Sukhdev Singh Kamboj

Purification and properties of four monocot lectins from the family araceae

Four new monocot lectins from the tubers of araceous plants, namely, Arisaema consanguineum Schott (ACA), A. curvatum Kunth (ACmA) and Sauromatum guttatum Schott (SGA) from the tribe Areae, and Gonatanthus pumilus D. Don (GPA) from the tribe Colocasieae have been purified by affinity chromatography on asialofetuin-linked amino activated silica beads. These lectins possess similar physicochemical and biological properties. All the lectins gave a single peak on HPLC size exclusion and cation exchange columns, and a single band on PAGE, (pH 4.5). In SDS-PAGE, all the lectins gave a single band corresponding to a subunit of M(r) 1,3000. All the lectins yielded multiple peaks on anion-exchange column, multiple bands on non-denatured PAGE (pH 8.3) and a family of bands on isoelectric focusing. The lectins agglutinate rabbit, rat and sheep red blood cells (RBCs) but are inactive towards human ABO erythrocytes. The haemagglutination activity of these lectins is inhibited by asialofetuin only, while simple sugars/derivatives including chitin, porcine mucin and fetuin did not react. In serological studies against rabbit anti-SGA serum, all four lectins produced immunoprecipitin lines. The lectins within each tribe were identical but the lectins belonging to the tribe Areae were only partially identical to the lectins from the tribe Colocasieae.

Evaluation of hydro-alcoholic extract of Eclipta alba for its anticancer potential: An in vitro study

ETHNOPHARMACOLOGICAL RELEVANCE Eclipta alba is traditionally used as hepatoprotective agent. The study was designed to explore its antiproliferative activity on liver and other related cancer. AIM OF THE STUDY The present study was designed to assess and establish the role of Eclipta alba as anti-cancer agent using HepG2, C6 glioma and A498 cell lines as model system. MATERIALS AND METHODS Antiproliferative and cytotoxic effects of the Eclipta alba hydroalcoholic extract (EAE) was determined using MTT assay. The expression level of NF-kB was analysed by western blotting and RT PCR. Gelatin zymography was done for gelatinase matrix metalloproteinases (MMP-2 and 9) analysis. RESULTS EAE inhibited the cell proliferation in dose dependent manner in HepG2, A498 and C6 glioma cell lines with an IC50 of 22±2.9, 25±3.6 and 50±8.7 μg/ml, respectively. The expression of MMP (2 and 9) was down-regulated with EAE treatment. DNA damage was observed following 72h of extract treatment, leading to apoptosis. Additionally, the expression level of NF-kB was evaluated with western blotting and RT-PCR and was found to be down-regulated/inactivated. CONCLUSIONS The data establish the existence of anti-proliferative, DNA damaging and anti-metastasis properties in EAE which is yet unexplored and hold high therapeutic impact.

A novel antiproliferative and antifungal lectin from Amaranthus viridis Linn seeds.

A lectin from the seeds of Amaranthus viridis Linn has been purified by affinity chromatography on asialofetuin-linked amino activated silica. Amaranthus viridis lectin (AVL) has a native molecular mass of 67 kDa. It is a homodimer composed of two 36.6 kDa subunits. The lectin gave a single band in non-denaturing PAGE at pH 4.5 and pH 8.3 and a single peak on HPLC size exclusion and cation exchange columns. The purified lectin was specific for both T-antigen and N-acetyl-D-lactosamine, markers for various carcinomas, in addition to N-acetyl-D-galactosamine, asialofetuin and fetuin. This lectin reacted strongly with red blood cells (RBCs) from human ABO blood groups and rat. It also reacted with rabbit, sheep, goat and guinea pig RBCs. The lectin is a glycoprotein having no metal ion requirement for its activity. Denaturing agents such as urea, thiourea and guanidine-HCl had no effect on its activity when treated for 15 minutes. AVL showed significant antiproliferative activity towards HB98 and P388D1 murine cancer cell lines. It also exerted antifungal activity against phytopathogenic fungi Botrytis cincerea and Fusarium oxysporum but not against Rhizoctonia solani, Trichoderma reesei, Alternaria solani and Fusarium graminearum.

Purification and characterization of a tuber lectin from Alocasia indica

Abstract A lectin from the tubers of Alocasia indica Schott has been purified by affinity chromatography on asialofetuin-linked amino activated silica beads. The bound lectin was eluted with 0.1 M glycine-HC1 buffer, pH 2.5. The purified lectin yielded a single band on SDS-PAGE, pH 8.3, corresponding to M 1 of 13 000. In polyacrylamide gel electrophoresis, pH 4.5, and gel exclusion chromatography, it also gave a single band and a single peak, respectively, with M 1 of 55 000. However, in polyacrylamide gel electrophoresis at pH 8.3, it revealed three bands. Three peaks were obtained when the affinity purified lectin was applied on a DEAE-5PW HPLC analytical column. As a haemagglutinin, this lectin was effective against animal but not human erythrocytes. The haemagglutination activity is inhibited by asialofetuin only. The purified lectin is a glycoprotein with 1.47% carbohydrate content and has no metal ion requirement for its haemagglutinating activity. Alocasia indica was found to be mi togenic for human peripheral blood lymphocytes.

New Lymphocyte Stimulating Monocot Lectins from Family Araceae. II

Two lectins purified from the tubers of Arisaema consanguineum Schott (ACA) and A. curvatum Kunth (ACmA) belonging to the monocot family Araceae were mitogenic for human peripheral blood mononuclear cells (PBMC) in the [3H]-thymidine uptake assay. ACA and ACmA had an optimum stimulatory concentration of 10-25 micrograms/ml and 50-100 micrograms/ml, respectively, as observed in PBMC from five different individuals. The mitogenic response of PBMC was inhibitable in a dose-dependent manner by asialofetuin. The lectins were T-cell specific, and stimulation kinetic studies using ACA and ACmA showed that they induce maximum thymidine uptake in PBMC at day 4 and 3, respectively.

Two novel lectins from Parkia biglandulosa and Parkia roxburghii: isolation, physicochemical characterization, mitogenicity and anti-proliferative activity.

Two mannose/glucose specific seed lectins were isolated from Parkia biglandulosa and Parkia roxburghii and were characterized w.r.t various physicochemical properties. Unlike other Parkia lectins a comparison of native and subunit molecular mass showed that both Parkia lectins were heterotetramers. Parkia biglandulosa lectin was found to be T-cell mitogen as revealed by IL-2 bioassay. These lectins showed anti-proliferative effect on two murine macrophage cancer cell lines i.e. P 388DI (50%) and J774 (70%). In addition Parkia roxburghii also inhibited proliferation of HB98 (65.47%), a B-cell hybridoma cell line.

Characterization of a Lectin from Gonatanthus pumilus D. Don Having Anti-Proliferative Effect Against Human Cancer Cell Lines

A monocot araceous lectin from tubers of Gonatanthus pumilus (GPL) wa earlier purified in our laboratory and reported as T-cell mitogen having homotetrameric structure with subunit molecular mass of 13 kDa. Besides asialofetuin as reported earlier, in the present study it was also inhibited by N-acetyl-D-lactosamine but was non-reactive towards mannose or its derivatives. The lectin is rich in acidic amino acids and cysteine is completely absent. Chemical modification of GPL revealed requirement of tryptophan and tyrosine for lectin sugar interaction. The secondary structure content of GPL, as estimated with CD spectrum in K2D programme, has 73% alpha-helix, 26% beta-sheet and 38% random contributions. Fluorescence spectrum of the lectin solution at 280 nm was typical for tryptophan residues buried inside the protein. Lectin activity decreased when treated with denaturants like guanidine-HCL, urea and thiourea. GPL inhibited the growth of three plant pathogenic fungi namely Colletotrichum lindemuthianum, Fusarium oxysporum and Botrytis cinerea. Out of 11 human cancer cell lines tested, GPL significantly inhibited proliferation of five lines viz. Colo-205, IMR-32, HCT-15, SK-N-SH and HT-29.

A comprehensive in silico analysis of non-synonymous and regulatory SNPs of human MBL2 gene

Mannose binding lectin (MBL) is a liver derived protein which plays an important role in innate immunity. Mannose binding lectin gene 2 (MBL2) polymorphisms are reported to be associated with various diseases. In spite of being exhaustively studied molecule, no attempt has been made till date to comprehensively and systematically analyze the SNPs of MBL2 gene. The present study was carried out to identify and prioritize the SNPs of MBL2 gene for further genotyping and functional studies. To predict the possible impact of SNPs on MBL structure and function SNP data obtained from dbSNP database were analyzed using various bioinformatics tools. Out of total 661 SNPs, only 37 validated SNPs having minor allele frequency ≥0.10 were considered for the present study. These 37 SNPs includes one in 3′ near gene, nine in 3′ UTR, one non-synonymous SNP (nsSNP), thirteen intronic SNPs and thirteen in 5′ near gene. From these 37 SNPs, 11 non-coding SNPs were identified to be of functional significance and evolutionary conserved. Out of these, 4 SNPs from 3′ UTR were found to play role in miRNA binding, 7 SNPs from 5′ near and intronic region were predicted to involve in transcription factor binding and expression of MBL2 gene. One nsSNP Gly54Asp (rs1800450) was found to be deleterious and damaging by both SIFT and Polyphen-2 servers and thus affecting MBL2 protein stability and expression. Protein structural analysis with this amino acid variant was performed by using I-TASSER, RAMPAGE, Swiss-PdbViewer, Chimera and I-mutant. Information regarding solvent accessibility, molecular dynamics and energy minimization calculations showed that this variant causes clashes with neighboring amino acids residues that must interfere in the normal triple helix formation of trimeric subunit and further with the normal assembly of MBL oligomeric form, hence decrease in stability. Thus, findings of the present study indicated 12 SNPs of MBL2 gene to be functionally important. Exploration of these variants may provide novel remedial markers for various diseases.

Affinity purification and characterization of lectins from two Amaranthus species

Abstract Two Amaranthus lectins, namely A. caudatus (ACL) and A. spinosus (ASL), were purified by affinity chromatography on asialofetuin-linked amino-activated silica. The lectins were eluted non-specifically by using 0.1 M glycine-HCl buffer, pH 2.5. Both the lectins showed similarities in biological and physico-chemical properties. These are dimeric proteins composed of subunits having Mr of 35 800 and 37 000 Da, respectively, which are not held together by disulphide linkages. Among the various hapten inhibitors, the lectins showed affinity for N- acetyl- d -galactosamine , fetuin and asialofetuin. The Amaranthus lectins were found to be non-specific and reacted with human and various animal erythrocytes. These are glycoproteins having no metal ion requirement for their activity.

Occurrence and characterization of lympho-agglutinins in Indian plants

A survey of the occurrence of lympho‐agglutinins was carried out on seeds of 150 species of wild and cultivated plants. Potent agglutinins were detected in 17 extracts by employing lymphocytes of man, guinea pig, goat and sheep in the bioassay. Except for one species each of Mimosoideae, Euphorbiaceae and Clusiaceae, all the agglutinins were found in fabaceous seeds. Two of the agglutinins, namely, Parkia biglandulosa and Erythrina arborescens, were found to be monospecific for guinea pig and human lymphocytes, respectively, while the others agglutinated lymphocytes of 2–4 different species. The agglutination inhibition tests revealed the predominance of galactose (or its derivatives) binding lectins over those specific for glucose‐mannose series.