Thiocarboxylation of molybdopterin synthase provides evidence for the mechanism of dithiolene formation in metal-binding pterins.

Molybdopterin (MPT) is a pyranopterin with a unique dithiolene group coordinating molybdenum (Mo) or tungsten (W) in all Mo- and W-enzymes except nitrogenase. In Escherichia coli, MPT is formed by incorporation of two sulfur atoms into precursor Z, which is catalyzed by MPT synthase. The recently solved crystal structure of MPT synthase (Rudolph, M. J., Wuebbens, M. M., Rajagopalan, K. V., and Schindelin, H. (2000) Nat. Struct. Biol. 8, 42-46) shows the heterotetrameric nature of the enzyme that is composed of two small (MoaD) and two large subunits (MoaE). According to sequence and structural similarities among MoaD, ubiquitin, and ThiS, a thiocarboxylation of the C terminus of MoaD is proposed that would serve as the source of sulfur that is transferred to precursor Z. Here, we describe the in vitro generation of carboxylated and thiocarboxylated MoaD. Both forms of MoaD are monomeric and are able to form a heterotetrameric complex after coincubation in equimolar ratios with MoaE. Only the thiocarboxylated MPT synthase complex was found to be able to convert precursor Z in vitro to MPT. Slight but significant differences between the carboxylated and the thiocarboxylated MPT synthase can be seen using size exclusion chromatography. A two-step reaction of MPT synthesis is proposed where the dithiolene is generated by two thiocarboxylates derived from a single tetrameric MPT synthase.

Atrial natriuretic peptide responses during anaesthesia in patients with refractory cardiomyopathies.

In patients with congestive heart failure, the release of atrial natriuretic peptide (ANP) is decreased. This study sought to determine the extent of ANP, sympathetic and haemodynamic responses to acutely increased atrial pressure in patients with cardiomyopathies undergoing orthotopic cardiac transplantation. Haemodynamic variables, plasma ANP, norepinephrine, and epinephrine concentrations were measured in 17 patients at five times before and after induction of anaesthesia using either ketamine 1.5 micrograms.kg-1 or sufentanil 3.6 +/- 0.3 micrograms.kg-1. Preinduction values in the ketamine and sufentanil groups were not significantly different. Compared with preinduction values, increases in mean arterial pressure (26%), pulmonary capillary wedge pressure (90%), right atrial pressure (107%), and heart rate (24%) occurred in the ketamine group while cardiac index decreased by 19% (P less than 0.05). Haemodynamic variables in the sufentanil group did not change at any of the times studied. Plasma concentrations of atrial natriuretic peptide were not different within or between treatment groups. Following tracheal intubation plasma norepinephrine levels increased by 116% in the ketamine group (P less than 0.05), but did not change in the sufentanil group. Plasma norepinephrine concentrations differed significantly between the ketamine and sufentanil groups. There were no differences in epinephrine concentrations in either group. Despite the anticipated haemodynamic and catecholamine differences found between the ketamine and sufentanil groups, the levels of plasma ANP were similar. Based upon these results, it is concluded that ANP exerts little influence in the control of fluid volume or blood pressure in patients with refractory cardiomyopathy.

Cardiac transplantation: a prospective comparison of ketamine and sufentanil for anesthetic induction.

The hemodynamic effects of ketamine, 1.5 mg/kg, or sufentanil, 3.4 +/- 0.3 micrograms/kg, were studied prospectively for the anesthetic induction of 20 patients with cardiomyopathies undergoing cardiac transplantation. Plasma epinephrine (EPI), norepinephrine (NE), and sufentanil levels were also obtained. Measurements were taken at various times before induction and following intubation. Following ketamine, progressive increases (P less than 0.05) in mean arterial pressure (28%, MAP), mean pulmonary artery pressure (56%, PAP), central venous pressure (109%, CVP), and pulmonary capillary wedge pressure (84%, PCWP) occurred over time, whereas the cardiac index (CI), stroke volume index (SVI), and stroke work index (SWI) remained unchanged or decreased. The use of sufentanil was associated with no significant changes in MAP, PAP, CVP, PCWP, CI, SVI, or SWI. The heart rate (HR) did not significantly change in either group. Plasma NE significantly increased (31%) in the ketamine group, peaking at 10 minutes; whereas EPI levels did not significantly change in either group. Plasma sufentanil did not reflect the microgram/kg or microgram/BSA administered dose, suggesting individualized distribution kinetics. Since perioperative morbidity and mortality did not differ between groups, both ketamine and sufentanil are acceptable drugs for the anesthetic induction for cardiac transplantation. However, the dissimilar hemodynamic effects caused by ketamine and sufentanil suggest that this conclusion may not be applicable to the patient with a cardiomyopathy undergoing noncardiac surgery.

DNA-cell-binding (DCB) assay for suspected carcinogens and mutagens.

This report describes a novel technique for screening potential carcinogens and mutagens. The DNA-cell-binding (DCB) assay is based on earlier observations which indicated that DNA and other nucleic acids exposed to active carcinogens strongly react with other macromolecules, producing nucleic acid--nucleic acid and nucleic acid--protein adducts. The latter group of adducts included complexes with proteins present in both prokaryotic and eukaryotic cell membranes. Increased attachment of DNA to the intact bacterial and animal cells was seen in the presence of active carcinogens or carcinogens activated by extracts from mouse and rat livers. We have conducted a survey of almost 280 chemicals including 130 with known carcinogenic potential (i.e., either known carcinogens or known non-carcinogens). The DCB test and animal assays agreed in abut 96% of cases. Thus, as a predictor of potential carcinogenicity, this assay compares favorably with other rapid methods currently in use. In this respect, the DCB assay is also superior to other techniques which measure the formation of macromolecular complexes, such as velocity centrifugation through sucrose gradients, gel electrophoresis, filtration through nitrocellulose filters, chromatography on methyl-esterified albumin, equilibrium density gradient centrifugation, etc. In the few cases for which the data were available, combining the results of DCB assays with the results of experiments in which the induction of DNA--protein adducts in living human cells in tissue culture has been measured by cold phenol extraction, the predictability was increased to 100%. We suggest that DCB assay should be used either alone or in combination with other rapid methods of carcinogen detection for screening industrial, environmental and other chemicals and chemical mixtures for their carcinogenic potential. Ways of further improving and simplifying the DCB tests are considered.

Effect of dietary methionine on the biopotency of selenite and selenomethionine in the rat.

The effect of dietary methionine (Met) on the Biopotency of selenium (Se) from selenite and selenomethionine (Se-Met) was studied in rats fed a 30% torula yeast-based diet containing 0.24% Met and less than 0.02 ppm Se. Biopotency was quantitated by assaying liver, plasma and heart for the glutathione peroxidase (GSH-Px) increase elicited by a given level of Se fed for 1 week to Se-deficient rats. At dietary Se levels up to 0.5 ppm Se, the level of dietary Met supplementation did not alter selenite biopotency. In contrast, dietary Met supplementation increased the biopotency of Se-Met. With basal Met intakes, the biopotency of Se-Met was 25% that of selenite below 0.5 ppm Se. The addition of 0.4% Met to the diet made Se-Met biopotency equivalent to selenite biopotency in one experiment, but in a second experiment with younger, faster growing rats, 0.4% Met did not completely restore the biopotency of Se-Met. These results indicate that low dietary Met decreases the biopotency of Se-Met but not of selenite. Altered Se metabolism at suboptimal dietary Met may occur because more Se-Met is incorporated into protein and thus less Se is available for GSH-Px synthesis. These results suggest that adequate dietary Met is required for optimal utilization of the Se in feedstuffs of plant origin, as Se-Met is presumably a major form of plant selenium.