Recombinant NAD-dependent SIR-2 protein of Leishmania donovani: immunobiochemical characterization as a potential vaccine against visceral leishmaniasis.

BACKGROUND: The development of a vaccine conferring long-lasting immunity remains a challenge against visceral leishmaniasis (VL). Immunoproteomic characterization of Leishmania donovani proteins led to the identification of a novel protein NAD+-dependent Silent Information regulatory-2 (SIR2 family or sirtuin) protein (LdSir2RP) as one of the potent immunostimulatory proteins. Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity. In the present study, an immunobiochemical characterization of LdSir2RP and further evaluation of its immunogenicity and prophylactic potential was done to assess for its possible involvement as a vaccine candidate against leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: LdSir2RP was successfully cloned, expressed and purified. The gene was present as a monomeric protein of ~45 kDa and further established by the crosslinking experiment. rLdSir2RP shown cytosolic localization in L. donovani and demonstrating NAD+-dependent deacetylase activity. Bioinformatic analysis also confirmed that LdSir2RP protein has NAD binding domain. The rLdSir2RP was further assessed for its cellular response by lymphoproliferative assay and cytokine ELISA in cured Leishmania patients and hamsters (Mesocricetus auratus) in comparison to soluble Leishmania antigen and it was observed to stimulate the production of IFN-γ, IL-12 and TNF-α significantly but not the IL-4 and IL-10. The naïve hamsters when vaccinated with rLdSir2RP alongwith BCG resisted the L. donovani challenge to the tune of ~75% and generated strong IL-12 and IFN-γ mediated Th1 type immune response thereof. The efficacy was further supported by remarkable increase in IgG2 antibody level which is indicative of Th1 type of protective response. Further, with a possible implication in vaccine design against VL, identification of potential T-cell epitopes of rLdSir2RP was done using computational approach. CONCLUSION/SIGNIFICANCE: The immunobiochemical characterization strongly suggest the potential of rLdSir2RP as vaccine candidate against VL and supports the concept of its being effective T-cell stimulatory antigen.

Single tryptophan of disordered loop from Plasmodium falciparum purine nucleoside phosphorylase: involvement in catalysis and microenvironment.

Among various tropical diseases, malaria is a major life-threatening disease caused by Plasmodium parasite. Plasmodium falciparum is responsible for the deadliest form of malaria, so-called cerebral malaria. Purine nucleoside phosphorylase from P. falciparum is a homohexamer containing single tryptophan residue per subunit that accepts inosine and guanosine but not adenosine for its activity. This enzyme has been exploited as drug target against malaria disease. It is important to draw together significant knowledge about inherent properties of this enzyme which will be helpful in better understanding of this drug target. The enzyme shows disorder to order transition during catalysis. The single tryptophan residue residing in conserved region of transition loop is present in purine nucleoside phosphorylases throughout the Plasmodium genus. This active site loop motif is conserved among nucleoside phosphorylases from apicomplexan parasites. Modification of tryptophan residue by N-bromosuccinamide resulted in complete loss of activity showing its importance in catalysis. Inosine was not able to protect enzyme against N-bromosuccinamide modification. Extrinsic fluorescence studies revealed that tryptophan might not be involved in substrate binding. The tryptophan residue localised in electronegative environment showed collisional and static quenching in the presence of quenchers of different polarities.

Lipid lowering and antioxidant effect of miglitol in triton treated hyperlipidemic and high fat diet induced obese rats.

Miglitol, an anti-diabetic drug, has been shown to reduce plasma lipids and inhibit free radical generation. The anti-hyperlipidemic and antioxidant effects of miglitol were studied in triton-induced hyperlipidemic rats and high fat diet-fed obese rats. Plasma cholesterol and triglycerides levels were significantly lowered by miglitol at 100 mg/kg body weight doses. Miglitol inhibited generation of superoxide anion and hydroxyl free radicals by 14 and 31 % in enzymatic systems and 19 and 25 % in non-enzymatic systems, respectively. The in-vitro effect of the drug on adipogenesis using 3T3-L1 preadipocytes at 2-, 5- and 10-µM concentrations showed significant inhibition of adipogenesis (34.2 %) at 10-µM concentration. High fat diet-fed rat model was used to investigate anti-hyperlipidemic, anti-obesity and antioxidant effect of miglitol. Miglitol increased the activities of lecithin-cholesterol-acyltransferase (19 %), post heparin lipolytic activity (26 %), lipoprotein lipase (26 %) and triglyceride lipase (31 %) which result in a decrease in plasma lipid levels. The antioxidant enzymes viz., catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase and thioredoxin reductase were increased by the drug in the treated animals. The antihyperlipidemic and antioxidant effect of miglitol can be correlated to its effect on different enzymes and it can be used for inhibiting the development of cardiovascular diseases.

Purification and characterization of Plasmodium yoelii adenosine deaminase.

Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed K(m) values of 41 µM and 34 µM for adenosine and 2'-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with K(i) value of 0.5 µM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional -SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA.

DNA binding and topoisomerase II inhibitory activity of water-soluble ruthenium(II) and rhodium(III) complexes.

Water-soluble piano-stool arene ruthenium complexes based on 1-(4-cyanophenyl)imidazole (CPI) and 4-cyanopyridine (CNPy) with the formulas [(eta6-arene)RuCl2(L)] (L = CPI, eta6-arene = benzene (1), p-cymene (2), hexamethylbenzene (3); L = CNPy, eta6-arene = benzene (4), p-cymene (5), hexamethylbenzene (6)) have been prepared by our earlier methods. The molecular structure of [(eta6-C6Me6)RuCl2(CNPy)] (6) has been determined crystallographically. Analogous rhodium(III) complex [(eta5-C5Me5)RhCl2(CPI)] (7) has also been prepared and characterized. DNA interaction with the arene ruthenium complexes and the rhodium complex has been examined by spectroscopic and gel mobility shift assay; condensation of DNA and B-->Z transition have also been described. Arene ruthenium(II) and EPh3 (E = P, As)-containing arene ruthenium(II) complexes exhibited strong binding behavior, however, rhodium(III) complexes were found to be Topo II inhibitors with an inhibition percentage of 70% (7) and 30% (7a). Furthermore, arene ruthenium complexes containing polypyridyl ligands also act as mild Topo II inhibitors (10%, 3c and 40%, 3d) in contrast to their precursor complexes. Complexes 4-6 also show significant inhibition of beta-hematin/hemozoin formation activity.

Synthesis and antifilarial evaluation of 7-O-acetamidyl-4-alkyl-2H-1-benzopyran-2-ones.

A series of 7-O-acetamidyl-4-alkyl-2H-1-benzopyran-2-ones (5-23) has been synthesized by amidation of 7-O-(carbethoxymethyl)-4-alkyl-2H-1-benzopyran-2-ones (2a, 2b) with different primary and secondary amines in fair to good yield. The resulting compounds were screened for their filarial DNA topoisomerase inhibitory activity under in vivo condition in Setaria cervi. The compounds were tested in vitro against Brugia malayi. A few of the compounds possess promising antifilarial activity.