Malaria Research Group

Delhi, India

Malaria Research Group

Delhi, India
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Bhattacharyya A.,University of Delhi | Bhattacharyya A.,Malaria Research Group | Babu C.R.,University of Delhi
International Journal of Biological Macromolecules | Year: 2017

Seeds of tropical legumes posses a repertoire of proteinase inhibitors (PI) and the current study highlights some structural/functional features of a strong serine PI from the seeds of Caesalpinia bonduc (CbTI–2). Following purification, N-terminal sequence of CbTI–2 revealed over 40% similarity with a few serine PIs of Caesalpinioideae subfamily. Upon exposure to metal ions and ionic/non ionic surfactants, CbTI–2 showed immense variation in the levels of antitryptic activity. Exposure of CbTI–2 to 1,4-Dithiothreitol, Guanidinium HCl, H2O2 and Dimethyl sulfoxide led to a steady loss of inhibitory activity. Chemical modification of amino acids suggested an arginine as the active site residue. Circular Dichroism spectrum of native CbTI–2 revealed an unordered state. Secondary structure composition of CbTI–2 following exposure to extreme conditions (heat, acidic/alkaline environment, Guanidine hydrochloride and DTT) showed considerable perturbations that caused severe loss of antiproteolytic activity. DLS studies yielded a hydrodynamic radius of ∼2.2 nm for CbTI–2 and also reconfirmed 1:1 stoichiometry for the trypsin-CbTI–2 complex. Initial studies indicated CbTI–2 to be a potent antiplasmodial agent by being highly toxic towards growth, schizont rupture process and erythrocytic invasion of Plasmodium falciparum. © 2017 Elsevier B.V.

Bag N.,National University of Singapore | Ali A.,Malaria Research Group | Chauhan V.S.,Malaria Research Group | Wohland T.,National University of Singapore | Mishra A.,Malaria Research Group
Chemical Communications | Year: 2013

Monomeric hIAPP significantly destabilizes both model and live cell membranes by increasing membrane fluidity. This interaction with membranes happens via carpet formation followed by lipid extraction in a concentration dependent manner and thus we propose that hIAPP aggregation prior to membrane interaction may not be necessary for its cytotoxicity. © 2013 The Royal Society of Chemistry.

Mishra A.,Jamia Millia Islamia University | Kaushik N.K.,Malaria Research Group | Sardar M.,Jamia Millia Islamia University | Sahal D.,Malaria Research Group
Colloids and Surfaces B: Biointerfaces | Year: 2013

In the present study silver nanoparticles (silvernp) were synthesized from AgNO3 through simple green routes using either purified Alpha Amylase or aqueous leaf extracts of Ashoka and Neem respectively. The use of plant extract/enzyme for synthesis of nanoparticles is a single-step, cost effective and eco-friendly process. The silvernp obtained by these three different ways were characterized using UV-visible spectroscopy, DLS, TEM, XRD and FTIR. These nanoparticles were found to be antiplasmodial with IC50 (μg/ml) 3.75 (Amylasenp), 8 (Ashokanp) and 30 (Neemnp) whereas plant extracts or amylase alone did not show any activity up to 40μg/ml. Although AgNO3 was also found to have intrinsic antiplasmodial activity (IC50 0.5μg/ml), the hemolytic tendencies appeared to be higher for AgNO3 (MHC10: 10μg/ml) against the nanoparticulate preparations (MHC10: >40μg/ml). © 2013 Elsevier B.V.

Kafuye-Mlwilo M.Y.,National Institute for Medical Research | Mukherjee P.,Malaria Research Group | Chauhan V.S.,Malaria Research Group
Infection and Immunity | Year: 2012

The 19-kDa carboxyl-terminal fragment of the merozoite surface protein-1 (MSP-1 19) has been shown to regulate antibody (Ab)- mediated protective immunity to blood-stage malaria infection. But the serological memory to this antigen tends to be shortlived, and little is known of the mechanisms that regulate the formation of B cell memory to MSP-1 19 antigen. We studied the formation of B cell memory response after immunization with the recombinant 19-kDa Plasmodium falciparum merozoite surface protein 1 (PfMSP-1 19). Immunization with PfMSP-1 19 resulted in delayed increase in germinal center (GC) B cell numbers. This poor GC reaction correlated with short-lived PfMSP-1 19-specific antibodies in serum and the short life of PfMSP- 1 19-specific plasma cells and memory B cells (MBCs) in spleen and bone marrow. PfMSP-1 19-specific MBCs were capable of producing antigen (Ag)-specific Ab-secreting cell (ASC) responses that were short-lived following challenge immunization of the immune mice with antigen or transgenic Plasmodium berghei parasite expressing PfMSP-1 19 in place of native P. berghei MSP- 1 19 at 8 weeks after the last immunization or following adoptive transfer into naive hosts. However, no protection was achieved in PfMSP-1 19 immune mice or recipient mice with PfMSP-1 19-specific MBCs following challenge with transgenic P. berghei. Our findings suggest that PfMSP-1 19-specific IgG production by short-lived plasma cells combined with the poor ability of the PfMSP-1 19-induced MBCs to maintain the anamnestic IgG responses failed to contribute to protection against infection. © 2012, American Society for Microbiology.

Chugh M.,Malaria Research Group | Sundararaman V.,Malaria Research Group | Kumar S.,Plant Transformation Group | Reddy V.S.,Plant Transformation Group | And 3 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2013

Malaria parasites use hemoglobin (Hb) as a major nutrient source in the intraerythrocytic stage, during which heme is converted to hemozoin (Hz). The formation of Hz is essential for parasite survival, but to date, the underlying mechanisms of Hb degradation and Hz formation are poorly understood. We report the presence of a ∼200-kDa protein complex in the food vacuole that is required for Hb degradation and Hz formation. This complex contains several parasite proteins, including falcipain 2/2', plasmepsin II, plasmepsin IV, histo aspartic protease, and heme detoxification protein. The association of these proteins is evident from coimmunoprecipitation followed by mass spectrometry, coelution from a gel filtration column, cosedimentation on a glycerol gradient, and in vitro protein interaction analyses. To functionally characterize this complex, we developed an in vitro assay using two of the proteins present in the complex. Our results show that falcipain 2 and heme detoxification protein associate with each other to efficiently convert Hb to Hz. We also used this in vitro assay to elucidate the modes of action of chloroquine and artemisinin. Our results reveal that both chloroquine and artemisinin act during the heme polymerization step, and chloroquine also acts at the Hb degradation step. These results may have important implications in the development of previously undefined antimalarials.

Alam A.,Malaria Research Group | Bhatnagar R.K.,Insect Resistance Group | Chauhan V.S.,Malaria Research Group
Molecular and Biochemical Parasitology | Year: 2012

PfSUB3 is the third subtilisin-like protease annotated in Plasmodium genome database "PlasmoDB". The other two members, PfSUB1 and PfSUB2 have been implicated in merozoite egress and invasion in asexual blood stages. In this study, we recombinantly expressed a region of PfSUB3 spanning from Asn 334 to Glu 769 (PfSUB3c) which encompassed the predicted catalytic domain with all the active site residues and predicted mature region spanning from Thr 516 to Glu 769 (PfSUB3m) in E. coli. PfSUB3m showed PMSF-sensitive proteolytic activity in in vitro assays. Replacement of active site serine with alanine in PfSUB3m resulted in inactive protein. We found that PfSUB3c and PfSUB3m undergo truncation to produce a 25-kDa species which was sufficient for proteolytic activity. Quantitative real-time PCR, immnufluorescence assay and Western blot analyses revealed that PfSUB3 is expressed at late asexual blood stages. Serine protease activity of PfSUB3 and its expression in the late stages of erythrocytic schizogony are indicative of some possible role of the protease in merozoite egress and/or invasion processes. © 2012 Elsevier B.V. All rights reserved.

Alam A.,Malaria Research Group | Chauhan V.S.,Malaria Research Group
PLoS ONE | Year: 2012

Plasmodium falciparum serine repeat antigen 5 (SERA5) is a target for both drug and vaccine intervention against malaria. SERA5 is secreted in the parasitophorous vacuole where it is proteolytically processed before schizont rupture. Among the processed products is a 50.8-kDa central domain of the protease, which possesses chymotrypsin-like activity and consists of a 28.9-kDa catalytic domain with a 21.9-kDa N-terminal prodomain, which remain attached together. Because SERA5 has been implicated in merozoite egress from host erythrocytes, the effect of the prodomain and a heptapeptide derived from its C-terminus spanning from D 560 to F 566 (DNSDNMF) on parasite growth was studied. When E. coli-expressed prodomain was incubated with parasite culture, a significant delay in transition from schizont to ring stages was observed up to nanomolar concentrations. The peptide, DNSDNMF also showed similar effects but at nearly 1000-fold higher concentrations. The peptide was also found to interact with the catalytic domain. These data demonstrate the crucial role of SERA5 prodomain for the egress process. Given the inhibitory potential of the prodomain for the parasite, we suggest that peptidomimetic inhibitors based on SERA5 prodomain sequences can be developed as future therapeutics against malaria. © 2012 Alam, Chauhan.

Singh P.K.,Malaria Research Group | Kanodia S.,Malaria Research Group | Dandin C.J.,Indian Institute of Science | Vijayraghavan U.,Indian Institute of Science | Malhotra P.,Malaria Research Group
Biochimica et Biophysica Acta - Gene Regulatory Mechanisms | Year: 2012

Large numbers of Plasmodium genes have been predicted to have introns. However, little information exists on the splicing mechanisms in this organism. Here, we describe the DExD/DExH-box containing Pre-mRNA processing proteins (Prps), PfPrp2p, PfPrp5p, PfPrp16p, PfPrp22p, PfPrp28p, PfPrp43p and PfBrr2p, present in the Plasmodium falciparum genome and characterized the role of one of these factors, PfPrp16p. It is a member of DEAH-box protein family with nine collinear sequence motifs, a characteristic of helicase proteins. Experiments with the recombinantly expressed and purified PfPrp16 helicase domain revealed binding to RNA, hydrolysis of ATP as well as catalytic helicase activities. Expression of helicase domain with the C-terminal helicase-associated domain (HA2) reduced these activities considerably, indicating that the helicase-associated domain may regulate the PfPrp16 function. Localization studies with the PfPrp16 GFP transgenic lines suggested a role of its N-terminal domain (1-80 amino acids) in nuclear targeting. Immunodepletion of PfPrp16p, from nuclear extracts of parasite cultures, blocked the second catalytic step of an in vitro constituted splicing reaction suggesting a role for PfPrp16p in splicing catalysis. Further we show by complementation assay in yeast that a chimeric yeast-Plasmodium Prp16 protein, not the full length PfPrp16, can rescue the yeast prp16 temperature-sensitive mutant. These results suggest that although the role of Prp16p in catalytic step II is highly conserved among Plasmodium, human and yeast, subtle differences exist with regards to its associated factors or its assembly with spliceosomes. © 2012 Elsevier B.V.

Saini A.,Malaria Research Group | Chauhan V.S.,Malaria Research Group
ChemBioChem | Year: 2011

Ubiquitinated cytoplasmic inclusions of TDP-43 and its C-terminal cleavage products are the pathological hallmarks of amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitinated inclusions. The C-terminal fragments (CTFs) of TDP-43 are increasingly considered to play an important role in its aggregation and in disease. Here, we employed a set of synthetic peptides spanning the length of the TDP-43 CTF (220-414) in order to find out its core aggregation domains. Two regions, one in the RRM-2 domain (246-255) and the other in the C-terminal domain (311-320) of TDP-43, stand out as highly aggregation prone. Studies done on recombinant purified TDP-43 CTF and its three mutants, in which these sequences were deleted individually and together, suggested that the 311-320 region has a more crucial role to play than the 246-255 in its aggregation. The study helps in defining specific peptide sequences that might form the core of TDP-43 aggregation. Identification of these sequences could help in designing peptide based inhibitors of TDP-43 aggregation. © 2011 Wiley-VCH Verlag GmbH& Co. KGaA, Weinheim.

Kaushik N.K.,Malaria Research Group | Sharma J.,Malaria Research Group | Sahal D.,Malaria Research Group
Malaria Journal | Year: 2012

Background: A lack of vaccine and rampant drug resistance demands new anti-malarials. Methods. In vitro blood stage anti-plasmodial properties of several de novo-designed, chemically synthesized, cationic, amphipathic, helical, antibiotic peptides were examined against Plasmodium falciparum using SYBR Green assay. Mechanistic details of anti-plasmodial action were examined by optical/fluorescence microscopy and FACS analysis. Results: Unlike the monomeric decapeptides {(Ac-GXRKXHKXWA-NH2) (X=F,ΔF) (Fm , ΔFm IC50 >100 μM)}, the lysine-branched, dimeric versions showed far greater potency {IC50 (μM) Fd 1.5, ΔFd 1.39}. The more helical and proteolytically stable ΔFd was studied for mechanistic details. ΔFq, a K-K2 dendrimer of ΔFm and (ΔFm)2 a linear dimer of ΔFm showed IC 50 (μM) of 0.25 and 2.4 respectively. The healthy/infected red cell selectivity indices were >35 (ΔFd), >20 (ΔFm)2 and 10 (ΔFq). FITC-ΔFd showed rapid and selective accumulation in parasitized red cells. Overlaying DAPI and FITC florescence suggested that ΔFd binds DNA. Trophozoites and schizonts incubated with ΔFd (2.5μM) egressed anomalously and Band-3 immunostaining revealed them not to be associated with RBC membrane. Prematurely egressed merozoites from peptide-treated cultures were found to be invasion incompetent. Conclusion: Good selectivity (>35), good resistance index (1.1) and low cytotoxicity indicate the promise of ΔFd against malaria. © 2012 Kaushik et al.; licensee BioMed Central Ltd.

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