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Histidase activity rather than uptake of L-histidine is the limiting factor for the utilization of histidine as the sole nitrogen source for Aspergillus nidulans. Histidine cannot act as the sole carbon source, and evidence is presented indicating that this is attributable to an inability to convert histidine to L-glutamate in vivo. It has been shown that this fungus lacks an active urocanase enzyme and that histidine is quantitatively converted to urocanate, which accumulates in the extracellular medium. The use of histidine as a nitrogen source is regulated by nitrogen metabolite repression control of histidase synthesis. In addition, evidence for a requirement for a carbon source for histidase synthesis and for a minor form of control by nitrate is presented. The activity of the histidase enzyme is inhibited by micromolar concentrations of the product urocanate and by physiological levels of L-glutamate and L-glutamine.

The kinetics of reduction of turkey liver xanthine dehydrogenase by substrates were investigated by stopped-flow spectrophotometry. The results may be explained in terms of the known redox potentials of the various centres in the enzyme [Barber, Bray, Cammack & Coughlan (1977) Biochem. J. 163, 279-289]. They are, morover, consistent with the scheme [Olson, Ballou, Palmer & Massey (1974) J. Biol. Chem. 249, 4363-4382] in which reduction occurs in three consecutive steps, one molecule of substrate reacting with the active site at each step. First-order rate constants believed to correspond respectively to the combined first and second steps and to the third step in the reduction by excess of xanthine and of NADH were determined. The rates of reaction with these substrates in the combined first and second steps are independent of the degree of enzyme functionality.

Quinelorane (LY163502) has the endocrine, neurochemical and behavioral profile of a potent and highly selective D2-dopaminergic agonist. The administration of quinelorane produced dose-related decreases in serum prolactin concentration of reserpinized, male rats and increases in serum corticosterone concentration of male rats. The minimum effective doses (MED) for these effects were 10 and 30 micrograms/kg i.p., respectively. Quinelorane induced increases in 3-methoxy-4-hydroxyphenylglycol-sulfate levels in the brain stem (MED, 30 micrograms/kg i.p.) and decreases in hypothalamic epinephrine levels (MED, 100 micrograms/kg i.p.) in male rats as determined by high-pressure liquid chromatography with electrochemical detection methods. Quinelorane induced increases in extracellular ascorbic acid as determined by in vivo voltammetry in the nucleus accumbens and striatum of male rats. Quinelorane produced concentration-dependent suppression of K+-evoked release of acetylcholine from superfused caudate slices, with an IC50 of approximately 10(-8)M. Quinelorane administration produced dose-related increases in compulsive, contralateral turning in male rats with unilateral nigrostriatal lesions and increases in locomotor activity and stereotypic behavior in male rats. In dogs, quinelorane administration produced dose-related increases in emetic response with an ED50 of 7 micrograms/kg i.v. Quinelorane administration also produced dose-related decreases in the striatal concentrations of the dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic (MED, 1 microgram/kg i.p. for both metabolites) as determined by high-pressure liquid chromatography with electrochemical detection methods and decreases in extracellular concentrations of homovanillic acid in the nucleus accumbens and striatum as determined by in vivo voltammetry., Quinelorane produced concentration-dependent decreases in K+-evoked dopamine release from superfused striatal slices (IC50 = 3 X 10(-9) M).(ABSTRACT TRUNCATED AT 250 WORDS)

A limitation of genetic studies of the rumen bacterium, Butyrivibrio fibrisolvens, has been the availability of suitable vectors and transfer systems. Using the conjugative tetracycline resistant transposon, Tn916, the Staphylococcus aureus plasmid, pUB110, and the pUB110-based shuttle vector, pUBLRS, a conjugative transfer system was developed for B. fibrisolvens. B. fibrisolvens donor strains H17c2 and H17c12, containing Tn916 and pUB110 or pUBLRS, respectively, were used in mating experiments with selected B. fibrisolvens strains. Kanamycin resistant transconjugants, containing pUB110, of strains 193, 194, and 195 were detected at a combined average frequency of 7.78 × 10^-7 per donor and 1.11 × 10^-5 per recipient. Transconjugants of strains 193 and 194, containing pUBLRS, were detected at an average frequency of 1.22 × 10^-6 per donor and 4.70 × 10^-8 per recipient. Southern hybridization analysis confirmed the presence of pUB110 and pUBLRS in transconjugants. Results indicated that Tn916 was necessary for mobilization of pUB110 as transconjugants were not detected when the transposon was absent from the donor strains. The ability to mobilize pUB110 and pUBLRS between B. fibrisolvens strains provides a conjugative transfer system that circumvents problems encountered with electroporation. Copyright 1994, 1999 Academic Press

Clonidine was found to possess dose-dependent analgesic and antiwithdrawal activity. In mice, clonidine prolonged the tail flick latency and inhibited phenylquinone-induced writhing. In rats, it inhibited tail withdrawal from hot water and a pain response to pressure application on an inflamed paw. The effective doses of clonidine were different in the different tests employed, but they were always smaller than those of morphine. Naloxone failed to antagonize the analgesic actions of clonidine but effectively antagonized those of morphine. Phenoxybenzamine also did not alter the inhibition of tail flick-induced by clonidine. Clonidine suppressed morphine withdrawal body shakes in a dose-dependent manner as does morphine. This action of clonidine was not reversed by naloxone. In usual laboratory tests, clonidine appears to be an effective analgesic which antagonizes signs of morphine withdrawal.

Loss-of-function mutations in the regulatory gene areA of Aspergillus nidulans prevent the utilization of a wide variety of nitrogen sources. The phenotypes of nit-2 mutants of Neurospora crassa suggest that this gene may be analogous to the areA gene. Transformation has been used to introduce a plasmid containing the nit-2 gene into A. nidulans. The nit-2 gene of Neurospora complemented mutations in the areA gene, restoring the ability to use a variety of nitrogen sources. This indicated that the activator function of nit-2 and areA gene products was retained across these two fungal species. Southern blot analysis revealed both single-copy and multicopy integrations and, in at least one case, integration appeared to generate a nit-2 mutation. Integration of the transforming plasmid appeared to be by nonhomologous events at a number of different sites in the Aspergillus genome. The transformants were less sensitive to nitrogen-metabolite repression of extracellular protease activity and nitrate reductase (EC 1.6.6.3) than were wild-type A. nidulans. This indicated that nitrogen control was not completely normal in the nit-2 transformants.

Somatomedin-C or insulin-like growth factor I (Sm-C/IGF-I) and insulin-like growth factor II (IGF-II) have been implicated in the regulation of fetal growth and development. In the present study 32P-labeled complementary DNA probes encoding human and mouse Sm-C/IGF-I and human IGF-II were used in Northern blot hybridizations to analyse rat Sm-C/IGF-I and IGF-II mRNAs in poly(A+) RNAs from intestine, liver, lung, and brain of adult rats and fetal rats between day 14 and 17 of gestation. In fetal rats, all four tissues contained a major mRNA of 1.7 kilobases (kb) that hybridized with the human Sm-C/IGF-I cDNA and mRNAs of 7.5, 4.7, 1.7, and 1.2 kb that hybridized with the mouse Sm-C/IGF-I cDNA. Adult rat intestine, liver, and lung also contained these mRNAs but Sm-C/IGF-I mRNAs were not detected in adult rat brain. These findings provide direct support for prior observations that multiple tissues in the fetus synthesize immunoreactive Sm-C/IGF-I and imply a role for Sm-C/IGF-I in fetal development as well as postnatally. The abundance of a 7.5-kb Sm-C/IGF-I mRNA in poly(A+) RNAs from adult rat liver was 10-50-fold higher than in other adult rat tissues which provides further evidence that in the adult rat the liver is a major site of Sm-C/IGF-I synthesis and source of circulating Sm-C/IGF-I. Multiple IGF-II mRNAs of estimated sizes 4.7, 3.9, 2.2, 1.75, and 1.2 kb were observed in fetal rat intestine, liver, lung, and brain. The 4.7- and 3.9-kb mRNAs were the major hybridizing IGF-II mRNAs in all fetal tissues. Higher abundance of IGF-II mRNAs in rat fetal tissues compared with adult tissues supports prior hypotheses, based on serum IGF-II concentrations, that IGF-II is predominantly a fetal somatomedin. IGF-II mRNAs are present, however, in some poly(A+) RNAs from adult rat tissues. The brain was the only tissue in the adult rat where the 4.7- and 3.9-kb IGF-II mRNAs were consistently detected. Some samples of adult rat intestine contained the 4.7- and 3.9-kb IGF-II mRNAs and some samples of adult liver and lung contained the 4.7-kb mRNA. These findings suggest that a role for IGF-II in the adult rat, particularly in the central nervous system, cannot be excluded.

Muscarinic receptors of the rabbit ear artery were characterized by observing the effect of the subtype selective antagonist pirenzepine on functional responses and radioligand binding. Pirenzepine has been shown to bind with high affinity to muscarinic receptors of certain brain regions and peripheral ganglia (M1 subtype) and with low affinity to receptors of the heart and upper gastrointestinal tract (M2 subtype). The affinity (pKB) of pirenzepine for the muscarinic sites of the endothelium was determined by the competitive antagonism of the relaxation response to methacholine. Schild analysis gave a pKB of 6.5 (320 nM) which is consistent with the low affinity, M2, subtype of muscarinic receptor. Removal of the endothelium eliminates any response to muscarinic agonists but does not decrease the density of muscarinic binding sites determined by binding of the specific ligand (-)-[3H]quinuclidinyl benzilate. This indicates a second group of muscarinic receptors most probably located on vascular smooth muscle cells for which there is no known function. The pKi for pirenzepine at these sites, as determined by the inhibition of (-)-[3H]quinuclidinyl benzilate binding, was 6.26 (550 nM) which is also consistent with a low affinity subtype. Thus, both types of vascular muscarinic binding sites, those on the endothelium which mediate relaxation and those on the vascular smooth muscle cells, are of the low affinity, M2, subtype.

Growth of Aspergillus nidulansin the presence of methylammonium leads to lowered levels of the enzymes, acetamidase, formamidase, benzamidase, histidase, nitrate reductase and urate oxidase. This phenomenon is not altered in strains that are insensitive to ammonium repression due to a lesion in the gdhA gene. Similarly repression of acetamidase, formamidase and histidase by high concentrations of caesium ion is not affected in these strains. The results indicate that caesium ion and methylammonium may not act as direct analogues of ammonium in repression of enzyme synthesis.

HeLa cells were irradiated at liquid nitrogen temperature with negative pions from Nimrod at the Rutherford Laboratory and then assayed for survival of colony-forming ability. Complete dose-response curves were obtained from repeated determinations at 14 different positions along the depth-dose profile and survival curves were fitted to the data by computer programme. A depth-damage profile was thus established in terms of the final slopes of these survival curves. This confirmed the expected RBE value of 1.84 at the ionization peak. Although a value of unity was found in the main plateau region, the “entrance” positions showed significantly higher values.

Previous analysis of the amdS gene of Aspergillus nidulans has identified multiple regulatory circuits mediated by trans-acting regulatory genes, cis-acting mutations have been identified and shown to specifically affect individual regulatory circuits. Fine-structure genetic mapping of the amdS regions showed that these cis-acting mutations occur in a complex controlling region adjacent to the amdS structural gene. The amdS gene was cloned by differential hybridization, using cDNA probes derived from a high-level-producing strain and from a strain with a large amdS deletion mutation. RNA blotting experiments showed that a single RNA species of 1,600 to 1,700 base pairs is transcribed from the amdS gene. DNA blotting experiments on a large number of amdS mutant strains, including deletions and translocations, allowed the genetic and physical maps of the gene to be correlated. The controlling region of the gene is situated at the 5' end of the gene and the direction of transcription is toward the centromere of chromosome III. The regulatory mutations in the controlling region were found to be due to small-scale alterations in the DNA rather than to large-scale rearrangements resulting in gene fusions.

Nitrous oxide, like morphine, stimulates locomotor activity in the CD-1, C-57 and C-3H mouse strains. This stimulation of locomotor activity is antagonized by the narcotic antagonist naltrexone (3 and 30 mg/kg). There are several strains (A/J and DBA/2J) of mice in which morphine does not stimulate locomotor activity. Mice resistant to this behavioral effect of morphine show a stimulation of locomotor activity in response to nitrous oxide which is not blocked by naltrexone. These results provide new data linking the effects of morphine and nitrous oxide. The opiate-like stimulation of activity produced by nitrous oxide may result from the release of endogenous opiate-like substances. Nitrous oxide causes a displacement of [3H]naloxone from brain opiate receptors labeled in vivo in the CD-1 mouse strain. In marked contrast to these results, nitrous oxide administration to the A/J and DBA/2J morphine-resistant strains failed to displace [3H]naloxone from opiate receptors labeled in vivo. Thus, variations in the response to nitrous oxide may not only be strain-dependent but possibly related to deficits in the response of or interaction with the endogenous opiate system.

LY141865, a dopamine agonist selective for D2 dopamine receptors, caused hypomotility at low doses (0.01 and 0.1 mg/kg) and hypermotility at high doses (1 and 10 mg/kg) after i.p. injection into normotensive rats (WKY). In age-matched hypertensive rats (SHR), LY141865 caused hypomotility (not hypermotility) at all of these doses. The basal locomotor activity was higher in SHR than in WKY, but striatal concentrations of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, and of serotonin and its metabolite, 5-hydroxyindoleacetic acid, were not different between the two groups of rats. The specific binding of a dopamine receptor radioligand, tritiated pergolide, in striatum and mesolimbic regions, did not differ in SHR compared with WKY. In contrast to the lack of locomotor stimulation in SHR, other dopaminergic responses to LY141865 occurred in SHR as well as WKY. For instance, LY141865 decreased striatal and mesolimbic concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid, increased striatal and mesolimbic concentrations of acetylcholine, decreased hypothalamic concentrations of epinephrine, increased serum corticosterone concentration and decreased serum prolactin concentration in SHR as in WKY. Because radioligand-labeled dopamine receptors and several LY141865 responses mediated by dopamine receptors did not differ appreciably in SHR compared with WKY, the lack of behavioral hypermotility in response to LY141865 in SHR may be due to abnormalities in some synaptic mechanisms beyond dopamine receptor activation that are involved in the expression of increased locomotion in response to the dopamine agonist.

In Aspergillus nidulans the acetamidase enzyme is inducible by omega-amino acids, sources of acetyl-coenzyme A, and benzoate. The amdR (or intA) gene is a positive-control gene involved in omega-amino acid induction only. A cis-acting mutation amdI93 located in a complex controlling region adjacent to the acetamidase structural gene was found to abolish induction by omega-amino acids but not induction by other sources of induction. As predicted, this mutation was epistatic to constitutive amdR alleles but did not affect the expression of mutations in other regulatory genes.

A cyclic nucleotide-binding phosphohydrolase that possesses both a phosphomonoesterase and a phosphodiesterase catalytic function has been partially purified from Aspergillus nidulans. The enzyme hydrolyzes both p-nitrophenylphosphate and bis-(p-nitrophenyl)-phosphate. o'-Nucleoside monophosphates are the best physiological phosphomonesterase substrates but 5'- and 2'-nucleoside monophosphates are also hydrolyzed. The enzyme catalyzes the hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, and 2',3'- and 3'5'-cyclic nucleotides, but not of ribonucleic acid, deoxyribonucleic acid, or nicotinamide adenine dinucleotide. The enzyme has acid pH optima and is not activated by divalent cations. Nucleosides and nucleotides inhibit the enzyme. Cyclic nucleotides are competitive inhibitors of the phosphodiesterase-phosphomonoesterase. The enzyme can occur extracellularly. The phosphodiesterase-phosphomonoesterase is present at high levels in nitrogen-starved mycelium, and it is strongly repressed during growth in media containing ammonium or glutamine and weakly repressed during growth in glutamate-containing medium. Experiments with various area mutants show that this regulatory gene is involved in the control of the enzyme. No evidence for regulation of the enzyme by carbon or phosphorus starvation has been found.

Aspergillus nidulansproduces acetamidase and formamidase enzymes. The acetamidase is produced in reduced amounts during growth on glucose, whereas the formamidase is not greatly affected. Mutations in a gene, amdT, which affect glucose repression of amidases are described. One of these, amdT102, causes the acetamidase to be no longer subject to glucose repression and also affects ability to synthesize formamidase. The other, amdT19, results in both the formamidase and the acetamidase being subject to abnormally strong glucose repression, and also in increased maximal acetamidase activities. The dominance relationships at the amdTlocus have been investigated. It is suggested that the amdTgene may play a positive role in controlling amidase synthesis.

Aspergillus nidulanscan grow on acetamide as both a carbon and nitrogen source and can also grow on formamide as a nitrogen source. Two distinct enzymes, an acetamidase and a formamidase, are produced. The control of the synthesis of these two enzymes in a wild-type strain was investigated. The formamidase is induced by acetamide and formamide and repressed by ammonia. The acetamidase is induced by formamide and acetamide, repressed by carbon metabolites derived from glucose and acetate, and repressed by ammonia. Repression of the acetamidase by ammonia depends on the carbon source; growth on glucose but not on acetate or acetamide allows repression to occur. The pattern of acetamidase repression is compared with that of histidine catabolic enzymes in various bacteria.