Amino acid sequences of alpha-helical segments from S-carboxymethylkerateine-A. Tryptic and chymotryptic peptides from a type-II segment.

1. Amino acid-sequence studies were done on a peptide of mol.wt. approx. 12500 that was isolated from the highly helical fragments obtained by partial chymotryptic digestion of the low-sulphur proteins (S-carboxymethylkerateine-A) from wool. 2. The peptides obtained by tryptic and chymotryptic digestion of this large peptide were separated by ion-exchange chromatography on DEAE-cellulose at pH8.5 with an (NH4)(2)CO(3) concentration gradient and, where necessary, purified further by paper electrophoresis. 3. Determination of the sequences of many of these peptides showed that a high proportion of the cationic residues occurs in pairs. 4. Although two of the four S-carboxymethylcysteine residues are located in what appears to be a non-helical region near the N-terminus the other two S-carboxymethylcysteine residues occur in or near sequences suggesting a helical conformation. 5. Some peptides were obtained, in low yields, that appeared to be homologues of more major ones. These suggest either homologies in the helical portions of the low-sulphur proteins or the presence of closely related amino acid sequences in helical regions of completely different origins. 6. A partial sequence of the complete peptide is proposed.     

Cyanide formation from glyoxylate and hydroxylamine catalysed by extracts of higher-plant leaves

Extracts of spinach, maize and barley contain an enzyme which catalyses the formation of hydrogen cyanide from glyoxylate and hydroxylamine. The enzyme is dependent upon ADP and a divalent cation such as manganese. Glyoxylicacid oxime is a poor substrate for the enzyme. Carbon dioxide is another product of the reaction and is probably produced in 1:1 stoichiometry with hydrogen cyanide. The possible relationship of this enzyme to the regulation of nitrate reduction is discussed.     

Phospholipid fatty acid and infra-red spectroscopic analysis of a sulphate-reducing consortium

Abstract In order to validate unusual fatty acids as biomarkers for sulphate-reducing bacteria, selective conditions were arranged for the enrichment of a marine glutamate-fermenting bacterium which made hydrogen and acetate available for oxidation via the respiration of sulphate. Under these conditions the complete oxidation of glutamate via sulphate reduction accounted for 84% of the available electron equivalents. Fatty acid biomarkers for hydrogen-oxidizing Desulfovibrio sp. (iso 17:1w7c and branched monoenoics) and for acetate-oxidizing Desulfobacter (10 methyl 16:0) were detected in the enrichment. These biomarkers were demonstrated in pure cultures of Desulfovibrio sp. and Desulfobacter sp. obtained from the enrichment. The predominant glutamate-fermenting bacterium isolated from the consortium contained no branched ester-linked phospholipid fatty acids, and produced acetate and hydrogen. With energy limitation the enriched consortium produced increased amounts of extracellular polysaccharide and the endogenous storage lipid poly-beta-hydroxybutyrate as detected with Fourier transform/infra-red (FT-IR) spectroscopy.     

Molecular and pharmacological properties of GABA-ρ subunits from white perch retina

Five γ-aminobutyric acid (GABA)-ρ subunits were cloned from a white perch retinal cDNA library and expressed in Xenopus oocytes. The deduced amino acid sequences indicated that all are highly homologous to the GABA-ρ subunits cloned from mammalian retinas; two clones (perch-ρ1A and perch-ρ1B) were in the ρ1 family, two (perch-ρ2A and perch-ρ2B) were in the ρ2 family, and one clone has been tentatively identified as a perch-ρ3 subunit. When expressed in Xenopus oocytes, all but one of the subunits (ρ3) formed functional homooligomeric receptors. However, the receptors expressed by each of the GABA-ρ subunits displayed unique response properties that distinguished one from the other. For example, receptors formed by perch-ρ1B subunits were more sensitive to GABA than the receptors formed by other GABA-ρ subunits, the dose–response curves for the various receptors revealed different Hill coefficients, and there were differences in the kinetics of the GABA-induced currents. In addition, the GABA-mediated current–voltage curve for ρ2 receptors was approximately linear, whereas the responses from ρ1 receptors showed outward rectification. A further division in the properties of the GABA-ρ subunits was revealed in their responses to imidazole-4-acetic acid (I4AA); the drug behaved as an antagonist on A-type ρ receptors and a partial agonist on the B-type ρ receptors. These results suggest that there is a large diversity of GABA_c receptors in the vertebrate retina, probably formed by homooligomeric and heterooligomeric combinations of GABA ρ subunits, that exhibit different functional properties. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 305–320, 1998     

Characterization of sulphate-reducing bacterial populations within marine and estuarine sediments with different rates of sulphate reduction

Viable counts of sulphate-reducing bacteria, able to use a range of different growth substrates were determined in sediments from two Sea Lochs (Etive and Eil) and an estuarine site (Tay), in Scotland. The composition of the sulphate-reducing bacterial population, in terms of substrate utilization, broadly corresponded to the in situ substrates for sulphate reduction and concentration of substrates at each site. Addition of acetate, lactate, propionate, butyrate, hydrogen and glutamate/serine (20 mM) to replicate slurries from each site resulted in stimulation of the corresponding population of sulphate-reducing bacteria and the in situ rates of sulphate reduction. The metabolism of the added substrates and changes in bacterial phospholipid fatty acids (PLFA) were quantified. With the exception of acetate and hydrogen, added substrates were incompletely oxidised, producing a mixture of further substrates, which predominantly were sequentially oxidised, and resulted in the stimulation of a mixed population of sulphate-reducing bacteria. There were significant changes in the PLFA of slurries with added substrate compared to controls. Acetate was completely removed at all sites and the small increase in even chain PLFA together with the absence of stimulation of any other biomarker, indicated that acetate was oxidised by sulphate-reducing bacteria distinctly different from those using other substrates. A biomarker for Desulfobacter, 10 Methyl 16:0, was not stimulated in any of the acetate slurries or in slurries where acetate was produced. Biomarkers for the propionate utilizing Desulfobulbus sp (17:1w6, 15:1w6) were always stimulated in propionate slurries and also in lactate slurries, where partial lactate fermentation produced propionate and acetate. In lactate and glutamate / serine slurries from the Tay estuary and lactate and hydrogen slurries from Loch Etive the biomarker for Desulfovibrio sp (i17:1w7) as well as those for Desulfobulbus were stimulated. This provides direct evidence for the significance of Desulfovibrio sp. within sediment slurries and demonstrates the competitive interaction between members of this genus and Desulfobulbus sp. for lactate, hydrogen and amino acid metabolism. At the estuarine site, sulphate reduction was limited at higher sulphate concentrations (about 3.5 mM) than the Sea Loch sites (<2 mM) and this had a significant effect on propionate and butyrate metabolism, as well as on methane production. These results demonstrate that although the sulphate-reducing bacterial population at each site could metabolise identical substrates, the types of sulphate-reducing bacteria involved and their sulphate thresholds were characteristically different.     

Complex regulation of the lactase-phlorizin hydrolase promoter by GATA-4

Lactase-phlorizin hydrolase (LPH), a marker of intestinal differentiation, is expressed in absorptive enterocytes on small intestinal villi in a tightly regulated pattern along the proximal-distal axis. The LPH promoter contains binding sites that mediate activation by members of the GATA-4, -5, and -6 subfamily, but little is known about their individual contribution to LPH regulation in vivo. Here, we show that GATA-4 is the principal GATA factor from adult mouse intestinal epithelial cells that binds to the mouse LPH promoter, and its expression is highly correlated with that of LPH mRNA in jejunum and ileum. GATA-4 cooperates with hepatocyte nuclear factor (HNF)-1alpha to synergistically activate the LPH promoter by a mechanism identical to that previously characterized for GATA-5/HNF-1alpha, requiring physical association between GATA-4 and HNF-1alpha and intact HNF-1 binding sites on the LPH promoter. GATA-4 also activates the LPH promoter independently of HNF-1alpha, in contrast to GATA-5, which is unable to activate the LPH promoter in the absence of HNF-1alpha. GATA-4-specific activation requires intact GATA binding sites on the LPH promoter and was mapped by domain-swapping experiments to the zinc finger and basic regions. However, the difference in the capacity between GATA-4 and GATA-5 to activate the LPH promoter was not due to a difference in affinity for binding to GATA binding sites on the LPH promoter. These data indicate that GATA-4 is a key regulator of LPH gene expression that may function through an evolutionarily conserved mechanism involving cooperativity with an HNF-1alpha and/or a GATA-specific pathway independent of HNF-1alpha.     

Steady-state kinetic analysis of human ubiquitin-activating enzyme (E1) using a fluorescently labeled ubiquitin substrate

We report the synthesis of fluorescently labeled ubiquitin (Ub) and its use for following ubiquitin transfer to various proteins. Using Oregon green (Og) succinimidyl ester, we prepared a population of Ub mainly labeled by a single Og molecule; greater than 95% of the Og label is associated with Lys 6 of Ub. We demonstrate that Og-Ub is efficiently accepted by Ub-utilizing enzymes, such as the human ubiquitin-activating enzyme (E1). We used this fluorescent substrate to follow the steady-state kinetics of human E1-catalyzed Ub-transfer to the ubiquitin-carrier enzyme Ubc4. In this reaction, E1 uses three substrates: ATP, Ubc4, and Ub. The steady-state kinetics of Og-Ub utilization by E1 is presented. We have also used analytical ultracentrifugation methods to establish that E1 is monomeric under our assay condition (low salt) as well as under physiological condition (150 mM NaCl).     

Evidence for multiple genetic forms with similar eyeless phenotypes in the blind cavefish, Astyanax mexicanus

A diverse group of animals has adapted to caves and lost their eyes and pigmentation, but little is known about how these animals and their striking phenotypes have evolved. The teleost Astyanax mexicanus consists of an eyed epigean form (surface fish) and at least 29 different populations of eyeless hypogean forms (cavefish). Current alternative hypotheses suggest that adaptation to cave environments may have occurred either once or multiple times during the evolutionary history of this species. If the latter is true, the unique phenotypes of different cave-dwelling populations may result from convergence of form, and different genetic changes and developmental processes may have similar morphological consequences. Here we report an analysis of variation in the mitochondrial NADH dehydrogenase 2 (ND2) gene among different surface fish and cavefish populations. The results identify a minimum of two genetically distinctive cavefish lineages with similar eyeless phenotypes. The distinction between these divergent forms is supported by differences in the number of rib-bearing thoracic vertebrae in their axial skeletons. The geographic distribution of ND2 haplotypes is consistent with roles for multiple founder events and introgressive hybridization in the evolution of cave-related phenotypes. The existence of multiple genetic lineages makes A. mexicanus an excellent model to study convergence and the genes and developmental pathways involved in the evolution of the eye and pigment degeneration.     

The evolution of ecosystem processes: growth rate and elemental stoichiometry of a key herbivore in temperate and arctic habitats

Understanding the reciprocal interactions between the evolved characteristics of species and the environment in which each species is embedded is a major priority for evolutionary ecology. Here we use the perspective of ecological stoichiometry to test the hypothesis that natural selection on body growth rate affects consumer body stoichiometry. As body elemental composition (nitrogen, phosphorus) of consumers influences nutrient cycling and trophic dynamics in food webs, such differences should also affect biogeochemical processes and trophic dynamics. Consistent with the growth rate hypothesis, body growth rate and phosphorus content of individuals of the Daphnia pulex species complex were lower in Wisconsin compared to Alaska, where the brevity of the growing season places a premium on growth rate. Consistent with stoichiometric theory, we also show that, relative to animals sampled in Wisconsin, animals sampled in Alaska were poor recyclers of P and suffered greater declines in growth when fed low‐quality, P‐deficient food. These results highlight the importance of evolutionary context in establishing the reciprocal relationships between single species and ecosystem processes such as trophic dynamics and consumer‐driven nutrient recycling.     

Comparison of synexin isotypes in secretory and non-secretory tissues

Three synexin isotypes were identified in bovine liver or adrenal medullary tissues by immune blotting of one- or two-dimensional SDS gels and by two-dimensional tryptic peptide mapping of gel bands or spots. These isotypes were: alpha-synexin, mass 47 kDa, pI 6.9; beta-synexin, mass 47 kDa, pI 6.5; and mu-synexin, mass 51 kDa, pI 6.1. A non-secretory tissue, bovine skeletal muscle, was found to contain only mu-synexin. The absence of alpha- and beta-synexins in a non-secretory tissue suggests these proteins may perform specific roles in the process of exocytosis.