Stress increases gene expression of phenylethanolamine N-methyltransferase in spleen of rats via pituitary-adrenocortical mechanism.

Gene expression of phenylethanolamine N-methyltransferase (PNMT), the enzyme catalyzing conversion of norepinephrine to epinephrine, has been detected in rat spleen using the reverse transcription polymerase chain reaction. PNMT identity was subsequently verified by Southern blots. Localization of the spleen cells responsible for the PNMT gene expression was investigated by the in situ hybridization and PNMT mRNA was found to be present in the white pulp. The hypothesis that stress may produce an increase in PNMT gene expression in rat spleen was tested and a robust rise in the relative abundance of PNMT mRNA levels was observed after a single or repeated immobilization (about 80%). Adrenalectomy or hypophysectomy completely prevented the immobilization-induced increase in spleen PNMT mRNA levels, suggesting that stress-induced PNMT gene expression in the spleen is regulated predominantly via pituitary-adrenocortical axis. In control animals, however, spleen PNMT was not significantly affected by the ectomies and therefore basal PNMT gene expression might be regulated by different mechanism(s).Thus, PNMT gene expression in the rat spleen is exaggerated by stress stimuli, suggesting its role in physiological regulations.

Existence of cardiac PNMT mRNA in adult rats: elevation by stress in a glucocorticoid-dependent manner.

Phenylethanolamine N-methyltransferase (PNMT) is the enzyme that synthesizes epinephrine from norepinephrine. The aim of this study was to determine potential PNMT gene expression in the cardiac atria and ventricles of adult rats and to examine whether the gene expression of this enzyme is affected by immobilization stress. PNMT mRNA levels were detected in all four parts of the heart, with the highest level in the left atrium. Both Southern blot and sequencing verified the specificity of PNMT detected by RT-PCR. Single immobilization for 2 h increased gene expression of PNMT in both atria and ventricles. In atria, this effect was clearly modulated by glucocorticoids, because either adrenalectomy or hypophysectomy prevented the increase in PNMT mRNA levels in response to immobilization stimulus. This study establishes, for the first time, that PNMT gene expression occurs in cardiac atria and also, to a small extent, in ventricles of adult rats. Immobilization stress increases gene expression in atria and ventricles. This increase requires an intact hypothalamus-pituitary-adrenocortical axis, indicating the involvement of glucocorticoids.

Effect of novel stressors on gene expression of tyrosine hydroxylase and monoamine transporters in brainstem noradrenergic neurons of long-term repeatedly immobilized rats.

Responses of central noradrenergic (NE) neurons to stressors like immobilization (IMO), cold exposure, insulin-induced hypoglycemia, and cellular glucoprivation caused by 2-deoxy-D-glucose (2-DG) were investigated in intact and long-term repeatedly immobilized (LTR, 2 h daily IMO for 41 days) rats. Expression of tyrosine hydroxylase (TH), norepinephrine transporter (NET) and vesicular monoamine transporter (VMAT2) genes were determined by using in situ hybridization histochemistry in brainstem A1, A2, A5 and locus coeruleus (LC) neurons. TH mRNA levels were increased by single IMO or 2-DG administration in all areas studied. Cold was effective only in LC and A2 neurons while insulin had no effect. LTR immobilization elevated TH mRNA levels in all investigated cell groups. These elevations were equally high to those elicited by a single IMO in each noradrenergic group, except the LC where LTR IMO was less effective than the single IMO. The levels of NET and VMAT2 mRNAs were elevated only in the A1 and A2 cell groups of LTR IMO rats. A newly applied IMO in LTR rats did not alter TH, NET, and VMAT2 mRNA levels in any NE cell group investigated. Novel stressors like cold and 2-DG exaggerated the increased TH mRNA levels only in the LC of LTR IMO rats, unlike in the other NE cell groups. The present data indicate that repeated exposure of rats to homotypic stressor induces an adaptation of NE neurons, whereas single exposure of such animals to heterotypic novel stressor produces an exaggerated response of the system at the level of TH (in LC) and NET (in A1, A2) gene expression.

Different effects of insulin and 2-deoxy-D-glucose administration on tyrosine hydroxylase gene expression in the locus coeruleus and the adrenal medulla in rats.

The major brain norepinephrinergic nucleus, locus coeruleus, is an important integrating element of extero- and interoceptive stimuli in organisms facing different physiological challenges. We investigated the effects of single and repeated (seven times) exposure to immobilization stress (120 min daily), insulin (5 IU/kg, i.p. daily) or 2-deoxy-D-glucose (500 mg/kg, i.p. daily) administration on tyrosine hydroxylase (TH) mRNA levels, the rate-limiting enzyme in catecholamine biosynthesis, by in situ hybridization in locus coeruleus and by Northern blot analysis in the adrenal medulla of rats. Both the single and repeated immobilization caused a significant increase in TH mRNA levels in the locus coeruleus (1.5-2-fold; p < 0.05) and in the adrenal medulla (about 4-fold; p < 0.05) when compared with unstressed controls. Hypoglycemia induced by a single or repeated insulin administration led to about fourfold (p < 0.01) elevation in adrenal medullary TH mRNA levels, whereas TH mRNA in locus coeruleus remained unchanged when compared with saline-treated controls. In contrast to the effect of insulin-induced hypoglycemia, cellular glucoprivation caused by a single or repeated 2-deoxy-D-glucose administration significantly elevated TH mRNA levels in both the adrenal medulla (fourfold; p < 0.01) and the locus coeruleus (twofold; p < 0.01). Our data suggest that in contrast to immobilization or cellular glucoprivation caused by 2-deoxy-D-glucose administration, insulin-induced hypoglycemia is not a specific or quantitatively sufficient stimulus for induction of TH gene expression in the locus coeruleus, although all these stressors strongly activate the process in the adrenal medulla.

Chronic blockade of nitric oxide synthesis elevates plasma levels of catecholamines and their metabolites at rest and during stress in rats.

Formation of nitric oxide, and endothelium-derived relaxing factor, can be inhibited by administration of N-nitro-L-arginine methylesther (L-NAME). In the present study, the activity of the sympathoadrenal system in rats with blood pressure (BP) elevation induced by L-NAME was investigated. L-NAME was administered in a dose of 50 mg/kg, i.p. every 12 h for 4 days. Blood samples were collected via chronically inserted arterial catheters in conscious, freely moving rats at rest and during immobilization stress. Plasma epinephrine (EPI), norepinephrine (NE), and dopamine (DA), as well as catecholamine metabolites dihydroxyphenylglycol (DHPG) and dihydroxyphenylacetic acid (DOPAC) were measured by HPLC method. In L-NAME treated animals, which slowed a significant increase in BP, plasma EPI levels were markedly elevated both before and during stress. Plasma NE levels were not significantly increased, however, DHPG levels, which indicate NE turnover and reuptake, were highly elevated. Plasma DA levels were not changed after L-NAME administration but DA metabolite DOPAC showed a significant elevation both under basal conditions and during stress. Thus, the present results indicate that the prolonged blockade of nitric oxide synthesis that causes arterial hypertension is associated with an activation of the sympathoadrenal system.

Features of structural zinc in mammalian alcohol dehydrogenase. Site-directed mutagenesis of the zinc ligands.

All four cysteine ligands to the structural zinc atom of human class-I and class-III alcohol dehydrogenase have been exchanged by site-directed mutagenesis in order to study the importance of the metal in the mammalian enzymes. The cysteine residues were replaced with Ala and Ser, residues that are not able to ligand zinc. All mutations resulted in inactive, unstable enzymes, in contrast to the non-mutated human alcohol dehydrogenases that are easily isolated. Northern-blot analysis revealed the presence of the expected mRNAs from expression plasmids constructed with the different mutated and non-mutated alcohol dehydrogenases, and Western-blot analysis gave faint signals for the mutated recombinant proteins from crude extracts. This verifies that the plasmid constructs are correct, but that the translated, mutated proteins lacking the zinc-stabilized local fold, are subject to rapid degradation. Hence, the results directly illustrate the importance of the structural zinc atom in mammalian alcohol dehydrogenase and confirm it as a component with 'structural' properties. The results are compatible with those from sensitivities to proteases and from the structures of other proteins within the super-family, indicating that the structural role of the zinc atom may involve conservation of interfaces regulating the enzyme quaternary structure.

Expression of Japanese quail ovalbumin in Saccharomyces cerevisiae.

A cDNA sequence coding for Japanese quail ovalbumin was used for the construction of expression plasmid under the ADH1 promoter of the yeast shuttle vector pVT101-U. The resulting recombinant expression vector pJK2 was used for the transformation of Saccharomyces cerevisiae. Expression of quail ovalbumin in yeast cells was demonstrated by Western blotting followed by immunochemical detection.

A rapid and simple method for DNA preparation from Candida utilis.

A method for the extraction of genomic DNA from the industrial yeast Candida utilis is described. The method is rapid, simple and produces DNA that is sufficiently pure for restriction analysis and should be suitable for Southern blotting and the construction of gene libraries.

Inhibition of bacterial DNA-dependent RNA polymerases and restriction endonuclease by UV-absorbing components from propolis.

Several UV-absorbing substances inhibiting the DNA-dependent RNA polymerases of Escherichia coli and Streptomyces aureofaciens, as well as the restriction endonuclease Eco RI have been isolated from the water-soluble extract of Propolis by two-dimensional paper chromatography. The inhibition of bacterial RNA-polymerases by the components of Propolis was probably due to the loss of their ability to bind to DNA. The general characteristic of the UV-absorbing component of Propolis with the most pronounced inhibitory effect upon transcription in vitro is described.