Mitogenic activity of edible mushroom lectins

A special group of lectins were isolated from three popular Asian edible mushrooms: Volvariella volvacea, Pleurotus flabellatus and Hericium erinacium, and their mitogenic activities towards mouse T cells were compared to the extensively investigated Agaricus bisporus lectin (ABL) and the Jack bean lectin, Concanavalin A (Con A). Among the four mushroom lectins tested, V. volvacea lectin (VVL) exhibited strong mitogenic activity as demonstrated by 3H-thymidine incorporation, which was at least 10-fold more effective than that of Con A, and the other mushroom lectins did not exhibit any proliferative activity. Treatment with VVL and ABL resulted in activation of the protein tyrosine kinase, p56lck, and expression of early activation markers, CD69 and CD25, but only VVL induced intracellular calcium influx while ABL triggered cell death. The calcium influx was sensitive to calcium channel antagonists such as nifedipine and verapamil. The P. flabellatus lectin (PFL) and H. erinacium lectin (HEL) did not stimulate p56lck expression and cell proliferation. Neither of these lectins interfered with Con A-mediated lymphocyte proliferation, which further indicated that both PFL and HEL were non-mitogenic. Taken all results together, VVL induced mitogenesis through T cell receptors and the subsequent calcium signaling pathway.     

NTDB: Thermodynamic Database for Nucleic Acids,Version 2.0

The second release of Thermodynamic Database for Nucleic Acids, NTDB 2.0, includes more than 4600 entries (250% increase over release 1.0). It contains sequence types and details of several thermodynamic parameters (enthalpy, DeltaH; entropy, DeltaS; Gibbs free energy, DeltaG; melting temperature, T(m)), experimental models and methods for extracting thermodynamic parameters, buffer conditions as well as all relevant literature information. In addition, the database statistics and references related to NTDB are included. Information on normal and modified nucleobases and nucleosides are collected in a new section 'Nucleoside' whereby data collected thus far will be release in NTDB 2.0. The NTDB is freely available at http://ntdb.chem.cuhk.edu.hk.     

An endoplasmic reticulum-bound Ca(2+)/Mn(2+) pump,ECA1, supports plant growth and confers tolerance to Mn(2+) stress

Plants can grow in soils containing highly variable amounts of mineral nutrients, like Ca(2+) and Mn(2+), though the mechanisms of adaptation are poorly understood. Here, we report the first genetic study to determine in vivo functions of a Ca(2+) pump in plants. Homozygous mutants of Arabidopsis harboring a T-DNA disruption in ECA1 showed a 4-fold reduction in endoplasmic reticulum-type calcium pump activity. Surprisingly, the phenotype of mutant plants was indistinguishable from wild type when grown on standard nutrient medium containing 1.5 mM Ca(2+) and 50 microM Mn(2+). However, mutants grew poorly on medium with low Ca(2+) (0.2 mM) or high Mn(2+) (0.5 mM). On high Mn(2+), the mutants failed to elongate their root hairs, suggesting impairment in tip growth processes. Expression of the wild-type gene (CAMV35S::ECA1) reversed these conditional phenotypes. The activity of ECA1 was examined by expression in a yeast (Saccharomyces cerevisiae) mutant, K616, which harbors a deletion of its endogenous calcium pumps. In vitro assays demonstrated that Ca(2+), Mn(2+), and Zn(2+) stimulated formation of a phosphoenzyme intermediate, consistent with the translocation of these ions by the pump. ECA1 provided increased tolerance of yeast mutant to toxic levels of Mn(2+) (1 mM) and Zn(2+)(3 mM), consistent with removal of these ions from the cytoplasm. These results show that despite the potential redundancy of multiple Ca(2+) pumps and Ca(2+)/H(+) antiporters in Arabidopsis, pumping of Ca(2+) and Mn(2+) by ECA1 into the endoplasmic reticulum is required to support plant growth under conditions of Ca(2+) deficiency or Mn(2+) toxicity.     

A Plasma Membrane-Type Ca2+-ATPase of 120 Kilodaltons on the Endoplasmic Reticulum from Carrot (Daucus carota) Cells (Properties of the Phosphorylated Intermediate)

Cytosolic Ca2+ levels are regulated in part by Ca2+-pumping ATPases that export Ca2+ from the cytoplasm; however, the types and properties of Ca2+ pumps in plants are not well understood. We have characterized the kinetic properties of a 120-kD phosphoenzyme (PE) intermediate formed during the reaction cycle of a Ca2+-ATPase from suspension-cultured carrot (Daucus carota) cells. Only one Ca2+-dependent phosphoprotein was formed when carrot membrane vesicles were incubated with [[gamma]-32P]ATP (W.L. Hsieh, W.S. Pierce, and H. Sze [1991] Plant Physiol 97: 1535-1544). Formation of this 120-kD phosphoprotein was inhibited by vanadate, enhanced by La3+, and decreased by hydroxylamine, confirming its identification as an intermediate of a phosphorylated-type Ca2+-translocating ATPase. The 120-kD Ca2+-ATPase was most abundant in endoplasmic reticulum-enriched fractions, in which the Ca2+-ATPase was estimated to be 0.1% of membrane protein. Direct quantitation of Ca2+-dependent phosphoprotein was used to examine the kinetics of PE formation. PE formation exhibited a Km for Ca2+ of 1 to 2 [mu]M and a Km for ATP of 67 nM. Relative affinities of substrates, determined by competition experiments, were 0.075 [mu]M for ATP, 1 [mu]M for ADP, 100 [mu]M for ITP, and 250 [mu]M for GTP. Thapsigargin and cyclopiazonic acid, specific inhibitors of animal sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, had no effect on PE formation; erythrosin B inhibited with 50% inhibition at <0.1 [mu]M. Calmodulin (1 [mu]M) stimulated PE formation by 25%. The results indicate that the carrot 120-kD Ca2+-ATPase is similar but not identical to animal plasma membrane-type Ca2+- ATPase and yet is located on endomembranes, such as the endoplasmic reticulum. This type of Ca2+ pump may reside on the cortical endoplasmic reticulum, which is thought to play a major role in anchoring the cytoskeleton and in facilitating secretion.     

Calcium-induced potassium pathway in sided erythrocyte membrane vesicles.

We have characterized the asymmetric effect of Ca2+ on passive K+ permeability in erythrocyte membranes, using inside out and right-side out vesicles. Ca2+, but not Mg2+, can induce an increase in K+ uptake in inside out vesicles. The half-maximal concentration of Ca2+ required to induce the K+ uptake is 0.2 mM, and the permeability increase is not specific for K+. Thus, the Ca2+- induced permeation process in inside out vesicles is changed from that in the energy-depleted intact cell which requires only micromolar concentrations of Ca2+ and is specific for K+. Removal of spectrin had no effect on the vesicle permeability increase due to Ca2+. Studies with N-ethylmaleimide show that the vesicle channel openings is mediated by a protein and passage is controlled by sulfhydryl groups; furthermore, the Ca2+-induced vesicle pathway is distinct from the normal channel for passive K+ leak in the absence of Ca2+. The protein is sensitive to its phospholipid environment since removal of easily accessible phospholipid head groups on the cytoplasmic face of the vesicles inhibits the Ca2+ -stimulated channel opening.     

Characterization of lipid composition in stimulated human lymphocytes by 1H-NMR

Recent in vivo NMR studies have raised interest in the structural changes of cellular lipids during proliferative activity. We investigated the changes in plasma membrane lipid and total cell lipid during mitogenically-stimulated proliferation of human peripheral blood lymphocytes by extraction of lipids and assay by 500 MHz 1H-NMR. Resonances were assigned using one- and two-dimensional spectroscopic techniques, and signals unique to certain species of lipid were identified. Choline and ethanolamine-containing lipids, glycerophospholipid backbones, sphingolipids, cholesterol, plasmalogens and triacylglycerols were readily detected. Resolution of a number of lipid species was not possible, despite the use of high-resolution techniques. NMR values for proliferation-induced changes in the most easily determined parameters, namely the total cholesterol to total phospholipid molar ratio, and phosphatidylcholine, phosphatidylethanolamine and sphingolipid composition, were found to agree with traditional methods. Differences in phospholipid and fatty acid profiles were found between plasma membranes and total cell lipid for resting values and for response to mitogen.