Brijesh Sisodia

Antimicrobial potential of Glycyrrhiza glabra roots.

The present study was aimed to investigate antimicrobial potential of Glycyrrhiza glabra roots. Antimycobacterial activity of Glycyrrhiza glabra was found at 500 microg/mL concentration. Bioactivity guided phytochemical analysis identified glabridin as potentially active against both Mycobacterium tuberculosis H(37)Ra and H(37)Rv strains at 29.16 microg/mL concentration. It exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. Our results indicate potential use of licorice as antitubercular agent through systemic experiments and sophisticated anti-TB assay.

Proteome mapping of overexpressed membrane‐enriched and cytosolic proteins in sodium antimony gluconate (SAG) resistant clinical isolate of Leishmania donovani

AIMS This study aimed to identify differentially overexpressed membrane-enriched as well as cytosolic proteins in SAG sensitive and resistant clinical strains of L. donovani isolated from VL patients which are involved in the drug resistance mechanism. METHODS The proteins in the membrane-enriched as well as cytosolic fractions of drug-sensitive as well as drug-resistant clinical isolates were separated using two-dimensional gel electrophoresis and overexpressed identified protein spots of interest were excised and analysed using MALDI-TOF/TOF. RESULTS Six out of 12 overexpressed proteins were identified in the membrane-enriched fraction of the SAG resistant strain of L. donovani whereas 14 out of 18 spots were identified in the cytosolic fraction as compared with the SAG sensitive strain. The major proteins in the membrane-enriched fraction were ABC transporter, HSP-83, GPI protein transamidase, cysteine-leucine rich protein and 60S ribosomal protein L23a whereas in the cytosolic fraction proliferative cell nuclear antigen (PCNA), proteasome alpha 5 subunit, carboxypeptidase, HSP-70, enolase, fructose-1,6-bisphosphate aldolase, tubulin-beta chain have been identified. Most of these proteins have been reported as potential drug targets, except 60S ribosomal protein L23a and PCNA which have not been reported to date for their possible involvement in drug resistance against VL. CONCLUSION This study for the first time provided a cumulative proteomic analysis of proteins overexpressed in drug resistant clinical isolates of L. donovani indicating their possible role in antimony resistance of the parasite. Identified proteins provide a vast field to be exploited for novel treatment strategies against VL such as cloning and overexpression of these targets to produce recombinant therapeutic/prophylactic proteins.

A promising anticancer and antimalarial component from the leaves of Bidens pilosa.

Bidens pilosa is used in folk medicine for various applications due to the presence of polyacetylenes, flavonoids, terpenoids, phenylpropanoids and others. Bioactivity-guided fractionation of different extracts of B. pilosa leaf showed potential in vitro anticancer and antimalarial activity and led to the identification of a potential marker compound, phenyl-1,3,5-heptatriyne. Erythrocyte osmotic fragility experiments revealed the various extracts as well as the marker components toxicity profiles on normal blood cells.

Low molecular weight proteins of outer membrane of Salmonella typhimurium are immunogenic in Salmonella induced reactive arthritis revealed by proteomics

In patients with reactive arthritis (ReA)/undifferentiated spondyloarthropathy (uSpA), synovial fluid mononuclear cells (SFMC) show proliferation to bacterial antigens that trigger ReA, i.e. Chlamydia, Yersinia, Campylobactor, Shigella and Salmonella species. We have shown previously that SFMC proliferate significantly to outer membrane proteins of S typhimurium in Salmonella induced ReA. In the present study we characterized the immunoreactive fractions of outer membrane protein (Omp) of S typhimurium in Salmonella induced ReA. Omp of Salmonella was isolated and fractionated by continuous elution sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) using Prep‐Cell into eight Omp fractions based on molecular weight. Twenty‐three patients with ReA were screened for the bacterial trigger using the SFMC proliferative response to crude lysates of Y enterocolitica, S flexneri, C jejuni and S typhimurium using thymidine uptake assay. SFMC from patients with salmonella induced ReA were tested against eight fractions. Seven of 23 patients with ReA had S typhimurium‐induced ReA. Of these seven patients, five patients SFMC had a significant stimulation index (SI) against < 22, 22–26, 25–35 and 28–40 kDa fractions of Omp. These fractions were analysed by SDS‐PAGE and matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry, which revealed 10 proteins. These proteins were 37 kDa OmpA, 33 kDa TsX, 28 kDa putative Omp, 28 kDa Vac J, 39 kDa OmpD, 18 kDa OmpX, 23 kDa OmpW, 43 kDa OmpS1 and 19 kDa peptidoglycan‐associated lipoprotein. In conclusion, for the first time we have identified some low molecular weight proteins in the Omps of Salmonella which are T cells immunoreactive in patients with salmonella induced ReA/uSpA.

Th1-stimulatory polyproteins of soluble Leishmania donovani promastigotes ranging from 89.9 to 97.1 kDa offers long-lasting protection against experimental visceral leishmaniasis

Our earlier studies identified a fraction (F2) of Leishmania donovani soluble promastigote antigen belonging to 97.4-68 kDa for its ability to stimulate Th1-type cellular responses in cured visceral leishmaniasis (VL) patients as well as in cured hamsters. A further fractionation of F2-fraction into seven subfractions (F2.1-F2.7) and re-assessment for their immunostimulatory responses revealed that out of these, only four (F2.4-F2.7) belonging to 89.9-97.1 kDa, stimulated remarkable Th1-type cellular responses either individually or in a pooled form (P4-7). In this study these potential subfractions were further assessed for their prophylactic potential in combination with BCG against L. donovani challenge in hamsters. Optimum parasite inhibition ( approximately 99%) was obtained in hamsters vaccinated with pooled subfractions and they survived for 1 year. The protection was further supported by remarkable lymphoproliferative, IFN-gamma and IL-12 responses along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody as observed on days 45, 90 and 120 post-challenge suggesting that a successful subunit vaccine against VL may require multiple Th1-immunostimulatory proteins. MALDI-TOF-MS/MS analysis of these subfractions further revealed that of the 19 identified immunostimulatory proteins, Elongation factor-2, p45, Heat shock protein-70/83, Aldolase, Enolase, Triosephosphate isomerase, Disulfideisomerase and Calreticulin were the major ones in these subfractions.

Immunization with inflammatory proteome of Brugia malayi adult worm induces a Th1/Th2-immune response and confers protection against the filarial infection

Mastomys coucha and jirds (Meriones unguiculatus) were immunized with four cytokine-stimulating SDS-PAGE resolved fractions F5 (68-84 kDa), F6 (54-68 kDa), F10 (38-42 kDa) and F14 (20-28 kDa) of Brugia malayi adult worm to determine which of these fractions has the potential to influence the establishment of subsequently introduced B. malayi infection in the animals. The proteins in the fractions were analyzed by 2DE and MALDI-TOF. Immunization with F6 suppressed the establishment of third stage larva (L(3)) initiated infection in M. coucha (64%; P<0.01) and jird (42%; P<0.01). Survival of intraperitoneally implanted adult worms in M. coucha was lowered by F6 (72%; P<0.01) and F14 (66%; P<0.05) but not by F5 and F10. Immunization with F6 intensely upregulated both Th1 (IFN-gamma, TNF-alpha, IL-1 beta, IL-2, IL-6, IgG1, IgG2a and lymphoproliferation) and Th2 (IgG2b and IL-10) responses and NO release. Immunostimulatory proteins HSP60, intermediate filament protein, and translation elongation factor EF-2 were identified in F6 fraction by 2DE and MALDI. The findings suggest that F6 protects the host from the parasite via Th1/Th2 type responses and thus holds promise for development as a vaccine.

Proteomics indicates modulation of tubulin polymerization by L-menthol inhibiting human epithelial colorectal adenocarcinoma cell proliferation†

Menthol is a naturally occurring cyclic monoterpene used in oral hygiene products, confectionary, pharmaceuticals, cosmetics, pesticides, and as a flavoring agent. In the present study, we analyzed the differentially expressing proteome in L‐menthol‐treated Caco‐2 cell line as it was found to inhibit cell proliferation. Interestingly, free tubulin proteins were observed to be limited after menthol treatment. Semiquantitative RT‐PCR with α‐tubulin primers showed no change in the level of RNA expression in menthol‐treated cell line. However, tubulin polymerization assay with menthol indicated a trend similar to taxol in promoting microtubule assembly. Further, physical counting of apoptotic nuclei and active caspase‐3 assays confirmed onset of apoptosis though the rate was slower as compared with that of taxol treatment. This study is the first report of a monoterpene L‐menthol modulating tubulin polymerization and apoptosis to inhibit cancer cell proliferation.

Antiplasmodial activity of steroidal chalcones: evaluation of their effect on hemozoin synthesis and the new permeation pathway of Plasmodium falciparum-infected erythrocyte membrane

Chalcone derivatives on an estradiol framework were evaluated for their ability to inhibit the growth and development of the malaria parasite Plasmodium falciparum. Out of twelve steroidal chalcones and one indanone derivative studied, three were found to have 50% growth inhibitory concentration less than 5 μm and minimum inhibitory concentration for parasite development from ring to schizont stage as ≤20 μm with best activity for gallic acid‐based chalcone derivative 1 as 2.07 and 10 μm, respectively. Two of the active derivatives 1 and 10 did not exhibit cytotoxicity against vero cells as evident by the good selectivity ratio. Study of structure–activity relationship indicated that increasing substitution in the benzoyl ring‐enhanced antiplasmodial activity. Hemozoin synthesis of the parasite remained unaffected by these derivatives. These derivatives were also investigated for their effect on parasite‐induced new permeation pathway in the erythrocyte membrane by sorbitol‐induced hemolysis, and four derivatives 1, 2, 9, and 10 exhibited significant inhibition (>70%) at 20 μm concentration. A positive correlation was also observed among the antiplasmodial activity and inhibition of new permeation pathway. These observations suggest that steroidal chalcones with selective activity for the parasite may be considered as antimalarial leads for further optimization and preclinical study.

Proteomic analyses of membrane enriched proteins of Leishmania donovani Indian clinical isolate by mass spectrometry

Visceral leishmaniasis (VL) is a major fatal disease prevalent in North-East India, caused by a protozoan parasite Leishmania donovani. The parasite multiplies and thrives inside mammalian macrophages and is transmitted by the bite of the sandfly. Due to the unsatisfactory treatment measures, increasing drug resistance and the advent of HIV-Leishmania co-infection there has been an urgent need to develop novel drug/vaccine targets against VL. Target identification is the key step in drug discovery and proteomics seems to be a suitable strategy for it due to the availability of Leishmania major, Leishmania infantum, Leishmania braziliensis, Leishmania donovani, Leishmania mexicana and Leishmania tarentolae genome sequence. Since, majority of proteome analyses of Leishmania have, so far, been performed on whole-cell extracts; this study is dealing with the sub-proteome analysis of the membrane-enriched protein (MEP) fractions of L. donovani. The analysis of 95 protein spots of the MEPs from two dimensional (2-D) gel image through matrix asserted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) endorsed the identification of various relevant functional proteins. Out of 95 the MEP spots 72 have been identified and were classified on the basis of their biological function. Several proteins of unknown function that belong to different classes like cell signaling, transmembrane receptors, and transporters have been identified which could be the new potential therapeutic targets against VL in future. The proteome array of the MEPs contributes to further elucidation of the biological system of L. donovani as well as host-parasite relationships which may be further investigated for their crucial biological role in L. donovani for disease management.

Mass spectrometry-based proteomic analysis of Leishmania donovani soluble proteins in Indian clinical isolate

Leishmania donovani, a causative organism of visceral leishmaniasis (VL), is responsible for high mortality throughout the world. Due to the unsatisfactory treatment measures and increasing drug resistance, there has been an urgent need to develop novel drug/vaccine targets against VL. The aim of this study was to identify novel targets in soluble L. donovani (SLD) protein. SLD protein was isolated and resolved by two-dimensional gel electrophoresis and analyzed through MALDI-TOF/TOF-based mass spectrometry. Proteomic results identified several proteins as drug targets, Th1 stimulatory, novel, and hypothetical proteins which could have crucial biological functions in Leishmania pathogenesis.