Purification, structure and immunobiological activity of an arabinan-rich pectic polysaccharide from the cell walls of Prunus dulcis seeds

The structure and bioactivity of a polysaccharide extracted and purified from a 4M KOH + H3BO3 solution from Prunus dulcis seed cell wall material was studied. Anion-exchange chromatography of the crude extract yielded two sugar-rich fractions: one neutral (A), the other acidic (E). These fractions contain a very similar monosaccharide composition: 5:2:1 for arabinose, uronic acids and xylose, respectively, rhamnose and galactose being present in smaller amounts. As estimated by size-exclusion chromatography, the acidic fraction had an apparent molecular mass of 762 kDa. Methylation analysis (from the crude and fractions A and E), suggests that the polysaccharide is an arabinan-rich pectin. In all cases, the polysaccharides bear the same type of structural Ara moieties with highly branched arabinan-rich pectic polysaccharides. The average relative proportions of the arabinosyl linkages is 3:2:1:1 for T-Araf:(1-->5)-Araf:(1-->3,5)-Araf:(1-->2,3,5)-Araf. The crude polysaccharide extract and fractions A and E induced a murine lymphocyte stimulatory effect, as evaluated by the in vitro and in vivo expression of lymphocyte activation markers and spleen mononuclear cells culture proliferation. The lymphocyte stimulatory effect was stronger on B- than on T-cells. No evidence of cytotoxic effects induced by the polysaccharide fractions was found.     

Entamoeba histolytica: correlation of assessment methods to measure erythrocyte digestion, and effect of cysteine proteinases inhibitors in HM-1:IMSS and HK-9:NIH strains

Entamoeba histolytica trophozoites are able to degrade human erythrocytes; the loss of erythrocyte cellular matrix and degradation of plasma membrane were observed, along with the decrease in the average size of digestive vacuoles. Ninety-six percent of hemoglobin ingested was hydrolyzed by trophozoites within 3h, as evidenced by electrophoresis. Accordingly, X-ray spectroscopy revealed the presence of iron inside vacuoles after erythrophagocytosis, the concentration of which decreased to control levels in a similar period. Quantification of erythrocyte digestion at the early and late periods was determined by a spectrophotometric procedure, with t(1/2)=1.67 h and 35-min for HM-1:IMSS and HK-9:NIH trophozoites, respectively. In the latter, activity was due to the combined action of intracellular enzymatic activity and exocytosis. E-64c and leupeptin totally inhibited erythrocyte digestion within a 3-h period, thereafter hydrolysis took place at lower rate. Our results suggest that erythrocyte digestion in E. histolytica proceeds in different ways in these two amebic strains, and can be blocked by proteinase inhibitors.     

On the Metropolis-Hastings acceptance probability to add or drop a quantitative trait locus in Markov chain Monte Carlo-based Bayesian analyses

The Metropolis-Hastings algorithm used in analyses that estimate the number of QTL segregating in a mapping population requires the calculation of an acceptance probability to add or drop a QTL from the model. Expressions for this acceptance probability need to recognize that sets of QTL are unordered such that the number of equivalent sets increases with the factorial of the QTL number. Here, we show how accounting for this fact affects the acceptance probability and review expressions found in the literature.     

The P2Y2 nucleotide receptor mediates vascular cell adhesion molecule-1 expression through interaction with VEGF receptor-2 (KDR/Flk-1)

UTP stimulates the expression of pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells through activation of the P2Y(2) nucleotide receptor P2Y(2)R. Here, we demonstrated that activation of the P2Y(2)R induced rapid tyrosine phosphorylation of vascular endothelial growth factor receptor (VEGFR)-2 in human coronary artery endothelial cells (HCAEC). RNA interference targeting VEGFR-2 or inhibition of VEGFR-2 tyrosine kinase activity abolishes P2Y(2)R-mediated VCAM-1 expression. Furthermore, VEGFR-2 and the P2Y(2)R co-localize upon UTP stimulation. Deletion or mutation of two Src homology-3-binding sites in the C-terminal tail of the P2Y(2)R or inhibition of Src kinase activity abolished the P2Y(2)R-mediated transactivation of VEGFR-2 and subsequently inhibited UTP-induced VCAM-1 expression. Moreover, activation of VEGFR-2 by UTP leads to the phosphorylation of Vav2, a guanine nucleotide exchange factor for Rho family GTPases. Using a binding assay to measure the activity of the small GTPases Rho, we found that stimulation of HCAEC by UTP increased the activity of RhoA and Rac1 (but not Cdc42). Significantly, a dominant negative form of RhoA inhibited P2Y(2)R-mediated VCAM-1 expression, whereas expression of dominant negative forms of Cdc42 and Rac1 had no effect. These data indicate a novel mechanism whereby a nucleotide receptor transactivates a receptor tyrosine kinase to generate an inflammatory response associated with atherosclerosis.     

AtDGK2, a novel diacylglycerol kinase from Arabidopsis thaliana, phosphorylates 1-stearoyl-2-arachidonoyl-sn-glycerol and 1,2-dioleoyl-sn-glycerol and exhibits cold-inducible gene expression

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DAG) to generate phosphatidic acid (PA). Both DAG and PA are implicated in signal transduction pathways. DGKs have been widely studied in animals, but their analysis in plants is fragmentary. Here, we report the cloning and biochemical characterization of AtDGK2, encoding DGK from Arabidopsis thaliana. AtDGK2 has a predicted molecular mass of 79.4 kDa and, like AtDGK1 previously reported, harbors two copies of a phorbol ester/DAG-binding domain in its N-terminal region. AtDGK2 belongs to a family of seven DGK genes in A. thaliana. AtDGK3 to AtDGK7 encode approximately 55-kDa DGKs that lack a typical phorbol ester/DAG-binding domain. Phylogenetically, plant DGKs fall into three clusters. Members of all three clusters are widely expressed in vascular plants. Recombinant AtDGK2 was expressed in Escherichia coli and biochemically characterized. The enzyme phosphorylated 1,2-dioleoyl-sn-glycerol to yield PA, exhibiting Michaelis-Menten type kinetics. Estimated K(m) and V(max) values were 125 microm for DAG and 0.25 pmol of PA min(-1) microg(-1), respectively. The enzyme was maximally active at pH 7.2. Its activity was Mg(2+)-dependent and affected by the presence of detergents, salts, and the DGK inhibitor R59022, but not by Ca(2+). AtDGK2 exhibited substrate preference for unsaturated DAG analogues (i.e. 1-stearoyl-2-arachidonoyl-sn-glycerol and 1,2-dioleoyl-sn-glycerol). The AtDGK2 gene is expressed in various tissues of the Arabidopsis plant, including leaves, roots, and flowers, as shown by Northern blot analysis and promoter-reporter gene fusions. We found that AtDGK2 is induced by exposure to low temperature (4 degrees C), pointing to a role in cold signal transduction.