Growth of Certain Aquatic Oomycetes on Amino Acids II. Apodachlya, Aphanomyces, and Pythium

Four aquatic fungi —Apodachlya brachynema and A. minima (Leptomitales), Aphanomyces laevis (Saprolegniales), and Pythium ultimum (Peronosporales) —were tested for growth in synthetic media containing one of a variety of carbon sources. Apodachlya brachynema readily utilized five amino acids — alanine, glutamate, aspartate, proline and leucine — as well as glucose and acetate. Growth on sucrose as a carbon source was slight. Apodachlya minima differed from A. brachynema in that it could not utilize proline and leucine. Aphanomyces laevis grew well on only three of the substrates tested — glucose, alanine and glutamate. Pythium ultimum utilized glucose, sucrose, maltose, cellobiose, alanine, glutamate, aspartate, proline, asparagine, ornithine, and serine, but not eight other amino acids. All of these fungi hydrolyzed gelatin. Radioactively labeled carbon dioxide was released during incubation of Aphanomyces laevis in media containing labeled leucine, proline, or phenylalanine. These data provide evidence of some catabolism of the three substrates although none of these substrates can support the growth of Aphanomyces laevis as a sole source of carbon and nitrogen.     

Purification and characterization of chymodenin. A hormone-like peptide from porcine duodenum

Chymodenin, a hormone-like duodenal peptide which rapidly alters the proportions of secreted pancreatic digestive enzymes to a mixture relatively richer in chymotrypsinogen than that found in basal secretion, has been purified to homogeneity. The starting material was an acidic methanol-soluble, neutral pH-insoluble fraction of an acetic acid extract of porcine duodenum; the purification consisted of cation-exchange chromatography on SP-Sephadex and CM-Sephadex in ammonium bicarbonate gradients, and gel filtration on Sephadex G-75 in dilute acetic acid. The yield of material was followed by radioimmunoassay. Homogeneity was determined from chymodenin's behavior in disc gel electrophoresis in an acidic counter-migration-of-dye system, sodium dodecyl sulfate-urea gels, gel filtration, dansyl-Edman reaction, reversed-phase high pressure liquid chromatography, isotachophoresis, and sedimentation equilibrium ultracentrifugation. The electrophoretic mobility, the molecular weight of 9,000-10,000, and the biological activity differed from those of other gastrointestinal peptide hormones. The amino acid composition was unique. Chymodenin is the first purified hormone-like substance reported capable of altering the composition of the mixture of secreted digestive enzymes, independent of the stimulation of massive pancreatic protein output.     

Effect of apolipoprotein A-V on plasma triglyceride, lipoprotein size, and composition in genetically engineered mice

Transgenic (Tg) mice that overexpress the human apolipoprotein A-V gene (APOA5) yet lack an endogenous mouse apoa5 gene (APOA5 Tg mice) were generated. Subsequently, the effect of human apoA-V expression on plasma triglyceride (TG) concentration and lipoprotein and apolipoprotein distribution was determined and compared with that in mice deficient in apoA-V (apoa5(-/-) mice). NMR analysis of plasma lipoproteins revealed that APOA5 Tg mice had a very low VLDL concentration (26.4 +/- 7.7 nmol/dl), whereas VLDL in apoa5(-/-) mice was 18- fold higher (467 +/- 152 nmol/dl). SDS-PAGE analysis of the d < 1.063 g/ml plasma fraction revealed that the apoB-100/apoB-48 ratio was 14-fold higher in APOA5 Tg versus apoa5(-/-) mice and that the apoE/total apoB ratio was 7-fold greater in APOA5 Tg versus apoa5(-/-) mice. It is anticipated that a reduction in apoB-100/apoB-48 ratio as well as that for apoE/apoB would impair the uptake of VLDL and remnants in apoa5(-/-) mice, thereby contributing to increased plasma TG levels. The concentration of apoA-V in APOA5 Tg mice was 12.5 +/- 2.9 microg/ml, which is approximately 50- to 100-fold higher than that reported for normolipidemic humans. ApoA-V was predominantly associated with HDL but was rapidly and efficiently redistributed to apoA- V-deficient VLDL upon incubation. Consistent with findings reported for human subjects, apoA-V concentration was positively correlated with TG levels in normolipidemic APOA5 Tg mice. It is conceivable that, in a situation in which apoA-V is chronically overexpressed, complex interactions among factors regulating TG homeostasis may result in a positive correlation of apoA-V with TG concentrations.     

Apolipoprotein A-V associates with intrahepatic lipid droplets and influences triglyceride accumulation

Apolipoprotein A-V (apoA-V), secreted solely by the liver, is a low abundance protein that strongly influences plasma triglyceride (TG) levels. In vitro, in transfected hepatoma cell lines apoA-V is largely retained within the cell in association with cytosolic lipid droplets (LD). To evaluate if this is true in vivo, in the present study the amount of apoA-V in the plasma compartment versus liver tissue was determined in APOA5 transgenic (Tg) mice. The majority of total apoA-V (∼ 80%) was in the plasma compartment. Injection of APOA5 Tg mice with heparin increased plasma apoA-V protein levels by ∼ 25% indicating the existence of a heparin-releasable pool. Intrahepatic apoA-V was associated with LD isolated from livers of wild type (WT) and APOA5 Tg mice. Furthermore, livers from APOA5 Tg mice contained significantly higher amounts of TG than livers from WT or apoa5 knockout mice suggesting that apoA-V influences intrahepatic TG levels.     

Cas-Br-E murine leukemia virus: sequencing of the paralytogenic region of its genome and derivation of specific probes to study its origin and the structure of its recombinant genomes in leukemic tissues.

The ecotropic Cas-Br-E murine leukemia virus (MuLV) and its molecularly cloned derivative pBR-NE-8 MuLV are capable of inducing hind-limb paralysis and leukemia after inoculation into susceptible mice. T1 oligonucleotide fingerprinting, molecular hybridization, and restriction enzyme analysis previously showed that the env gene of Cas-Br-E MuLV diverged the most from that of other ecotropic MuLVs. To analyze proviruses in leukemic tissues, we derived DNA probes specific to Cas-Br-E sequences: two from the env region and one from the U3 long terminal repeat. With these probes, we found that this virus induced clonal (or oligoclonal) tumors and we documented the presence of typical mink cell focus-forming-type proviruses in leukemic tissues and the possible presence of other recombinant MuLV proviruses. Since the region harboring the determinant of paralysis was mapped within the pol-env region of the virus (L. DesGroseillers, M. Barrette, and P. Jolicoeur, J. Virol. 52:356-363, 1984), we performed the complete nucleotide sequence of this region covering the 3' end of the genome. We compared the deduced amino acid sequences of the pol carboxy-terminal domain and of the env gene products with those of other nonparalytogenic, ecotropic, and mink cell focus-forming MuLVs. This amino acid comparison revealed that this part of the pol gene product and the p15E diverged very little from homologous proteins of other MuLVs. However, the Cas-Br-E gp70 sequence was found to be quite divergent from that of other MuLVs, and the amino acid changes were distributed all along the protein. Therefore, gp70 remains the best candidate for harboring the determinant of paralysis.     

The amino acid sequence of chymodenin, a hormone-like peptide from porcine duodenum, is identical to cytochrome C-oxidase, peptide VII

The amino acid sequence of chymodenin, a hormone-like peptide from porcine duodenum is reported. The molecule is known to rapidly alter the proportions of digestive enzymes secreted by the rabbit pancreas in vivo and in vitro, by selection of the specific intra-pancreatic source from which the preset mixture of digestive enzymes is secreted. The sequence is identical to that of cytochrome C-oxidase peptide VII (cCoVII) from bovine heart, with the exception of a substitution of threonine for alanine at position 6 and a second substitution of alanine for threonine at position 71. Disulfide bridges link positions 29-64 and 39-53. cCoVII-chymodenin has a pentapeptide (-Ala-Glu-Gly-Thr-Phe-) near the carboxy-terminus which is immediately preceded by an -Arg-Arg- sequence in the porcine and bovine sequences of cCoVII. This peptide is identical to a pentapeptide found close to the amino terminus of the hormones gastric inhibitory peptide (GIP) and glucagon-like peptide I. The identity to cCoVII means chymodenin as isolated is itself unlikely to be a gastrointestinal hormone. However, the partial commonality of sequence with the glucagon-secretin family immediately adjacent to a pro-hormone-like activation site, and the specific actions on the exocrine pancreas, means that the molecule probably mimics the natural actions of an as-yet uncharacterized member of the glucagon family, which exerts a unique action on exocrine pancreatic secretion.