Thermostable Pyrococcus furiosus DNA ligase catalyzes the synthesis of (di)nucleoside polyphosphates

DNA ligase from the hyperthermophilic marine archaeon Pyrococcus furiosus (Pfu DNA ligase) synthesizes adenosine 5'-tetraphosphate (p4A) and dinucleoside polyphosphates by displacement of the adenosine 5'-monophosphate (AMP) from the Pfu DNA ligase-AMP (E-AMP) complex with tripolyphosphate (P3), nucleoside triphosphates (NTP), or nucleoside diphosphates (NDP). The experiments were performed in the presence of 1-2 microM [alpha-32P]ATP and millimolar concentrations of NTP or NDP. Relative rates of synthesis (%) of the following adenosine(5')tetraphospho(5')nucleosides (Ap4N) were observed: Ap4guanosine (Ap4G) (from GTP, 100); Ap4deoxythymidine (Ap4dT) (from dTTP, 95); Ap4xanthosine (Ap4X) (from XTP, 94); Ap4deoxycytidine (Ap4dC) (from dCTP, 64); Ap4cytidine (Ap4C) (from CTP, 60); Ap4deoxyguanosine (Ap4dG) (from dGTP, 58); Ap4uridine (Ap4U) (from UTP, <3). The relative rate of synthesis (%) of adenosine(5')triphospho(5')nucleosides (Ap3N) were: Ap3guanosine (Ap3G) (from GDP, 100); Ap3xanthosine (Ap3X) (from XDP, 110); Ap3cytidine (Ap3C) (from CDP, 42); Ap3adenosine (Ap3A) (from ADP, <1). In general, the rate of synthesis of Ap4N was double that of the corresponding Ap3N. The enzyme presented optimum activity at a pH value of 7.2-7.5, in the presence of 4 mM Mg2+, and at 70 degrees C. The apparent Km values for ATP and GTP in the synthesis of Ap4G were about 0.001 and 0.4mM, respectively, lower values than those described for other DNA or RNA ligases. Pfu DNA ligase is used in the ligase chain reaction (LCR) and some of the reactions here reported [in particular the synthesis of Ap4adenosine (Ap4A)] could take place during the course of that reaction.     

Postnatal maturation in nitric oxide-induced pulmonary artery relaxation involving cyclooxygenase-1 activity

The maturation in the vasodilator response to nitric oxide (NO) in isolated intrapulmonary arteries was analyzed in newborns and 15- to 20-day-old piglets. The vasodilator responses to NO gas but not to the NO donor sodium nitroprusside increased with age. The inhibitory effects of the superoxide dismutase inhibitor diethyldithiocarbamate and xanthine oxidase plus hypoxanthine and the potentiation induced by superoxide dismutase and MnCl(2) of NO-induced vasodilatation were similar in the two age groups. Diphenyleneiodonium (NADPH oxidase inhibitor) potentiated the response to NO, and this effect was more pronounced in the older animals. The nonselective cyclooxygenase inhibitors indomethacin and meclofenamate and the preferential cyclooxygenase-1 inhibitor aspirin augmented NO-induced relaxation specifically in newborns, whereas the selective cycloxygenase-2 inhibitor NS-398 had no effect. The expressions of alpha-actin, cycloxygenase-1, and cycloxygenase-2 proteins were similar, whereas Cu,Zn-superoxide dismutase decreased with age. Therefore, the present data suggest that the maturational increase in the vasodilatation of NO in the pulmonary arteries during the first days of extrauterine life involves a cycloxygenase-dependent inhibition of neonatal NO activity.     

Heavy metals and arsenic uptake by wild vegetation in the Guadiamar river area after the toxic spill of the Aznalcóllar mine

On 25 April 1998, approximately 4.5 hm(3) of pyritic sludge, containing 5000 mg of As kg(-1) among other pollutants, was spilled into the Agrio and Guadiamar rivers and the surrounding agricultural areas (Aznalcóllar, Seville, Southern Spain). Many trace metals such as Pb, Cu, Zn, Cd, Tl, Sb and As reached the Do?ana National Park, the largest wetland area in Europe, affecting soils, different plant and animal species. In order to recuperate the affected lands by employing plants capable of accumulating high levels of contaminants in shoots, periodical field surveys have been made to identify the metal-tolerant species that are spontaneously growing in the polluted soils, and are able to uptake one or various of the contaminants. Among the 99 different plant species studied, Anchusa azurea, Beta vulgaris, Chamaemelum fuscatum, Convolvulus arvensis, Cynodon dactylon, Diplotaxis virgata, Erodium aethiopicum, Lavatera cretica, Malva nicaeensis, Silybum marianum and, above all, Amaranthus blitoides highlight as the most promising to be used in the remediation of the affected area.     

Effect of pre-adsorbed proteins on attachment,proliferation, and function of endothelial cells

As certain proteins control cell adhesion, it has been hoped that cell transplantation and tissue engineering could be augmented by pre-adsorption of specific proteins to biological or synthetic surfaces. The questions that remain, however, are whether such proteins can affect cell production as well as adhesion, and if so, whether in a protein-specific manner. We examined the adhesion and the biochemical secretion of bovine aortic endothelial cells (BAEC) on tissue culture polystyrene (TCPS) discs coated with fibronectin (Fn), laminin (Ln), or gelatin. The three coating proteins nonspecifically promote sub-confluent and post-confluent endothelial cell production of total protein up to 2.5-fold of the reference value. Total soluble glycosaminoglycan (GAG) production slightly increased with the different coatings only at low cell density. In contrast, Ln and Fn, not gelatin, drastically enhanced post-confluent BAEC production of prostaglandin (PGI2). However, antibody-blockage of the alpha5 integrin, constituent of the Fn receptor in BAEC, appeared to inhibit the upregulation of PGI2 production observed on Fn-coated surfaces. The results indicate that the cell adhesion mediators used as coating agents dictate cell biological production as well as adhesion and proliferation.     

Changes in mitochondrial electron partitioning in response to herbicides inhibiting branched-chain amino acid biosynthesis in soybean

The adaptation of the respiratory metabolism in roots of soybean (Glycine max L. Merr. cv Ransom) treated with herbicides that inhibit the enzyme acetolactate synthase (ALS) was analyzed. A new gas phase dual-inlet mass spectrometry system for simultaneous measurement of 34O2 to 32O2 and O2 to N2 ratios has been developed. This system is more accurate than previously described systems, allows measurements of much smaller oxygen gradients, and, as a consequence, works with tissues that have lower respiration rates. ALS inhibition caused an increase of the alternative oxidase (AOX) protein and an accumulation of pyruvate. The combination of these two effects is likely to induce the activation of the alternative pathway and its participation in the total respiration. Moreover, the start of the alternative pathway activation and the increase of AOX protein were before the decline in the activity of cytochrome pathway. The possible role of AOX under ALS inhibition is discussed.     

Influence of maternal stress on uranium-induced developmental toxicity in rats

It has been demonstrated that uranium is an embryo/fetal toxicant when given orally or subcutaneously to pregnant mice. On the other hand, maternal stress has been shown to enhance the developmental toxicity of a number of metals. In this study, maternal toxicity and developmental effects of a concurrent exposure to uranyl acetate dihydrate (UAD) and restraint stress were evaluated in rats. Four groups of pregnant animals were given subcutaneous injections of UAD at 0.415 and 0.830 mg/kg/day on Days 6 to 15 of gestation. Animals in two of these groups were also subjected to restraint for 2 hr/day during the same gestational days. Control groups included restrained and unrestrained pregnant rats not exposed to UAD. Cesarean sections were performed on gestation Day 20, and the fetuses were weighed and examined for malformations and variations. Maternal toxicity and embryotoxicity were noted at 0.830 mg/kg/day of UAD, while fetotoxicity was evidenced at 0.415 and 0.830 mg/kg/day of UAD by significant reductions in fetal body weight and increases in the total number of skeletally affected fetuses. No teratogenic effects were noted in any group. Maternal restraint enhanced uranium-induced embryo/fetal toxicity only at 0.830 mg/kg/day, a dose that was also significantly toxic to the dams. As in previous studies with other metals, maternal stress enhances uranium-induced developmental toxicity at uranium doses that are highly toxic to the dams; however, at doses that are less acutely toxic the role of maternal stress would not be significant.     

PF 9601N [N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine], a new MAO-B inhibitor,attenuates MPTP-induced depletion of striatal dopamine levels in C57/BL6 mice

Monoamine oxidase isoform B (MAO-B) is involved in Parkinson's disease (PD) induced by the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxin (MPTP) in human and non-human-primate. MAO-B inhibitors, such as L-deprenyl have shown to prevent against MPTP-toxicity in different species, and it has been used in Parkinson therapy, however, the fact that it is metabolized to (-)-methamphetamine and (-)-amphetamine highlights the need to find out new MAO-B inhibitors without a structural amphetaminic moiety. In this context we herein report, for the first time, anywhere a novel non-amphetamine-like MAO-B inhibitor, PF 9601N, N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine. This attenuates the MPTP-induced striatal dopamine depletion in young-adult and adult-old C57/BL mice, using different schedules of administration, and which behave "ex vivo" as a slightly more potent and selective MAO-B inhibitor than L-deprenyl, assayed for comparative purposes in the same experimental conditions. The MAO-B ID(50) values were calculated from the total MAO-B activity measured against [14C] phenylethylamine (22 microM) as substrate, at each inhibitor concentration. The MAO-B ID(50) values resulted to be 381 and 577 nmol/kg for PF 9601N and L-deprenyl, respectively. The intraperitoneally (i.p.) co-administration to young-adult C57/BL6 mice of MPTP (30 mg/kg), with different concentrations of PF 9601N or L-deprenyl (29.5-0.357 micromol/kg) showed a dose-dependent protective effect against striatal dopamine depletion, measuring the dopamine contents and its metabolites by HPLC. The ED(50) value proved to be 3.07 micromol/kg without any significant differences between either MAO-B inhibitor. Nevertheless, lower doses of PF 9601N (1.5 micromol/kg) were necessary to get almost total protection, without any change in the DOPAC and HVA content, when administered 2 h before MPTP (30 mg/kg), whereas partial protection (45%) against dopamine depletion was observed in the case of L-deprenyl. In both cases, MAO-B inhibition was a necessary condition in order to observe the protective effect. When adult-old (8-10 months) C57/BL6 mice were used, MPTP (25 mg/kg) administration induced 25 days later, an irreversible dopamine depletion. In these conditions, chronic administration with 0.15 micromol/kg of PF 9601N, before the toxin, every 24 h for 10 days, rendered almost total protection of dopamine depletion, whereas L-deprenyl yielded only 50% protection of the dopamine content, assayed in the same conditions. It is worth remarking, that in both cases MAO-B was not affected. From these results, it can be concluded that PF 9601N attenuates MPTP neurotoxicity "in vivo" better than L-deprenyl through different mechanisms, with special relevance to the protective effect, independent of MAO-B inhibition, observed in the irreversibly MPTP-lesioned adult-old mice. Therefore, this novel non-amphetamine MAO-B inhibitor could be potentially effective in PD therapy.