alemtuzumab schedule of 1 mg/kg starting on day -14. The cumulative incidences (CIs) of mixed chimerism, upfront acute GVHD grades II-IV, and receipt of additional hematopoietic cell products after HCT were compared in patients treated with a distal alemtuzumab schedule (n = 15) and those treated with a proximal alemtuzumab schedule (n = 33). All patients received fludarabine and melphalan. The CI of mixed chimerism was 31% in the intermediate group, 72% in the proximal group (P < .01), and 75% in the distal group patients who received ≥2 mg/kg alemtuzumab (P = .03). The CI of acute GVHD grades II-IV before the development of mixed chimerism was 4% in the intermediate group, 0% in the proximal group, and 13% in the distal group (P = .04, proximal versus distal). The 1-year CI of administration of additional hematopoietic cell products for mixed chimerism (donor lymphocyte infusion ± hematopoietic stem cell boost ± repeat HCT) was 14% in the intermediate group, 53% in the proximal group (P = .01), and 38% in the distal ≥2 mg/kg alemtuzumab group (P = .02). Our findings indicate that intermediate RIC reduces the incidence of mixed chimerism, is associated with a low incidence of upfront acute GVHD, and decreases the need for additional hematopoietic cell products after HCT.
PMCID: PMC4167781  PMID: 24035782
Hemophagocytic; lymphohistiocytosis; Familial hemophagocytic; lymphohistiocytosis; Bone marrow transplantation; Hematopoietic cell; transplantation; XIAP deficiency; SAP deficiency; X-linked lymphoproliferative; disease; Reduced-intensity conditioning; Alemtuzumab
3.  Molecular Characterization of an rsmD-Like rRNA Methyltransferase from the Wolbachia Endosymbiont of Brugia malayi and Antifilarial Activity of Specific Inhibitors of the Enzyme 
The endosymbiotic organism Wolbachia is an attractive antifilarial drug target. Here we report on the cloning and expression of an rsmD-like rRNA methyltransferase from the Wolbachia endosymbiont of Brugia malayi, its molecular properties, and assays for specific inhibitors. The gene was found to be expressed in all the major life stages of B. malayi. The purified enzyme expressed in Escherichia coli was found to be in monomer form in its native state. The activities of the specific inhibitors (heteroaryl compounds) against the enzyme were tested with B. malayi adult and microfilariae for 7 days in vitro at various concentrations, and NSC-659390 proved to be the most potent compound (50% inhibitory concentration [IC50], 0.32 μM), followed by NSC-658343 (IC50, 4.13 μM) and NSC-657589 (IC50, 7.5 μM). On intraperitoneal administration at 5 mg/kg of body weight for 7 days to adult jirds into which B. malayi had been transplanted intraperitoneally, all the compounds killed a significant proportion of the implanted worms. A very similar result was observed in infected mastomys when inhibitors were administered. Docking studies of enzyme and inhibitors and an in vitro tryptophan quenching experiment were also performed to understand the binding mode and affinity. The specific inhibitors of the enzyme showed a higher affinity for the catalytic site of the enzyme than the nonspecific inhibitors and were found to be potent enough to kill the worm (both adults and microfilariae) in vitro as well as in vivo in a matter of days at micromolar concentrations. The findings suggest that these compounds be evaluated against other pathogens possessing a methyltransferase with a DPPY motif and warrant the design and synthesis of more such inhibitors.
PMCID: PMC3719755  PMID: 23733469
4.  Molecular modelling, docking and interaction studies of human-plasmogen and salmonella enolase with enolase inhibitors 
Bioinformation  2012;8(4):185-188.
Salmonella enteric serovar Typhi Ty2 is a human specific pathogen and an etiological agent for typhoid fever. Most of Salmonella serotypes produce glycogen which has a comparatively minor role in virulence and colonization, but has a more significant role in survival. Enzymes present in glycolytic pathway of bacteria help bacteria to survive by activating other factors inside host. Numerous pathogenic bacteria species intervene with the plasminogen system, and this plasminogen–enolase association may play a critical role in the virulence of S. Typhi by causing direct damage to the host cell extracellular matrix, possibly by enzymic degradation of extracellular matrix proteins or other protein constituents. In this study, molecular modelling of enolase of Salmonella has been accomplished in silico by comparative modelling; we have then analyzed Human alpha enolase which is a homodimer and serves on epithelial cells with our model. Both Structures were docked by D-tartronate semialdehyde phosphate (TSP) and 3-aminoenolpyruvate phosphate (AEP) enolase inhibitors. Our study shows that salmonella enolase and human enolase have different active sites in their structure. This will help in development of new ligands, more suitable for inhibiting bacterial survival inside host as vaccines for typhoid fever are not fully protective. The study also confirmed that enolase Salmonella and Human Plasminogen suggested direct physical interaction between both of them as the activation loop of plasminogen residues showed conformational changes similar to the tissue type plasminogen activator. Various computational biology tools were used for our present study such as Modeller, Molegro Virtual Docker, Grommacs.
PMCID: PMC3301999  PMID: 22419838
Salmonella Ty2; TSP (D-tartronate semialdehyde phosphate); AEP (3-aminoenolpyruvate phosphate); Modelling; Docking
5.  Prediction and characterization of T-cell epitopes for epitope vaccine design from outer membrane protein of Neisseria meningitidis serogroup B 
Bioinformation  2010;5(4):155-161.
Neisseria meningitidis serogroup B (MC58) is a leading cause of meningitis and septicaemia, principally infects the infants and adolescents. No vaccine is available for the prevention of these infections because the serogroup B capsular polysaccharide is unable to stimulate an immune response, due to its similarity with polysialic acid. To overcome these obstacles, we proposed to develop a peptide based epitope vaccine from outer membrane protein contained in outer membrane vesicles (OMV) based on our computational analysis. In OMV a total of 236 proteins were identified, only 15 (6.4%) of which were predicted to be located in outer membrane. The major requirement is the identification and selection of T-cell epitopes that act as a vaccine target. We have selected 13 out of 15 outer membrane proteins from OMV proteins. Due to similarity of the fkpA and omp85 with the human FKBP2 and SAMM50 protein, we removed these two sequences from the analysis as their presence in the vaccine is likely to elicit an autoimmune response. ProPred and ProPred1 were used to predict promiscuous helper T Lymphocytes (HTL) and cytotoxic T Lymphocytes (CTL) epitopes and MHCPred for their binding affinity in N. meningitidis serogroup B (MC58), respectively. Binding peptides (epitopes) are distinguished from nonbinding peptides in properties such as amino acid preference on the basis of amino acid composition. By using this dataset, we compared physico-chemical and structural properties at amino acid level through amino acid composition, computed from ProtParam server. Results indicate that porA, porB, opc, rmpM, mtrE and nspA are more suitable vaccine candidates. The predicted peptides are expected to be useful in the design of multi-epitope vaccines without compromising the human population coverage
PMCID: PMC3040476  PMID: 21364778
Outer membrane vesicles; epitope vaccine; epitopes; Neisseria meningitides serogroup B
6.  Maternal Footprints of Southeast Asians in North India 
Human heredity  2008;66(1):1-9.
We have analyzed 7137 samples from 125 different caste, tribal and religious groups of India and 99 samples from three populations of Nepal for the length variation in the COII/tRNALys region of mtDNA. Samples showing length variation were subjected to detailed phylogenetic analysis based on HVS-I and informative coding region sequence variation. The overall frequencies of the 9-bp deletion and insertion variants in South Asia were 1.8% and 0.5%, respectively. We have also defined a novel deep-rooting haplogroup M43 and identified the rare haplogroup H14 in Indian populations carrying the 9bp-deletion by complete mtDNA sequencing. Moreover, we redefined haplogroup M6 and dissected it into two well-defined subclades. The presence of haplogroups F1 and B5a in Uttar Pradesh suggests minor maternal contribution from Southeast Asia to Northern India. The occurrence of haplogroup F1 in the Nepalese sample implies that Nepal might have served as a bridge for the flow of eastern lineages to India. The presence of R6 in the Nepalese, on the other hand, suggests that the gene flow between India and Nepal has been reciprocal.
PMCID: PMC2588665  PMID: 18223312
South Asia; 9bp indel; mtDNA; Haplogroup
7.  Angiogenic growth factors augment K–Cl cotransporter expression in erythroid cells via hypoxia-inducible factor-1α 
American Journal of Hematology  2013;89(3):273-281.
The potassium chloride cotransporters (KCC) family of proteins are widely expressed and are involved in the transepithelial movement of potassium and chloride ions and the regulation of cell volume. KCC activity is high in reticulocytes, and contributes to the dehydration of sickle red blood cells. Because plasma levels of both vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are elevated in sickle cell individuals, and VEGF has been shown to increase KCC expression in other cells, we hypothesized that VEGF and PlGF influence KCC expression in erythroid cells. Both VEGF and PlGF treatment of human erythroid K562 cells increased both mRNA and protein levels of KCC1, KCC3b, and KCC4. VEGF- and PlGF-mediated cellular signaling involved VEGF-R1 and downstream effectors, specifically, PI-3 kinase, p38 MAP kinase, mTOR, NADPH-oxidase, JNK kinase, and HIF-1α. VEGF and PlGF-mediated transcription of KCC3b and KCC4 involved hypoxia response element (HRE) motifs in their promoters, as demonstrated by promoter analysis, EMSA and ChiP. These results were corroborated in vivo by adenoviral-mediated overexpression of PlGF in normal mice, which led to increased expression of mKCC3 and mKCC4 in erythroid precursors. Our studies show that VEGF and PlGF regulate transcription of KCC3b and KCC4 in erythroid cells via activation of HIF-1α, independent of hypoxia. These studies provide novel therapeutic targets for regulation of cell volume in RBC precursors, and thus, amelioration of dehydration in RBCs in sickle cell disease. Am. J. Hematol. 89:273–281, 2014. © 2013 Wiley Periodicals, Inc.
PMCID: PMC4223994  PMID: 24227191

Results 1-7 (7)