Selection and biochemical studies of pyrimidine-requiring mutants of Lactobacillus plantarum

Mutagenesis and mutant enrichment in Lactobacillus plantarum, a lactic acid bacterium used in silage, sauerkraut and sausage fabrication, were studied. In optimal conditions, auxotrophic mutants were obtained that permitted investigation of the de novo pyrimidine biosynthesis. Uracil-requiring mutants were characterized for their enzymatic defect, in aspartate transcarbamylase (ATCase), dihydro-orotase (DHOase), dihydro-orotate dehydrogenase (DHOdehase), orotidine monophosphate pyrophosphorylase (OMPppase) or in orotidine monophosphate decarboxylase (OMPdecase). The five enzymatic activities are totally repressed by uracil in the growth medium.     

Effects of guanine nucleotides on cns neuropeptide receptors

The effect of nucleotides on central nervous system neuropeptide receptor binding was investigated. The guanine nucleotides, guanosine-5′-triphosphate and guanylyl-5′-imidodiphosphate, significantly inhibited the binding of radiolabeled vasoactive intestinal polypeptide but not that of [Tyr⁴]bombesin to rat brain membranes. Vasoactive intestinal polypeptide binding was inhibited by guanine nucleotides in a dose-dependent manner. Using a 20 μM dose, 60% of the specific vasoactive intestinal polypeptide binding was inhibited by guanylyl-5′-imidodiphosphate, which was more potent than guanosine-5′-triphosphate, whereas other nucleotides were not effective. This reduction in binding was a consequence of lower affinity of the receptor for vasoactive intestinal polypeptide, which in turn resulted from an increased rate of dissociation.     

Naltrexone reduces weight gain, alters “β-endorphin”, and reduces insulin output from pancreatic islets of genetically obese mice

Naltrexone, an opiate antagonist, was administered to young obese (ob/ob) and lean mice for five weeks. Animals had continuous access to food and received 10 mg/kg SC twice daily with equivalent volumes of saline given to controls. The effects on body weight, and pituitary and plasma levels of β-endorphin-like material were measured. Naltrexone-injected obese animals gained weight more slowly over the first three weeks while the weight gain of lean animals was not affected by naltrexone. Plasma levels of β-endorphin were shown to be significantly higher in untreated ob/ob mice and this difference increased with age (4–20 weeks). With naltrexone treatment, plasma levels in +/? mice rose and exceeded those in ob/ob. Saline treatment appeared to be a stress, and pituitary β-endorphins rose 4–6 fold in ob/ob compared with +/?. While naltrexone reduced the levels in ob/ob pituitary towards normal, no effect on β-endorphin levels in pituitary of lean mice was obtained. In vitro studies of effects of the opiate antagonists, naloxone, on insulin secretion by isolated islets provided additional evidence of resistance of lean mice to naloxone relative to ob/ob. (IRI secretion fell only in naloxone treated ob/ob islets.) These observations support the contention that this form of genetic obesity is characterized by elevated endogenous opiate levels and an increased sensitivity to opiate antagonists such as naltrexone or naloxone.     

Visualization and solubilization of rat brain opiate receptors with a “κ” ligand selectivity pattern

1. Specific binding of [3H]ethylketocyclazocine (EkappaC), a prototype kappa-opiate agonist, to slide-mounted rat striatal sections is increased in the presence of 100 mM NaCl at 4 degrees C. 2. Under similar incubation conditions, binding of mu and delta prototype opiates is reduced to almost undetectable levels. 3. Correlation (P less than 0.01) of the ligand selectivity pattern of [3H]EKC displacement with the potencies of various opiate drugs in inhibiting the contractions of the rabbit vas deferens, a kappa-opiate receptor bioassay, suggests that the binding site under study represents the pharmacologically relevant kappa-opiate receptor. 4. Visualization of these kappa-opiate receptors with tritium-sensitive film reveals a striking, highly discrete brain distribution pattern (e.g., striatal patches, habenular stripe) which is similar to that of [3H]dihydromorphine and [3H]naloxone. 5. Soluble [3H]EKC binding sites obtained from rat membranes also possess a kappa-like ligand selectivity pattern, with bremazocine being a potent displacer while mu and delta ligands are almost inactive. 6. A possible explanation of these data is that the "kappa"-opiate binding site in rat brain is one transitional state of an opiate receptor capable of assuming distinct conformations with characteristic ligand selectivity patterns. Other possibilities such as pre and post-synaptic locations should also be considered.     

Determination of 3-methoxy-4-hydroxyphenylglycol in urine by high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method has been used for the quantitative determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in urine. After incubation with glusulase, free MHPG is extracted into ethyl acetate and further isolated by a combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The addition of amperometric detection provides increased sensitivity to a highly specific assay.     

The biotransformation of α-(2, 4, 6-trimethylphenyl) ethylamine by rabbit liver preparations

Microsomal incubation of the parent amine, α-(2, 4, 6-trimethylphenyl) ethylamine, VI, produced imine, II, alcohol, IV, oxime, V, and several unknowns. The isolation of imine, II, produces evidence that imines may be involved in the microsomal deamination of primary amines. Separate incubations of the imine, II, produced oxime, V, and several unknowns, one of which is tentatively identified as the nitro derivative. Incubation of the oxime, V, gave the alcohol, IV, the “nitro” metabolite as above and 2 unknowns. Incubation of the hydroxylamine, VII, gave oxime, V, alcohol, IV, 2 unknowns and the “nitro” metabolite.     

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