High metal reactivity and environmental risks at a site contaminated by glass waste.

This study addresses the reactivity and risks of metals (Ba, Cd, Co, Cr, Cu, Ni, Pb, Zn, As and Sb) at a Swedish site with large glass waste deposits. Old glassworks sites typically have high total metal concentrations, but as the metals are mainly bound within the glass waste and considered relatively inert, environmental investigations at these kinds of sites are limited. In this study, soil and landfill samples were subjected to a sequential chemical extraction procedure. Data from batch leaching tests and groundwater upstream and downstream of the waste deposits were also interpreted. The sequential extraction revealed that metals in <2 mm soil/waste samples were largely associated with geochemically active fractions, indicating that metals are released from pristine glass and subsequently largely retained in the surrounding soil and/or on secondary mineral coatings on fine glass particles. From the approximately 12,000 m(3) of coarse glass waste at the site, almost 4000 kg of Pb is estimated to have been lost through corrosion, which, however, corresponds to only a small portion of the total amount of Pb in the waste. Metal sorption within the waste deposits or in underlying soil layers is supported by fairly low metal concentrations in groundwater. However, elevated concentrations in downstream groundwater and in leachates of batch leaching tests were observed for several metals, indicating on-going leaching. Taken together, the high metal concentrations in geochemically active forms and the high amounts of as yet uncorroded metal-rich glass, indicate considerable risks to human health and the environment.

Clue to damage recognition by UvrB: residues in the beta-hairpin structure prevent binding to non-damaged DNA.

UvrB, the ultimate damage-recognizing component of bacterial nucleotide excision repair, contains a flexible beta-hairpin rich in hydrophobic residues. We describe the properties of UvrB mutants in which these residues have been mutated. The results show that Y101 and F108 in the tip of the hairpin are important for the strand-separating activity of UvrB, supporting the model that the beta-hairpin inserts between the two DNA strands during the search for DNA damage. Residues Y95 and Y96 at the base of the hairpin have a direct role in damage recognition and are positioned close to the damage in the UvrB-DNA complex. Strikingly, substituting Y92 and Y93 results in a protein that is lethal to the cell. The mutant protein forms pre- incision complexes on non-damaged DNA, indicating that Y92 and Y93 function in damage recognition by preventing UvrB binding to non-damaged sites. We propose a model for damage recognition by UvrB in which, stabilized by the four tyrosines at the base of the hairpin, the damaged nucleotide is flipped out of the DNA helix.

Pharmacokinetics and pharmacodynamics of clevidipine in healthy volunteers after intravenous infusion.

OBJECTIVE: To determine the pharmacokinetics and pharmacodynamics of clevidipine, a new ultrashort-acting calcium antagonist, in healthy male volunteers following a constant rate infusion. METHODS: Eight healthy male volunteers received 1030 nmol x min(-1) of clevidipine together with a tracer dose of 3[H]-clevidipine for 1 h as an i.v. infusion. Frequent venous blood samples and effect recordings were obtained during ongoing infusion and up to 32 h following termination of the infusion. The excretion of radioactivity in urine and faeces was followed for 7 days. RESULTS: A two-compartment model gave the best fit to the individual clevidipine blood levels, resulting in a mean blood clearance of 0.14 (0.03) l x min(-1) x kg(-1) and a mean volume of distribution at steady state of 0.6 (0.1) l x kg(-1). The initial half-life was 1.6 (0.3) min, and the terminal half-life was 15 (5) min. The maximum concentration of the metabolite H 152/81 was reached 2.2 (1.3) min following termination of the infusion. The mean terminal half-life of the inactive primary metabolite was 9.5 (0.8) h and the mean recovery of the radioactive dose reached 83 (3)%. Following termination of the 1 h infusion, the effect on blood pressure (BP) and heart rate was back to pre-dose values within 15 min. CONCLUSION: Clevidipine is a high clearance drug, which is rapidly metabolized to the corresponding inactive acid. The tmax value of the primary metabolite, and a virtually identical value of the initial half-life and the half-life for elimination from the central compartment, indicate that the initial rapid decline of the post-infusion blood levels is mainly due to elimination rather than distribution. The duration of action of clevidipine is short.

Deoxyribonucleotide metabolism in hydroxyurea-resistant V79 hamster cells.

V79 hamster cells were made resistant against hydroxyurea by continuous culture at stepwise increasing drug concentrations. Two cell lines were cloned, resistant to 0.4 mM (V79/H0.4) and 4 mM (V79/H4) hydroxyurea, with a fivefold and a 20-fold increase in soluble ribonucleotide reductase activity. We investigated how the increased amount of enzyme affected the in situ activity of ribonucleotide reductase and deoxyribonucleotide metabolism, in particular substrate cycles between pyrimidine deoxyribonucleosides and their 5'-phosphates. The in situ activity of the reductase was only moderately elevated (1.3-fold in V79/H4 cells). In the fully resistant line, the steady-state level of dATP was increased fourfold, and that of dTTP twofold. These nucleotides are negative allosteric effectors of the reductase and we propose that the increased pools inhibit the enzyme and thereby maintain the in situ activity of the reductase at only a slightly increased level. The surplus deoxyribonucleotides was excreted from the cells as thymidine and deoxycytidine via substrate cycles. The data support and extend our previous model for the regulation of deoxyribonucleotide synthesis via the allosteric properties of ribonucleotide reductase and substrate cycles that link salvage and degradation of deoxyribonucleotides.

Nucleotidase activities in soluble and membrane fractions of three different mammalian cell lines.

Soluble cytoplasmic and membrane fractions were prepared from three cultured mammalian cell lines: 3T3 mouse fibroblasts, V79 hamster lung cells, and human "Cherry" B-lymphoblastoid cells. By using relatively specific nucleotidase assays, together with a phosphotransferase assay, the activities of three different enzymes (low-Km nucleotidase, high-Km nucleotidase, and 5'(3')-nucleotidase) capable of dephosphorylating deoxyribonucleoside 5'-monophosphates were determined in these fractions. The three nucleotidases exist simultaneously in all cell lines, but their relative amounts showed large variations. The 5'(3')-nucleotidase dominated Cherry and 3T3 cells, while in V79 cells equal amounts of this enzyme and the high-Km nucleotidase were recovered. In the membrane fractions, the low-Km nucleotidase was the predominant enzyme. We found no evidence for cell-cycle control of any nucleotidase. We postulated earlier that substrate cycles, involving 5'-nucleotidases and deoxyribonucleoside kinases, provide a mechanism for the regulation of deoxyribonucleotide pools. We suggest that both the low-Km nucleotidase and the 5'(3)-nucleotidase are candidate enzymes for such cycles.

Cytoplasmic 5'(3')-nucleotidase from human placenta.

The 5'(3')-nucleotidase earlier partially purified from rat liver by Fritzson and Smith [1971) Biochim. Biophys. Acta 235, 128-141) was purified 15,000-fold to apparent homogeneity from human placenta. The soluble enzyme is a homodimer with a native molecular mass of 44-45 kDa. It has a pH optimum between 6.0 and 6.5 and is absolutely dependent on Mg2+ ions. The enzyme dephosphorylates certain 2'-, 3'-, and 5'-nucleotides. Km values for 2'- and 3'-nucleotides are around 0.3 mM with no preference for either ribo- or deoxyribonucleotides. 5'-Deoxyribonucleotides are 10-fold better substrates than the corresponding ribonucleotides, with dIMP greater than dUMP greater than dGMP greater than dTMP. dAMP is a poor substrate, dCMP is essentially insert. In all cases the Km values are in the millimolar range. Of the different forms of nucleotidases characterized in animal cells, the 5'(3')-nucleotidase is unique in its preference for 5'-deoxyribonucleotides. In intact cells, a portion of de novo synthesized deoxyribonucleotides is degraded as part of a homeostatic mechanism regulating the size of deoxyribonucleotide pools. This requires the participation of one or several 5'-nucleotidases. The 5'(3')-nucleotidase may be one such enzyme.

Effects of deoxycytidine and thymidine kinase deficiency on substrate cycles between deoxyribonucleosides and their 5'-phosphates.

Substrate cycles constructed from a deoxyribonucleoside kinase and a deoxyribonucleotidase contribute to the metabolism of deoxyribonucleotides in cultured cells. The two enzymes catalyze in opposite directions the irreversible interconversion between a deoxyribonucleoside and its 5'-phosphate. Depending on the balance between the two reactions the net result of the cycle's activity will be synthesis or degradation of the deoxyribonucleotide, and favor import or export of the deoxyribonucleoside. With genetically changed hamster cells (V79 and CHO) deficient in either deoxycytidine or thymidine kinase we now quantify by kinetic isotope flow experiments the contributions of the two kinases to the function of the respective cycles. For each, loss of the relevant kinase was accompanied by an increased degradation of the deoxynucleotide, a slower rate of DNA synthesis, and a longer generation time for the mutant cells. The size of the corresponding deoxyribonucleoside triphosphate pool was apparently not decreased.

Action potential, force and function of the sarcoplasmic reticulum in the anaerobic trout heart.

The electromechanical coupling mechanism was studied in anaerobic heart ventricle strips of rainbow trout which had been electrically stimulated to contraction at 12 contractions/min. The twitch force fell immediately upon the onset of anaerobiosis (5 mM sodium cyanide and N2). The duration and overshoot of the action potential were reduced, the duration only transiently however, since a recovery to the pre-anaerobic level occurred later on. A change of extracellular Ca2+ from 1.25 to 5 mM during anaerobiosis caused a prompt increase in twitch force, whereas the action potential duration and overshoot decreased, the former, again only for a short time. The action potential, measured in 14 mM K+ to block the initial sodium current, was not significantly affected by anaerobiosis, although the accompanying twitch force values were more than halved. The negative effects of anaerobiosis on contractility were not influenced by either ryanodine or caffeine, whereas they were reduced at an elevated extracellular concentration of Ca2+. The results suggest that the regulation of the cytoplasmic Ca activity of the trout heart is well maintained under anaerobic conditions although the contractility is strongly reduced.

Electrical and mechanical activity in heart tissue of flounder and rainbow trout during acidosis.

1. Twitch force and voltage across the sarcolemma were measured in heart tissue of flounder and rainbow trout. 2. For the trout heart, hypercapnia was followed by a loss of force and an action potential prolongation. 3. This was also observed for the flounder heart, but only initially. 4. About 5 min after the onset of hypercapnia, an increase in force and a shortening of the action potential occurred in the flounder heart. 5. After about 30 min of hypercapnia a decrease in force and a prolongation of the action potential slowly appeared. 6. These results can be interpreted in terms of a species-dependent effect of acidosis on the cellular Ca2+ handling and the influence of intracellular Ca2+ on the action potential.

Carbonic anhydrase activity in the blood and the gills of rainbow trout during long-term hypercapnia in hard, bicarbonate-rich freshwater.

Carbonic anhydrase (CA) activities in gills and venous blood, acid-base balance, and haematological variables were studied during environmental hypercapnia in rainbow trout (Salmo gairdneri). Batches of 8-10 fish were exposed to about 3 or 13 mmHg Pco2 in flow-through tests of various duration from 4 h to 80 days. After initial acidosis, blood pH rose above pre-experimental values. At 3 mmHg it became normal again within 21 days, while at 13 mmHg the overshoot lasted for 80 days. In fish acclimated for 3 weeks or more to 13 mmHg Pco2, blood HCO-3 increased four to five times while plasma Cl- levels were lower and K+ higher. Na+ levels did not show any consistent trend associated with exposure to hypercapnia. After an initial acidaemia, Hct, Hb, and RBC remained relatively constant. Patterns of change in CA activity differed between gills and erythrocytes. Initially, blood CA decreased at both Pco2 levels. It then began rising after about 3 weeks and tended to reach pre-experimental values by 80 day's hypercapnia. At 13 mmHg Pco2, gill CA increased to twice the pre-experimental level. Compared with blood CA, gill CA appeared to be more specifically involved in fish acclimation to hypercapnia, which demands an increase in blood bicarbonate to provide a sufficient buffering capacity. Increased CA indicates that the gill enzyme may play a more important role than blood CA in acid-base regulation in fish during hypercapnia.

Affinity chromatography on anti-B1 monoclonal gels for purification and characterization of protein B1 from Escherichia coli ribonucleotide reductase.

Affinity gels were prepared from four monoclonal antibodies against the B1 protein of ribonucleotide reductase of Escherichia coli. The gels were used to purify protein B1 and also to study some of its properties. Gels from the nonneutralizing monoclonal anti-B1-k bound as much as 2 mg of B1/mL and were employed to prepare essentially pure B1 protein in a single step from extracts of wild-type E. coli and strains overproducing the subunit. However, B1 prepared from wild-type extracts had a lowered specific activity, suggesting some denaturation during elution of the protein from the column. Addition of the allosteric effector dATP during affinity chromatography changed the chromatographic pattern. Some protein B2, the second subunit of the reductase, remained in all cases bound to the gels together with B1. The gel prepared from anti-B1-c retained two additional proteins. In other experiments involving binding of proteolytic fragments of B1 to various antibodies, we also found a striking effect of dATP, suggesting that dATP made protein B1 less accessible to proteolysis. In these experiments fragments around 15K still had the ability to bind monoclonals, making possible more detailed investigations of the structural contacts between B1 and the monoclonals.

Mycoplasma pneumoniae infection of intact guinea pig tracheas cultured in a unique matrix-embed/perfusion system.

A new method for the in vitro culture of entire, intact tracheas from adult guinea pigs is described. Matrix-embed/perfusion (MEP) culture is based on an immobilization of the tissue in nutrient agar The tubular piece of agar-embedded organ was contained in a special perfusion block with two wells for liquid medium at either end. When incubated on a rocker platform, liquid medium flows through the trachea and supplies oxygen an nutrients. In this configuration, tracheas maintain near-normal metabolism (ATP content and dehydrogenase activity), structure (as determined by light and electron microscopy), and function (ciliary motion). Tissues could be maintained in vitro in a normal for at least 4 wk, with reduced ciliary motion and cell metabolism detectable for at least 6 wk. Agar-embedded tissues from the MEP cultures were nearly identical to those cultivated with standard tracheal ring explant techniques. Tracheas in the MEP cultures were infected with Mycoplasma pneumoniae. Attachment was neuraminidase-sensitive. Mycoplasma attachment was lowest on the epithelium along the dorsal ridge, but was uniform along the length of the trachea. Ciliostasis and cytonecrosis induced by M. pneumoniae was dose dependent. The matrix-embed/perfuse technique appears to have considerable potential for several types of in vitro studies on trachea or other tubular organs.

Autoradiography on erythrokinesis and multihemoglobins in juvenile Salmo salar L. at various respiratory gas regimes.

The kinetics of erythropoiesis in 5--6 month old Salmo salar L. was correlated to 4 combinations of environmental PO2 and pH/PCO2. Given single doses of 55Fe at the start of continuous flow bioassays, the incorporation was examined in circulating red blood cells (RBC) by autoradiography on blood smears. The proportions of immature, labelled immature and labelled mature erythrocytes were calculated on samples taken at intervals up to 52 days. Compared to control fish kept at a PO2 corresponding to 90% air saturation and pH/PCO2 at 7.6/8 pH unit/mmHg, oxygen depletion to 50% air saturation stimulated the proliferation of RBC stem cells and enhanced RBC maturation. Ceteris paribus, sustained hypercapnia at a PCO2 in a respiratory water raised to 23 mm Hg (pH 7.1--7.2), stimulated proliferation but did not affect the output of mature RBC. Simultaneously lowered PO2 and raised PCO2 to the levels mentioned above obviously stressed the fish, as the effect of lowered PO2 per se was not manifested. Radioactivity was traced to all electrophoretically separable protein fractions from RBC hemolysates. The relevance of blood physiological criteria for probing the fitness or well being of fish in environmental respiratory stress conditions has been discussed.