Molecular characterization of nitrate reductase gene and its expression in the marine red alga Gracilaria tenuistipitata (Rhodophyta)

The enzyme nitrate reductase (NR) responsible for the conversion of nitrate to nitrite is considered to be the rate-limiting step in nitrogen assimilation. The economically important marine macroalga Gracilaria tenuistipitata presents a circadian oscillation in NR protein content and activity. In order to identify if the regulation of NR in G. tenuistipitata happens at transcriptional levels, the NR cDNA and gene were sequenced and the NR mRNA expression was studied. Analysis of the sequenced gene revealed absence of introns which is unusual for NR genes. The transcriptional profiling revealed a circadian rhythm for NR; furthermore, a rhythm was observed in constant light condition, suggesting a possible regulation by the biological clock at the mRNA levels for NR in G. tenuistipitata.

Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

Os autores padronizaram métodos para a avaliação da atividade da glicose-6-fosfato desidrogenase e glutationa redutase. O princípio geral do primeiro método baseou-se na formação de metahemoglobina pelo nitrito de sódio, seguido da estimulação da via das pentoses pelo azul de metileno. Foram estudados 46 indivíduos adultos, sendo 23 do sexo masculino e 23 do feminino, não deficientes em glicose-6-fosfato desidrogenase (G6PD), com idades variando entre 20 e 30 anos. Os resultados revelaram que a redução da metahemoglobina pelo azul de metileno para sangue total, foram de 154.50 e 139.90 mg/min (p<0.05) respectivamente para o sexo masculino e feminino. Para hemácias lavadas os valores foram de 221.10 e 207.85 mg/min (n.s.) respectivamente. Estas observações permitiram concluir que ao se empregar hemácias lavadas e 0.7 g% de concentração de nitrito de sódio, por um lado não houve diferença entre os sexos e por outro, abreviou o tempo de leitura da quantidade residual de metahemoglobina para 90 minutos. A avaliação da atividade da glutationa redutase foi feita baseado no fato de que a cistamina (agente tiol) liga-se aos grupos SH da hemoglobina formando complexos. Estes complexos são revertidos pela ação da glutationa redutase, ocorrendo conjuntamente nesta reação a redução da metahemoglobina. Foram estudados 32 indivíduos adultos, sendo 16 do sexo masculino e 16 do feminino, não deficientes em G6PD, com idades variando entre 20 e 30 anos. Os resultados revelaram valores de redução da metahemoglobina pela cistamina de 81.27 e 91.13 mg/min (p<0.01) respectivamente para o sexo masculino e feminino. Estas observações permitiram concluir que o emprego de hemácias lavadas e 0.1 molar de concentração de cistamina torna possível a leitura da quantidade residual de metahemoglobina aos 180 minutos de incubação. A atividade da glutationa redutase avaliada por meio da redução da metahemoglobina pela cistamina, foi estudada em 14 indivíduos do sexo feminino antes e após o tratamento com 10 mg por dia de riboflavina durante 8 dias. Os resultados foram de 73.69 e 94.26 mg/min (p<0.01) antes e após o tratamento. Estas observações permitiram concluir que a oferta de riboflavina, mesmo para indivíduos normais, aumenta a atividade da glutationa redutase. Foram ainda avaliados 3 indivíduos da raça negra e deficientes em G6PD, sendo 2 do sexo masculino e 1 do feminino. Houve ativação parcial da G6PD e glutationa redutase, sendo estas alterações mais intensas nos indivíduos do sexo masculino. Considerando-se a raça e as características laboratoriais observadas, foi possível sugerir que a deficiência em G6PD verificada é do tipo Africano, bem como, permitiu considerar os indivíduos do sexo feminino coin o sendo heterozigoto para esta deficiência. Por fim, a análise dos resultados em seu conjunto permitiu concluir que os métodos propostos se mostraram eficientes para avaliar a atividade da G6PD e glutationa redutase. Esta última é dependente da via das pentoses, geradora de NADPH e da riboflavina, vitamina precursora de FAD.

Antihypertensive and antioxidant effects of a single daily dose of sodium nitrite in a model of renovascular hypertension

Dietary nitrite and nitrate have been reported as alternative sources of nitric oxide (NO). In this regard, we reported previously that sodium nitrite added to drinking water was able to exert antihypertensive effects in an experimental model of hypertension in a dose-dependent manner. Taking into consideration that nitrite is continuously converted to nitrate in the bloodstream, here we expanded our previous report and evaluate whether a single daily dose of sodium nitrite could exert antihypertensive effects in 2 kidney-1 clip (2K1C) hypertensive rats. Sham-operated and 2K1C rats were treated with vehicle or sodium nitrite (15 mg/kg/day) for 4 weeks. We evaluated the effects induced by sodium nitrite treatment on systolic blood pressure (SBP) and NO markers such as plasma nitrite, nitrite + nitrate (NOx), cGMP, and blood levels of nitrosyl-hemoglobin. In addition, we also evaluated effects of nitrite on oxidative stress and antioxidant enzymes. Dihydroethidium (DHE) was used to evaluate aortic reactive oxygen species (ROS) production by fluorescence microscopy, and plasma levels of thiobarbituric acid-reactive species (TBARS) were measured in plasma samples from all experimental groups. Red blood cell superoxide dismutase (SOD) and catalase activity were evaluated with commercial kits. Sodium nitrite treatment reduced SBP in 2K1C rats (P < 0.05). We found lower plasma nitrite and NOx levels in 2K1C rats compared with normotensive controls (both P < 0.05). Nitrite treatment restored the lower levels of nitrite and NOx. While no change was found in the blood levels of nitrosyl-hemoglobin (P > 0.05), nitrite treatment increased the plasma levels of cGMP in 2K1C rats (P < 0.05). Higher plasma TBARS levels and aortic ROS levels were found in hypertensive rats compared with controls (P < 0.05), and nitrite blunted these alterations. Lower SOD and catalase activities were found in 2K1C hypertensive rats compared with controls (both P < 0.05). Nitrite treatment restored SOD activity (P < 0.05), whereas catalase was not affected. These data suggest that even a single daily oral dose of sodium nitrite is able to lower SBP and exert antioxidant effects in renovascular hypertension.

GENETIC ORGANIZATION OF THE STRUCTURAL GENES FOR NITRATE REDUCTASE (TN5)

Nitrate reductase in Escherichia coli is a membrane-bound anaerobic enzyme that is repressed by oxygen and induced by nitrate. The genetic organization of the structural genes for the two larger subunits of nitrate reductase ((alpha) and (beta)) was determined by immunoprecipitation analysis of the formation of these proteins in nitrate reductase-deficient mutants resulting from transposon Tn5 mutagenesis. The results suggested that the genes encoding the (alpha) and (beta) subunits (narG and H) were arranged in an operon with transcription in the direction promoter(--->)(alpha)(--->)(beta). Segments of the chromosome containing the Tn5 inserts from several of the mutants were cloned into plasmid pBR322 and the positions of the transposons determined by restriction mapping. The Tn5 insertion sites were localized on two contiguous EcoRI fragments spanning about 6.6 kilobases of DNA. The narI gene (proposed to encode the (gamma) subunit) was positioned immediately downstream from the (beta)-gene (narH) by Southern analysis of Tn10 insertions into the narI locus. A Tn10 insertion into the narK locus, proposed to encode a nitrate-sensitive repressor of other anaerobic enzymes, was located about 1.5 kilobases upstream from the narGHI operon promoter. The narL locus, proposed to encode a nitrate-sensitive positive regulator of the narGHI operon and known to be genetically linked to the other nar genes, was demonstrated to lie outside a 19.3-kilobase region of the chromosome which encompasses the other nar genes. The physical limit of the narGHI promoter was defined by studying the effect of Tn5 insertions into a hybrid plasmid containing the functional operon. The points of origin of the coding regions for the (alpha) and (beta) genes were deduced by alignment of the chromosomal map of Tn5 insertion sites with the sizes of (alpha) and (beta) subunit fragments produced by plasmids carrying these Tn5 inserts in the nar operon. The coding region for the (alpha) subunit (143,000 daltons) begins about 250 nucleotides downstream from the deduced limit of the promoter region and includes about 4.0 kilobases of DNA; the region encoding (beta) (60,000 daltons) lies immediately downstream from the (alpha)-gene and is approximately 1.6 kilobases in length. The adjacent region encoding the (gamma) subunit (19,000 daltons) is approximately 0.5 kilobase in length. ^

Experiment: Elevated CO2 levels affect the activity of nitrate reductase and carbonic anhydrase in the calcifying rhodophyte Corallina officinalis

The concentration of CO2 in global surface ocean waters is increasing due to rising atmospheric CO2 emissions, resulting in lower pH and a lower saturation state of carbonate ions. Such changes in seawater chemistry are expected to impact calcification in calcifying marine organisms. However, other physiological processes related to calcification might also be affected, including enzyme activity. In a mesocosm experiment, macroalgal communities were exposed to three CO2 concentrations (380, 665, and 1486 µatm) to determine how the activity of two enzymes related to inorganic carbon uptake and nutrient assimilation in Corallina officinalis, an abundant calcifying rhodophyte, will be affected by elevated CO2 concentrations. The activity of external carbonic anhydrase, an important enzyme functioning in macroalgal carbon-concentrating mechanisms, was inversely related to CO2 concentration after long-term exposure (12 weeks). Nitrate reductase, the enzyme responsible for reduction of nitrate to nitrite, was stimulated by CO2 and was highest in algae grown at 665 µatm CO2. Nitrate and phosphate uptake rates were inversely related to CO2, while ammonium uptake was unaffected, and the percentage of inorganic carbon in the algal skeleton decreased with increasing CO2. The results indicate that the processes of inorganic carbon and nutrient uptake and assimilation are affected by elevated CO2 due to changes in enzyme activity, which change the energy balance and physiological status of C. officinalis, therefore affecting its competitive interactions with other macroalgae. The ecological implications of the physiological changes in C. officinalis in response to elevated CO2 are discussed.

Selenocysteine, identified as the penultimate C-terminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene.

The possible relationship of selenium to immunological function which has been suggested for decades was investigated in studies on selenium metabolism in human T cells. One of the major 75Se-labeled selenoproteins detected was purified to homogeneity and shown to be a homodimer of 55-kDa subunits. Each subunit contained about 1 FAD and at least 0.74 Se. This protein proved to be thioredoxin reductase (TR) on the basis of its catalytic activities, cross-reactivity with anti-rat liver TR antibodies, and sequence identities of several tryptic peptides with the published deduced sequence of human placental TR. Physicochemical characteristics of T-cell TR were similar to those of a selenocysteine (Secys)-containing TR recently isolated from human lung adenocarcinoma cells. The sequence of a 12-residue 75Se-labeled tryptic peptide from T-cell TR was identical with a C-terminal-deduced sequence of human placental TR except that Secys was present in the position corresponding to TGA, previously thought to be the termination codon, and this was followed by Gly-499, the actual C-terminal amino acid. The presence of the unusual conserved Cys-Secys-Gly sequence at the C terminus of TR in addition to the redox active cysteines of the Cys-Val-Asn-Val-Gly-Cys motif in the FAD-binding region may account for the peroxidase activity and the relatively low substrate specificity of mammalian TRs. The finding that T-cell TR is a selenoenzyme that contains Se in a conserved C-terminal region provides another example of the role of selenium in a major antioxidant enzyme system (i.e., thioredoxin-thioredoxin reductase), in addition to the well-known glutathione peroxidase enzyme system.

A new selenoprotein from human lung adenocarcinoma cells: purification, properties, and thioredoxin reductase activity.

We report the isolation and characterization of a new selenoprotein from a human lung adenocarcinoma cell line, NCI-H441. Cells were grown in RPMI-1640 medium containing 10% (vol/vol) fetal bovine serum and 0.1 microM [75Se]selenite. A 75Se-labeled protein was isolated from sonic extracts of the cells by chromatography on DE-23, phenyl-Sepharose, heparin-agarose, and butyl-Sepharose. The protein, a homodimer of 57-kDa subunits, was shown to contain selenium in the form of selenocysteine; hydrolysis of the protein alkylated with either iodoacetate or 3-bromopropionate yielded Se-carboxymethyl-selenocysteine or Se-carboxyethyl-selenocysteine, respectively. The selenoprotein showed two isoelectric points at pH 5.2 and pH 5.3. It was distinguished from selenoprotein P by N-glycosidase assay and by the periodate-dansylhydrazine test, which indicated no detectable amounts of glycosyl groups on the protein. The selenoprotein contains FAD as a prosthetic group and catalyzes NADPH-dependent reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and reduction of insulin in the presence of thioredoxin (Trx). The specific activity was determined to be 31 units/mg by DTNB assay. Apparent Km values for DTNB, Escherichia coli Trx, and rat Trx were 116, 34, and 3.7 microM, respectively. DTNB reduction was inhibited by 0.2 mM arsenite. Although the subunit composition and catalytic properties are similar to those of mammalian thioredoxin reductase (TR), the human lung selenoprotein failed to react with anti-rat liver TR polyclonal antibody in immunoblot assays. The selenocysteine-containing TR from the adenocarcinoma cells may be a variant form distinct from rat liver TR.

Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer.

Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H4B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared from H4B-deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H4B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNOS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-translational mechanism by which intracellular H4B can regulate iNOS expression.

A potassium channel beta subunit related to the aldo-keto reductase superfamily is encoded by the Drosophila hyperkinetic locus.

Genetic and physiological studies of the Drosophila Hyperkinetic (Hk) mutant revealed defects in the function or regulation of K+ channels encoded by the Shaker (Sh) locus. The Hk polypeptide, determined from analysis of cDNA clones, is a homologue of mammalian K+ channel beta subunits (Kv beta). Coexpression of Hk with Sh in Xenopus oocytes increases current amplitudes and changes the voltage dependence and kinetics of activation and inactivation, consistent with predicted functions of Hk in vivo. Sequence alignments show that Hk, together with mammalian Kv beta, represents an additional branch of the aldo-keto reductase superfamily. These results are relevant to understanding the function and evolutionary origin of Kv beta.

Influence Of The Major Nitrite Transporter Nirc On The Virulence Of A Swollen Head Syndrome Avian Pathogenic E. Coli (apec) Strain.

Avian Pathogenic Escherichia coli (APEC) strains are extra-intestinal E. coli that infect poultry and cause diseases. Nitrite is a central branch-point in bacterial nitrogen metabolism and is used as a cytotoxin by macrophages. Unlike nitric oxide (NO), nitrite cannot diffuse across bacterial membrane cells. The NirC protein acts as a specific channel to facilitate the transport of nitrite into Salmonella and E. coli cells for nitrogen metabolism and cytoplasmic detoxification. NirC is also required for the pathogenicity of Salmonella by downregulating the production of NO by the host macrophages. Based on an in vitro microarray that revealed the overexpression of the nirC gene in APEC strain SCI-07, we constructed a nirC-deficient SCI-07 strain (ΔnirC) and evaluated its virulence potential using in vivo and in vitro assays. The final cumulative mortalities caused by mutant and wild-type (WT) were similar; while the ΔnirC caused a gradual increase in the mortality rate during the seven days recorded, the WT caused mortality up to 24h post-infection (hpi). Counts of the ΔnirC cells in the spleen, lung and liver were higher than those of the WT after 48 hpi but similar at 24 hpi. Although similar number of ΔnirC and WT cells was observed in macrophages at 3 hpi, there was higher number of ΔnirC cells at 16 hpi. The cell adhesion ability of the ΔnirC strain was about half the WT level in the presence and absence of alpha-D-mannopyranoside. These results indicate that the nirC gene influences the pathogenicity of SCI-07 strain.

Ensaio in vitro da enzima nitrato redutase e efeito da disponibilidade de nitrato e fosfato em variantes pigmentares de Hypnea musciformis (Wulfen) J. V. Lamour. (Gigartinales, Rhodophyta)

A enzima nitrato redutase (NR) catalisa a redução do nitrato a nitrito e controla a taxa de assimilação do nitrato. O ensaio in vitro da nitrato redutase foi otimizado para a linhagem selvagem (marrom, MA) e para a linhagem deficiente em ficoeritrina (verde-clara, VC) de Hypnea musciformis. As duas linhagens foram cultivadas em temperatura de 23 ± 2°C, fotoperíodo de 14 horas, irradiância de 60-90µmol fótons m-2s-1, e meio composto por água do mar esterilizada (30ups) enriquecida com a solução de von Stosch na concentração de 50% (VSES/2). As condições ótimas de ensaio para ambas as linhagens foram: 40µM de NADH; 10min de incubação do extrato bruto (EB) e 100µL de EB. A atividade ótima da NR ocorreu em 4 e 2mM de nitrato para a linhagem VC e MA, respectivamente. As linhagens VC e MA apresentaram, respectivamente, constante aparente de Michaelis-Menten (K M) para NADH de 0,2068 e 0,0837 µM, e K M para nitrato de 0,0492 e 0,0294mM. Os resultados indicam que a NR da linhagem MA tem maior afinidade pelo substrato do que a NR da linhagem VC de H. musciformis. Os experimentos para avaliar os efeitos da disponibilidade de nitrato (5 a 105µM) e nitrato e fosfato (0,5 a 25,5µM, com a relação N:P de 4:1) mostraram que a atividade da NR das linhagens VC e MA não aumentou com a adição de nitrato no meio, o que pode estar relacionado com o estado nutricional dessas algas. A atividade da NR foi maior nos tratamentos com adição de fosfato do que naqueles com adição de apenas nitrato, indicando que esse nutriente é importante para os processos metabólicos relacionados a atividade da NR.

The inducing NO-vasodilation by chemical reduction of coordinated nitrite ion in cis-[Ru(NO(2))L(bpy)(2)](+) complex

The synthesis of [Ru(NO(2)) L(bpy)(2)](+) (bpy = 2,2'-bipyridine and L = pyridine (py) and pyrazine (pz)) can be accomplished by addition of [Ru(NO) L(bpy) 2](PF(6))(3) to aqueous solutions of physiological pH. The electrochemical processes of [Ru(NO2) L(bpy) 2]+ in aqueous solution were studied by cyclic voltammetry and differential pulse voltammetry. The anodic scan shows a peak around 1.00 V vs. Ag/AgCl attributed to the oxidation process centered on the metal ion. However, in the cathodic scan a second peak around-0.60 V vs. Ag/AgCl was observed and attributed to the reduction process centered on the nitrite ligand. The controlled reduction potential electrolysis at-0.80 V vs. Ag/AgCl shows NO release characteristics as judged by NO measurement with a NO-sensor. This assumption was confirmed by ESI/MS(+) and spectroelectrochemical experiment where cis-[Ru(bpy)(2)L(H(2)O)](2+) was obtained as a product of the reduction of cis-[Ru(II)(NO(2)) L(bpy)(2)](+). The vasorelaxation observed in denuded aortic rings pre-contracted with 0.1 mu mol L(-1) phenylephrine responded with relaxation in the presence of cis-[RuII(NO2) L(bpy) 2]+. The potential of rat aorta cells to metabolize cis-[RuII(NO(2)) L(bpy)(2)](+) was also followed by confocal analysis. The obtained results suggest that NO release happens by reduction of cis-[RuII(NO(2)) L(bpy)(2)](+) inside the cell. The maximum vasorelaxation was achieved with 1 x 10(-5) mol L(-1) of cis-[RuII(NO(2)) L(bpy)(2)](+) complex.

Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and high plasma homocysteine in chronic hepatitis C (CHC) infected patients from the Northeast of Brazil

Background/Aim: Hyperhomocysteinemia due to Methylenetetrahydrofolate Reductase (MTHFR) gene, in particular the C677T (Ala222Val) polymorphism were recently associated to steatosis and fibrosis. We analyzed the frequency of MTHFR gene in a cross-sectional study of patients affected by Chronic Hepatitis C (CHC) from Northeast of Brazil. Method: One hundred seven-four untreated patients with CHC were genotyped for the C677T MTHFR. Genomic DNA was extracted from peripheral blood cells and the C677T MTHFR polymorphism was identified by PCR-RFLP. The homocysteine (Hcy) levels were determined by chemiluminescence method. All patients were negative for markers of Wilson's disease, hemochromatosis and autoimmune diseases and have current and past daily alcohol intake less than 100 g/week. Results: Among subjects infected with CHC genotype non-1 the frequency of MTHFR genotypes TT was 9.8% versus 4.4% genotype 1 (p = 0.01). Nevertheless, association was found between the MTHFR genotype TT x CT/CC polymorphism and the degree of steatosis and fibrosis in both hepatitis C genotype (p < 0.05). A significant difference was found on plasma Hcy levels in patients with steatosis regardless of HCV genotype (p = 0.03). Conclusion: Our results indicate that plasma Hcy levels is highly prevalent in subjects with chronic hepatits C with steatosis regardless of HCV genotype and vitamin deficiency. The presence of genotype TT of MTHFR C677T polymorphism was more common in CHC genotype non-1 infected patient regardless of histopathological classification and genotype TT+CT frequencies were significant in the presence of fibrosis grade 1+2 and of steatosis in CHC infected patients from the northeast of Brazil regardless of HCV genotype. The genetic susceptibility of MTHFR C677T polymorphism should be confirmed in a large population.

Profiles of xylose reductase, xylitol dehydrogenase and xylitol production under different oxygen transfer volumetric coefficient values

BACKGROUND: Xylitol is a sugar alcohol (polyalcohol) with many interesting properties for pharmaceutical and food products. It is currently produced by a chemical process, which has some disadvantages such as high energy requirement. Therefore microbiological production of xylitol has been studied as an alternative, but its viability is dependent on optimisation of the fermentation variables. Among these, aeration is fundamental, because xylitol is produced only under adequate oxygen availability. In most experiments with xylitol-producing yeasts, low oxygen transfer volumetric coefficient (K(L)a) values are used to maintain microaerobic conditions. However, in the present study the use of relatively high K(L)a values resulted in high xylitol production. The effect of aeration was also evaluated via the profiles of xylose reductase (XR) and xylitol clehydrogenase (XD) activities during the experiments. RESULTS: The highest XR specific activity (1.45 +/- 0.21 U mg(protein)(-1)) was achieved during the experiment with the lowest K(L)a value (12 h(-1)), while the highest XD specific activity (0.19 +/- 0.03 U mg(protein)(-1)) was observed with a K(L)a value of 25 h(-1). Xylitol production was enhanced when K(L)a was increased from 12 to 50 h(-1), which resulted in the best condition observed, corresponding to a xylitol volumetric productivity of 1.50 +/- 0.08 g(xylitol) L(-1) h(-1) and an efficiency of 71 +/- 6.0%. CONCLUSION: The results showed that the enzyme activities during xylitol bioproduction depend greatly on the initial KLa value (oxygen availability). This finding supplies important information for further studies in molecular biology and genetic engineering aimed at improving xylitol bioproduction. (C) 2008 Society of Chemical Industry