Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.     

Effect of temperature on the saccharide composition obtained after alpha-amylolysis of starch

The hydrolysis of starch to low-molecular-weight products (normally characterised by their dextrose equivalent (DE), which is directly related to the number-average molecular mass) was studied at different temperatures. Amylopectin potato starch, lacking amylose, was selected because of its low tendency towards retrogradation at lower temperatures. Bacillus licheniformis alpha-amylase was added to 10% [w/w] gelatinised starch solutions. The hydrolysis experiments were done at 50, 70, and 90 degrees C. Samples were taken at defined DE values and these were analysed with respect to their saccharide composition. At the same DE the oligosaccharide composition depended on the hydrolysis temperature. This implies that at the same net number of bonds hydrolysed by the enzyme, the saccharide composition was different. The hydrolysis temperature also influenced the initial overall molecular-weight distribution. Higher temperatures led to a more homogenous molecular weight distribution. Similar effects were observed for alpha-amylases from other microbial sources such as Bacillus amyloliquefaciens and Bacillus stearothermophilus. Varying the pH (5.1, 6.2, and 7.6) at 70 degrees C did not significantly influence the saccharide composition obtained during B. licheniformis alpha-amylase hydrolysis. The underlying mechanisms for B. licheniformis alpha-amylase were studied using pure linear oligosaccharides, ranging from maltotriose to maltoheptaose as substrates. Activation energies for the hydrolysis of individual oligosaccharides were calculated from Arrhenius plots at 60, 70, 80, and 90 degrees C. Oligosaccharides with a degree of polymerisation exceeding that of the substrate could be detected. The contribution of these oligosaccharides increased as the degree of polymerisation of the substrate decreased and the temperature of hydrolysis increased. The product specificity decreased with increasing temperature of hydrolysis, which led to a more equal distribution between the possible products formed. Calculations with the subsite map as determined for the closely related alpha-amylase from B. amyloliquefaciens reconfirmed this finding of a decreased substrate specificity with increased temperature of hydrolysis. Copyright 1999 John Wiley & Sons, Inc.     

Independent segregation of human immunodeficiency virus type 1 Gag protein complexes and lipid rafts

Formation of human immunodeficiency virus type 1 (HIV-1) particles takes place at the plasma membrane of cells and is directed by the Pr55Gag polyprotein. A functional assembly domain (the M domain) within the N-terminal portion of Pr55Gag mediates the interaction of Gag with cellular membranes. However, the determinants that provide specificity for assembly on the plasma membrane, as opposed to intracellular membranes, have not been identified. Recently, it was reported that Pr55Gag interacts with lipid raft microdomains of the plasma membrane. We sought to identify the domains within Pr55Gag that contribute to lipid raft association of Gag. Here we demonstrate that the I domain is required for interaction with detergent-resistant membrane fractions (DRMs). Mutation of key I-domain residues or loss of myristylation abrogated the association of Gag with DRMs. Thus, the I domain and the M domain combine to mediate Gag-lipid raft interactions as defined by these biochemical criteria. However, Gag protein complexes defined by flotation studies were much denser than classical lipid rafts, failed to incorporate classical lipid raft marker proteins, and were not disrupted by cholesterol extraction. Large sheets of Gag protein were identified in DRM fractions upon examination by electron microscopy. These results indicate that HIV-1 Pr55Gag forms detergent-resistant complexes at the cellular periphery that are distinct from lipid raft microdomains.     

Modulation of the zinc(II) center in protein farnesyltransferase by mutagenesis of the zinc(II) ligands

Protein farnesyltransferase (PFTase) is a zinc-containing metalloenzyme that catalyzes the alkylation of cysteine (C) in protein substrates containing a C-terminal "CaaX" motif by farnesyl diphosphate (FPP). In yeast PFTase Zn(II) is coordinated to D307, C309, and H363 in the beta-subunit. The inner coordination sphere of the metal also contains a water molecule to give a net charge of 0 for the tetracoordinate Zn(II) center. When the protein substrate binds, the water molecule is replaced by the thiol of the cysteine residue, and the thiol is deprotonated to generate a Zn(II)-stabilized thiolate in the PFTase.FPP.protein ternary complex for the ensuing prenyl transfer reaction. An expression system was constructed for yeast PFTase containing a His(6) tag at the C-terminus of the beta-subunit to facilitate purification of the wild-type enzyme and site-directed mutants. The amino acids that coordinate Zn(II) were substituted to give a series of mutant PFTases with net charges of +1, 0, -1, and -2 at the Zn(II) center of the ternary enzyme.substrate complexes. Wild-type PFTase and the site-directed mutants were purified as alpha,beta-heterodimers, and each was found to contain an equivalent of Zn(II). All of the mutants were less reactive than wt PFTase (net charge of -1), with the greatest losses of activity seen for the mutants with net charges of 0 and +1. Equilibrium binding experiments with dGCVIA peptide and an unreactive analogue of FPP, (E,E)-2-[2-oxo-2-[[(3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy]amino]ethyl]phosphonate (FNP), established that all of the mutants bound an equivalent of the peptide substrate. Like wt PFTase, the pH dependence of K(D) for the mutants did not change significantly between pH 5 and pH 9, indicating that pK(A)s for the thiol moiety in the (mutant PFTase).FNP.peptide complexes were <5. dGSVIA and dG(beta-NH2-Ala)VIA, where the sulfhydryl moiety was replaced by hydroxyl and amino groups, respectively, were not substrates. These experiments suggest a direct relationship between the net charge of the Zn(II) center in PFTase and the reactivity of the peptide thiolate that is alkylated by FPP.     

POCUS: mining genomic sequence annotation to predict disease genes

Here we present POCUS (prioritization of candidate genes using statistics), a novel computational approach to prioritize candidate disease genes that is based on over-representation of functional annotation between loci for the same disease. We show that POCUS can provide high (up to 81-fold) enrichment of real disease genes in the candidate-gene shortlists it produces compared with the original large sets of positional candidates. In contrast to existing methods, POCUS can also suggest counterintuitive candidates.