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PubMed1995

Signaling pathways regulating ornithine decarboxylase activity in the embryonic chicken.

The pathways regulating ornithine decarboxylase (ODC) activity in the chick embryo were studied to determine which kinase-signaling pathways regulate ODC activity levels during development. Insulin-dependent tyrosine kinase, protein kinase C and cAMP-dependent protein kinase were activated by the addition of insulin, tetradecanoylphorbol-12,13-acetate, and forskolin, respectively. All three drugs increased ODC activity and forskolin combined with insulin increased ODC activity above the increase caused by either drug alone. These results suggest that all three signaling pathways regulate ODC activity during development and that common intermediates exist among the pathways downstream of the kinases.

Animals↗
PubMed1993

Effect of nerve growth factor and fibroblast growth factor on PC12 cells: inhibition by orthovanadate.

Sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, causes increased levels of tyrosine phosphorylation and blocks, at noncytotoxic concentrations, the differentiative response of rat pheochromocytoma (PC12) cells to beta-nerve growth factor (beta NGF) and basic fibroblast growth factor (bFGF) in a reversible manner. It also prevents growth factor-induced neurite proliferation in primed cells and causes the retraction of previously formed neurites, even in the presence of beta NGF or bFGF. It is equally effective in blocking neurite proliferation by 8-Br-cAMP. Zinc chloride and ammonium molybdate, two other inhibitors of tyrosine phosphatases, also cause parallel decreases in neurite proliferation. Orthovanadate generally reduces the transcription of immediate early response genes (TIS 8 and c-fos) and secondary response genes (ornithine decarboxylase (ODC), acetyl-cholinesterase (AChE) and SCG 10) induced by beta NGF, bFGF, EGF, and PMA, albeit in a variable fashion. There was no observed effect on the kinetics of expression as judged by TIS 8 induction by beta NGF and protein kinase C (PKC) downregulation did not change the levels of inhibition by orthovanadate seen in control cells. Orthovanadate does not affect the production of diacylglycerol induced by beta NGF or bFGF. These observations are consistent with the view that growth factor stimulation of differentiation in PC12 cells involves at least one other PKC independent pathway, and that cAMP and PMA (and their active analogs) activate tyrosine kinases (albeit probably secondarily), which are at least partially responsible for their actions. Although the exact site(s) of action of orthovanadate that lead to the inhibition of growth factor-induced neurite proliferation are unknown, the results presented suggest that it prolongs tyrosine phosphorylations by nonreceptor tyrosine kinases that act downstream from the receptor kinases.

Acetylcholinesterase↗
PubMed1982

Vitamin B6 metabolism in liver and liver-derived tumors.

Vitamin B6 metabolism has been investigated in several highly and well-differentiated Morris hepatomas. Comparisons have been made with two poorly differentiated Morris hepatomas, with host livers obtained from tumor-bearing animals, and with fetal, neonatal, and adult rat liver. The pyridoxal phosphate content and the activities of pyridoxine kinase and pyridoxine phosphate oxidase of all Morris hepatomas examined were significantly less than those in adult host or control livers and generally fell in the range determined for fetal and neonatal liver. A similar pattern was not evident for the activity of pyridoxine phosphate phosphatase. Relative to control and host livers, the activity in hepatomas of the pyridoxal phosphate (PLP)-dependent enzyme, ornithine decarboxylase, was generally elevated. Dexamethasone, at a dose which caused an elevation in the activity of PLP-dependent tumor tyrosine aminotransferase, had no effect on PLP metabolism. The data indicate that tumor progression in the Morris hepatoma spectrum in relation to vitamin b6 metabolism falls into an onco-developmental pattern characterized by a diminished amount of tissue PLP and a diminished capability to metabolize precursor vitamer forms to PLP.

Animals↗
PubMed1962

Decarboxylase inhibitors affect convulsion thresholds to hexafluorodiethyl ether.

Alpha-methyl dihydroxyphenylalanine (alpha-MeDOPA) and alpha-methyl meta-tyrosine (alpha-MMT) do not lower significantly the convulsion thresholds to hexafluorodiethyl ether in the mouse, whereas reserpine and tetrabenazine produce marked lowering of these thresholds. Other workers have shown that alpha-MeDOPA and alpha-MMT decrease brain norepinephrine concentrations much more than brain 5-hydroxytryptamine concentrations, while reserpine and tetrabenazine decrease the brain level of these two amines in a parallel fashion. Thus, the fact that alpha-MeDOPA and alpha-MMT, even in very large doses, do not lower convulsion thresholds and in some cases raise them suggests that the decline in 5-hydroxytryptamine may be implicated in the lowering of convulsion thresholds produced by reserpine and other, similar agents.

Amines↗
PubMed1993

Stimulation of dopamine biosynthesis in cultured PC 12 phaeochromocytoma cells by the coenzyme nicotinamide adeninedinucleotide (NADH).

The activity of the tyrosine hydroxylase, the enzyme which is diminished in the brains of Parkinson patients, has been measured in cultured PC 12 rat phaeochromocytoma cells. In the same way dopamine content in the medium after incubating these cells with or without NADH was assayed. The experiment shows that NADH is able to increase the activity of the tyrosine hydroxylase and dopamine - production in PC 12 cells up to 6 times. The results provide evidence that NADH is able to stimulate dopamine - biosynthesis directly.

Animals↗
PubMed1995

Investigation of intracellular signals mediating the anti-apoptotic action of prolactin in Nb2 lymphoma cells.

Studies were undertaken to identify intracellular mediators of prolactin inhibition of glucocorticoid-induced apoptosis in Nb2 lymphoma cells. A short-term assay was implemented that quantitates fragmented DNA released from the genome by reaction with diphenylamine. Induction and inhibition of internucleosomal DNA cleavage (indicative of apoptosis) was verified by agarose gel electrophoresis of extracted cellular DNA. Synchronized Nb2 cells (G0/G1) exhibited increased DNA fragmentation after 4-hr incubation with dexamethasone (DEX) (25-100 nM) which was inhibited by ovine prolactin (oPRL) (0.1-1 ng/ml), the glucocorticoid receptor antagonist, RU486 (500 nM), and the nuclease inhibitor, aurintricarboxylic acid (100 microM). Signals previously implicated in prolactin induction of mitogenesis in Nb2 cells were investigated for their role in prolactin inhibition of apoptosis including: protein kinase C activation, arachidonic acid metabolism, polyamine production, tyrosine phosphorylation, and extracellular calcium. Protein kinase C agonists, phorbol-12-myristate-13-acetate, and 1,2-dioctanoyl-sn-glycerol, +/- the calcium ionophore, A23187 (200 nM), did not mimic oPRL inhibition of DEX-induced DNA fragmentation. Protein kinase C inhibitors, gossypol and quercetin, did not block prolactin action. Arachidonic acid did not mimic prolactin protection against DEX-induced DNA fragmentation. Inhibitors of arachidonic acid metabolism, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and indomethacin did not block prolactin action. The polyamine, spermine, inhibited DEX-induced DNA fragmentation at 1.5 to 2.5 mM. However, inhibition of polyamine synthesis with alpha-difluoromethyl ornithine or methylglyoxal bis(guanylhydrazone) did not inhibit prolactin action. Prolactin action was not blocked by inhibitors of tyrosine kinase activation, genistein and tyrphostin-47. On the other hand, pervanadate, a potent tyrosine phosphatase inhibitor, consistently inhibited DEX-induced DNA fragmentation. Prolactin action and DEX-induced apoptosis both occurred in calcium-free PBS. In summary, protein kinase C activation and eicosanoid production do not appear to mediate this prolactin action. Although spermine could block DNA fragmentation, blockade of the polyamine cascade did not inhibit prolactin action, suggesting that polyamines do not mediate this prolactin effect. While inhibitors of tyrosine kinase activation did not block prolactin action, tyrosine phosphatase inhibition in the presence of basal tyrosine kinase activity mimicked prolactin action, suggesting tyrosine phosphorylation participation in the anti-apoptotic effect. Extra-cellular calcium was not required for prolactin or DEX action.

Animals↗
PubMed1994

Peripherally administered (6R)-tetrahydrobiopterin increases in vivo tyrosine hydroxylase activity in the striatum measured by microdialysis both in normal mice and in transgenic mice carrying human tyrosine hydroxylase.

The intraperitoneal administration of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4), the natural cofactor of tyrosine hydroxylase (TH), increased the accumulation of L-3,4-dihydroxyphenylalanine (DOPA) measured using microdialysis under the inhibition of aromatic L-amino acid decarboxylase by NSD-1015 (in vivo TH activity) in the striatum both of transgenic mice carrying human TH gene and of non-transgenic mice, to a similar extent by about 4-fold. The results indicate that the peripherally administered 6R-BH4 activates in vivo TH activity in the nigrostriatal dopamine neurons in both non-transgenic and transgenic mice.

Animals↗
PubMed2003

Mesangial cells are able to produce catecholamines in vitro.

Mesangial cells (MC) participate in the control of the glomerular function due to their ability to synthesize hormones and induce cell contraction. Since MC can produce various kinds of hormones, the purpose of the present study was to determine if they are able to synthesize catecholamines. For this evaluation, the levels of norepinephrine, epinephrine, dopamine, and biopterin, the enzymatic cofactor of tyrosine hydroxylase (TH), were analyzed by HPLC in the intracellular compartment and in the medium of primary cultured MC. To identify and locate the enzymes responsible for monoamine synthesis, TH, dopa decarboxylase, and dopamine beta-hydroxylase, Western blotting and immunocytochemistry were employed using monoclonal and polyclonal antibodies. Concentrations of NE = 57 +/- 8, EPI = 82 +/- 10, and DA = 52 +/- 9 pg/mg protein (X +/- SEM) were found in the cell homogenate. The culture medium showed concentrations of NE = 25 +/- 3, EPI = 33 +/- 3, and DA = 62 +/- 15 pg/mg protein. Western blotting analysis and immunocytochemistry evidenced the presence of all enzymes. Moreover, biopterin was also detected in the intracellular compartment and in the medium (0.28 +/- 0.03 and 5.70 +/- 2 nmol/mg cell protein, respectively). Overall, the data indicate that MC have the biosynthetic machinery necessary to produce catecholamines, suggesting that they can act as a paracrine/autocrine hormone system, contributing to the regulation of glomerular hemodynamic and renal microcirculation.

Animals↗
PubMed1981

The site of dopamine formation in rat striatum after L-dopa administration.

Unilateral nigrostriatal lesions produced by injecting 6-hydroxydopamine stereotaxically into both the substantia nigra and the medial forebrain bundle reduced striatal tyrosine hydroxylase activity and dopamine (DA) concentrations by 95% (compared with the intact, contralateral striata) but lowered dopa decarboxylase (DDC) activity by only 80%. L-Dopa administration increased DA concentrations in both lesioned and unlesioned sides; absolute increases were higher in control striata and pretreatment with carbidopa (an inhibitor of peripheral DDC) amplified the increases on both sides. Animals given both of the above lesions plus intrastriatal kainic acid injections exhibited a further reduction in DDC activity, i.e., to only 6% of the activity measured in intact, contralateral striata. Kainic acid lesions alone reduced striatal DDC activity by 20%, without affecting striatal tyrosine hydroxylase activity or DA concentrations, and diminished DA formation from exogenous L-dopa. These observations indicate that DA formation from exogenous L-dopa within the striatum occurs mainly, but not exclusively, within DA terminals. Some DA formation persists after most DA neurons have been destroyed; it may occur within kainic acid-sensitive striatal interneurons or efferent neurons. The DA formed outside DA neurons is apparently able to stimulate postsynaptic DA receptors and to mediate some of the behavioral effects of L-dopa, since L-dopa continued to induce circling behavior in animals with unilateral nigrostriatal lesions, even when these lesions approached totality.

Animals↗
PubMed1995

Synaptic connections and putative functions of the dopaminergic innervation of the guinea pig cochlea.

We report our findings in the guinea pig involving dopamine in postsynaptic regulation of the activity of glutamatergic inner hair cells (IHCs) and in protection of primary auditory neurons during transient ischemia. Seven days after intracochlear perfusion of 6-hydroxydopamine, no immunoreactivity to tyrosine hydroxylase (TH) was demonstrable within the organ of Corti. TH and aromatic amino acid decarboxylase were immunolocalized at an ultrastructural level within lateral olivocochlear varicosities synapsing with radial auditory dendrites postsynaptic to the IHCs. The D2 agonist piribedil induced a dose-dependent decrease in the amplitude of the compound action potential of the auditory nerve. Piribedil also prevented appearance of ischemia-induced swelling of the radial dendrites.

Animals↗
PubMed1991

Effect of the NMDA receptor antagonist, MK-801, on locomotor activity and on the metabolism of dopamine in various brain areas of mice.

Various doses (0.1-0.5 mg/kg i.p.) of the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, produced a dose-dependent increase in well-coordinated locomotor activity of NMRI mice. Higher doses (greater than 0.5 mg/kg) produced a typical motor syndrome characterized by head weaving, body rolling, ataxia and salivation. MK-801, 0.2 mg/kg i.p., a dose which produced marked locomotor stimulation, increased the rate of disappearance of dopamine in the striatum and in the limbic forebrain of the animals, whereas the rate of disappearance of noradrenaline remained unchanged in the limbic forebrain and in the hippocampus. MK-801 increased the rate of tyrosine hydroxylation (measured as the accumulation of 3,4-dihydroxyphenylalanine (DOPA) after inhibition of DOPA decarboxylase) in the striatum with no change in DOPA formation in the limbic forebrain. The levels of 3,4-dihydroxyphenylacetic acid (DOPAC) remained unchanged both in the striatum and in the limbic forebrain following the administration of MK-801. It is concluded that MK-801 may facilitate the activation of dopaminergic mechanisms through an indirect (perhaps by reducing glutamatergic activity) rather than a direct effect on dopamine neurons.

Animals↗
PubMedDate not supplied

Prenatal stress, moderate fetal alcohol, and dopamine system function in rhesus monkeys.

This study examined the striatal dopamine system integrity and associated behavior in 5- to 7-year-old rhesus monkeys born from mothers that experienced stress and/or consumed moderate levels of alcohol during pregnancy. Thirty-one young adult rhesus monkeys were derived from females randomly assigned to one of four groups: (1) control group that consumed isocaloric sucrose solution throughout gestation; (2) stress group that experienced prenatal stress (10-min removal from home cage and exposure to three random loud noise bursts, gestational days 90 through 145); (3) alcohol group that consumed alcohol (0.6 g/kg/day) throughout gestation; or (4) combined alcohol plus stress group that received both treatments. The subjects were assessed for striatal dopamine system function using positron emission tomography (PET), in which the dopamine (DA)-rich striatum was evaluated in separate scans for the trapping of [(18)F]-Fallypride (FAL) and 6-[(18)F]fluoro-m-tyrosine (FMT) to assess dopamine D2 receptor binding potential (BP) and DA synthesis via dopa decarboxylase activity, respectively. Subjects were previously assessed for non-matching-to-sample (NMS) task acquisition, with ratings of behavioral inhibition, stereotypies, and activity made after each NMS testing session. Subjects from prenatal stress conditions (Groups 2 and 4) showed an increase in the ratio of striatal dopamine D2 receptor BP and DA synthesis compared to controls (Group 1). An increase in the radiotracer distribution volume ratios (DVRs), which is used to evaluate the balance between striatal DA synthesis and receptor availability, respectively, was significantly correlated with less behavioral inhibition. The latter supports a hypothesis linking striatal function to behavioral inhibitory control.

Analysis of Variance↗
PubMed1991

Trabulsiella guamensis, a new genus and species of the family Enterobacteriaceae that resembles Salmonella subgroups 4 and 5.

In 1985 the vernacular name Enteric Group 90 was coined for a small group of strains that had been referred to our laboratory as probable strains of Salmonella but did not agglutinate in Salmonella typing antisera. By DNA-DNA hybridization (hydroxyapatite method, 32P), seven strains of Enteric Group 90 were found to be closely related (98 to 100% at 60 degrees C and 94 to 100% at 75 degrees C) to the first strain received (0370-85). The relatedness of Enteric Group 90 to 62 strains of other species of the family Enterobacteriaceae was only 6 to 41%, with the highest values obtained with strains of Salmonella, Kluyvera, Shigella, Klebsiella, Enterobacter, and Citrobacter. We propose a new genus, Trabulsiella, with a single new species, Trabulsiella guamensis, for the highly related group of eight strains formerly known as Enteric Group 90. The type strain is designated ATCC 49490 (CDC 0370-85). T. guamensis strains grew well at 36 degrees C and had positive reactions in the following tests: methyl red, citrate utilization (Simmons) (38% positive at day 1, 88% positive at 2 days), H2S production, lysine decarboxylase, arginine dihydrolase (50% positive at 2 days, 100% positive at 7 days), ornithine decarboxylase, motility, growth in KCN medium, mucate fermentation, acetate utilization, nitrate reduction to nitrite, weak tyrosine hydrolysis (88% positive at 2 days, 100% positive at 7 days), and ONPG (o-nitrophenyl-beta-D-galactopyranoside) test. The strains fermented D-glucose with gas production and fermented L-arabinose, cellobiose, D-galactose, D-galacturonate, maltose, D-mannitol, D-mannose, L-rhamnose, D-sorbitol, trehalose, and D-xylose. T. guamensis strains had negative reactions in the following tests: indole production (13% positive), Voges-Proskauer, urea hydrolysis, phenylalanine deaminase, malonate utilization, lipase (corn oil), DNase, oxidase, pigment production, and acid production from adonitol, D-arabitol, dulcitol, erythritol, myo-inositol, melibiose, alpha-methyl-D-glucoside, raffinose, and sucrose. There were delayed positive reactions for gelatin liquefaction (22 degrees C), which was positive at 12 to 23 days, esculin hydrolysis (13% positive at day 1, 50% positive at 7 days), lactose fermentation (13% positive at 3 to 7 days, 100% positive at 8 to 10 days), glycerol fermentation (88% positive at 7 days), and salicin fermentation (13% positive at day 1, 88% positive at 7 days). All strains were susceptible by the disk diffusion method to colistin, nalidixic acid, gentamicin, streptomycin, kanamycin, chloramphenicol, and trimethoprim-sulfamethoxazole, and most strains were susceptible to sulfadiazine (75% susceptible), tetracycline (88%), and carbenicillin (75%). The strains were resistant to penicillin, cephalothin, and ampicillin. The strains were isolated from vacuum cleaner dust (five strains), soil (one strain), and human feces (two strains). Although T. guamensis can occur in human diarrheal stools, there is no evidence that it actually causes diarrhea. Its main interest to clinical microbiologists may be its possible misidentification as a strain Salmonella.

DNA, Bacterial↗
PubMed1981

Differentiation of fetal mouse hypothalamic cells in serum-free medium.

We show here that cells dissociated from fetal mouse hypothalamus and cerebral hemispheres can be grown in primary culture in a serum-free medium (SFM). We describe several properties of these cultures and compare them to those in serum-supplemented medium (SSM). The SFM used is a modification of that described for neuroblastoma cells: neuronal survival is improved when 17 beta-estradiol is added. Initial events in culture development were similar to those observed in SSM. However, after 1 week, several differences could be noted: in SFM, the proportion of neuron-like cells was increased while the basal glial layer was noticeably reduced, and the neurite network remained less developed than in SSM. These findings demonstrate that the use of SFM permits manipulation of the types and proportions of cells in these primary cultures. This point has been already made. Several neuronal activities were studied. In cultures from both hypothalamus and cerebral hemispheres, thyroliberin (TRH)-immunoreactive cells were visualized by immunohistochemistry, and TRH was radioimmunoassayed in cell extracts and in the medium. In hypothalamic cultures, tyrosine hydroxylase was shown to remain stable for 1 week, and then declined. Glutamic acid decarboxylase disappeared very quickly in vitro, whereas choline acetyltransferase activity increased more rapidly in SFM than in SSM. It is concluded that the use of an SFM for growing normal fetal hypothalamic cells offers a promising model for studying neuroendocrine regulatory mechanisms in culture.

Animals↗
PubMed1985

Production and excretion of dopamine by the isolated perfused rat kidney.

Renal catecholamine concentrations and urinary dopamine excretion from the isolated perfused kidney were measured in intact and peripherally sympathectomized rats. Urinary dopamine excretion was not diminished by sympathectomy, was increased by l-dopa (but not tyrosine or dopamine 4-O-sulphate) in the perfusate and was virtually abolished by prior treatment with the dopa decarboxylase inhibitor, carbidopa. These results confirm the importance of renal extraneuronal dopamine production, from circulating l-dopa, as a contributor to urinary dopamine excretion.

Animals↗
PubMed2003

Induction of diabetes-related autoantibodies below cutoff for "positivity" in young nondiabetic children.

The aim was to study the natural course of diabetes-related autoantibodies at low concentrations, below "positivity", in a nondiabetic population followed up from infancy. Blood samples were taken from 205 children at 6 weeks, 6 months, 18 months, and 5 years of age. Autoantibodies against GAD(65) (GADA), tyrosine phosphatase (IA-2A), and insulin (IAA) were determined by radioligand-binding assays. All children had detectable levels of GADA and approximately half had IA-2A, but only approximately 10% had detectable levels of IAA during the follow-up period. Many children developed IA-2A already at 6 months of age, similar concentrations were seen at 18 months, and then the levels of IA-2A decreased until 5 years of age. GADA were induced less often at 6 months of age, increased up to 18 months, and fluctuated at similar levels up to 5 years of age. IAA were detectable in so few children and at low levels, so no trend in natural course could be revealed. We conclude that there is a natural induction of humoral immune response to beta cell autoantigens early in life. Our results suggest that the mechanisms of beta cell tolerance to GAD and IA-2 differ in healthy children.

Autoantibodies↗
PubMed1973

Stimulation of lysine decarboxylase production in Escherichia coli by amino acids and peptides.

A commercial hydrolysate of casein stimulated production of lysine decarboxylase (EC 4.1.1.18) by Escherichia coli B. Cellulose and gel chromatography of this hydrolysate yielded peptides which were variably effective in this stimulation. Replacement of individual, stimulatory peptides by equivalent amino acids duplicated the enzyme levels attained with those peptides. There was no indication of specific stimulation by any peptide. The peptides were probably taken up by the oligopeptide transport system of E. coli and hydrolyzed intracellularly by peptidases to their constituent amino acids for use in enzyme synthesis. Single omission of amino acids from mixtures was used to screen them for their relative lysine decarboxylase stimulating abilities. Over 100 different mixtures were evaluated in establishing the total amino acid requirements for maximal synthesis of lysine decarboxylase by E. coli B. A mixture containing all of the common amino acids except glutamic acid, aspartic acid, and alanine increased lysine decarboxylase threefold over an equivalent weight of casein hydrolysate. The nine most stimulatory amino acids were methionine, arginine, cystine, leucine, isoleucine, glutamine, threonine, tyrosine, and asparagine. Methionine and arginine quantitatively were the most important. A mixture of these nine was 87% as effective as the complete mixture. Several amino acids were inhibitory at moderate concentrations, and alanine (2.53 mM) was the most effective. Added pyridoxine increased lysine decarboxylase activity 30%, whereas other B vitamins and cyclic adenosine 5'-monophosphate had no effect.

Amino Acids↗