A variety of resistance mechanisms to SAG, most of them consistent with a model based on the study of resistance in vitro, were present in clinical isolates from the same geographical region.

Although both sequences exhibit an overall conservation of key residues, DHSL20 protein lacks a critical lysine residue, and the recombinant protein showed no DHS activity in vitro. However, DHS34 contains the critical lysine residue, and the recombinant DHS34 effectively catalyzed deoxyhypusine synthesis. Furthermore, in vivo labeling confirmed that hypusination of eukaryotic initiation factor 5A occurs in intact Leishmania parasites. Interestingly, the DHS34 is much longer, with 601 amino acids, compared with the human DHS enzyme (369 amino acids) and contains several unique insertions. To study the physiological role of DHS34 in Leishmania, gene deletion mutations were attempted via targeted gene replacement. However, chromosomal null mutants of DHS34 could only be obtained in the presence of a DHS34-containing episome. The present data provide evidence that DHS34 is essential for L. donovani and that structural differences in the human and leishmanial DHS enzyme may be exploited for designing selective inhibitors against the parasite.

Antimonial-containing drugs are the first line of treatment against Leishmaniasis. Resistance to antimonials in Leishmania is proposed to be due to reduced uptake of trivalent antimony (SbIII) through the aquaglyceroporin (AQP1). We investigated the uptake of SbIII and involvement of aquaglyceroporin in developing antimony resistance phenotype in Leishmania donovani clinical isolates. SbIII accumulation, copy number of AQP1 gene, and transcript levels were compared in antimony-sensitive versus -resistant isolates. Antimony-resistant field isolates showed reduced uptake of SbIII. The copy number of AQP1 gene showed higher copy number in the antimony-resistant isolates when compared with the sensitive isolates and did not correlate to the reduced uptake of SbIII. Downregulation of AQP1 RNA levels was not consistently found in the antimony-resistant isolates. Our studies indicate that while downregulation of AQP1 may be one of the mechanisms of antimony resistance, it is however not an invariable feature.

Chronic myeloid leukemia is a myeloproliferative disorder with a unique rearrangement, the Philadelphia chromosome. Oxidative stress, a pervasive condition of an increased number of reactive oxygen species, is now recognized to be prominent feature of various diseases and their progression. Thus antioxidants, which control the oxidative stress state, represent a major line of defense regulating overall true state of health. The relationship between antioxidants status and levels of well-known markers of oxidative stress that are measured as lipid peroxides and oxidized proteins reflect better health indices and postures. The aim of this study was to evaluate the role of oxidative stress in pathophysiology of Chronic myeloid leukemia by measuring the circulating plasma lipid peroxide levels in terms of malonyldialdehyde, total lipid hydroperoxide and oxidized proteins as protein carbonyl whereas antioxidant status were estimated in terms of reduced glutathione and total thiol in plasma of Chronic myeloid leukemia patients. The present study included 47 Chronic myeloid leukemia patients and 20 age-and sex-matched healthy subjects. Out of 47 Chronic myeloid leukemia patients, 31 were in chronic phase (CML-CP) and 16 in accelerated phase (CML-AP). The median age of Chronic myeloid leukemia patients was 33 years and that of controls was 32 years. Oxidative stress and antioxidant status in plasma were evaluated by spectrophotometric procedures. There was a significant increase (p<0.05) in plasma malonyldialdehyde, total lipid hydroperoxide and protein carbonyl levels in Chronic myeloid leukemia patients as compared to healthy subjects. Our results also showed that plasma malonyldialdehyde and protein carbonyl levels were markedly elevated (p<0.05) in both chronic phase (CML-CP) and accelerated phase (CML-AP) as compared to healthy volunteers. Antioxidant status was found to be significantly decreased (p<0.05) in Chronic myeloid leukemia patients and its phases as compared to healthy participants. It could be concluded that oxidative stress may be associated with the pathophysiology of Chronic myeloid leukemia.

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The hypolipidemic action of Anthocephalus indicus (family, Rubiaceae: Hindi name, Kadam) fruit extract has been studied in hyperlipidemic rats fed a triton-and cholesterol-rich high-fat diet. In triton WR-1339-induced hyperlipidemic rats, feeding with the fruit extract (500 mg/kg b.w.) exerted a lipid-lowering effect as assessed by reversal of plasma levels of total cholesterol, phospholipids, and triglyceride following reactivation of the post-heparin lipolytic activity. In another model, chronic feeding of this natural product (500 mg/kg, b.w.) to animals simultaneously fed a high-fat diet for 30 days caused lowering of lipid levels in plasma and liver accompanied with stimulation of hepatic lipolytic activity. The hypolipidemic activity of Anthocephalus indicus fruit extract iscompared with guggulipid, a known lipid-lowering drug, in both models.

Visceral leishmaniasis, a lethal parasitic disease, is caused by the protozoan parasite Leishmania donovani. The absence of an effective vaccine, drug toxicity and parasite resistance necessitates the identification of novel drug targets. Reconstruction of genome-scale metabolic models and their simulation has been established as an important tool for systems-level understanding of a microorganism's metabolism. In this work, amalgamating the tools and techniques of computational systems biology with rigorous manual curation, a constraint-based metabolic model for Leishmania donovani BPK282A1 has been developed. New functional annotations for 18 formerly hypothetical or erroneously annotated genes (encountered during iterative refinement of the model) have been proposed. Further, to formulate an accurate biomass objective function, experimental determination of previously uncharacterized biomass constituents was performed. The developed model is a highly compartmentalized metabolic model, comprising 1159 reactions, 1135 metabolites and 604 genes. The model exhibited around 76% accuracy for the prediction of experimental phenotypes of gene knockout studies and drug inhibition assays. Employing in silico gene knockout studies, we identified 28 essential genes with negligible sequence identity to the human proteins. Moreover, by dissecting the functional interdependencies of metabolic pathways, 70 synthetic lethal pairs were identified. Finally, in order to delineate stage-specific metabolism, gene-expression data of the amastigote stage residing in human macrophages were integrated into the model. By comparing the flux distribution, we illustrated the stage-specific differences in metabolism and environmental conditions that are in good agreement with the experimental findings. The developed model can serve as a highly enriched knowledgebase of legacy data and an important tool for generating experimentally verifiable hypotheses.

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