Influence of δ-Opioid Receptors on Production of Labeled Methionine5-Enkephalin in Murine Neuroblastoma Cells

Synthesis of methionine5-enkephalin by intact cells of murine neuroblastoma clone N1E-115 has been demonstrated both immunocytochemically and biochemically. In addition, N1E-115 cells possess homogeneous enkephalin (delta) receptors which inhibit prostaglandin E1-induced intracellular cyclic AMP formation. An assay was developed for measuring de novo synthesis of methionine5-enkephalin by pulsing cells in culture with radioactive methionine and isolating this pentapeptide to radiochemical purity by a procedure that included immunoaffinity chromatography specific for oxidized methionine5-enkephalin. This assay indicated that production of radiolabeled-methionine5-enkephalin was increased upon lengthy exposure of intact N1E-115 cells in the late logarithmic phase of growth to a nonproteolyzable analog of methionine5-enkephalin. This increase in synthesis of intracellular methionine5-enkephalin relative to control cells was prevented by prior incubation of the clone with naloxone, indicating that the response was mediated by the delta receptor.     

Effect of β-FNA on Opiate δ Receptor Binding

Abstract: (β-FNA, the β -fumaramate methyl ester of naltrexone, has been shown to antagonize irreversibly the actions of morphine on the guinea pig ileum and mouse vas deferens bioassays but does not affect the actions of δ-receptor ligands on the mouse vas deferens bioassay, suggesting that the compound does not irreversibly bind to the S receptor. In this paper we examine the effect of (β -FNA on the binding of the prototypic δ agonists, Leuenkephalin and d -Ala²- d -Leu⁵-enkephalin, its metabolically stable analogue, and show that treatment of membranes with β -FNA does lead to alterations in the in vitro properties of δ receptors.     

Influence of dietary supplementation with thiamine on lead intoxication in rats

The influence of dietary supplementation with thiamine on lead (Pb) contents in blood and tissues, blood δ-aminolevulinic acid dehydratase (δ-ALAD) activity, and urinary excretion of δ-aminolevulinic acid (δ-ALA) was evaluated in male Sprague-Dawley rats. Groups of randomly selected animals were given a thiamine-deficient diet, a diet containing normal thiamine (20 mg/kg), or a thiamine-supplemented diet (50 mg/kg), along with control drinking water or water containing 100 ppm Pb, for 4 mo. Animals fed the thiamine-supplemented diet (50 mg/kg) and Pb showed decreased urinary excretion of δ-ALA and a decreased inhibition of δ-ALAD activity in blood compared to those given Pb with normal thiamine diet. The liver, kidney, and blood of rats receiving supplemental thiamine also contained significantly less Pb than the other two treatment groups given Pb-containing water. The protective effect of thiamine against Pb toxicity may be attributed to its interference with retention of the metal in body tissue, possibly resulting from the formation of excretable thiamine-lead complexes.     

Diversity,metabolic types and δ13C carbon isotope ratios in the grass flora of Namibia in relation to growth form,precipitation and habitat conditions

The grass flora of Namibia (374 species in 110 genera) shows surprisingly little variation in 948.gif" alt="delta" align="BASELINE" BORDER="0">¹³C values along a rainfall gradient (50–600 mm) and in different habitat conditions. However, there are significant differences in the 948.gif" alt="delta" align="BASELINE" BORDER="0">¹³C values between the metabolic types of the C4 photosynthetic pathway. NADP-ME-type C4 species exhibit the highest 948.gif" alt="delta" align="BASELINE" BORDER="0">¹³C values (–11.7 ) and occur mainly in regions with high rainfall. NAD-ME-type C4 species have significantly lower 948.gif" alt="delta" align="BASELINE" BORDER="0">¹³C values (–13.4 ) and dominate in the most arid part of the precipitation regime. PCK-type C4 species play an intermediate role (–12.5 ) and reach a maximum abundance in areas of intermediate precipitation. This pattern is also evident in genera containing species of different metabolic types. Within the same genus NAD species reach more negative 948.gif" alt="delta" align="BASELINE" BORDER="0">¹³C values than PCK species and 948.gif" alt="delta" align="BASELINE" BORDER="0">¹³C values decreased with rainfall. Also in Aristida, with NADP-ME-type photosynthesis, 948.gif" alt="delta" align="BASELINE" BORDER="0">¹³C values decreased from –11 in the inland region (600 mm precipitation) to –15 near the coast (150 mm precipitation), which is a change in discrimination which is otherwise associated by a change in metabolism. The exceptional C3 species Eragrostis walteri and Panicum heterostachyum are coastal species experiencing 50 mm precipitation only. Many of the rare species and monotypic genera grow in moist habitats rather than in the desert, and they are not different in their carbon isotope ratios from the more common flora. The role of species diversity with respect to habitat occupation and carbon metabolism is discussed.     

Trends in plant carbon concentration and plant demand for N throughout this century

 Atmospheric CO₂ concentration has increased by 25% over the preindustrial level. A parallel increase in C concentration and decreases in N concentration and δ¹³C of plants grown throughout this century have been observed in plant specimens stored in herbaria. We tested our previous results in a study of 12 more species collected in the western Mediterranean throughout this century (1920–1930, 1945–1955, and 1985–1990) and tree rings of Quercus pubescens from the same area. These changes were accompanied by apparent increases in condensed tannin concentration. A decreasing trend in δ¹⁵N both in herbarium material and tree rings was also found, indicating that ecosystems might cope with higher plant N demand by decreasing N losses and increasing N fixation and mineralization. These results may contribute to a better understanding of the effects of global change on carbon and nitrogen cycling. Received: 12 November 1995 / Accepted: 17 May 1996     

Production of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine by entrapped ACV-synthetase fromStreptomyces clavuligerus

Summary 948.gif" alt="delta" align="BASELINE" BORDER="0">-(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV)-synthetase fromStreptomyces clavuligerus was studied under conditions that enabled the reuse of the enzyme. Coupling of ACV-synthetase to DEAE-Trisacryl and aminopropyl-glass resulted in an immobilized enzyme product of little or no catalytic activity. However, an enzyme reactor was designed by physical confinement of partially-purified ACV-synthetase in an ultrafiltration cell. This system was stimulated by phosphoenolpyruvate at lower concentrations of ATP, an effect not observed with purified enzyme. Up to 30% conversion of the limiting substrate, cysteine, to ACV occurred under semi-continuous conditions. Reaction products were investigated as potential inhibitors: AMP was the most inhibitory, but only when used at concentrations in excess of those produced in reaction mixtures. Under a nitrogen atmosphere, both product and enzyme stabilities were greatly improved and the enzyme retained 45–46% of its initial activity after five uses at room temperature during a 24-h period. Extrapolations based on these data suggest that 1.3 g partially purified enzyme (0.13 U g^–1) would be capable of producing 411 mg of ACV in a 1-L reaction mixture in this period.     

Development of insect resistance in tomato plants expressing the δ-endotoxin gene ofBacillus thuringiensis subsp.tenebrionis

A crystal 948.gif" alt="delta" align="BASELINE" BORDER="0">-endotoxin gene ofBacillus thuringiensis subsp.tenebrionis (B.t.t.) encoding a coleopteran insect-specific toxin was used to construct a chimeric gene which expressed the toxin in plant cells. Via anAgrobacterium tumefaciens binary vector system, the toxin gene was transferred into tomato cells. From leaf disks recombinant plants were regenerated. Hybridization experiments demonstrated that these plants synthesized toxin-specific mRNA of the expected size. Transgenic tomato plants with the chimericB.t.t. toxin gene contained a 74 kDa protein which cross-reacted with toxin antibodies. The expression caused a significant insecticidal activity of the transgenic tomato plants against Colorado potato beetle larvae.     

Biosynthesis of δ-aminolevulinic acid from glutamate by Sulfolobus solfataricus

The extremely thermophilic, obligately aerobic bacterium Sulfolobus solfataricus forms the tetrapyrrole precursor, 948.gif" alt="delta" align="BASELINE" BORDER="0">-aminolevulinic acid (ALA), from glutamate by the tRNA-dependent five-carbon pathway. This pathway has been previously shown to occur in plants, algae, and most prokaryotes with the exception of the -group of proteobacteria (purple bacteria). An alternative mode of ALA formation by condensation of glycine and succinyl-CoA occurs in animals, yeasts, fungi, and the -proteobacteria. Sulfolobus and several other thermophilic, sulfur-dependent bacteria, have been variously placed within a subgroup of archaea (archaebacteria) named crenarchaeotes, or have been proposed to comprise a distinct prokaryotic group designated eocytes. On the basis of ribosomal structure and certain other criteria, eocytes have been proposed as predecessors of the nuclear-cytoplasmic descent line of eukaryotes. Because aplastidic eukaryotes differ from most prokaryotes in their mode of ALA formation, and in view of the proposed affiliation of eocytes to eukaryotes, it was of interest to determine how eocytes form ALA. Sulfolobus extracts were able to incorporate label from [1-¹⁴C]glutamate, but not from [2-¹⁴C]glycine, into ALA. Glutamate incorporation was abolished by preincubation of the extract with RNase. Sulfolobus extracts contained glutamate-1-semialdehyde aminotransferase activity, which is indicative of the five-carbon pathway. Growth of Sulfolobus was inhibited by gabaculine, a mechanism-based inhibitor of glutamate-1-semialdehyde aminotransferase, an enzyme of the five-carbon ALA biosynthetic pathway. These results indicate that Sulfolobus uses the five-carbon pathway for ALA formation.Abbreviations AHA 4-amino-5-hexynoic acid - ALA 948.gif" alt="delta" align="BASELINE" BORDER="0">-aminolevulinic acid, Gabaculine, 3-amino-2,3-dihydrobenzoic acid - GSA glutamate 1-semialdehyde