Chloroplastic glutamine synthetase from normal and water stressed safflower (Carthamus tinctorius L.) leaves

The chloroplastic isoform of glutamine synthetase (GS(2), EC 6.3.1.2) from normal and water stressed safflower (Carthamus tinctorius L. cv.A-300) leaves has been purified to apparent electrophoretic homogeneity by a procedure involving anion-exchange, hydrophobic and size-exclusion chromatography followed by electroelution of the protein from preparative polyacrylamide gels. The observed molecular weight of the native protein varied from 305-330 kDa depending on the sizing column employed. The native protein is composed of 44 kDa subunits. Under conditions of saturating ammonium and at ATP levels of 0.1-10 mM, double-reciprocal plots with respect to glutamate are biphasic and concave downward at high concentrations of the varied substrate for normal enzyme but are linear for enzyme from water-stressed plants. Under subsaturating ATP levels, K-Glu is over 18-fold lower for enzyme from stressed leaves. The K-m, (ATP) varies with Mg2+ levels in the assay mixture. Double-reciprocal plots of initial velocity with respect to ATP at changing fixed levels of NH4+ are linear for normal enzyme but are curved upwards for enzyme from stressed leaves. Initial velocity data of 1/v vs. 1/ammonium for the enzyme from both the sources are non-linear (curved upwards) when ATP is saturating. At subsaturating ATP levels, the data are linear for normal enzyme but are still non-linear for the enzyme from stressed leaves. The results obtained suggest positively cooperative binding of NH4+ A V-max(/2) value of 3.6 mM for Mg2+ was obtained at 5 mM ATP. The isoelectric point of the native protein from normal and stressed leaves was determined to be, respectively, 5.6 and 6.1. The mixed competitive and competitive inhibitors, methionine sulfoximine and ADP and K-i values of 0.086 mM (0.017 for the enzyme from stressed leaves) and 2.15 mM (1.70 for the enzyme from stressed leaves), respectively. Enzyme from stressed leaves is not inhibited by 5 mM proline. The observed kinetic constants of GS(2) from normal and water stressed safflower seedlings are discussed in relation to the known water-stress tolerance of this crop plant.

Cross-Hetero-Dehydrogenative Coupling Reaction of Phosphites: A Catalytic Metal-Free Phosphorylation of Amines and Alcohols

Phosphorylation of amines, alcohols, and sulfoximines are accomplished using molecular iodine as a catalyst and H2O2 as the sole oxidant under mild reaction conditions. This method provides an easy route for synthesizing a variety of phosphoramidates, phosphorus triesters and sulfoximine-derived phosphoramidates which are of biological importance.

谷氨酰胺合成酶在固氮鱼腥藻固氮调节中的作用

在MSX(methlonine sulfoximine,谷氨酰胺合成酶的不可逆抑制剂)存在下,固氮鱼腥藻(Anabaena azotica)所分泌的氨量和谷氨酰胺合成酶(GS)活力有较好的负相关性,证明谷氨酰胺合成酶-谷氨酸合成酶(GS-GOGAT)是固氮鱼腥藻氨同化的重要途径。在蛋白质合成受到氯霉素拟制时,NH_4~+对固氮酶的失活是迅速的,同时GS活力有较大下降,表明NH_4~+的调控酶的失活或降解。在氮固定条件下,固氮酶活力半衰期小于4小时,GS活力半衰期大于10小时,则GS并不是固氮酶的正调节因子

路易斯碱催化还原胺化反应研究

胺及其衍生物是很多重要生物活性分子的结构单元，是合成天然产物和手性药物的重要中间体。 直接还原胺化由于其合成步骤简单而成为制备二级胺和三级胺的简便方法。为了发展一种较为简便的直接还原胺化反应，我们把研究的重点放在开发一种简便实用的有机小分子催化方法上。由文献调研可知，现已报道的直接还原胺化方法大多是催化醛或酮与一级胺或者脂肪二级胺的直接还原胺化，而醛或酮与芳香二级胺的直接还原胺化却尚无报道。在本文中，我们发现用简单的四甲基乙二胺(TEMED)在室温下以二氯甲烷为溶剂即可催化三氯氢硅对酮和芳香二级胺之间的直接还原胺化反应，并取得了高达92%的收率。该反应条件温和，底物普适性广，各种类型的酮均可以与芳香二级胺进行直接还原胺化，并且得到比较满意的收率。 同时，我们从手性Sulfoximine出发，设计和合成了一系列的Sulfoximine类新衍生物，并将其应用于间接还原胺化反应中。遗憾的是我们并没有得到预期的不对称催化效果。 Amines and their derivatives are basic structural motifs in natural products and pharmaceuticals and highly versatile building blocks for various organic substrates. Direct reductive amination (DRA) is a convenient method for the preparation of secondary and tertiary amines owing to its operational simplicity. In an effort to develop a simple and convenient procedure for direct reductive amination reaction, we focused our study on search for a mild and efficient organocatalytic system. In the literature, there are many reports concerning DRA between aldehydes or ketones and either primary amines or secondary aliphatic amines. But there are no reports concerning DRA between aldehydes or ketones and secondary aromatic amines. In this study, we have developed a highly practical method for the synthesis of tertiary amines by the direct reductive amination of ketones and secondary aromatic amines with tetramethylethylenediamine (TEMED) as the catalyst using HSiCl3 as the reducing agent in dichloromethane (affording up to 92% yield). This method can be carried out under mild conditions and is compatible with many functional groups. A variety of ketones were efficiently aminated with secondary aromatic amines to afford the corresponding amines in good to excellent yields. Starting from chiral sulfoximine, we designed and synthesized a series of new sulfoximine derivatives and tested their efficiencies as asymmetric organocatalysts for the reduction of imines, which, unfortunately, only exhibited low catalytic activity and enantioselectivity.

Simultaneous and ultrarapid determination of reactive oxygen species and reduced glutathione in apoptotic leukemia cells by microchip electrophoresis

A microchip electrophoresis method coupled with laser-induced fluorescence (LIF) detection was established for simultaneous determination of two kinds of intracellular signaling molecules (reactive oxygen species, ROS, and reduced glutathione, GSH) related to apoptosis and oxidative stress. As the probe dihydrorhodamine-123 (DHR123) can be converted intracellularly by ROS to the fluorescent rhodamine-123 (Rh123), and the probe naphthalene-2,3-dicarboxaldehyde (NDA) can react quickly with GSH to produce a fluorescent adduct, rapid determination of Rh-123 and GSH was achieved on a glass microchip within 27 s using a 20 mm borate buffer (pH 9.2). The established method was tested to measure the intracellular ROS and GSH levels in acute promyelocytic leukemia (APL)-derived NB4 cells. An elevation of intracellular ROS and depletion of GSH were observed in apoptotic N134 cells induced by arsenic trioxide (AS(2)O(3)) at low concentration (1-2 mu m). Buthionine sulfoximine (BSO), in combination with AS(2)O(3) enhanced the decrease of reduced GSH to a great extent. The combined treatment of AS(2)O(3) and hydrogen peroxide (H2O2) led to an inverse relationship between the concentrations of ROS and GSH obtained, showing the proposed method can readily evaluate the generation of ROS, which occurs simultaneously with the consumption of the inherent antioxidant.

Genome-wide hydroxymethylcytosine pattern changes in response to oxidative stress

The TET enzymes convert methylcytosine to the newly discovered base hydroxymethylcytosine. While recent reports suggest that TETs may play a role in response to oxidative stress, this role remains uncertain, and results lack in vivo models. Here we show a global decrease of hydroxymethylcytosine in cells treated with buthionine sulfoximine, and in mice depleted for the major antioxidant enzymes GPx1 and 2. Furthermore, genome-wide profiling revealed differentially hydroxymethylated regions in coding genes, and intriguingly in microRNA genes, both involved in response to oxidative stress. These results thus suggest a profound effect of in vivo oxidative stress on the global hydroxymethylome.

Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants

A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.

Is differential phytochelatin production related to decreased arsenate influx in arsenate tolerant Holcus lanatus?

Phytochelatins (PCs) are required for arsenic (As) detoxification in nontolerant plants. In addition, a role for PCs in arsenate tolerance has recently been proven, with tolerant plants able to accumulate significantly higher concentrations of As-PC complexes at equivalent levels of stress than nontolerant plants. The relationship between arsenate influx and PC production in tolerant and non-tolerant Holcus lanatus plants was determined in this study, along with an investigation of the effect of inhibition of PC synthesis by buthionine sulfoximine (BSO) on arsenate tolerance. A strong correlation between PC production and arsenate influx was demonstrated in arsenate-tolerant plants. In addition, inhibition of PC synthesis by BSO in tolerant plants increased arsenate sensitivity to that of the nontolerant clone. This dramatic reduction in tolerance proves that PC production is an essential component of the arsenate tolerance mechanism in H. lanatus. This study proposes that while there is a single major gene for arsenate tolerance, hypostatic modifiers are also in operation, affecting the expression of the tolerance character. © New Phytologist (2002).

Efeito modulador da glutationa na liberação de gaba induzida por glutamato em retinas de embrião de galinha

O ácido γ-aminobutírico (GABA) e o glutamato são, respectivamente, os principais neurotransmissores inibitório e excitatório no Sistema Nervoso Central (SNC) e são fundamentais para o processamento visual. Estudos revelam que o glutamato induz liberação de GABA na retina. Trabalhos prévios também apontam que compostos tióis regulam a liberação de GABA, mas ainda não são totalmente esclarecidos os efeitos de tióis (-SH) sobre os níveis endógenos deste neurotransmissor na retina. Neste intermédio, a glutationa (GSH) além de ser o mais importante dos compostos tióis, vem demonstrando exercer um papel neuromodulador na liberação de neurotransmissores. Desta forma, o objetivo deste trabalho foi avaliar um possível efeito modulador de GSH sobre a liberação de GABA mediada por glutamato em retinas de embrião de galinha. Para isso, utilizamos como modelo experimental tecido retiniano íntegro de embrião de galinha, com sete ou oito dias de desenvolvimento. Nos ensaios de liberação de GABA, as retinas foram tratadas com GSH (100 e 500 μM); glutamato (50 e 500 μM) e Butionina Sulfoximina (BSO), inibidor da síntese de glutationa, (50 μM) por 15 minutos, e os níveis de GABA liberado para o meio extracelular foram quantificados por Cromatografia Líquida de Alta Eficácia (CLAE). Para experimentos de liberação de compostos tióis (–SH), as retinas foram incubadas com glutamato (100 μM) com ou sem Na+ por 15 minutos, e os seus níveis extracelulares foram determinados pela reação com DTNB e quantificados por espectrofotometria (412 nm). Os resultados revelam que o glutamato, assim como GSH, liberam GABA. Nossos dados também demonstram que BSO atenua a liberação de GABA promovida por glutamato. Além disso, demonstramos que glutamato induz liberação de compostos tióis independentemente de sódio. Sendo assim, é sabido que glutamato é capaz de liberar GABA e tióis; dentre estes, GSH é o mais abundante e responsável por também liberar GABA. Sabe-se também que uma vez inibida a síntese de GSH por BSO, a liberação de GABA induzida por glutamato é atenuada. Então, se sugere uma possível modulação de GSH na liberação de GABA induzida por glutamato, em retinas íntegras de embrião de galinha.

1,4-Diamino-2-butanone, a putrescine analogue, promotes redox imbalance in Trypanosoma cruzi and mammalian cells

The putrescine analogue 1,4-diamino-2-butanone (DAB) is highly toxic to various microorganisms, including Trypanosoma cruzi. Similar to other a-aminocarbonyl metabolites. DAB exhibits pro-oxidant properties. DAB undergoes metal-catalyzed oxidation yielding H2O2, NH4+ ion, and a highly toxic alpha-oxoaldehyde. In vitro. DAB decreases mammalian cell viability associated with changes in redox balance. Here, we aim to clarify the DAB pro-oxidant effects on trypomastigotes and on intracellular T. cruzi amastigotes. DAB (0.05-5 mM) exposure in trypomastigotes, the infective stage of T. cruzi, leads to a decline in parasite viability (IC50 c.a. 0.2 mM DAB; 4 h incubation), changes in morphology, thiol redox imbalance, and increased TcSOD activity. Medium supplementation with catalase (2.5 mu M) protects trypomastigotes against DAB toxicity, while host cell invasion by trypomastigotes is hampered by DAB. Additionally, intracellular amastigotes are susceptible to DAB toxicity. Furthermore, pre-treatment with 100-500 mu M buthionine sulfoximine (BSO) of LLC-MK2 potentiates DAB cytotoxicity, whereas 5 mM N-acetyl-cysteine (NAC) protects cells from oxidative stress. Together, these data support the hypothesis that redox imbalance contributes to DAB cytotoxicity in both T. cruzi and mammalian host cells. (C) 2012 Elsevier Inc. All rights reserved.

Fructose-1,6-bisphosphate preserves intracellular glutathione and protects cortical neurons against oxidative stress

Fructose-1,6-bisphosphate (FBP), an endogenous intermediate of glycolysis, protects the brain against ischemia-reperfusion injury. The mechanisms of FBP protection after cerebral ischemia are not well understood. The current study was undertaken to determine whether FBP protects primary neurons against hypoxia and oxidative stress by preserving reduced glutathione (GSH). Cultures of pure cortical neurons were subjected to oxygen deprivation, a donor of nitric oxide and superoxide radicals (3-morpholinosydnonimine), an inhibitor of glutathione synthesis (L-buthionine-sulfoximine) or glutathione reductase (1,3-bis(2-chloroethyl)-1-nitrosourea) in the presence or absence of FBP (3.5 mM). Neuronal viability was determined using an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. FBP protected neurons against hypoxia-reoxygenation and oxidative stress under conditions of compromised GSH metabolism. The efficacy of FBP depended on duration of hypoxia and was associated with higher intracellular GSH concentration, an effect partly mediated via increased glutathione reductase activity.

The antioxidant glutathione in the fish cell lines EPC and BCF-2: response to model pro-oxidants as measured by three different fluorescent dyes

Reduced glutathione (GSH) protects cells against injury by oxidative stress and maintains a range of vital functions. In vitro cell cultures have been used as experimental models to study the role of GSH in chemical toxicity in mammals; however, this approach has been rarely used with fish cells to date. The present study aimed to evaluate sensitivity and specificity of three fluorescent dyes for measuring pro-oxidant-induced changes of GSH contents in fish cell lines: monochlorobimane (mBCl), 5-chloromethylfluorescein diacetate (CMFDA) and 7-amino-4-chloromethylcoumarin (CMAC-blue). Two cell lines were studied, the EPC line established from a skin tumour of carp Cyprinus carpio, and BF-2 cells established from fins of bluegill sunfish Lepomis macrochirus. The cells were exposed for 6 and 24 h to low cytotoxic concentrations of pro-oxidants including hydrogen peroxide, paraquat (PQ), copper and the GSH synthesis inhibitor, L-buthionine-SR-sulfoximine (BSO). The results indicate moderate differences in the GSH response between EPC and BF-2 cells, but distinct differences in the magnitude of the GSH response for the four pro-oxidants. Further, the choice of GSH dye can critically affect the results, with CMFDA appearing to be less specific for GSH than mBCl and CMAC-blue.

The cellular and molecular involvement of glutathione in cisplatin-induced apoptosis

The goal of this study was to investigate the cellular and molecular mechanisms by which glutathione (GSH) is involved in the process of apoptosis induced by cisplatin [cis-diamminedichloroplatinum(II), cis-DDP] in the HL60 human promyelocytic leukemia cell line. The data show that during the onset or induction of apoptosis, GSH levels in cisplatin-treated cells increased 50% compared to control cells. The increase in intracellular GSH was associated with enhanced expression of γ-glutamylcysteine synthetase (γ-GCS), the enzyme that catalyzes the rate- limiting step in the biosynthesis of glutathione. After depletion of intracellular GSH with D,L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of γ-GCS, biochemical and morphological analysis revealed that the mechanism of cell death had switched from apoptosis to necrosis. In contrast, when intracellular GSH was elevated by exposure of cells to a GSH-ethyl-ester and then treatment with cisplatin, no change in the induction and kinetics of apoptosis were observed. However, when cells were exposed to cisplatin before intracellular GSH levels were increased, apoptosis was observed to occur 6 hours earlier compared to cells without GSH elevation. To further examine the molecular aspects of these effects of GSH on the apoptotic process, changes in the expression of bcl-2 and bax, were investigated in cells with depleted and elevated GSH. Using reverse transcription polymerase chain reaction, no significant change in the expression of bcl-2 gene transcripts was observed in cells in either the GSH depleted or elevated state; however, a 75% reduction in GSH resulted in a 40% decrease in the expression of bax gene transcripts. In contrast, a 6-fold increase in GSH increased the expression of bax by 3-fold relative to controls. Similar results were obtained for bax gene expression and protein synthesis by northern analysis and immunoprecipitation, respectively. These results suggest that GSH serves a dual role in the apoptotic process. The first role which is indirect, involves the protection of the cell from extensive damage following exposure to a specific toxicant so as to prevent death by necrosis, possibly by interacting with the DNA damaging agent and/or its active metabolites. The second role involves a direct involvement of GSH in the apoptotic process that includes upregulation of bax expression. ^

Increasing the Glutathione Content in a Chilling-Sensitive Maize Genotype Using Safeners Increased Protection against Chilling-Induced Injury

With the aim of analyzing their protective function against chilling-induced injury, the pools of glutathione and its precursors, cysteine (Cys) and gamma -glutamyl-Cys, were increased in the chilling-sensitive maize (Zea mays) inbred line Penjalinan using a combination of two herbicide safeners. Compared with the controls, the greatest increase in the pool size of the three thiols was detected in the shoots and roots when both safeners were applied at a concentration of 5 muM. This combination increased the relative protection from chilling from 50% to 75%. It is interesting that this increase in the total glutathione (TG) level was accompanied by a rise in glutathione reductase (GR; EC 1.6.4.2) activity. When the most effective safener combination was applied simultaneously with increasing concentrations of buthionine sulfoximine, a specific inhibitor of glutathione synthesis, the total gamma -glutamyl-Cys and TG contents and GR activity were decreased to very low levels and relative protection was lowered from 75% to 44%. During chilling, the ratio of reduced to oxidized thiols first decreased independently of the treatments, but increased again to the initial value in safener-treated seedlings after 7 d at 5 degreesC. Taking all results together resulted in a linear relationship between TG and GR and a biphasic relationship between relative protection and GR or TG, thus demonstrating the relevance of the glutathione levels in protecting maize against chilling-induced injury.

Evaluation of cell responses toward adhesives with different photoinitiating systems

OBJECTIVES The photoinitiator diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) is more reactive than a camphorquinone/amine (CQ) system, and TPO-based adhesives obtained a higher degree of conversion (DC) with fewer leached monomers. The hypothesis tested here is that a TPO-based adhesive is less toxic than a CQ-based adhesive. METHODS A CQ-based adhesive (SBU-CQ) (Scotchbond Universal, 3M ESPE) and its experimental counterpart with TPO (SBU-TPO) were tested for cytotoxicity in human pulp-derived cells (tHPC). Oxidative stress was analyzed by the generation of reactive oxygen species (ROS) and by the expression of antioxidant enzymes. A dentin barrier test (DBT) was used to evaluate cell viability in simulated clinical circumstances. RESULTS Unpolymerized SBU-TPO was significantly more toxic than SBU-CQ after a 24h exposure, and TPO alone (EC50=0.06mM) was more cytotoxic than CQ (EC50=0.88mM), EDMAB (EC50=0.68mM) or CQ/EDMAB (EC50=0.50mM). Cultures preincubated with BSO (l-buthionine sulfoximine), an inhibitor of glutathione synthesis, indicated a minor role of glutathione in cytotoxic responses toward the adhesives. Although the generation of ROS was not detected, a differential expression of enzymatic antioxidants revealed that cells exposed to unpolymerized SBU-TPO or SBU-CQ are subject to oxidative stress. Polymerized SBU-TPO was more cytotoxic than SBU-CQ under specific experimental conditions only, but no cytotoxicity was detected in a DBT with a 200μm dentin barrier. SIGNIFICANCE Not only DC and monomer-release determine the biocompatibility of adhesives, but also the cytotoxicity of the (photo-)initiator should be taken into account. Addition of TPO rendered a universal adhesive more toxic compared to CQ; however, this effect could be annulled by a thin dentin barrier.