Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene.

Precorrin-6x reductase, which catalyzes the NADPH-dependent reduction of precorrin-6x to a dihydro derivative named precorrin-6y, was purified 14,300-fold to homogeneity with an 8% yield from extracts of a recombinant strain of Pseudomonas denitrificans. Precorrin-6y was identified by fast atom bombardment-mass spectrometry. It was converted in high yield (90%) to hydrogenobyrinic acid by cell-free protein preparations from P.

Multiple benzodiazepine receptors: no reason for anxiety.

Since the introduction of the benzodiazepines into clinical practice in 1960, these drugs have been widely employed as anxiolytics, sedative/hypnotics and anticonvulsants. In recent years, concern has been expressed about their side-effects, and their use has declined. During this latter period many advances have been made in understanding the molecular mechanisms by which these drugs produce their effects.

Measurement of nerve growth factor-like immunoreactivity in human brain using an anti-mouse-NGF enzyme immunoassay.

Nerve growth factor (NGF) like immunoreactivity, expressed as mouse 2.5S nerve growth factor equivalents, was evaluated in three structures of the human brain post-mortem using a commercially available enzyme immunoassay. Regional differences in NGF-like immunoreactivity were observed.

Pharmacological characterization of RP 62203, a novel 5-hydroxytryptamine 5-HT2 receptor antagonist.

1. RP 62203 (2-[3-(4-(4-fluorophenyl)-piperazinyl)propyl]naphto[1,8- ca]isothiazole-1,1-dioxide) is a novel naphtosultam derivative which shows very high affinity for 5-HT2 receptors in the rat cerebral cortex (Ki = 50.0 pM).

Potassium markedly potentiates the effect of veratridine on dopamine release from rat superfused striatal ribbons.

The effects of veratridine-induced depolarization on [3H]dopamine ([3H]DA) release in the presence of a physiological (5 mM) or a depolarizing (25 mM) concentration of K+ were studied in-vitro in rat superfused striatal ribbons. A combination of the two depolarizing agents induced a marked potentiation in the overflow of [3H]DA, giving an overall 3- to 5-fold increase in veratridine activity. This potentiation was completely antagonized by tetrodotoxin (100 nM).

Comparison of the effects of propylthiouracil, amiodarone, diphenylhydantoin, phenobarbital, and 3-methylcholanthrene on hepatic and renal T4 metabolism and thyroid gland function in rats.

We studied the effects of propylthiouracil (PTU), amiodarone (AMIO), diphenylhydantoin (DPH), phenobarbital (PB), and 3-methylcholanthrene (MC) on thyroid histomorphology, on the hepatic and renal enzymes involved in endogenous and exogenous metabolism, and on the plasma levels and pharmacokinetics of thyroid hormones after 7 and 14 days of treatment. PTU and PB, by decreasing both serum tetraiodothyronine (T4) and triiodothyronine (T3), induced a massive increase in serum thyrotropin (TSH) and thus induced thyroid hypertrophy. AMIO and MC, by decreasing respectively serum T3 and T4, also induced an increase of TSH, but to a lesser extent, not sufficient to induce thyroid hypertrophy.

Purification, cloning, and primary structure of a new enantiomer-selective amidase from a Rhodococcus strain: structural evidence for a conserved genetic coupling with nitrile hydratase.

A new enantiomer-selective amidase active on several 2-aryl propionamides was identified and purified from a newly isolated Rhodococcus strain. The characterized amidase is an apparent homodimer, each molecule of which has an Mr of 48,554; it has a specific activity of 16.5 mumol of S(+)-2-phenylpropionic acid formed per min per mg of enzyme from the racemic amide under our conditions.

Differential effects of potassium channel blockers on dopamine release from rat striatal slices.

The effects of different potassium channel blockers on tritiated dopamine [( 3H]DA) release were investigated in rat striatal slices in the presence of pargyline and nomifensine (10 microM each). 4-Aminopyridine (4-AP; 10 and 30 microM) and 3,4-diaminopyridine (3,4-DAP; 30 microM) markedly increased the basal tritium outflow, whereas tetraethylammonium (TEA; 100-1000 microM) was without effect. The facilitating effect of 4-AP (10 microM) on spontaneous release was Ca(2+)- and K(+)-dependent.

Nicorandil: differential contribution of K+ channel opening and guanylate cyclase stimulation to its vasorelaxant effects on various endothelin-1-contracted arterial preparations. Comparison to aprikalim (RP 52891) and nitroglycerin.

In endothelium-denuded rat aortic rings, the sustained contractile effects produced by endothelin-1 (ET-1; 3.2 nM) were concentration-dependently overcome by nicorandil, aprikalim (RP 52891), a specific K+ channel opener, and nitroglycerin, a stimulant of guanylate cyclase (EC50: 2.55 +/- 0.

Inhibition of eukaryotic DNA topoisomerase I and II activities by indoloquinolinedione derivatives.

With the aim of obtaining new inhibitors of topoisomerases, we have evaluated various heterocyclic quinone derivatives for their ability to induce topoisomerase I (Topo I)- or Topo II-associated DNA breaks, using P388 cell nuclear extract. Several compounds belonging to the indolo[3,2-c]quinoline-1,4-dione series have been shown to possess DNA-cleavage activity. Further analysis using purified Topo I and II preparations has indicated that the members of the series stimulate cleavable complex formation of both Topo I and II.

Purification and partial characterization of Cob(I)alamin adenosyltransferase from Pseudomonas denitrificans.

Cob(I)alamin adenosyltransferase (EC 2.5.1.

Experimental antitumor activity of taxotere (RP 56976, NSC 628503), a taxol analogue.

Taxotere (RP 56976; NSC 628503; N-debenzoyl-N-tert-butoxycarbonyl-10-deacetyl taxol) is a new microtubule stabilizing agent. It is obtained by semisynthesis from a noncytotoxic precursor extracted from the needles of the tree, Taxus baccata L. Taxotere was evaluated for antitumor activity against a variety of transplantable tumors of mice.

Cross tachyphylaxis to endothelin isopeptide-induced hypotension: a phenomenon not seen with proendothelin.

1. In anaesthetized rats, an i.v.

The platelet activating factor receptor antagonist, RP 59227, blocks platelet activating factor receptors mediating liberation of reactive oxygen species in guinea pig macrophages and human polymorphonuclear leukocytes.

In elicited (mineral oil) peritoneal guinea pig macrophages, there was a specific [3H]platelet activating factor (PAF) binding which displayed concentration dependency, saturability, high affinity (Kd = 2.2 +/- 0.2 nM, n = 15), elevated capacity (Bmax = 122,808 +/- 10,234 sites/cell, n = 15) and irreversibility.

Effects of the K+ channel activators, RP 52891, cromakalim and diazoxide, on the plasma insulin level, plasma renin activity and blood pressure in rats.

In normo- or hyperglycemic (i.v. infusion of 50 mg/kg/min glucose over 30 min) pithed rats, diazoxide (1 mg/kg/min i.

Optimum associations of tester strains for maximum detection of mutagenic compounds in the Ames test.

The Ames test is now widely used as a short-term test for the detection of mutagens. Different strains are available with various genetic characteristics, and in the past decade various authors have recommended different associations of strains to give maximum detection potential. However, few studies have been done to compare the sensitivity of individual strains towards a wide range of compounds in a single study.

Determination of 125I-labelled thyroxine glucuronide by reversed-phase high-performance liquid chromatography using on-line radiochemical detection to determine UDPglucuronosyltransferase activity.

A convenient, fast and highly sensitive high-performance liquid chromatographic method, using on-line radiochemical detection, is described for the determination of [125I]thyroxine glucuronide. The method involves direct injection of the supernatant, a total analysis time of 30 min and a detection limit of 1 pmol. The results demonstrate that the method is suitable for the determination of UDPglucuronosyltransferase activity with thyroxine as substrate in native hepatic microsomes.

Pig Leydig cell culture: a useful in vitro test for evaluating the testicular toxicity of compounds.

In vivo studies have shown that the testis is a target organ for drugs and chemicals. In order to evaluate the testicular toxicity of compounds and to identify the mechanisms of their toxicity, we have developed a miniaturized primary culture of immature pig Leydig cells. Five well-known drugs with differing mechanisms of toxicity on testicular functions were tested to validate the model.

SK&F 87516, a close analog of fenoldopam, is a partial agonist at dopamine-1 and alpha-2 receptors and produces stimulation of 5-hydroxytryptamine-2 receptors in the cardiovascular system of the rat.

In pentobarbital-anesthetized rats, SK&F 87516 (1.25-80 micrograms/kg/min intravenously over 15 min), the fluoro analog of the selective DA-1 dopamine receptor agonist fenoldopam produced dose-related decreases in carotid artery blood pressure that faded during the infusion period. These effects were abolished by SCH 23390, prolonged by ritanserin, but unchanged by bilateral vagotomy, atenolol, ICI 118,551, idazoxan, methylatropine or S-sulpiride.

K+ channel opening mediates the vasorelaxant effects of nicorandil in the intact vascular system.

Nicorandil and cromakalim relaxed rat aortic rings denuded of endothelium and precontracted with a low concentration of KCl (25 mM). Glibenclamide (1 microM) strongly antagonized only the effects of cromakalim while those of nicorandil were inhibited by methylene blue, an inhibitor of the soluble form of guanylate cyclase. High concentrations of nicorandil also produced vasorelaxation in aortic preparations contracted with 55 mM KCl, whereas cromakalim did not.

Flow cytometric detection of erythropoietic cytotoxicity in mouse bone marrow.

Erythroblast proliferation and maturation in bone marrow are the processes leading to the formation of polychromatic erythrocytes (PE) and normochromatic erythrocytes (NE), respectively. PE contain RNA but no DNA, and can therefore be distinguished both from NE (which lack both RNA and DNA) and from nucleated cells (which contain both DNA and RNA). Cytotoxic agents that induce impairment of the maturation process change the PE:NE ratio.

Characterization of Peroxidase Secretion and Subcellular Organization of Phanerochaete chrysosporium INA-12 in the Presence of Various Soybean Phospholipid Fractions.

Stimulation of lignin peroxidase production by exogenous phospholipids depends on the composition of the phospholipid fraction prepared by using the Nattermann process. The fraction composed mainly of negatively charged phospholipids (NAT 89) was the most efficient source for exoprotein secretion by Phanerochaete chrysosporium INA-12. The results of biochemical marker assays and ultrastructural morphology determination by electron microscopy were correlated.