Protein variation in wild Atlantic salmon, with particular reference to southern Ireland

The extent of genetic variation in wild Atlantic salmon parr, Sulmo salur L., from river systems in Ireland, Iceland and eastern Canada, was investigated using starch gel electrophoresis. Within Ireland, seven polymorphic enzyme loci ( sAAT-4 *, GPI-1 *, IDDH-1 *, IDDH-2 *, IDHP-3 *, MDH-3 * and mMEP-2 *) were screened in nine different rivers and nine tributaries from the River Blackwater. Significant heterogeneity in gene frequencies occurred between riverine samples and between samples from tributaries of the River Blackwater. Variation between tributaries was as great as between rivers elsewhere in the country. Levels of population differentiation were comparable to those found in other regions throughout the range of the species, and temporal stability in gene frequencies was apparent when the results were compared with previously published data. Screening of riverine samples from Iceland and eastern Canada (Newfoundland and New Brunswick) allowed the Irish results to be considered in a broader context. Irish salmon cluster in the western European group, to which may be added Icelandic populations. Salmon from eastern Canada show a high level of genetic distinctiveness from the European group.     

Interaction of nitrogen dioxide with human plasma. Antioxidant depletion and oxidative damage.

Nitrogen dioxide (NO2.) is often present in inhaled air and may be generated in vivo from nitric oxide. Exposure of human blood plasma to NO2. caused rapid losses of ascorbic acid, uric acid and protein thiol groups, as well as lipid peroxidation and depletions of alpha-tocopherol, bilirubin and ubiquinol-10. No increase in protein carbonyls was detected. Supplementation of plasma with ascorbate decreased the rates of lipid peroxidation, alpha-tocopherol depletion and loss of uric acid. Uric acid supplementation decreased rates of lipid peroxidation but not the loss of alpha-tocopherol. We conclude that ascorbic acid, protein -SH groups, uric acid and alpha-tocopherol may be important agents protecting against NO2. in vivo. If these antioxidants are depleted, peroxidation of lipids occurs and might contribute to the toxicity of NO2..     

Galactose-containing glycosylphosphatidylinositols in Trypanosoma brucei.

Many eukaryotic surface glycoproteins, including the variant surface glycoproteins (VSGs) of Trypanosoma brucei, are synthesized with a carboxyl-terminal hydrophobic peptide extension that is cleaved and replaced by a complex glycosylphosphatidylinositol (GPI) membrane anchor within 1-5 min of the completion of polypeptide synthesis. We have reported the purification and partial characterization of candidate precursor glycolipids (P2 and P3) from T. brucei. P2 and P3 contain ethanolamine-phosphate-Man alpha 1-2Man alpha 1-6Man alpha 1-GlcN linked glycosidically to an inositol residue, as do all the GPI anchors that have been structurally characterized. The anchors on mature VSGs contain a heterogenously branched galactose structure attached alpha 1-3 to the mannose residue adjacent to the glucosamine. We report the identification of free GPIs that appear to be similarly galactosylated. These glycolipids contain diacylglycerol and alpha-galactosidase-sensitive glycan structures which are indistinguishable from the glycans derived from galactosylated VSG GPI anchors. We discuss the relevance of these galactosylated GPIs to the biosynthesis of VSG GPI anchors.     

Molecular dynamics computations and solid state nuclear magnetic resonance of the gramicidin cation channel.

This paper reports on a coupled approach to determining the structure of the gramicidin A ion channel, utilizing solid state nuclear magnetic resonance (NMR) of isotopically labeled gramicidin channels aligned parallel to the magnetic field direction, and molecular dynamics (MD). MD computations using an idealized right-handed beta-helix as a starting point produce a refined molecular structure that is in excellent agreement with atomic resolution solid state NMR data. The data provided by NMR and MD are complementary to each other. When applied in a coordinated manner they provide a powerful approach to structure determination in molecular systems not readily amenable to x-ray diffraction.     

Sequences of junction fragments in the deletion-prone region of the dystrophin gene

The Duchenne muscular dystrophy locus is remarkable in that it shows a high mutation rate and the majority of mutations found are deletions. These deletions are generated as meiotic as well as mitotic events and occur preferentially in the central region of the gene. Nothing is known so far about the mechanisms involved. This paper reports the first sequencing of deletion junctions in the dystrophin gene. The data from a study of two patients with deletions in the central region of dystrophin show the breakpoints to lie in regions of introns in which stretches of dA-dT are seen. The relationship between these observations and possible mechanisms for the mutations is discussed.     

The pattern of X-chromosome inactivation in the embryonic and extra-embryonic tissues of post-implantation digynic triploid LT/Sv strain mouse embryos

Spontaneously cycling LT/Sv strain female mice were mated to hemizygous Rb(X.2)2Ad males in order to facilitate the distinction of the paternal X chromosome, and the pregnant females were autopsied at about midday on the tenth day of gestation. Out of a total of 222 analysable embryos recovered, 165 (74.3%) were diploid and 57 (25.7%) were triploid. Of the triploids, 26 had an XXY and 31 an XXX sex chromosome constitution. Both embryonic and extra-embryonic tissue samples from the triploids were analysed cytogenetically by G-banding and by the Kanda technique to investigate their X-inactivation pattern. The yolk sac samples were separated enzymatically into their endodermally-derived and mesodermally-derived components, and these were similarly analysed, as were similar samples from a selection of control XmXp diploid embryos. In the case of the XmXmY digynic triploid embryos, a single darkly-staining Xm chromosome was observed in 485 (82.9%) out of 585, 304 (73.3%) out of 415, and 165 (44.7%) out of 369 metaphases from the embryonic, yolk sac mesodermally-derived and yolk sac endodermally-derived tissues, respectively. The absence of a darkly staining X-chromosome in the other metaphase spreads could either indicate that both X-chromosomes present were active, or that the Kanda technique had failed to differentially stain the inactive X-chromosome(s) present. In the case of the XmXmXp digynic triploid embryos, virtually all of the tissues analysed comprised two distinct cell lineages, namely those with two darkly-staining X-chromosomes, and those with a single darkly staining X-chromosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimizing and characterizing alignment of oriented lipid bilayers containing gramicidin D.

31P NMR spectroscopy and optical microscopy have been used to characterize samples of gramicidin D in oriented lipid bilayers. Correlations have been made between the defect structures observed under crossed polarizers by optical microscopy and characteristic features of 31P NMR spectra. The sample preparation protocol has been improved using these techniques to achieve minimal dispersion of the bilayer normal and minimal amounts of unoriented sample. The molar ratio of gramicidin to dimyristoyl-phosphatidylcholine, the extent of hydration, and the cosolubilizing solvent system were used as the protocol variables. While hydration level and solvent system had profound effects on the sample orientation the molar ratio did not. However, the 31P chemical shift anisotropy is very sensitive to the molar ratio and can be used as an in situ method for determining the molar ratio.     

Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. II. Lipid structures of phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids

A common diagnostic feature of glycosylinositol phospholipid (GPI)-anchored proteins is their release from the membrane by a phosphatidylinositol-specific phospholipase C (PI-PLC). However, some GPI-anchored proteins are resistant to this enzyme. The best characterized example of this subclass is the human erythrocyte acetylcholinesterase, where the structural basis of PI-PLC resistance has been shown to be the acylation of an inositol hydroxyl group(s) (Roberts, W. L., Myher, J. J., Kuksis, A., Low, M. G., and Rosenberry, T. L. (1988) J. Biol. Chem. 263, 18766-18775). Both PI-PLC-sensitive and resistant GPI-anchor precursors (P2 and P3, respectively) have been found in Trypanosoma brucei, where the major surface glycoprotein is anchored by a PI-PLC-sensitive glycolipid anchor. The accompanying paper (Mayor, S., Menon, A. K., Cross, G. A. M., Ferguson, M. A. J., Dwek, R. A., and Rademacher, T. W. (1990) J. Biol. Chem. 265, 6164-6173) shows that P2 and P3 have identical glycans, indistinguishable from the common core glycan found on all the characterized GPI protein anchors. This paper shows that the single difference between P2 and P3, and the basis for the PI-PLC insusceptibility of P3, is a fatty acid, ester-linked to the inositol residue in P3. The inositol-linked fatty acid can be removed by treatment with mild base to restore PI-PLC sensitivity. Biosynthetic labeling experiments with [3H]palmitic acid and [3H]myristic acid show that [3H]palmitic acid specifically labels the inositol residue in P3 while [3H]myristic acid labels the diacylglycerol portion. Possible models to account for the simultaneous presence of PI-PLC-resistant and sensitive glycolipids are discussed in the context of available information on the biosynthesis of GPI-anchors.     

Partial aldolase B gene deletions in hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is an autosomal recessive condition caused by a deficiency of aldolase B. We have recently shown that three point mutations in this gene account for approximately 85% of HFI alleles in Europe and the United States and are thus of diagnostic importance. In this paper we define three new lesions in the aldolase B gene: two are large deletions, one of 1.65 kb and one of 1.4 kb; the third is a small deletion of 4 bp. We have determined the breakpoints of these deletions and have demonstrated that the presence of such lesions may complicate the genotyping of individuals for diagnosis of HFI.     

The association of FAD with the cytochrome b−245 of human neutrophils

A plasma membrane fraction prepared from human neutrophils had a fluorescence resembling that of a fluorescent flavoprotein, with emission maximum near 520nm and excitation maxima near 380 and 460nm. The fluorescence emission and excitation properties of Triton N-101-solubilized membrane fraction resembled those of FAD. FAD was present in the membranes at a concentration of 417pmol/mg of protein and cytochrome b₋₂₄₅ at a concentration of 407pmol/mg of protein. In a 110-fold purified preparation of cytochrome b₋₂₄₅ the ratio of FAD:cytochrome b was 1:1. Analytical gradient centrifugation of neutrophil homogenates shows a coincidence of two cytochrome b peaks and two peaks of fluorescence, corresponding with plasma membrane and specific granule fractions; most of the FAD was non-fluorescent and located in fractions lighter than the plasma membrane. Plasma membrane fractions prepared from neutrophils of patients suffering from the X-linked form of chronic granulomatous disease lacked cytochrome b and contained 194pmol of FAD/mg of protein; plasma membrane fractions prepared from neutrophils of patients with the autosomal recessive form of chronic granulomatous disease contained both cytochrome b₋₂₄₅ and FAD in the normal range of concentrations in a ratio of 1:1. Phagocytic vesicles were prepared from normal neutrophils and found to contain FAD and cytochrome b in a ratio 2.22:1, suggesting that activation of neutrophils many involve the incorporation of an additional flavin into the membrane. Under anaerobic conditions in the presence of EDTA to act as an electron donor to a flavin, the cytochrome b₋₂₄₅ of neutrophil membranes was partly (12%) photoreducible, an effect increased to 100% by the addition of FMN. The extent of reduction of cytochrome b in an anaerobic neutrophil homogenate containing NADH increased from 30% to 70% on illumination. We suggest that these results indicate a close association between FAD and cytochrome b₋₂₄₅ and support a scheme for electron transport thus: [Formula: see text]