Subrata Laskar

Neem leaf glycoprotein restores the impaired chemotactic activity of peripheral blood mononuclear cells from head and neck squamous cell carcinoma patients by maintaining CXCR3/CXCL10 balance

Interaction between CXCL10 and CXCR3 is dysregulated in head and neck squamous cell carcinoma (HNSCC) and hampers chemotaxis of cytotoxic cells at tumor site. In continuation of the demonstration of significant immunomodulatory effects of neem leaf preparation (NLP), the active ingredient of NLP is characterized as a glycoprotein (NLGP). NLGP is responsible for in vivo immunomodulation to restrict the growth of mice tumors. Effect of NLGP in rectification of the dysregulated IFN gamma dependent chemokine and its receptor CXCR3 splice variants was investigated. Upregulated expression of CXCR3B in HNSCC-PBMC were downregulated following in vitro NLGP treatment. Unchanged expression of CXCR3A+B by NLGP with downregulation of the CXCR3B indirectly suggests the upregulation of the CXCR3A, responsible for cellular migration. However, stimulation of healthy-PBMC with NLGP maintains physiological homeostasis of CXCL10 and increases IFN gamma secretion. The suppressed chemotaxis of HNSCC-PBMC could be restored either by in vitro treatment with NLGP or during use of NLGP stimulated PBMC supernatant as a chemoattractant. Neutralization studies confirmed that the chemoattraction process is guided by both receptor (CXCR3A) and its ligand (CXCL10). Neutralization of the IFN gamma in PBMC culture in presence of NLGP unexpectedly increases the intracellular release of CXCL10, suggesting the NLGP mediated IFN gamma independent release of CXCL10. Interestingly, downregulation of the CXCL10 release was detected after IFN gamma neutralization in absence of NLGP and IFN gamma receptor neutralization in presence of NLGP. Efficacy of NLGP in restoration of the dysregulation of the chemokine signaling may be utilized to design new immunotherapeutic protocol.

Mosquito larvicidal and antimicrobial activity of protein of Solanum villosum leaves

BackgroundMosquitoes are associated with the transmission of malaria, dengue, Japanese encephalitis, filariasis and other viral diseases throughout the globe, apart from being a nuisance pest. Biological control alone or as a part of integrated vector management stands to be a better alternative to the chemical controls aimed against pest mosquitoes. At the same time it is necessary to control bacteria by synthetic or natural means (plant products). Hence the present study was designed to screen the effect of mosquito larvicidal and antimicrobial activitiy of protein isolated from matured leaves of Solanum villosum against mosquito immatures and some pathogenic bacteria.MethodsAqueous solvent extract of fresh mature leaves of S. villosum was tested against 3rd instar larvae of Anopheles stephensi, Culex quinquefasciatus and Stegomyia aegypti mosquitoes and against four pathogenic bacteria. The protein fraction was isolated and tested for mosquitocidal and antibacterial activities. Amino acid analysis was performed on isolated protein using PICO.TAG amino acid system. SDS-PAGE was also done to detect the bands of amino acid on the basis of their molecular weights.ResultsProteins isolated from mature leaves of S. villosum were found to have larvicidal and antimicrobial properties. Analysis of the isolated protein identified fifteen amino acids of which eight were essential amino acids. SDS-PAGE detected seven bands corresponding to different molecular weights in the range of 69–109 KDa.ConclusionProteins of mature leaves of S. villosum exhibited moderate larvicidal and antimicrobial activities. The study provides considerable scope in exploiting local indigenous resources for isolation of antimicrobial and mosquito larvicidal proteins.

Neem leaf glycoprotein helps to generate carcinoembryonic antigen specific anti-tumor immune responses utilizing macrophage-mediated antigen presentation

In an objective to generate effective carcinoembryonic antigen (CEA) specific anti-tumor immune response in Swiss mice, CEA was presented using macrophages with adjuvant help from neem leaf glycoprotein (NLGP). Such vaccination generates significantly higher antibody (IgG2a) and T cell response than immunization protocol without NLGP. NLGP controls the function of both B cells and macrophages by altering the expressions of various regulatory molecules, like, CD19, CD11b, etc. NLGP also directs CEA vaccination towards Th1 bias, by modulating cytokine secretion. This NLGP-generated anti-CEA immune response would be effective as a vaccine to lyse CEA(+) tumors in vitro and in vivo.

Neem leaf glycoprotein directs T-bet–associated type 1 immune commitment

Neem leaf glycoprotein (NLGP)-mediated immune activation and associated immune polarization was studied. NLGP-induced activation is reflected in upregulation of early activation marker CD69 on lymphocytes, monocytes, and dendritic cells. Activation is also denoted by CD45RO enhancement, with a decrease in CD45RA phenotype and CD62L (L-selectin). NLGP-activated T cells secrete greater amount of signature T-helper (Th)1 cytokines interferon-gamma and a lower amount of the Th2 cytokine interleukin (IL)-4. Similar type 1 directiveness is also observed in antigen-presenting monocytes and dendritic cells by upregulation of IL-12, tumor necrosis factor -alpha and downregulation of IL-10. Creation of the type 1 microenvironment is also assisted by NLGP-induced downregulation of FoxP3(+) T-Reg cells. A type 1-specific transcription factor, T-bet, is upregulated in circulating immune cells after their stimulation with NLGP. In the creation of type 1 immune network, increased phosphorylation of STAT1 and STAT4 with decreased phosphorylation of STAT3 might have significance. We conclude that NLGP may be effective in maintaining normal immune homeostasis by upregulating type 1 response in immunosuppressed hosts, which may have significant role in the induction of host protective antitumor functions.

A brief resume on the genus Ailanthus: chemical and pharmacological aspects

Over the past decade, herbal medicine has become an item of global importance, with both medicinal and economic implications. Thus, accurate scientific assessment has become a prerequisite for acceptance of herbal health claims. The plants of genus Ailanthus, belong to the family Simaroubaceae, is widely used in Ayurveda and evidence-based phytotherapy. Several species of the genus Ailanthus are widely distributed over Asia and north Australia and studied extensively over the last few decades. A compilation of the constituents isolated from different Ailanthus species covering the literature up to December 2008 is presented in the review. The botanical classification and ethno-pharmacology of Ailanthus plants, as well as the biological activities and pharmacological applications of both distinct phytochemicals and active plant materials (formulations, extracts etc.), are discussed in detail.

A New Reagent for Identification of Amino Acids on Thin-Layer Chromatography Plates

A variety of selective and non-selective reagents [1–18] are used in thin-layer chromatography for identification of amino acids. Such identification is very useful because of the occurrence of the amino acids as the structural units of proteins, in the free state in natural products, and for determination of the C-terminal amino acids of degraded proteins. Of the reagents generally used, ninhydrin, a non-selective reagent, is popular because of its remarkably high sensitivity [2], even though it produces the same purple color with all the amino acids except proline and hydroxyproline. To solve this problem a new reagent, 4hydroxyacetophenone–isatin-5-sulfonic acid (sodium salt) is proposed. This communication deals with this reagent, which produces distinguishable colors for most of the amino acids. With the proposed reagent detection limits for the amino acids range between 0.1 and 2.0 μg.

New reagent for detection of amino acids on TLC plates

Determination of protein structure is very dependent on amino acid detection, because these compounds form the backbones of proteins, and also for determination of the C-terminal units of degraded proteins. Several selective and nonselective reagents have been used for detection and identification of amino acids on thin-layer chromatograms [1–20]. Among the reagents used, ninhydrin is the most well-known nonselective reagent. It is widely used, because of its high sensitivity [2], but produces same color, purple–violet, for all amino acids except proline and hydroxyproline (yellow). To overcome this problem 2,3dichloro-1,4-naphthaquinone and isatin have been introduced as a new reagent which produces distinguishable and stable colors (more or less stable for 24 to 48 h) with many amino acids and enables convenient and easy detection of these compounds on silica gel G TLC plates with very high sensitivity (detection limit between 0.01 and 0.3 μg).

Efficacy of Limonia acidissima L. (Rutaceae) leaf extract on larval immatures of Culex quinquefasciatus Say 1823.

OBJECTIVE To investigate the role of leaf extract of Limonia acidissima L. (Rutaceae) as a biocontrol agent against the larval form of Culex quinquefasciatus, and characterization of bioactive component responsible for larvicidal activity. METHODS Larval mortality of mosquito species was observed after 24, 48 and 72 hours of exposure to different concentrations of aqueous extract, solvent extract and subsequently bioactive compound. The bioactive compound was subjected to IR and GC-MS analysis. RESULTS Mortality rate at 3% concentration of crude extract were highest (90%) amongst all concentrations tested and subsequently highest (95%) mortality was achieved in chloroform: methanol extract at 100 ppm concentrations. IR and GC-MS analysis of bioactive compound revealed the presence of steroid compound which may act as larvicide. CONCLUSIONS The chloroform: methanol extract of mature leaves of Limonia acidissima was found to exhibit considerable mosquito larvicidal activity against Culex quinquefasciatus.

Neem leaf glycoprotein is nontoxic to physiological functions of Swiss mice and Sprague Dawley rats: histological, biochemical and immunological perspectives.

We have evaluated the toxicity profile of a unique immunomodulator, neem leaf glycoprotein (NLGP) on different physiological systems of Swiss mice and Sprague Dawley rats. NLGP injection, even in higher doses than effective concentration caused no behavioral changes in animals and no death. NLGP injection increased the body weights of mice slightly without any change in organ weights. NLGP showed no adverse effect on the hematological system. Moreover, little hematostimulation was noticed, as evidenced by increased hemoglobin content, leukocyte count and lymphocyte numbers. Histological assessment of different organs revealed no alterations in the organ microstructure of the NLGP treated mice and rats. Histological normalcy of liver and kidney was further confirmed by the assessment of liver enzymes like alkaline phosphatase, SGOT, SGPT and nephrological products like urea and creatinine. NLGP has no apoptotic effect on immune cells but induces proliferation of mononuclear cells collected from mice and rats. Number of CD4(+), CD8(+) T cells, DX5(+) NK cells, CD11b(+) macrophages and CD11c(+) dendritic cells is upregulated by NLGP without a significant change in CD4(+)CD25(+)Foxp3(+) regulatory T cells. Type 1 cytokines, like IFNγ also increased in serum with a decrease in type 2 cytokines. Total IgG content, especially IgG2a increased in NLGP treated mice. These type 1 directed changes help to create an anti-tumor immune environment that results in the restriction of carcinoma growth in mice. Accumulated evidence strongly suggests the non-toxic nature of NLGP. Thus, it can be recommended for human use in anti-cancer therapy.

Extraction and chemical investigation of Kulthi (Macrotylona uniflorus, Lam.) seed protein

Studies have been carried out on the protein solubility profile of Kulthi (Macrotylona uniflorus, Lam.) seed in aqueous solution over various pHs and at different concentrations of NaCl, Na2SO3, CaCl2, and MgCl2 at pH 8.0. Amino acid analysis of isolated protein identified 17 amino acids, 9 of which are essential. Gel-permeation chromatography on Sephadex G-200 revealed the presence of seven components in the protein fraction. Their molecular weights were determined by two comparable standard methods. Extractable Kulthi seed proteins in salt solutions were separated electrophoretically into eight fractions whose molecular weights were found to be 186,200, 131,800, 108,400, 91,200, 53,700, 44,700, 38,000, and 27,500.