Probing the structure of Leishmania donovani chagasi DHFR-TS: comparative protein modeling and protein–ligand interaction studies

Dihydrofolate reductase (DHFR) has been used successfully as a drug target in the area of anti-bacterial, anti-cancer and anti-malarial therapy. It also acts as a drug target for Leishmaniasis. Inhibition of DHFR leads to cell death through lack of thymine (nucleotide metabolism). Although the crystal structures of Leishmania major and Trypanosoma cruzi DHFR-thymidylate synthase (TS) have been resolved, to date there is no three-dimensional (3D)-structural information on DHFR-TS of Leishmania donovani chagasi, which causes visceral leishmaniasis. Our aim in this study was to model the 3D structure of L. donovani chagasi DHFR-TS, and to investigate the structural requirements for its inhibition. In this paper we describe a highly refined homology model of L. donovani chagasi DHFR-TS based on available crystallographic structures by using the Homology module of Insight II. Structural refinement and minimization of the generated L. donovani chagasi DHFR-TS model employed the Discover 3 module of Insight II and molecular dynamic simulations. The model was further validated through use of the PROCHECK, Verify_3D, PROSA, PSQS and ERRAT programs, which confirm that the model is reliable. Superimposition of the model structure with the templates L. major A chain, L. major B chain And T. cruzi A chain showed root mean square deviations of 0.69 Å, 0.71 Å and 1.11 Å, respectively. Docking analysis of the L. donovani chagasi DHFR-TS model with methotrexate enabled us to identify specific residues, viz. Val156, Val30, Lys95, Lys75 and Arg97, within the L. donovani chagasi DHFR-TS binding pocket, that play an important role in ligand or substrate binding. Docking studies clearly indicated that these five residues are important determinants for binding as they have strong hydrogen bonding interactions with the ligand.     

Immobilization of β-amylase from Bacillus megaterium B6 into gelatin film by cross-linking

β-Amylase (EC 3.2.1.2) from Bacillus megaterium B₆ was immobilized in gelatin by covalent cross-linking with organic hardeners, formaldehyde and gluteraldehyde, of which gluteraldehyde was proved to be better. The effect of temperature, pH, incubation time, substrate concentration, different surfactants and thiol inhibitors on immobilized β-amylase had been investigated. Reuse efficiency and cross-linker concentration was also studied. The data indicated that enhanced resistance to thermal and chemical denaturation, increased temperature optima (from 60° to 65°C) and rapid rate of substrate saturation were achieved after immobilization. The gelatin-bound enzyme could also be reutilized for repeated batch experiments.     

Particle Simulation of Oxidation Induced Band 3 Clustering in Human Erythrocytes

Oxidative stress mediated clustering of membrane protein band 3 plays an essential role in the clearance of damaged and aged red blood cells (RBCs) from the circulation. While a number of previous experimental studies have observed changes in band 3 distribution after oxidative treatment, the details of how these clusters are formed and how their properties change under different conditions have remained poorly understood. To address these issues, a framework that enables the simultaneous monitoring of the temporal and spatial changes following oxidation is needed. In this study, we established a novel simulation strategy that incorporates deterministic and stochastic reactions with particle reaction-diffusion processes, to model band 3 cluster formation at single molecule resolution. By integrating a kinetic model of RBC antioxidant metabolism with a model of band 3 diffusion, we developed a model that reproduces the time-dependent changes of glutathione and clustered band 3 levels, as well as band 3 distribution during oxidative treatment, observed in prior studies. We predicted that cluster formation is largely dependent on fast reverse reaction rates, strong affinity between clustering molecules, and irreversible hemichrome binding. We further predicted that under repeated oxidative perturbations, clusters tended to progressively grow and shift towards an irreversible state. Application of our model to simulate oxidation in RBCs with cytoskeletal deficiency also suggested that oxidation leads to more enhanced clustering compared to healthy RBCs. Taken together, our model enables the prediction of band 3 spatio-temporal profiles under various situations, thus providing valuable insights to potentially aid understanding mechanisms for removing senescent and premature RBCs.     

Relative Abundance and Plasmodium Infection Rates of Malaria Vectors in and around Jabalpur,a Malaria Endemic Region in Madhya Pradesh State,Central India

BackgroundThis study was undertaken in two Primary Health Centers (PHCs) of malaria endemic district Jabalpur in Madhya Pradesh (Central India).MethodsIn this study we had investigated the relative frequencies of the different anopheline species collected within the study areas by using indoor resting catches, CDC light trap and human landing methods. Sibling species of malaria vectors were identified by cytogenetic and molecular techniques. The role of each vector and its sibling species in the transmission of the different Plasmodium species was ascertained by using sporozoite ELISA.ResultsA total of 52,857 specimens comprising of 17 anopheline species were collected by three different methods (39,964 by indoor resting collections, 1059 by human landing and 11,834 by CDC light trap). Anopheles culicifacies was most predominant species in all collections (55, 71 and 32% in indoor resting, human landing and light trap collections respectively) followed by An. subpictus and An. annularis. All five sibling species of An. culicifacies viz. species A, B, C, D and E were found while only species T and S of An. fluviatilis were collected. The overall sporozoite rate in An. culicifacies and An. fluviatilis were 0.42% (0.25% for P. falciparum and 0.17% for P. vivax) and 0.90% (0.45% for P. falciparum and 0.45% for P. vivax) respectively. An. culicifacies and An. fluviatilis were found harbouring both P. vivax variants VK-210 and VK-247, and P. falciparum. An. culicifacies sibling species C and D were incriminated as vectors during most part of the year while sibling species T of An. fluviatilis was identified as potential vector in monsoon and post monsoon season.ConclusionsAn. culicifacies species C (59%) was the most abundant species followed by An. culicifacies D (24%), B (8.7%), E (6.7%) and A (1.5%). Among An. fluviatilis sibling species, species T was common (99%) and only few specimens of S were found. Our study provides crucial information on the prevalence of An. culicifacies and An. fluviatilis sibling species and their potential in malaria transmission which will assist in developing strategic control measures against these vectors.     

An Escherichia coli Strain,PGB01, Isolated from Feral Pigeon Faeces,Thermally Fit to Survive in Pigeon,Shows High Level Resistance to Trimethoprim

In this study, of the hundred Escherichia coli strains isolated from feral Pigeon faeces, eighty five strains were resistant to one or more antibiotics and fifteen sensitive to all the antibiotics tested. The only strain (among all antibiotic-resistant E. coli isolates) that possessed class 1 integron was PGB01. The dihydrofolate reductase gene of the said integron was cloned, sequenced and expressed in E. coli JM109. Since PGB01 was native to pigeon’s gut, we have compared the growth of PGB01 at two different temperatures, 42°C (normal body temperature of pigeon) and 37°C (optimal growth temperature of E. coli; also the human body temperature), with E. coli K12. It was found that PGB01 grew better than the laboratory strain E. coli K12 at 37°C as well as at 42°C. In the thermal fitness assay, it was observed that the cells of PGB01 were better adapted to 42°C, resembling the average body temperature of pigeon. The strain PGB01 also sustained more microwave mediated thermal stress than E. coli K12 cells. The NMR spectra of the whole cells of PGB01 varied from E. coli K12 in several spectral peaks relating some metabolic adaptation to thermotolerance. On elevating the growth temperature from 37°C to 42°C, susceptibility to kanamycin (both strains were sensitive to it) of E. coli K12 was increased, but in case of PGB01 no change in susceptibility took place. We have also attempted to reveal the basis of trimethoprim resistance phenotype conferred by the dfrA7 gene homologue of PGB01. Molecular Dynamics (MD) simulation study of docked complexes, PGB01-DfrA7 and E. coli TMP-sensitive-Dfr with trimethoprim (TMP) showed loss of some of the hydrogen and hydrophobic interaction between TMP and mutated residues in PGB01-DfrA7-TMP complex compared to TMP-sensitive-Dfr-TMP complex. This loss of interaction entails decrease in affinity of TMP for PGB01-DfrA7 compared to TMP-sensitive-Dfr.     

Na-H Exchanger Isoform-2 (NHE2) Mediates Butyrate-dependent Na+ Absorption in Dextran Sulfate Sodium (DSS)-induced Colitis

Diarrhea associated with ulcerative colitis (UC) occurs primarily as a result of reduced Na⁺ absorption. Although colonic Na⁺ absorption is mediated by both epithelial Na⁺ channels (ENaC) and Na-H exchangers (NHE), inhibition of NHE-mediated Na⁺ absorption is the primary cause of diarrhea in UC. As there are conflicting observations reported on NHE expression in human UC, the present study was initiated to identify whether NHE isoforms (NHE2 and NHE3) expression is altered and how Na⁺ absorption is regulated in DSS-induced inflammation in rat colon, a model that has been used to study UC. Western blot analyses indicate that neither NHE2 nor NHE3 expression is altered in apical membranes of inflamed colon. Na⁺ fluxes measured in vitro under voltage clamp conditions in controls demonstrate that both HCO₃⁻-dependent and butyrate-dependent Na⁺ absorption are inhibited by S3226 (NHE3-inhibitor), but not by HOE694 (NHE2-inhibitor) in normal animals. In contrast, in DSS-induced inflammation, butyrate-, but not HCO₃⁻-dependent Na⁺ absorption is present and is inhibited by HOE694, but not by S3226. These observations indicate that in normal colon NHE3 mediates both HCO₃⁻-dependent and butyrate-dependent Na⁺ absorption, whereas DSS-induced inflammation activates NHE2, which mediates butyrate-dependent (but not HCO₃⁻-dependent) Na⁺ absorption. In in vivo loop studies HCO₃⁻-Ringer and butyrate-Ringer exhibit similar rates of water absorption in normal rats, whereas in DSS-induced inflammation luminal butyrate-Ringer reversed water secretion observed with HCO₃⁻-Ringer to fluid absorption. Lumen butyrate-Ringer incubation activated NHE3-mediated Na⁺ absorption in DSS-induced colitis. These observations suggest that the butyrate activation of NHE2 would be a potential target to control UC-associated diarrhea.