Changes in nitrogen content and protein profiles following <Emphasis Type="Italic">in vitro</Emphasis> selection of NaCl resistant mung bean and tomato

Mung bean and tomato were in vitro selected on media containing 0, 25, 50, 100 and 150 mM NaCl. Two types of media (hormone supplemented media, CB and hormone free media, MS) were used for mung bean using cotyledon explants whereas two types of explants (cotyledons and shoot apices) were used for tomato on MS media. Total-N, protein content, nitrite reductase (NiR) activity and protein protein profiles were checked in selected plants and compared to original non selected ones. NaCl at low concentrations slightly increased total-N in shoots and roots of in vitro selected mung bean and tomato whereas higher concentrations induced significant reductions. Similar increases in protein content were detected at lower concentrations with no significant effects thereover. On the contrary, NaCl gradually inhibited NiR activity. Similar responses of total-N, protein and NiR activity, but with greater magnitudes, were detected in original plants. In addition, NaCl significantly reduced dry weights of shoots and roots of either in vitro selected or, in particular, original intact plants. Moreover, electrophoresis (SDS-PAGE) of protein from shoots of either in vitro selected or intact plants showed that NaCl induced new protein bands while some others were concomitantly disappeared. The induction of one or more of the 86.4, 79, 77.6, 77 and 71.5 kDa bands following in vitro selection and/or the disappearance of the 86 kDa band from intact plants seemed necessary for mung bean resistance. Also, the presence of 86.2 kDa band and/or the loss of the 85.8 and 57.5 kDa bands might be included in tomato resistance. Of these induced bands in mung bean selected on CB media, only two bands were detected in plants selected on MS media. In tomato, two bands lost following selection from cotyledons but only one band lost following selection from shoot apices. These changes in protein pattern therefore might serve as adaptive regulators for resistance to NaCl.     

Dissimilatory Oxidation and Reduction of Elemental Sulfur in Thermophilic Archaea

The oxidation and reduction of elemental sulfur and reduced inorganic sulfur species are some of the most important energy-yielding reactions for microorganisms living in volcanic hot springs, solfataras, and submarine hydrothermal vents, including both heterotrophic, mixotrophic, and chemolithoautotrophic, carbon dioxide-fixing species. Elemental sulfur is the electron donor in aerobic archaea like Acidianus and Sulfolobus. It is oxidized via sulfite and thiosulfate in a pathway involving both soluble and membrane-bound enzymes. This pathway was recently found to be coupled to the aerobic respiratory chain, eliciting a link between sulfur oxidation and oxygen reduction at the level of the respiratory heme copper oxidase. In contrast, elemental sulfur is the electron acceptor in a short electron transport chain consisting of a membrane-bound hydrogenase and a sulfur reductase in (facultatively) anaerobic chemolithotrophic archaea Acidianus and Pyrodictium species. It is also the electron acceptor in organoheterotrophic anaerobic species like Pyrococcus and Thermococcus, however, an electron transport chain has not been described as yet. The current knowledge on the composition and properties of the aerobic and anaerobic pathways of dissimilatory elemental sulfur metabolism in thermophilic archaea is summarized in this contribution.     

Coenzyme Q10 inhibits mitochondrial complex-1 down-regulation and nuclear factor-kappa B activation

We have used control-homozygous weaver mutant, and -heterozygous weaver mutant mice in order to explore the basic molecular mechanism of neurodegeneration and the neuroprotective potential of coenzyme Q(10). Homozygous weaver mutant mice exhibited progressive neurodegeneration in the hippocampus, striatum, and cerebellum, and a reduction in the striatal levels of dopamine and coenzyme Qs (Q(9) and Q(10)) without any significant changes in norepinephrine and serotonin. Mitochondrial complex-1 was down regulated; whereas nuclear factor-kappa B was up regulated in homozygous weaver mutant mice. Rotenone inhibited complex-1, enhanced nuclear factor-kappa B, and caused apoptosis in human dopaminergic (SK-N-SH) neurons; whereas nuclear factor-kappa B antibody suppressed rotenone-induced apoptosis, suggesting that enhancing coenzyme Q(10) synthesis and suppressing the induction of NF-kappa B, may provide neuroprotection.     

A path from primary protein sequence to ligand recognition

A novel method to organize protein structural information based solely on sequence is presented. The method clusters proteins into families that correlate with the three-dimensional protein structure and the conformation of the bound ligands. This procedure was applied to nicotinamide adenine dinucleotide [NAD(P)]-utilizing enzymes to identify a total of 94 sequence families, 53 of which are structurally characterized. Each of the structurally characterized proteins within a sequence family correlates to a single protein fold and to a common bound conformation of NAD(P). A wide range of structural folds is identified that recognize NAD(P), including Rossmann folds and beta/alpha barrels. The defined sequence families can be used to identify the type and prevalence of NAD(P)-utilizing enzymes in the proteomes of sequenced organisms. The proteome of Mycobacterium tuberculosis was mined to generate a proteome-wide profile of NAD(P)-utilizing enzymes coded by this organism. This enzyme family comprises approximately 6% of the open reading frames, with the largest subgroup being the Rossmann fold, short-chain dehydrogenases. The preponderance of short-chain dehydrogenases correlates strongly with the phenotype of M. tuberculosis, which is characterized as having one of the most complex prokaryotic cell walls.     

Pyruvate:NADP+ oxidoreductase is stabilized by its cofactor,thiamin pyrophosphate,in mitochondria of Euglena gracilis

Pyruvate:NADP(+) oxidoreductase (PNO) is a thiamin pyrophosphate (TPP)-dependent enzyme that plays a central role in the respiratory metabolism of Euglena gracilis, which requires thiamin for growth. When thiamin was depleted in Euglena cells, PNO protein level was greatly reduced, but its mRNA level was barely changed. In addition, a large part of PNO occurred as an apoenzyme lacking TPP in the deficient cells. The PNO protein level increased rapidly, without changes in the mRNA level, after supplementation of thiamin into its deficient cells. In the deficient cells, in contrast to the sufficient ones, a steep decrease in the PNO protein level was induced when the cells were incubated with cycloheximide. Immunofluorescence microscopy indicated that most of the PNO localized in the mitochondria in either the sufficient or the deficient cells. These findings suggest that PNO is readily degraded when TPP is not provided in mitochondria, and consequently the PNO protein level is greatly reduced by thiamin deficiency in E. gracilis.     

Crystal structure of a NO-forming nitrite reductase mutant: an analog of a transition state in enzymatic reaction

I257E was obtained by site directed mutagenesis of nitrite reductase from Achromobacter cycloclastes. The mutant has no enzyme activity. Its crystal structure determined at 1.65A resolution shows that the side-chain carboxyl group of the mutated residue, Glu257, coordinates with the type 2 copper in the mutant and blocks the contact between the type 2 copper and its solvent channel, indicating that the accessibility of the type 2 copper is essential for maintaining the activity of nitrite reductase. The carboxylate is an analog of the substrate, nitrite, but the distances between the type 2 copper and the two oxygen atoms of the side-chain carboxyl group are reversed in comparison to the binding of nitrite to the native enzyme. In the mutant, both the type 2 copper and the N epsilon atom on the imidazole ring of its coordinated residue His135 move in the substrate binding direction relative to the native enzyme. In addition, an EPR study showed that the type 2 copper in the mutant is in a reduced state. We propose that mutant I257E is in a state corresponding to a transition state in the enzymatic reaction.     

Control of biogenic H(2)S production with nitrite and molybdate

The effects of the metabolic inhibitors, sodium nitrite and ammonium molybdate, on production of H₂S by a pure culture of the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6 and a consortium of SRB, enriched from produced water of a Canadian oil field, were investigated. Addition of 0.1 mM nitrite or 0.024 mM molybdate at the start of growth prevented the production of H₂S by strain Lac6. With exponentially growing cultures, higher levels of inhibitors, 0.25 mM nitrite or 0.095 mM molybdate, were required to suppress the production of H₂S. Simultaneous addition of nitrite and molybdate had a synergistic effect: at time 0, 0.05 mM nitrite and 0.01 mM molybdate, whereas during the exponential phase, 0.1 mM nitrite and 0.047 mM molybdate were sufficient to stop H₂S production. With an exponentially growing consortium of SRB, enriched from produced water of the Coleville oil field, much higher levels of inhibitors, 4 mM nitrite or 0.47 mM molybdate, were needed to stop the production of H₂S. The addition of these inhibitors had no effect on the composition of the microbial community, as shown by reverse sample genome probing. The results indicate that the efficiency of inhibitors in containment of SRB depends on the composition and metabolic state of the microbial community. Journal of Industrial Microbiology & Biotechnology (2001) 26, 350–355. Received 02 August 2000/ Accepted in revised form 17 April 2001     

Measurement of ATP production and respiratory chain enzyme activities in mitochondria isolated from small muscle biopsy samples

A set of methods suitable for assessment of respiratory chain function in mitochondria isolated from 25mg of muscle is described. This set of methods includes determination of the mitochondrial ATP production rate (MAPR) and the activities of the respiratory chain complexes I, I+III, II+III, and IV and citrate synthase. MAPR is determined with an optimized version of a luminometric method previously described. The optimized method measures 50-220% higher activities than the original method. The highest MAPRs are recorded using the substrate combinations glutamate+succinate and N,N,N(1),N(1)-tetramethyl-1,4-phenyldiamine+ascorbate. The respiratory chain complex activities are determined with standard spectrophotometric methods, adapted to an automated photometer. The sensitivity in the determination of complex I, I+III, and II+III activities was increased considerably by pretreating the samples with saponin. The set of methods was evaluated on double biopsy samples from five healthy volunteers and showed coefficients of variation between 7 and 14% when citrate synthase was used as reference base. All of the various measures of mitochondrial function showed high correlation coefficients to each other (r=0.84-0.98; p<0.01). It is concluded that the set of methods is suitable for diagnosis of mitochondrial disorders in adults and small children.     

Spectrophotometric determination of serum nitrite and nitrate by copper-cadmium alloy

A macro and micro assay for the spectrophotometric determination of serum nitrite and nitrate was developed. Nitrite/nitrate in biological samples can be estimated in a single step by this method. The principle of the assay is the reduction of nitrate by copper-cadmium alloy, followed by color development with Griess reagent (sulfanilamide and N-naphthylethylenediamine) in acidic medium. This assay is sensitive to 1 microM nitrate and is suitable for different biological fluids, including sera with a high lipid concentration. The copper-cadmium alloy used in the present method is easy to prepare and can completely reduce nitrate to nitrite in an hour. The present method provides a simple, cost-effective assay for the estimation of stable oxidation products of nitric oxide in biological samples.     

高效液相色谱法测定体液中硝酸盐及亚硝酸盐

目的和方法 :应用高效液相色谱技术建立一种灵敏的检测不同体液中硝酸盐和亚硝酸盐的方法。结果 :唾液、血清及尿液经过不同方法处理后应用高效液相色谱ODS反相柱分离 ,紫外检测器于 2 10nm检测其中的硝酸盐和亚硝酸盐的含量。整个分离过程少于 7min ,硝酸盐和亚硝酸盐的测定线性范围分别为 0 .7～ 10 0ng、5～ 10 0ng ,最低检测极限分别为 0 .3ng和 2ng。硝酸盐回收率为 99%～ 10 2 % ,亚硝酸盐回收率为 99%～ 10 4 %。测定硝酸盐及亚硝酸盐的精密度分别为 0 .8%和 1.7%。结论 :本法测定硝酸盐和亚硝酸盐简便、灵敏度高、特异性好