Purification of 3 monomeric monocot mannose-binding lectins and their evaluation for antipoxviral activity: potential applications in multiple viral diseases caused by enveloped viruses.

Three monomeric monocot lectins from Zephyranthes carinata, Zephyranthes candida, and Gloriosa superba with carbohydrate specificity towards mannose derivatives and (or) oligomannose have been isolated and purified from their storage tissues. The lectins were purified by anion-exchange chromatography on DEAE-Sephacyl and by gel filtration chromatography on Biogel P-200 followed by high-performance liquid chromatography. The purified lectins, Z. carinata, Z. candida, and G. superba had molecular masses of 12, 11.5, and 12.5 kDa, respectively, as determined by gel filtration and SDS-PAGE, indicating that they are monomers. In a hapten inhibition assay, methyl-alpha-D-mannopyranoside inhibited agglutination of both Z. candida and Z. carinata; the latter was also inhibited by Man(alpha1-2)Man and Man(alpha1-3)Man. Gloriosa superba showed inhibition only with Man(alpha1-4)Man of all of the sugars and glycoproteins tested. All purified lectins agglutinated red blood cells from rabbit, whereas G. superba was also reactive towards erythrocytes from guinea pig. All of the lectins were nonglycosylated and did not require metal ions for their activity. They were labile above 60 degrees C and were affected by denaturing agents such as urea, thiourea, and guanidine-HCl. The lectins were virtually nonmitogenic, like other members of Amaryllidaceae and Liliaceae. Of the 3 lectins, G. superba was found to be highly toxic to the BSC-1 cell line (African green monkey kidney epithelial cells), while both of the Zephyranthes species showed significant in vitro inhibition of poxvirus replication in BSC-1 cells without any toxic effects to the cells. In addition, Z. candida also exhibited significant anticancer activity against SNB-78, a CNS human cancer 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.     

Insights into the Molecular Mechanism of Arsenic Phytoremediation

Journal of Plant Growth Regulation - Arsenic (As) is a widespread carcinogenic pollutant. Phytoremediation is the most suited technology for alleviating the As contamination of soil. In this...     

Novel cis-acting regulatory elements in wild Oryza species impart improved rice bran quality by lowering the expression of phospholipase D alpha1 enzyme (OsPLDα1)

Molecular Biology Reports - Rice bran oil is good quality edible oil, rich in antioxidants and comprised typically of oleic-linoleic type fatty acids. However, presence of a highly lipolytic enzyme...     

BST2 regulates interferon gamma-dependent decrease in invasion of HTR-8/SVneo cells via STAT1 and AKT signaling pathways and expression of E-cadherin

ABSTRACT

The mechanism by which interferon-gamma (IFN-γ) downregulates trophoblast invasion needs further investigation. Treatment of HTR-8/SVneo cells with IFN-γ led to a decrease in their invasion concomitant with an increased expression of BST2. Silencing of BST2 by siRNA showed a significant increase in their invasion and spreading after treatment with IFN-γ as well as downregulated expression of E-cadherin. Further, STAT1 silencing inhibited the IFN-γ-dependent increase in the expression of BST2 and E-cadherin. Treatment of HTR-8/SVneo cells with IFN-γ led to the activation of AKT, and its inhibition with PI3K inhibitor abrogated IFN-γ-mediated decrease in invasion/spreading and downregulated BST2 and E-cadherin expression. Collectively, IFN-γ decreases the invasion of HTR-8/SVneo cells by STAT1 and AKT activation via increased expression of BST2 and E-cadherin.     

Discovery of Mycobacterium tuberculosis α-1,4-Glucan Branching Enzyme (GlgB) Inhibitors by Structure- and Ligand-based Virtual Screening

GlgB (α-1,4-glucan branching enzyme) is the key enzyme involved in the biosynthesis of α-glucan, which plays a significant role in the virulence and pathogenesis of Mycobacterium tuberculosis. Because α-glucans are implicated in the survival of both replicating and non-replicating bacteria, there exists an exigent need for the identification and development of novel inhibitors for targeting enzymes, such as GlgB, involved in this pathway. We have used the existing structural information of M. tuberculosis GlgB for high throughput virtual screening and molecular docking. A diverse database of 330,000 molecules was used for identifying novel and efficacious therapeutic agents for targeting GlgB. We also used three-dimensional shape as well as two-dimensional similarity matrix methods to identify diverse molecular scaffolds that inhibit M. tuberculosis GlgB activity. Virtual hits were generated after structure and ligand-based screening followed by filters based on interaction with human GlgB and in silico pharmacokinetic parameters. These hits were experimentally evaluated and resulted in the discovery of a number of structurally diverse chemical scaffolds that target M. tuberculosis GlgB. Although a number of inhibitors demonstrated in vitro enzyme inhibition, two compounds in particular showed excellent inhibition of in vivo M. tuberculosis survival and its ability to get phagocytosed. This work shows that in silico docking and three-dimensional chemical similarity could be an important therapeutic approach for developing inhibitors to specifically target the M. tuberculosis GlgB enzyme.     

Computer aided identification of sodium channel blockers in the clinical treatment of epilepsy using molecular docking tools

AbbreviationsAEDs - Antiepileptic drugs, BLAST - Basic Local Alignment Search Tool, CBZ - Carbamazepine, GEFS+ - Generalized Epilepsy with Febrile Seizures Plus, GPCR - G Protein Coupled Receptor, Nav - Sodium channel with specific voltage conduction, PDB - Protein Data Bank, PHT - Phenytoin, PIR - Protein Information resources, SAVES - Structural Analysis and Verification Server, VGSC - Voltage-gated Sodium channels.     

Natural IgM anti-leukocyte autoantibodies attenuate excess inflammation mediated by innate and adaptive immune mechanisms involving Th-17

Little is known about the function of natural IgM autoantibodies, especially that of IgM anti-leukocyte autoantibodies (IgM-ALA). Natural IgM-ALA are present at birth and characteristically increase during inflammatory and infective conditions. Our prior clinical observations and those of other investigators showing fewer rejections in renal and cardiac allografts transplanted into recipients with high levels of IgM-ALA led us to investigate whether IgM-ALA regulate the inflammatory response. In this article, we show that IgM, in physiologic doses, inhibit proinflammatory cells from proliferating and producing IFN-γ and IL-17 in response to alloantigens (MLR), anti-CD3, and the glycolipid α-galactosyl ceramide. We showed in an IgM knockout murine model, with intact B cells and regulatory T cells, that there was more severe inflammation and loss of function in the absence of IgM after renal ischemia reperfusion injury and cardiac allograft rejection. Replenishing IgM in IgM knockout mice or increasing the levels of IgM-ALA in wild-type B6 mice significantly attenuated the inflammation in both of these inflammatory models that involve IFN-γ and IL-17. The protective effect on renal ischemia reperfusion injury was not observed using IgM preadsorbed with leukocytes to remove IgM-ALA. We provide data to show that the anti-inflammatory effect of IgM is mediated, in part, by inhibiting TLR-4-induced NF-κB translocation into the nucleus and inhibiting differentiation of activated T cells into Th-1 and Th-17 cells. These observations highlight the importance of IgM-ALA in regulating excess inflammation mediated by both innate and adaptive immune mechanisms and where the inflammatory response involves Th-17 cells that are not effectively regulated by regulatory T cells.     

In Vitro Regeneration of a High Oil-yielding Variety of Safflower (Carthamus tinctorius var HUS-305)

A protocol for regular in vitro regeneration of Carthamus tinctorius var HUS-305 is described. The morphogenic response of seed explants and explants from seedlings of different ages were studied on Murashige and Skoog’s medium (MS) supplemented with different growth regulators. The protocol finally standardized involves culture of cotyledonary explants from 2 cm long, 2- to 6-day-old seedlings on MS supplemented with 1.0 mg l^?1 BAP and 0.1 mg l^?1 kinetin. Various other adjuvants viz., adenine sulphate, glutamine and casein hydrolysate were also tested. None of these promoted the caulogenic response significantly. The microshoots could be rooted on medium supplemented with different auxins. The highest rhizogenic response was on 1/2 MS supplemented with 0.2 mg l^?1 NAA.