Sanjay S. Ingle

Aminopeptidase-N from the Helicoverpa armigera (Hubner) Brush Border Membrane Vesicles as a Receptor of Bacillus thuringiensis Cry1Ac δ-Endotoxin

Brush border membrane vesicles (BBMVs) were prepared from the 2nd instar larvae of Helicoverpa armigera. Binding of the activated Cry1Ac of Bacillus thuringiensis (Bt) toxin was shown by immunoblot. A 120-kDa protein was identified as a receptor for the Cry1Ac type δ-endotoxin. The aminopeptidase-N activity of BBMVs was measured as the hydrolysis of L-leucine p-nitroanilide. The specific activity was 35 units/mg protein. The BBMV preparation also showed low level of alkaline phosphatase activity. Zn++ chelating agents 2,2′-dipyridyl and 1,10-phenanthroline inhibited aminopeptidase activity at 10 mM concentration, indicating the presence of zinc-dependent aminopeptidase in the brush border of H. armigera. The aminopeptidase activity was increased with increasing concentration of δ-endotoxin. The purified 120-kDa binding protein was N-terminally sequenced. The first 10-amino-acid sequence showed 60–77% similarity with human cysteine-rich secretory protein-1 precursor, inhibin alpha chain precursor. Salmonella flagellar hook protein and yeast carboxypeptidase S.

Distribution and diversity analysis of Bacillus thuringiensis cry genes in different soil types and geographical regions of India.

Molecular characterization of 117 Bacillus thuringiensis (Bt) isolates from various geographical locations was previously done by PCR amplification of cry genes. In present investigation, diversity of cry genes from different soil types and climatic environments was studied using rarefaction method. Presence of cry1, cry2, cry3, 7, 8, cry4, cry5, 12, 14, 21, cry11, cry13 and cyt1 genes from Bt strains isolated from various regions of India was determined by PCR amplification. A varied distribution of cry genes and their profiles was found in four soil types. The cry1 gene was the most abundant in the isolates from four soil types and geographical regions. A higher degree of cry gene diversity was observed in isolates from alluvial soil. Rarefaction analysis indicated that more cry genes could be found from various soil types. Distribution of cry genes in semi arid, subtropical humid and tropical dry regions was varied but the degree of cry gene diversity determined by rarefaction analysis was similar. No major difference in distribution and diversity of cry genes was found in agricultural and non-agricultural samples except the absence of cry3 and cry13 genes in isolates of non-agricultural samples. We report the utility of rarefaction analysis to compare cry gene diversity from different geographical regions.

Ethnopharmacology guided screening of traditional Indian herbs for selective inhibition of Plasmodium specific lactate dehydrogenase

ETHNOPHARMACOLOGICAL RELEVANCE Medicinal plants traditionally used to treat malaria can provide quality leads towards identifying novel anti-malarial drugs. Here we combined this approach with target based drug discovery and explored Plasmodium specific lactate dehydrogenase (LDH) inhibitory activity of 8 Indian plants which are ethnically used to treat malaria. METHODS LDH from Indian Plasmodium falciparum and Plasmodium vivax strains, were cloned and expressed in Escherichia coli, followed by purification of recombinant enzymes (rPfLDH and rPvLDH respectively). Extracts of 8 plants in different organic and aqueous solvents, were screened for their inhibitory activity on rPfLDH, rPvLDH and mammalian LDHs. Phyllanthus amarus aqueous extract was further tested for in vitro parasiticidal activity. RESULTS Aqueous extract of Phyllanthus amarus Schum. and Thonn. and chloroform extract of Murraya koenigii (L.) Spreng. exhibited profound and exclusive inhibitory effect on Plasmodium falciparum LDH (IC(50)=11.2 μg/ml ± 0.4) and Plasmodium vivax LDH (IC(50)=6.0 μg/ml ± 0.6) respectively. Moreover, Phyllanthus amarus aqueous extract also demonstrated antiplasmodial activity in vitro, on Chloroquine sensitive and resistant strains of Plasmodium falciparum (IC(50)=7.1 μg/ml ± 0.5 and 6.9 μg/ml ± 0.7 respectively). CONCLUSION Target specific screening of traditional herbs used in malaria treatment has proffered Phyllanthus amarus and Murraya koenigii extracts as hits which can optimistically provide novel antimalarial drugs.

Molecular characterization of Bacillus thuringiensis isolated from diverse habitats of India.

Bacillus thuringiensis (Bt) strains were isolated from 94 samples from different geographical regions. Novel types of crystalline inclusion bodies were observed from some of the isolates. Crystalline inclusions of bipyramidal, spherical and cuboidal morphology were found produced by most of the isolates. Isolate GS12 showed crystal on one side of spore while isolate GM108 formed crystals on both termini of spore. Isolate GN31 produced large sized bipyramidal crystals. SDS‐PAGE analysis of the spore crystal suspension showed major protein bands in the range of 29 and 140 kDa. Two new serovars of Bt viz. GS4 and GN24 having H3abce and H3ab serotype respectively were isolated. Toxicity comparable to the reference strain Bacillus thuringiensis subs. kurstaki (Btk) HD‐1 was observed for the isolates GM20, GM17 and MP3 against larvae of Helicoverpa armigera. Some of the isolates harboring cry genes like cry1Ac and cry2 did not show any toxicity towards H. armigera while most of the isolates were harboring cry1, cry1Ac and cry2 gene. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Antimalarial activity of Evolvulus alsinoids Linn.-an in vitro Plasmodium falciparum specific lactate dehydrogenase enzyme inhibition assay

Abstract Objective To investigate the effect of standardized methanol extract of Evolvulus alsinoids Linn. (E. alsinoids) on Plasmodium falciparum specific lactate dehydrogenase (PfLDH) enzyme inhibition. Methods To carry out enzyme inhibition studies, lactate dehydrogenase was cloned from Plasmodium falciparum 3D7 strain using expression vector pET28a and expressed in Escherichia coli BL21 (DE3). Protein purification was carried out by Ni-affinity chromatography. This protein was used for the inhibition study. Methanol extract of E. alsinoids was standardized by high performance thin layer chromatography described by previous literature and screened for PfLDH enzyme inhibitory activity and compared with gossypol (a known PfLDH enzyme inhibitor). Results It was found that E. alsinoids possesses a compond scopoletin and potentially inhibits PfLDH [(25.04依0.51)%]. Conclusions Methanol extract of E. alsinoids showed significant PfLDH inhibition as evidenced from the experiments performed. The activity may be attributed to the presence of various polyphenolic and flavanoids compounds.

Plasmodium falciparum and Plasmodium vivax specific lactate dehydrogenase: Genetic polymorphism study from Indian isolates

Control and eradication of malaria is hindered by the acquisition of drug resistance by Plasmodium species. This has necessitated a persistent search for novel drugs and more efficient targets. Plasmodium species specific lactate dehydrogenase is one of the potential therapeutic and diagnostic targets, because of its indispensable role in endoerythrocytic stage of the parasite. A target molecule that is highly conserved in the parasite population can be more effectively used in diagnostics and therapeutics, hence, in the present study polymorphism in PfLDH (Plasmodiumfalciparum specific LDH) and PvLDH (Plasmodiumvivax specific LDH) genes was analyzed using PCR-single strand confirmation polymorphism (PCR-SSCP) and sequencing. Forty-six P. falciparum and thirty-five P. vivax samples were screened from different states of India. Our findings have revealed presence of a single PfLDH genotype and six PvLDH genotypes among the studied samples. Interestingly, along with synonymous substitutions, nonsynonymous substitutions were reported to be present for the first time in the PvLDH genotypes. Further, through amino acid sequence alignment and homology modeling studies we observed that the catalytic residues were conserved in all PvLDH genotypes and the nonsynonymous substitutions have not altered the enzyme structure significantly. Evolutionary genetics studies have confirmed that PfLDH and PvLDH loci are under strong purifying selection. Phylogenetic analysis of the pLDH gene sequences revealed that P. falciparum compared to P. vivax, has recent origin. The study therefore supports PfLDH and PvLDH as suitable therapeutic and diagnostic targets as well as phylogenetic markers to understand the genealogy of malaria species.

A New Enrichment Method for Isolation of Bacillus thuringiensis from Diverse Sample Types

New or more efficient methodologies having different principles are needed, as one method could not be suitable for isolation of organisms from samples of diverse types and from various environments. In present investigation, growth kinetics study revealed a higher germination rate, a higher growth rate, and maximum sporulation of Bacillus thuringiensis (Bt) compared to other Bacillus species. Considering these facts, a simple and efficient enrichment method was devised which allowed propagation of spores and vegetative cells of Bt and thereby increased Bt cell population proportionately. The new enrichment method yielded Bt from 44 out of 58 samples. Contrarily, Bt was isolated only from 16 and 18 samples by sodium acetate selection and dry heat pretreatment methods, respectively. Moreover, the percentages of Bt colonies isolated by the enrichment method were higher comparatively. Vegetative whole cell protein profile analysis indicated isolation of diverse population of Bt from various samples. Bt strains isolated by the enrichment method represented novel serovars and possibly new cry2 gene.

Engineered Cry1Ac-Cry9Aa hybrid Bacillus thuringiensis delta-endotoxin with improved insecticidal activity against Helicoverpa armigera

Recombinant Bt construct was prepared by exchange of pore forming domain I with cry1Ac to cry9Aa gene by overlap extension PCR (OE-PCR) technique. Construction of cry1Ac-cry9Aa was accomplished by six base pair homology at 3′ ends of PCR products of domain I of cry1Ac and domain II and III of cry9Aa. The recombinant toxin was also modified by deletion of N-terminal alpha helix-1 of recombinant toxin. Both Cry toxins were expressed in E. coli BL21(DE3) plysS and purified by His-tag purification. Upon insect bioassay analysis against devastating crop pest Helicoverpa armigera, toxicity of recombinant toxin was found around fivefold higher than native Cry1Ac while alpha helix-1 deleted N-terminal modified toxin did not resulted in significant increase in toxicity. The recombinant Cry toxins such as Cry1Ac-Cry9Aa and Cry1Ac-Cry9AaMod may be used for insect pest control.

Inhibition kinetics of Plasmodium Lactate Dehydrogenase with herbal extracts suggest possible enzyme inhibitor molecular interaction

Background Resistance acquired by Plasmodium species (especially P. falciparum )t o most of the present antimalarial drugs is the principal hindrance in controlling malaria. Thus, one of the major challenges towards elimination of Malaria is development of novel and sustainable antimalarial drugs. In this milieu, herbs traditionally used to treat malaria are promising repertoires of antimalarial drugs. Earlier, lab studies have reported selective inhibition of P. falciparum and P. vivax specific Lactate Dehydrogenase (PfLDH and PvLDH) by Phyllanthus amarus aqueous extract and Murraya koenigii chloroform extract respectively. In the present investigation, we studied inhibition kinetics of PfLDH and PvLDH to explore molecular interactions between enzyme and inhibitor. Materials and methods Recombinant PfLDH and PvLDH, expressed in E. coli, were used in enzyme assay. LDH activity was measured in the direction of pyruvate to L-lactate conversion [1]. Steady state kinetic constants for substrate and cofactor as well as inhibition constants for plant extracts were measured by double reciprocal plot (Lineweaver and Burk plot). The enzyme inhibitor interactions were determined based on variations in the kinetic constants in presence of inhibitors, compared to control. Results Enzyme inhibition kinetics results are summarised in Table 1. Conclusion