Isolation and characterization of a MADS-box type gene from apple (Malusx domestica).
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Amino Acid Sequence↗
Biomedical subjects
R Dreesen
Publications and source records attributed to R Dreesen.
Quantitative cDNA-AFLP analysis for genome-wide expression studies.
An improved cDNA-AFLP method for genome-wide expression analysis has been developed. We demonstrate that this method is an efficient tool for quantitative transcript profiling and a valid alternative to microarrays. Unique transcript tags, generated from reverse-transcribed messenger RNA by restriction enzymes, were screened through a series of selective PCR amplifications. Based on in silico analysis, an enzyme combination was chosen that ensures that at least 60% of all the mRNAs were represented by an informative sequence tag. The sensitivity and specificity of the method allows one to detect poorly expressed genes and distinguish between homologous sequences. Accurate gene expression profiles were determined by quantitative analysis of band intensities, and subtle differences in transcriptional activity were revealed. A detailed screen for cell cycle-modulated genes in tobacco demonstrates the usefulness of the technology for genome-wide expression analysis.
Base Sequence↗
Leafy gall formation is controlled by fasR, an AraC-type regulatory gene in Rhodococcus fascians.
Rhodococcus fascians can interact with many plant species and induce the formation of either leafy galls or fasciations. To provoke symptoms, R. fascians strain D188 requires pathogenicity genes that are located on a linear plasmid, pFiD188. The fas genes are essential for virulence and constitute an operon that encodes, among other functions, a cytokinin synthase gene. Expression of the fas genes is induced by extracts of infected plant tissue only. We have isolated an AraC-type regulatory gene, fasR, located on pFiD188, which is indispensable for pathogenesis and for fas gene expression. The combined results of our experiments show that in vitro expression of the fas genes in a defined medium is strictly regulated and that several environmental factors (pH, carbon and nitrogen sources, phosphate and oxygen content, and cell density) and regulatory proteins are involved. We further show that expression of the fas genes is controlled at both the transcriptional and the translational levels. The complex expression pattern probably reflects the necessity of integrating a multitude of signals and underlines the importance of the fas operon in the pathogenicity of R. fascians.
Amino Acid Sequence↗
Tetrahydrobiopterin and total biopterin content of neuroblastoma (N1E-115, N2A) and pheochromocytoma (PC-12) clones and the dependence of catecholamine synthesis on tetrahydrobiopterin concentration in PC-12 cells.
Tetrahydrobiopterin content was determined in several clonal cell lines by reversed-phase HPLC and subsequent electrochemical detection. The same chromatography system was used to determine the total biopterin (tetrahydrobiopterin and 7,8-dihydrobiopterin) by fluorescence detection. The catecholamine-producing clones neuroblastoma N1E-115 and pheochromocytoma PC-12 contained 96 and 60 ng tetrahydrobiopterin/mg protein, respectively. The corresponding amount for the neuroblastoma clone N2A was 36 ng/mg protein. The tetrahydrobiopterin content in C-6 glioma cells was below the limit of detection. The total biopterin is about 20% above the tetrahydrobiopterin content. Tetrahydrobiopterin and biopterin from the cells were identified by coelution with standard solutions and by potential-current relationship or emission and excitation spectra, respectively. Addition of 2,4-diamino-6-hydroxypyrimidine, an inhibitor of biopterin synthesis from GTP, to the culture medium of PC-12 cells resulted in a dose-dependent decrease of tetrahydrobiopterin and total biopterin content within 4 h, suggesting that the cells are capable of synthesising the biopterin which was found. A decrease in intracellular tetrahydrobiopterin levels by different concentrations of 2,4-diamino-6-hydroxypyrimidine reduces the cellular production of dihydroxyphenylalanine after inhibition of aromatic L-amino acid decarboxylase, indicating that the concentration of tetrahydrobiopterin might be a limiting factor for catecholamine synthesis in catecholamine-producing cells.
Adrenal Gland Neoplasms↗
Mouse neuroblastoma clone N1E-115: a suitable model for studying the action of dopamine agonists of tyrosine hydroxylase activity.
The DOPA-content in neuroblastoma clone N1E-115 is higher than the dopamine or noradrenaline content. Blockade of tyrosine hydroxylase by alpha-methyl-p-tyrosine (1 X 10(-3) M) resulted in a decrease of cellular DOPA-content to 24.9% after 4 hr. The accumulation of DOPA in these cells which is probably due to limited activity of l-aromatic amino acid decarboxylase led us to use DOPA-content as a measure of tyrosine hydroxylase (TH) activity. Dopamine and especially apomorphine were effective at low concentrations (dopamine IC50 1 X 10(-5) M, apomorphine 2 X 10(-7) M); lisuride had no effect on TH-activity. The low effective dose of apomorphine and the failure of lisuride to influence TH-activity are comparable to the observations in striatal synaptosomal preparations and make the N1E-115 clone a suitable model for studying the mechanism of TH-regulation. However, since haloperidol (1 X 10(-5) M) did not reverse the apomorphine-induced blockade of TH, a receptor-mediated blockade of TH seems to be improbable.
Animals↗
Dopa-release from mouse neuroblastoma clone N 1 E-115 into the culture medium. A test for tyrosine hydroxylase activity.
Due to a lack of L-Dopa-decarboxylase, the mouse neuroblastoma clone N 1 E-115 contains a high intracellular Dopa-content compared to a low noradrenaline- and dopamine-content. Because of this decarboxylase deficiency, the N 1 E-115 clone releases more than 95% of the produced Dopa into the culture medium. After renewal of the culture medium, Dopa production of the cells can be measured by the increase of Dopa in the medium. Dopa production was linear during 2 h and varied from 50-180 micrograms/mg prot-1 x h-1 between different subcultures. Dopa release into the medium was used as an indirect measure for the tyrosine-hydroxylase activity. Several dopaminergic agonist and antagonists were tested. Dopa production could be blocked dose-dependent by apomorphine (1 X 10(-7)-1 x 10(-6) M), but not by lisuride hydrogen maleate and bromocryptine. Several dopaminergic and adrenergic antagonists failed to reverse the apomorphine induced blockade of the tyrosine-hydroxylase activity.
Animals↗
Determination of L-erythro-tetrahydrobiopterin in biological tissues by high pressure liquid chromatography and electrochemical detection.
L-erythro-Tetrahydrobiopterin (H4-biopterin) was determined in rat tissue (liver, striatum, brain stem) by reversed phase high pressure liquid chromatography and subsequent electrochemical detection. Due to the low oxidation potential (+ 250 mV) necessary for the analysis of H4 biopterin, the electrochemical detector is a highly selective system for this compound. For analysis of H4 biopterin in biological tissues, only one extraction step with 0.2M perchloric acid and 10mM ascorbic acid is necessary prior to analysis. The H4 biopterin content of rat tissue determined with this method corresponds well with the values reported elsewhere.
Animals↗
Determination of reduced biopterins by high pressure liquid chromatography and subsequent electrochemical detection.
L-erythro-Tetrahydrobiopterin plays an important role as the cofactor of various hydroxylation reactions. We report here the separation of biopterin and its reduced forms by reversed-phase high pressure liquid chromatography. Subsequent electrochemical detection yields a sensitivity at the picomole level for dihydrobiopterin and tetrahydrobiopterin.
Biopterins↗
Action of lisuride on reserpine-induced muscular rigidity in rats after local application into the striatum, ventricular space or the spinal subarachnoid space.
Muscular rigidity was induced in rats by reserpine (10 mg/kg) and the tonic activity of the gastrocnemic muscle was recorded in the electromyogram. Systemic administration of lisuride, an ergoline, resulted in a dose-dependent depression of rigidity. To examine the site of action of lisuride, we injected lisuride into the striatum, the ventricular space, and the spinal subarachnoid space of intact reserpinized rats. Lisuride is effective if injected into either the striatum or the spinal subarachnoid space, the spinal effect being more pronounced. These results suggest that the spinal cord is an important site of action of lisuride.
Animals↗
Dopamine effects on the microcirculation and veins of the skin after local application and their changes by antagonistic drugs.
Dopamine causes reflex erythema, central blanching, and piloerection depending on the dose and the type of application wheal reaction. Intracutaneous application shows from 1.5 gamma/0.1 ml wheal formation, erythema, piloerection, and blanching combined with increased heat radiation from the skin surface (AGA thermovision). Epicutaneous application from 500 ml (occlusive patch test) following horny layer stripping, causes marked blanching with weak piloerection. Iontophoretic application of dopamine 1/1,000 (60 s, 0.5 mA) causes only blanching and weak surrounding erythema; application of dopamine 1/100 additionally causes piloerection. This application shows no changing of infrared radiation. Iontophoretic application of dopamine 1/100 or 50 gamma/0.1 ml i.c. in a blanched area after locally applied corticosteroids (McKenzie test) shows diminution of infrared radiation proved by AGA thermovision thermography. Antihistaminics, applied externally, decrease reddening, wheal development as well as blanching by dopamine. Guanethedine (1% in eucerin) increases the blanching phenomenon (false transmitter effect of dopamine). Phentolamine 1% in W/O emulsion is without effect on dopamine reaction. Caffeine ointment (4%) reduces erythema and accentuates the degree of blanching. Oral haloperidol has no influence on the dopamine skin reaction, but increases the blanching in areas of antihistamine treatment. Skin veins and varices show marked vasoconstriction within 10 min after iontophoretic (1 : 100, 3.5 mA, 60 s) or i.c. application (50 gamma/0.2 ml).
Adult↗