Genetic studies of water buffalo blood markers. I. Red cell acid phosphatase,albumin, catalase,red cell α-esterase-3, group-specific component,and protease inhibitor

We have developed the methodologies for typing and family studies to establish the modes of inheritance of water buffalo red cell acid phosphatase (Acp), protease inhibitor (Pi), and group-specific component (Gc) on isoelectric focusing and albumin (Alb), red cell -esterase-3 (Est-3), and catalase (Cat) on polyacrylamide gel electrophoresis. Family studies showed that Pi, Gc, Alb, and Cat are coded by autosomal genes with two codominant alleles, while Est-3 is autosomal with two codominant alleles and a recessive null allele and Acp exhibits three codominant alleles.This project was funded by the Australian Centre for International Agricultural Research through Grant PN 8364 and the Malaysian programme for Intensification of Research in Priority Areas through Grant IRPA 1-07-05-057.     

Amino acid neurotransmitter transporters: Structure,function, and molecular diversity

Many biologically active compounds including neurotransmitters, metabolic precursors, and certain drugs are accumulated intracellularly by transporters that are coupled to the transmembrane Na⁺ gradient. Amino acid neurotransmitter transporters play a key role in the regulation of extracellular amino acid concentrations and termination of neurotransmission in the CNS

1 Abbreviations: CNS, central nervous system; GABA, γ-aminobutyric acid; cDNA, complementary deoxyribonucleic acid; mRNA, messenger ribonucleic acid; NMDA, N-methyl-D-aspartate; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; DAG, diacyl glycerol; R59022, DAG kinase inhibitor; AA, arachidonic acid; ACHC, cis-3-aminocyclohexanecarboxylic acid; GAT-A, ACHC-sensitive GABA transporter; GAT-B, β-alanine-sensitive GABA transporter; GLY-1 and GLYT-1, glycine transporters; PROT-1, proline transporter; BGT-1, betaine transporter.

. Transporters for the major amino acid neurotransmitters glutamate, GABA, and glycine are found in both neurons and glial cells. Recent work has resulted in the identification of cDNAs encoding several amino acid neurotransmitter transport proteins, all of which belong to the Na⁺-and Cl^?-dependent transporter gene family. The diversity of this family suggests a degree of transporter heterogeneity that is greater than that indicated by biochemical and pharmacological studies.     

Synthetic glycosylation of peptides using unprotected saccharide β-glycosylamines

Glycopeptides can be valuable tools in determining the influence of carbohydrate moieties on the intrinsic properties of glycoproteins. However, glycopeptides of sufficient quantity and purity are as yet not readily available from biological sources. The chemical coupling of a -glycosylamino group of an unprotected carbohydrate with an activated aspartic acid residue of an unprotected peptide is a simple method for synthesizing asparagine-linked glycopeptides. In this report we demonstrate that the use of this method is not restricted to -glycosylamines of simple monosaccharides or short aspartic acid-containing pentapeptides. This is illustrated by the syntheses of several glycopentapeptides containingN,N-diacetylchitobiose, a glutamine-linked glycopentapeptide containing a biantennary complex oligosaccharide, and glycosylated variants of two analogs of a polypeptide hormone, atriopeptin, containingN,N-diacetylchitobiose.Abbreviations Ac acetyl - Bzl benzyl - DMF dimethylformamide - Fmoc 9-fluorenylmethoxycarbonyl - Fuc fucose - Gal galactose - GlcNAc N-acetylglucosamine - HBTU O-benzotriazol-1-yl-N,N,N,N-tetramethyluroniumhexa-fluorophosphate - HOBt 1-hydroxybenzotriazole - Man mannose - m/z mass/charge - NMR nuclear magnetic resonance - Xyl xylose - Z benzyloxycarbonyl; unless otherwise specified, amino acids are abbreviated using their one-letter codes.     

HNF-4 increases activity of the rat Apo A1 gene.

Apolipoprotein A1 (Apo A1) is the major protein component of high density lipoprotein (HDL) particles. HDL particles mediate the removal of cholesterol from extra-hepatic tissues via a process known as reverse cholesterol transport. Augmented production of Apo A1 will likely be beneficial to those who suffer from the consequences of hypercholesterolemia. One approach to increase expression of the protein is to identify nuclear factor(s) that enhance Apo A1 promoter activity. Therefore, we have used transient transfection to study a limited portion (-474 to -7) of the gene and showed that a cis-regulatory element, site C had a permissive effect on the ability of an adjacent site B to increase promoter activity by 30-fold. The importance of element C prompted us to identify the factor(s) that interact with this site. Results showed that HNF-4, a new member of the thyroid/steroid hormone receptor superfamily interacts with site C to enhance activity of the promoter. Based on this observation and that of the known inhibitory effects of ARP-1 on site C, we postulate a model which may account for the tissue-specific expression of the rat Apo A1 gene.     

Epidermal growth factor stimulates vascular endothelial growth factor production by human malignant glioma cells: a model of glioblastoma multiforme pathophysiology.

Hypervascularity, focal necrosis, persistent cerebral edema, and rapid cellular proliferation are key histopathologic features of glioblastoma multiforme (GBM), the most common and malignant of human brain tumors. By immunoperoxidase and immunofluorescence, we definitively have demonstrated the presence of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFr) in five out of five human glioma cell lines (U-251MG, U-105MG, D-65MG, D-54MG, and CH-235MG) and in eight human GBM tumor surgical specimens. In vitro experiments with glioma cell lines revealed a consistent and reliable relation between EGFr activation and VEGF production; namely, EGF (1-20 ng/ml) stimulation of glioma cells resulted in a 25-125% increase in secretion of bioactive VEGF. Conditioned media (CM) prepared from EGF-stimulated glioma cell lines produced significant increases in cytosolic free intracellular concentrations of Ca2+ ([Ca2+]i) in human umbilical vein endothelial cells (HUVECs). Neither EGF alone or CM from glioma cultures prepared in the absence of EGF induced [Ca2+]i increases in HUVECs. Preincubation of glioma CM with A4.6.1, a monoclonal antibody to VEGF, completely abolished VEGF-mediated [Ca2+]i transients in HUVECs. Likewise, induction by glioma-derived CM of von Willebrand factor release from HUVECs was completely blocked by A4.6.1 pretreatment. These observations provide a key link in understanding the basic cellular pathophysiology of GBM tumor angiogenesis, increased vascular permeability, and cellular proliferation. Specifically, EGF activation of EGFr expressed on glioma cells leads to enhanced secretion of VEGF by glioma cells. VEGF released by glioma cells in situ most likely accounts for pathognomonic histopathologic and clinical features of GBM tumors in patients, including striking tumor angiogenesis, increased cerebral edema and hypercoagulability manifesting as focal tumor necrosis, deep vein thrombosis, or pulmonary embolism.     

Identification of Products from Oxidation of Uric Acid Induced by Hydroxyl Radicals

The aim of the present study was to separate and characterise products formed by oxidation of uric acid by hydroxyl radicals with a view to probing for these products in vivo in clinical contexts. Aerated solutions of 200 μM uric acid, or its oxidation products, allantoin or parabanic acid, were exposed to gamma radiolysis, (52.0 Gy/min), as a source of HO- radicals, at pH 3.4 and 7.4. Aliquots were taken every 5 minutes for 20 minutes and oxidation products were separated by HPLC and analysed with a diode array detector. Identities of oxidation products were confirmed on the basis of similarity of retention times and absorbance spectra and peak purity parameters of known standards. Hydroperoxides were measured by tri-iodide formation in the 20 minute sample. Exposure of uric acid to such HO fluxes produced a net loss of the parent compound with formation of a complex mixture of products with allantoin and parabanic acid being the predominant products at pH 3.4. The rate of uric acid degradation at physiological pH was slower and the distribution of oxidation products was different. A small but significant amount of uric acid hydroperoxide was detected at both pHs. A mechanism for uric acid oxidation under these conditions is presented.