Herbicide Safener-Binding Protein of Maize1 : Purification, Cloning, and Expression of an Encoding cDNA

Dichloroacetamide safeners protect maize (Zea mays L.) against injury from chloroacetanilide and thiocarbamate herbicides. Etiolated maize seedlings have a high-affinity cytosolic-binding site for the safener [3H](R,S)-3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazol-idine ([3H]Saf), and this safener-binding activity (SafBA) is competitively inhibited by the herbicides. The safener-binding protein (SafBP), purified to homogeneity, has a relative molecular weight of 39,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 5.5. Antiserum raised against purified SafBP specifically recognizes a 39-kD protein in etiolated maize and sorghum (Sorghum bicolor L.), which have SafBA, but not in etiolated wheat (Triticum aestivum L.), oat (Avena sativa L.), barley (Hordeum vulgare L.), tobacco (Nicotiana tabacum L.), or Arabidopsis, which lack SafBA. SafBP is most abundant in the coleoptile and scarcest in the leaves, consistent with the distribution of SafBA. SBP1, a cDNA encoding SafBP, was cloned using polymerase chain reaction primers based on purified proteolytic peptides. Extracts of Escherichia coli cells expressing SBP1 have strong [3H]Saf binding, which, like binding to the native maize protein, is competitively inhibited by the safener dichlormid and the herbicides S-ethyl dipropylthiocarbamate, alachlor, and metolachlor. SBP1 is predicted to encode a phenolic O-methyltransferase, but SafBP does not O-methylate catechol or caffeic acid. The acquisition of its encoding gene opens experimental approaches for the evaluation of the role of SafBP in response to the relevant safeners and herbicides.

Synergy between chronic corticosterone and sodium azide treatments in producing a spatial learning deficit and inhibiting cytochrome oxidase activity.

Previously, we developed a rat model of persistent mitochondrial dysfunction based upon the chronic partial inhibition of the mitochondrial enzyme cytochrome oxidase (EC 1.9.3.1). Continuous systemic infusion of sodium azide at approximately 1 mg/kg per hr inhibited cytochrome oxidase activity and produced a spatial learning deficit. In other laboratories, glucocorticoids have been reported to exacerbate neuronal damage from various acute metabolic insults. Therefore, we tested the hypothesis that corticosterone, the primary glucocorticoid in the rat, would potentiate the sodium azide-induced learning deficit. To this end, we first identified nonimpairing doses of sodium azide (approximately 0.75 mg/kg per hr) and corticosterone (100-mg pellet, 3-week sustained-release). We now report that chronic co-administration of these individually nonimpairing treatments produced a severe learning deficit. Moreover, the low dose of corticosterone, which did not elevate serum corticosterone, acted synergistically with sodium azide to inhibit cytochrome oxidase activity. The latter result represents a previously unidentified effect of glucocorticoids that provides a candidate mechanism for glucocorticoid potentiation of neurotoxicity induced by metabolic insult. These results may have the clinical implication of expanding the definition of hypercortisolism in patient populations with compromised oxidative metabolism. Furthermore, they suggest that glucocorticoid treatment may contribute to pathology in disease or trauma conditions that involve metabolic insult.

Rapid acquisition of dendritic spines by visual thalamic neurons after blockade of N-methyl-D-aspartate receptors.

N-Methyl-D-aspartate (NMDA) receptors play an important role in the development of retinal axon arbors in the mammalian lateral geniculate nucleus (LGN). We investigated whether blockade of NMDA receptors in vivo or in vitro affects the dendritic development of LGN neurons during the period that retinogeniculate axons segregate into on-center and off-center sublaminae. Osmotic minipumps containing either the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV) or saline were implanted in ferret kits at postnatal day 14. After 1 week, LGN neurons were intracellularly injected with Lucifer yellow. Infusion of D-APV in vivo led to an increase in the number of branch points and in the density of dendritic spines compared with age-matched normal or saline-treated animals. To examine the time course of spine formation, crystals of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate were placed in the LGN in brain slices from 14- to 18-day-old ferrets. Labeled LGN cell dendrites were imaged on-line in living slices by confocal microscopy, with slices maintained either in normal perfusion medium or with the addition of D-APV or NMDA to the medium. Addition of D-APV in vitro at doses specific for blocking NMDA receptors led to a > 6-fold net increase in spine density compared with control or NMDA-treated slices. Spines appeared within a few hours of NMDA receptor blockade, indicating a rapid local response by LGN cells in the absence of NMDA receptor activation. Thus, activity-dependent structural changes in postsynaptic cells act together with changes in presynaptic arbors to shape projection patterns and specific retinogeniculate connections.

Role of sodium and potassium ions in regulation of glucose metabolism in cultured astroglia.

Effects of increasing extracellular K+ or intracellular Na+ concentrations on glucose metabolism in cultures of rat astroglia and neurons were examined. Cells were incubated in bicarbonate buffer, pH 7.2, containing 2 mM glucose, tracer amounts of [14C]deoxyglucose ([14C]dGlc), and 5.4, 28, or 56 mM KCl for 10, 15, or 30 min, and then for 5 min in [14C]dGlc-free buffer to allow efflux of unmetabolized [14C]dGlc. Cells were then digested and assayed for labeled products, which were shown to consist of 96-98% [14C]deoxyglucose 6-phosphate. Increased K+ concentrations significantly raised [14C]deoxyglucose 6-phosphate accumulation in both neuronal and mixed neuronal-astroglial cultures at 15 and 30 min but did not raise it in astroglial cultures. Veratridine (75 microM), which opens voltage-dependent Na+ channels, significantly raised rates of [14C]dGlc phosphorylation in astroglial cultures (+20%), and these elevations were blocked by either 1 mM ouabain, a specific inhibitor of Na+,K(+)-ATPase (EC 3.6.1.37), or 10 microM tetrodotoxin, which blocks Na+ channels. The carboxylic sodium ionophore, monensin (10 microM), more than doubled [14C]dGlc phosphorylation; this effect was only partially blocked by ouabain and unaffected by tetrodotoxin. L-Glutamate (500 microM) also stimulated [14C]dGlc phosphorylation in astroglia--not through N-methyl-D-aspartate or non-N-methyl-D-aspartate receptor mechanisms but via a Na(+)-dependent glutamate-uptake system. These results indicate that increased uptake of Na+ can stimulate energy metabolism in astroglial cells.

Evolução das doses no ambiente do Reator IEA-R1 e tendências com base nos resultados atuais

O Ipen/Cnen-SP possui um Reator de Pesquisa(IEA-R1) em operação desde 1957. Ele utiliza água leve como blindagem, moderador e como fluido refrigerante, o volume desta piscina é de 273m3. Até 1995 a operação do Reator era descontinua, ou seja, operava diariamente sendo desligado no final do dia, a uma potência de 2,0 MW. A partir daquele ano, após algumas modificações de segurança, o Reator passou a operar de forma continua, ou seja, de segunda-feira a quarta-feira sem ser desligado, totalizando 64 horas semanais. A potência também foi aumentando até 4,5 MW em 2012. Em virtude dessas alterações, a saber, operação contínua e do aumento da potência, as doses dos trabalhadores aumentaram e por isso foram realizados vários estudos para diminui-las. Estudos demonstraram que uma das principais limitações para operação de um reator em potência elevada, provém das radiações gama emitidas pelo sódio-24. Outros elementos como magnésio-27, Alumínio-28, Argônio-51, contribuem de forma considerável para a atividade da água da piscina. A introdução de uma camada de água quente em sua superfície, estável e isenta de elementos radioativos com 1,5m a 2m de espessura constituiria uma blindagem às radiações provenientes dos elementos radioativos dissolvidos na água. Estudos de otimização provaram que a instalação da camada quente não era necessária para o regime e potência atual de operação do Reator, pois outros procedimentos adotados eram mais eficazes. A partir desta decisão o serviço de Proteção Radiológica do Reator IEA-R1, montou um programa de avaliação das doses para certificar-se de que elas se mantinham em valores razoáveis baseados em princípios estabelecidos em normas nacionais e internacionais. O intuito deste trabalho é realizar uma análise das doses individuais dos IOE (Individuo Ocupacionalmente Expostos), considerando as mudanças no regime de operação do Reator e sugerir opções de proteção e segurança, viáveis em primeira instância, para reduzir as doses analisadas, visando se chegar aos níveis de referencia de 3 mSv/ano adotados pela instalação em apreço.

Noxious Weed Management: A Review of Herbicide Use at Colorado State Parks

Weed control in recreation areas is a complex public land management issue. Colorado State Parks are managed for both recreation and conservation, which often conflict. Noxious weeds affect both recreation and conservation efforts, and Parks often use herbicides to control these weeds. While herbicides are effective, they can be harmful to the environment and human health. Herbicide use at State Parks was reviewed to determine if chemical control is effective, safe, and efficient. The results revealed that many Parks are not using effective herbicides to treat noxious weeds and that some weed management plans lack pertinent information on chemical control. The results and recommendations provided can be used to improve the success of noxious weed control and create healthier Parks.

Critical relationship between sodium valproate dose and human teratogenicity: results of the Australian register of anti-epileptic drugs in pregnancy

To compare the incidence of foetal malformations (FMs) in pregnant women with epilepsy treated with different anti-epileptic drugs (AED) and doses, and the influence of seizures, family and personal history, and environmental factors. A prospective, observational, community-based cohort study. Methods. A voluntary, Australia-wide, telephone-interview-based register prospectively enrolling three groups of pregnant women: taking AEDs for epilepsy; with epilepsy not taking AEDs; taking AEDs for a non-epileptic indication. Four hundred and fifty eligible women were enrolled over 40 months. Three hundred and ninety six pregnancies had been completed, with 7 sets of twins, for a total of 403 pregnancy outcomes. Results. 354 (87.8%) pregnancy outcomes resulted in a healthy live birth, 26 (6.5%) had a FM, 4 (1%) a death in utero, 1 (0.2%) a premature labour with stillbirth, 14 (3.5%) a spontaneous abortion and 4 lost to follow-up. The FM rate was greater in pregnancies exposed to sodium valproate (VPA) in the first trimester (116.0%) compared with those exposed to all other AEDs (16.0% vs. 2.4%, P < 0.01) or no AEDs (16.0% vs. 3.1 %, P < 0.01). The mean daily dose of VPA taken in pregnancy with FMs was significantly greater than in those without (11975 vs: 1128 mg, P < 0.01). The incidence of FM with VPA doses greater than or equal to 1100 mg was 30.2% vs. 3.2% with doses < 1100 mg (P < 0.01). Conclusions. There is a dose-effect relationship for FM and exposure to VPA during the first trimester of pregnancy, with higher doses of VPA associated with a significantly greater risk than with lower doses or with other AEDs. These results highlight the need to limit, where possible, the dose of VPA in pregnancy. (C) 2004 Elsevier Ltd. All rights reserved.

Nitric oxide donors inhibit 5-hydroxytryptamine (5-HT) uptake by the human 5-HT transporter (SERT)

1 The aim was to test the hypothesis that nitric oxide ( NO) donor drugs can inhibit the 5-hydroxytryptamine (5-HT) transporter, SERT. 2 The NO donors, MAHMA/NO ( a NONOate; (Z)-1-[N-methyl-N-[6-(N-methylammoniohexyl)amino]]diazen- 1-ium-1,2-diolate), SIN-1 ( a sydnonimine; 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride), FK409 ( an oxime; (+/-)-(4-ethyl-2E-(hydroxyimino)-5-nitro-3E-hexenamide)) and peroxynitrite, but not Angeli's salt ( source of nitroxyl anion) or sodium nitrite, caused concentration-dependent inhibition of the specific uptake of [H-3]- 5-HT in COS-7 cells expressing human SERT. 3 Superoxide dismutase (150 U ml(-1)) plus catalase ( 1200 U ml(-1)), used to remove superoxide and hence prevent peroxynitrite formation, prevented the inhibitory effect of SIN-1 ( which generates superoxide) but not of MAHMA/NO or FK409. 4 The inhibitory effects of the NO donors were not affected by the free radical scavenger, hydroxocobalamin (1 mM) or the guanylate cyclase inhibitor, ODQ (1H-[ 1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one; 3 muM). 5 L-Cysteine ( 1 mM; source of excess thiol residues) abolished or markedly reduced the inhibitory effects of MAHMA/NO, SIN-1, FK409 and peroxynitrite. 6 It is concluded that inhibition of SERT by the NO donors cannot be attributed exclusively to NO free radical nor to nitroxyl anion. It does not involve guanosine-3',5'-cyclic monophosphate, but may involve nitrosation of cysteine residues on the SERT protein. Peroxynitrite mediates the effect of SIN-1, but not the other drugs. 7 Data in mice with hypoxic pulmonary hypertension suggest that SERT inhibitors may attenuate pulmonary vascular remodelling. Thus, NO donors may be useful in pulmonary hypertension, not only as vasodilators, but also because they inhibit SERT, provided they display this effect in vivo at appropriate doses.

Dissolution of porcine incisor pulps in sodium hypochlorite solutions of varying compositions and concentrations

Background: The solubility of dental pulp tissue in sodium hypochlorite has been extensively investigated but results have been inconsistent; due most likely to variations in experimental design, the volume and/or rate of replenishment of the solutions used and the nature of the tissues assessed. Traditionally, the sodium hypochlorite solutions used for endodontic irrigation in Australia have been either Milton or commercial bleach, with Milton being the most common. Recently, a range of Therapeutic Goods Administration (TGA) approved proprietary sodium hypochlorite solutions, which contain surfactant, has become available. Some domestic chlorine bleaches now also contain surfactants. The purpose of this study was to perform new solubility assessments, comparing Milton with new TGA approved products, Hypochlor 1% and Hypochlor 4% forte, and with a domestic bleach containing surfactant (White King). Methods: Ten randomly assigned pulp samples of porcine dental pulp of approximately equal dimensions were immersed in the above solutions, as well as representative concentrations of sodium hydroxide. Time to complete dissolution was measured and assessed statistically. Results: White King 4% showed the shortest dissolution time, closely followed by Hypochlor 4% forte. White King 1% and Hypochlor 1% each took around three times as long to completely dissolve the samples of pulp as their respective 4% concentrations, while Milton took nearly 10 times as long. The sodium hydroxide solutions showed no noticeable dissolution of the pulp samples. Conclusions: The composition and content of sodium hypochlorite solutions had a profound effect on the ability of these solutions to dissolve pulp tissue in vitro. Greater concentrations provided more rapid dissolution of tissue. One per cent solutions with added surfactant and which contained higher concentrations of sodium hydroxide were significantly more effective in dissolution of pulp tissue than Milton.

Herbicide-binding Sites Revealed in the Structure of Plant Acetohydroxyacid Synthase

The sulfonylureas and imidazolinones are potent commercial herbicide families. They are among the most popular choices for farmers worldwide, because they are nontoxic to animals and highly selective. These herbicides inhibit branched-chain amino acid biosynthesis in plants by targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This report describes the 3D structure of Arabidopsis thaliana AHAS in complex with five sulfonylureas (to 2.5 angstrom resolution) and with the imidazolinone, imazaquin (IQ; 2.8 angstrom). Neither class of molecule has a structure that mimics the substrates for the enzyme, but both inhibit by blocking a channel through which access to the active site is gained. The sulfonylureas approach within 5 angstrom of the catalytic center, which is the C2 atom of the cofactor thiamin diphosphate, whereas IQ is at least 7 angstrom from this atom. Ten of the amino acid residues that bind the sulfonylureas also bind IQ. Six additional residues interact only with the sulfonylureas, whereas there are two residues that bind IQ but not the sulfonylureas. Thus, the two classes of inhibitor occupy partially overlapping sites but adopt different modes of binding. The increasing emergence of resistant weeds due to the appearance of mutations that interfere with the inhibition of AHAS is now a worldwide problem. The structures described here provide a rational molecular basis for understanding these mutations, thus allowing more sophisticated AHAS inhibitors to be developed. There is no previously described structure for any plant protein in complex with a commercial herbicide.

mu O-conotoxin MrVIB selectively blocks Na(v)1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits

The tetroclotoxin-resistant voltage-gated sodium channel (VGSC) Na(v)1.8 is expressed predominantly by damage-sensing primary afferent nerves and is important for the development and maintenance of persistent pain states. Here we demonstrate that mu O-conotoxin MrVIB from Conus marmoreus displays substantial selectivity for Na(v)1.8 and inhibits pain behavior in models of persistent pain. In rat sensory neurons, submicromolar concentrations of MrVIB blocked tetroclotoxin-resistant current characteristic of Na(v)1.8 but not Na(v)1.9 or tetroclotoxin-sensitive VGSC currents. MrVIB blocked human Nav1.8 expressed in Xenopus oocytes with selectivity at least 10-fold greater than other VGSCs. In neuropathic and chronic inflammatory pain models, allodynia and hyperalgesia were both reduced by intrathecal infusion of MrVIB (0.03-3 nmol), whereas motor side effects occurred only at 30-fold higher doses. In contrast, the nonselective VGSC blocker lignocaine displayed no selectivity for allodynia and hyperalgesia versus motor side effects. The actions of MrVIB reveal that VGSC antagonists displaying selectivity toward Na(v)1.8 can alleviate chronic pain behavior with a greater therapeutic index than nonselective antagonists.

The in vivo susceptibility of Leishmania donovani to sodium stibogluconate is drug specific and can be reversed by inhibiting glutathione biosynthesis

Resistance to pentavallent antimonial (Sb-v) agents such as sodium stibogluconate (SSG) is creating a major problem in the treatment of visceral leishmaniasis. In the present study the in vivo susceptibilities of Leishmania donovani strains, typed as SSG resistant (strain 200011) or SSG sensitive (strain 200016) on the basis of their responses to a single SSG dose of 300 mg of Sb-v/kg of body weight, to other antileishmanial drugs were determined. In addition, the role of glutathione in SSG resistance was investigated by determining the influence on SSG treatment of concomitant treatment with a nonionic surfactant vesicle formulation of buthionine sulfoximine (BSO), a specific inhibitor of the enzyme gamma-glutamylcysteine synthetase which is involved in glutathione biosynthesis, and SSG, on the efficacy of SSG treatment. L. donovani strains that were SSG resistant (strain 200011) and SSG sensitive (strain 200016) were equally susceptible to in vivo treatment with miltefosine, paromomycin and amphotericin B (Fungizone and AmBisome) formulations. Combined treatment with SSG and vesicular BSO significantly increased the in vivo efficacy of SSG against both the 200011 and the 200016 L. donovani strains. However, joint treatment that included high SSG doses was unexpectedly associated with toxicity. Measurement of glutathione levels in the spleens and livers of treated mice showed that the ability of the combined therapy to inhibit glutathione levels was also dependent on the SSG dose used and that the combined treatment exhibited organ-dependent effects. The SSG resistance exhibited by the L. donovani strains was not associated with cross-resistance to other classes of compounds and could be reversed by treatment with an inhibitor of glutathione biosynthesis, indicating that clinical resistance to antimonial drugs should not affect the antileishmanial efficacies of alternative drugs. In addition, it should be possible to identify a treatment regimen that could reverse antimony resistance.

Novel 5-HT7 ligands as antidepressants:automated synthesis of N-substituted-N-[1-methyl-3-(4-methylpiperidin-1-yl)propyl]-arylsulfon amides

The 5-HT7 receptor is linked with various CNS disorders. Using an automated solution phase synthesis a combinatorial library of 384 N-substituted N-[1-methyl-3-(4-methylpiperidin-1-yl)propyl]-arylsulfonamides was prepared with 24 chemically diverse amines 1-24 and 16 sulfonyl chlorides A-P. The chemical library of alkylated sulfonamides was evaluated in a receptor binding assay with [3]H-5-CT as ligand. The key synthetic step was the alkylation of a sulfonamide with iodide E, which was prepared from butanediol in 4 synthetic steps. The target compounds 1A, 1B .....24A ... 24P were purified by solvent extraction on a Teacan liquid handling system. Sulfonamide J20, B23, D23, G23, G23, J23 , I24 and O24 displayed a binding affinity IC50 between 100 nM and 10 nM. The crystalline J20 (IC50=39 nM) and O24 (IC50=83 nM) were evaluated further in the despair swimming test and the tail suspension assay. A significant antidepressant activity was found in mice of a greater magnitude than imipramine and fluoxetine at low doses. © 2006 Bentham Science Publishers Ltd.

Molecular mechanisms of salt effects on carbon nanotube dispersions in an organic solvent (N-methyl-2-pyrrolidone)

We consider the effects of salt (sodium iodide) on pristine carbon nanotube (CNT) dispersions in an organic solvent, N-methyl-2-pyrrolidone (NMP). We investigate the molecular-scale mechanisms of ion interactions with the nanotube surface and we show how the microscopic ion-surface interactions affect the stability of CNT dispersions in NMP. In our study we use a combination of fully atomistic Molecular Dynamics simulations of sodium and iodide ions at the CNT-NMP interface with direct experiments on the CNT dispersions. In the experiments we analyze the effects of salt on the stability of the dispersions by photoluminescence (PL) and optical absorption spectroscopy of the samples as well as by visual inspection. By fully atomistic Molecular Dynamics simulations we investigate the molecular-scale mechanisms of sodium and iodide ion interactions with the nanotube surface. Our simulations reveal that both ions are depleted from the CNT surface in the CNT-NMP dispersions mainly due to the two reasons: (1) there is a high energy penalty for the ion partial desolvation at the CNT surface; (2) NMP molecules form a dense solvation layer at the CNT surface that prevents ions to come close to the CNT surface. As a result, an increase of the salt concentration increases the "osmotic" stress in the CNT-NMP system and, thus, decreases the stability of the CNT dispersions in NMP. Direct experiments confirm the simulation results: addition of NaI salt into the NMP dispersions of pristine CNTs leads to precipitation of CNTs (bundle formation) even at very small salt concentration (∼10 -3 mol L -1). In line with the simulation predictions, the effect increases with the increase of the salt concentration. Overall, our results show that dissolved salt ions have strong effects on the stability of CNT dispersions. Therefore, it is possible to stimulate the bundle formation in the CNT-NMP dispersions and regulate the overall concentration of nanotubes in the dispersions by changing the NaI concentration in the solvent. © 2012 The Royal Society of Chemistry.