Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis

We validated the nitrate reductase assay (NRA) for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) using sodium nitrate (NaNO3) in replacement of potassium nitrate (KNO3) as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB.

Método de nitrato-reductasa (GRIESS) para la detección rápida de la susceptibilidad a isoniacida y rifampicina / Method of nitrate reductase (GRIESS) for the rapid detection of susceptibility to isoniazid and rifampicin

La publicación describe el funcionamiento del método rápido, confiable, sencillo y económico al laboratorio que permita examinar un gran número de muestras a fin de seleccionar a aquellos pacientes que pudieran estar potencialmente infectados con Mycobacterium tuberculosis multirresistente (MDR) a los medicamentos. El método será aplicado en los laboratorios donde haya sido validado. La versión directa del método Griess debe ser usada por los laboratorios de la DISA como un método rápido de tamizaje para la detección de resistencia a isoniacida (INH) y rifampicina (RIF). Dicho método se aplicará a muestras respiratorias de pacientes primarios

Effects of NH4(+)-N/NO3(-)-N ratio in applied supplementary fertilizer on nitrogen metabolism, photosynthesis and growth of Isatis indigotica / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of NH4(+)-N/NO3(-)-N ratios in the applied supplementary fertilizer on the growth, nitrogen metabolis related enzymes activity and photosynthetic characteristics of Isatis indigotica.</p><p><b>METHOD</b>The sand culture experiment was conducted, and seedling of I. indigotica was fertilized with the mixed nutrition that containing the Hoagland's macro elements and the Aron's micro elements, the additional 63 mmol N was supplementary with the NH4(+)-N/NO3(-)-N ratio of 100:0, 75:25, 50:50, 25:75 and 0:100.</p><p><b>RESULT</b>The biomass of I. indigotica increased at first when the supplementary N of NH4(+)-N/NO3(-)-N ratio changed from 100:0 to 50:50 and decreased afterwards. The maximum value was at 50:50 and the minimum at 100: 0. With increasing the ratio of NO3(-)-N, the activity of nitrate reductase and glutamine synthetase increased and then decreased and the relationship between the activity and the ratio could be described with an approximate parabola curve. The net photosynthetic rate of I. indigotica was the highest at the NH4(+)-N/NO3(-)-N ratio of 75:25 and the lowest at 100:0.</p><p><b>CONCLUSION</b>Increasing the NO3(-)-N ratio properly was beneficial to promote the growth and improve the activity of nitrate reductase and glutamine synthetase and net photosynthetic rate of I. indigotica.</p>

Development of a transformation system for Penicillium brevicompactum based on the Fusarium oxysporum nitrate reductase gene

Penicillium brevicompactum é um fungo filamentoso que apresenta um potencial para a aplicacão industrial devido a sua eficiente producão de enzimas do complexo pectinolítico. Neste trabalho foi desenvolvido um sistema de transformacão heterólogo para P. brevicompactum baseado na complementacão de um mutante nitrato redutase. Mutantes nitrato redutase foram obtidos pela resistência ao clorato de sódio em uma taxa de 23,24per center. O mutante denominado 4457-18X foi escolhido para os experimentos de transformacão com o vetor pNH24, que contém o gene da nitrato redutase de Fusarium oxysporum. Uma freqüência de cerca de 3 transformantes/mg de DNA foi obtida utilizando-se o vetor pNH24 na forma circular e um aumento de cerca de 10 vezes nessa freqüência foi alcancado com a utilizacão desse vetor linearizado com a enzima de restricão Xba I. A análise dos transformantes pela técnica de hibridizacão revelou uma tendência do vetor linearizado diminuir o número de integracões em relacão ao vetor circular. A integracão foi aleatória e estável nos transformantes analisados. O estabelecimento de um sistema de transformacão para P. brevicompactum é essencial para a manipulacão genética desse microrganismo.

Nitrate reductase activty: a solution to nitrate problems tested in free and immobilized algal cells in presence of heavy metals

Every organism has different potential to accumulate NO3- from the environment. Nitrate reduction processes are perhaps most significant in maintaining water quality by alteration of nitrate to nitrite. A comparative study between the nitrate reductase NR activity of green and blue green algae in presence of heavy metals is being conducted to present a situation where nitrate reductase process may be affected in presence of heavy metals. Metals interacted negatively with the nitrate reductase activity of a blue green alga, Anacystis nidulans and green algae, Chlorella vulgaris in both free and immobilized state. The activity was more repressed in C. vulgaris in presence of Ni compared to Zn and Cd. However, Cd was more toxic to NR activity in A. nidulans [free state]. Metal dependent variation between free and immobilized cells were found to be significant [P< 0.01] however, the concentration dependent pattern in the activity between free and immobilized state was non significant in both the test organisms. C.vulgaris is more efficient in conversion of nitrate to nitrite compared to A.nidulans in presence of heavy metals

Drug susceptibility of Mycobacterium tuberculosis to primary antitubercular drugs by nitrate reductase assay.

BACKGROUND & OBJECTIVES: Rapid susceptibility testing of Mycobacterium tuberculosis strains is imperative for therapy selection but traditional drug susceptibility tests take weeks or are expensive. In this study we evaluated nitrate reductase assay which utilizes the detection of nitrate reduction as an indication of growth and therefore results can be obtained faster than by visual detection of colonies. METHODS: One hundred clinical isolates of M. tuberculosis were tested for four first line antitubercular drugs by nitrate reductase assay (NRA) and were compared with standard proportion method. The bacteria were inoculated on Lowenstein-Jensen (LJ) medium with primary antitubercular drugs and potassium nitrate was incorporated. After incubation for 7- 14 days, nitrate reduction indicating growth could be detected by colour change when reagents were added. RESULTS: Resistance of isolates as determined by both methods for isoniazid, rifampicin, streptomycin and ethambutol was 32, 35, 62 and 15 per cent respectively. Agreement between NRA and proportion method was 99 per cent for isoniazid and ethambutol. Complete agreement (100%) was found for rifampicin and streptomycin. Results were available in 7-14 days by NRA as compared to proportion method which takes 4-6 wk. INTERPRETATION & CONCLUSION: Nitrate reductase assay is a rapid and inexpensive method for susceptibility testing of M. tuberculosis for primary antitubercular drugs and could be an appropriate alternative to existing methods, particularly in resource-poor settings.

Physiological and biochemical alterations in Anabaena 7120 under iron stress.

Various physiological and biochemical process like growth, NO3- -uptake, nitrate reductase, glutamine synthetase and ATPases (Mg2+ and Ca2+ dependent) in the cyanobacterium Anabaena 7120 were observed under iron stress. Growth was found to be maximum in 50 microM Fe3+ added cells however, 20 microM Fe3+ (the Fe3+ concentration generally used for routine culturing of cyanobacterial cell in Chu 10 medium) incubation resulted in lower growth. Fe3+ starvation on the other hand showed very poor growth up to 4th day but once the growth started it reached at significant level on 7th day. Higher Fe3+ concentration reflected reduced growth with lethality at 500 microM Fe3+. Chlorophyll a fluorescence under Fe3+ stress reflected almost the similar results as in case of growth. However, the pigment was found to be more sensitive as compared to protein under Fe3+ stress. Similar results have been observed in case of NO3-uptake with only 80% reduction in nutrient uptake in 500 microM Fe3+ incubated cells. Nitrate reductase activity was lower in Fe3+ starved cells as compared to significant enzyme activity in 20 and 50 microM Fe3+ incubated cells. Similar to nitrate reductase, glutamine synthetase also showed maximum level in 50 microM Fe3+ added cells, however, higher Fe3+ concentration (300-500 microM ) resulted in reduced enzymatic activity. Glutamine synthetase activity was less sensitivity as compared to nitrate reductase activity under Fe3+ stress. ATPase (Mg2+ and Ca2+ dependent) always showed higher level with increasing Fe3+ concentration.

Transformation system of Beauveria bassiana and Metarhizium anisopliae using nitrate reductase gene of Aspergillus nidulans.

An heterologous transformation system for entomopathogenic fungi B. bassiana and M. anisopliae was developed based on the use of A. nidulans nitrate reductase gene (niaD). B. bassiana and M. anisopliae niaD stable mutants were selected by treatment of protoplast with ethane methane sulphonate (EMS) and regenerated on chlorate medium. The cloned gene was capable of transforming B. bassiana and M. anisopliae at a frequency of 5.8 to 20 transformants per microg of DNA. Most of them were mitotically stable.

Nitrate levels and stages of growth in hypernodulating mutants of Lupinus Albus. II.Enzymatic activity and transport of N in the xylem SAP

The enzymatic study and transport of N in the xylem sap was carried out with a view to observing the influence of different nitrate levels and growth stages of the plant in chemically treated mutants of Lupinus albus. Several stresses induce a reduction in plant growth, resulting in the accumulation of free amino acids, amides or ureides, not only in the shoot, but also in the roots and nodules. Although enzyme activity is decisive in avoiding products that inhibit nitrogenase by ammonium, little is known about the mechanism by wich the xylem carries these products. However, this process may be the key to the function of avoiding the accumulation of amino acids in the cells of infected nodules. The behaviour of the enzymes nitrate reductase (NR), phosphoenolpyruvate carboxylase (PEPC), glutamine synthetase (GS) and nitrogen compounds derived from fixation, such as N-Ó-amino, N-ureides and N-amide in mutant genotypes were observed. The NR enzyme was highly influenced by the application of nitrate showing much higher values than those in the non-application of nitrate, independently of genotype, being that the NR, the best evaluation period was in the tenth week. The L-62 genotype characterized with nitrate-resistance, clearly showed that the enzyme PEPC is inhibited by presence of nitrate. The L-135 genotype (nor fix) showed GS activity extremely low, thus demonstrating that GS is an enzyme highly correlated with fixation. With regard to the best growth stage for GS, Lupinus albus should be evaluated in the seventh week.

Effect of combined nitrogen on the expression of nitrate reductase and nitrite reductase in Azorhizobium caulinodans.

In aerobically grown Azorhizobium caulinodans strain IRBG 46, in vivo expression of nitrate reductase (NR) and nitrite reductase (NiR) requires the presence of either nitrate or nitrite. On the contrary mere microaerobic conditions are sufficient for the expression of NR and NiR, however, addition of nitrate to the growth medium enhanced the activities of the enzymes. Optimum concentration of nitrate for maximum expression of NR and NiR activities was different in aerobic and microaerobic conditions. Nitrite was released into the medium both in aerobic and microaerobic conditions beyond a particular concentration of nitrate in the medium. Dissimilatory nitrate reduction was affected to a lesser extent by ammonium compared to assimilatory nitrate reduction.

Imaging oscillations in Gonyzaulax: a chloroplast rhythm of nitrate reductase visualized by immunocytochemistry

Gonyaulax polyedra is a unicellular marine photosynthetic dinoflagellate known to display numerous circadian rhythms, including bioluminescence, motility, cell division and several chloroplast-related rhythms. Due to this, Gonyaulax has become a widely used model organism for studying the cellular biological clock. In this work we describe another rhythm for Gonyaulax cells also associated with the cell's chloroplasts, a rhythm in localization of the enzyme nitrate reductase (NR). A polyclonal antibody was raised against NR purified from G. polyedra cells and used as a probe in immunogold labelling experiments on cell thin sections, comparing day- and night-phase cells. The enzyme localizes to chloroplasts in day-phase cells, while the enzyme is active, and is largely absent in night-phase cells. Counts of gold particle distribution in day- versus night-phase cells show an approximate three-fold increase in enzyme labelling in day-phase plastids. These results closely approximate the four-fold differences shown for NR activity between day and night Gonyaulax cells by biochemical studies. We conclude from the diurnal difference in labelling that NR is localized in Gonyaulax chloroplasts during the day phase and is absent (broken down) in night-phase cells. Thus NR in Gonyaulax is compartmentalized in the chloroplasts and is therefore subject to similar circadian control mechanisms exhibited for other plastid rhythms.

Caracterización de una mutación nar y su efecto sobre la expresión transcripcional de una fusión moa: lac en escherichia coli K12 / Characterization of a nar mutation and its effect on the trascriptional expression of a moa: lac operon fision in escherichia coli K12

Dos grupos de genes, narC (GHJI) y mol (moa, mob, mod, moe y molR), codifican para los componentes estructurales de la enzima inducible Nitrato Reductasa Respiratoria A (NRA). En estudios previos, se observó que la mutación narC-8 bloqueaba la manifestación del caracter Lac + conferido por la fusión transcripcional moa::lac. Para determinar el posible papel controlar del operón narC, se caracterizó otra mutación independiente y fue clasificada como narC-MM78. Cepas isogénicas mutantes, doble y simple, fueron construídas y se observó que la referida mutación parece ejercer el mismo efecto que narC-MM78. Cepas isogénicas mutantes, doble y simple, fueron construídas y se observó que la referida mutación parece ejercer el mismo efecto que narC-8. Las condiciones de cultivo que controlan la biosíntesis de la NRA, no lograron suprimirlo fenotípicamente aún adicionando concentraciones variables de nitrato, molibdato, nitrito, azida o clorato. Sin embargo cuando la cepa de función era marC+, esas condiciones modularon la manifestación del carácter Lac+. Los resultados obtenidos permiten proponer un papel controlador de narC+ activando la expresión de moa. Esa acción sería ejercida dentro de un sistema coordinado de circuitos de control fisiológico y genético, el cual mantendría un balance adecuado en la biosíntesis de la NRA durante la respiración anaeróbica del nitrato, en E. coli K12

Influence of ammonium ions on the activity of nitrate reductase in the fresh water fern, azole Carolinian

The effect of NH[4]Cl either as a sole nitrogen source or together with NO[3], on nitrate reductase activity [NR] of the water fern, Azolla caroliniana was studied. Ammonium ions induced slightly the NR activity until 1.0 mM, then the activity decreased. When NH[+4]was added to the induction medium [2/5 N[2-]free Hoagland solution plus 5.0 mM N0[3]] a marked decrease in NR activity was -recorded. Nitrate uptake, assimilation and NR activity of Azolla plants pretreated with 1.0 mM NH[4]for 0,1,2,3 or 4 days were also studied. The uptake of NO[-3]was roughly the same, while NO[3]assimilation was greatly suppressed and, accordingly, accumulation of NO[3]was markedly increased. Maximum NR activity was shown with the non-treated Azolla plants. Ammonium uptake and NTR activity of previously induced or non-induced plants were also investigated. Induced Azolla plant showed a gradual increase in NH[-4]uptake and a marked decrease in NR activity throughout the whole experimental period. However, the non-induced plants absorbed much greater amounts of ammonia than did the induced ones

Interactive effects of salinity with some hormonal and non-hormonal growth substances on chlorella fusca II- nitrate reductase activity, proline and protein contents

Sodium chloride at 320 mM increased proline content but suppressed protein accumulation and nitrate reductase activity of Chlorella fusca. 10-5 M, 10-6 M of GA3, IAA, kinetin and thiourea increased protein, proline and NRA of the algal cells relevant to control. The four used growth substances reacted in antagonism with NaCl in the same parameters. In counteracting, the adverse effect of salinity, IAA was more effective, while thiourea was the least effective one

Modification of the redox state of cytochrome c oxidase of rice due to certain stress treatments.

The redox state of cytochrome alpha 3 during in situ respiration of leaves of 20-day-old rice seedlings was assessed by in vivo aerobic assay of nitrate reductase, after 1 min exposure to carbon monoxide. Different stress treatments like water and salt stresses, disintegration of leaf tissues and darkness modified the redox state of cytochrome c oxidase. The dark treatment altered the redox state of cytochrome oxidase from reduced to the oxidized state, as judged by its reaction with CO in CO-sensitive rice cultivar. The water and salt stresses as well as the disintegration of leaf tissue on the contrary altered cytochrome oxidase from the oxidized to its reduced state in CO-insensitive cultivars; probably by changing the cellular integrity, turgidity and structure of mitochondrial membrane, and also due to decreased mitochondrial energization.

Interactive effects of molybdenum and vanadium on the nitrogenase and nitrate reductase activities of aulosira fertilissima

The effects of molybdenum and vanadium interactions on the nitrogenase and nitrate reductase enzymatic systems in the cyano-bacterium Aulosira fertilissima were investigated at different time intervals. Addition.of Va to + Mo cultures up to 0.015 ppm increased both dry weight gain and pigment biosynthesis. Beyond this concentration significant inactivation of such enzymes was observed at all growth periods. Nitrogenase and nitrate reductase activities were significantly increased by addition of Va at different levels to the cultures augmented with Mo, a phenomenon that was reversed by increasing Va concentrations. Maximum nitrogenase activity was recorded after 48 hr of incubation, whereas the highest enzymatic potential was maintained after 96 hr incubation period. Nitrogenase and nitrate reductase activities in + Mo cultures were significantly higher than in -Mo cultures. Addition of Va to + Mo Aulosira cultures resulted in a marked elevation in heterocyst frequency particularly after 48 hr incubation

Isolation and characterization of mutants affected in the expression of the nar operon Escherichia coli

En el sistema nitrato reductasa de E. Coli, la máxima expresión del operon nar se obtiene bajo condiciones de anaerobiosis y en presencia de nitrato. Las mutaciones en las cepas obtenidas con el fago Mudl (Ap,lac), las cuales crecían anaeróbicamente sobre lactosa únicamente si se añadían nitrato am medio, mapearon en el locus (chlC), en el minuto 27 del mapa de E. Coli. En tales cepas que carecían de actividad nitrato reductasa medidas con benzilviologeno o con formiato, la síntesis de ß-galactosidasa refleja la regulación a nivel transcripcional del operon nar intacto. A partir de esas cepas se aislaron expontáneamente dos clases de mutantes regulatorias. Aquellas pertenecientes a la clase I sintetizaban ß-galactosidasa anaeróbicamente en ausencia de nitrato, mientras que en las cepas de la clase II tal síntesis era parcialmente independiente del nitrato pero no era reprimida por el oxígenio. Resultados obtenidos por transducción con el fago Pa, revelaron que la mutación presente en las cepas de la clase I estaba muy cercana al opron nar. La mutación probablemente afectó un elemento que actúa en cis o, alternativamente, un gen que codifica para una proteína que afecta negativamente la expresión del operon nar GHJI

Interets et signification ecophysiologique de l'estimation de l'activite nitrate reductase phytoplanctonique en milieu lacustre eutrophe

As part of the study of the eutrophic lake Aydat [Massif Central. FRANCE], we estimated the phytoplanktonic nitrate reductase activity [NRA] over 1 year [1985]. We have also looked for the ecophysiological significance of the relationships established between the NRA and several biotic and abiotic parameters of the lake. Weekly samples were gathred from several depths in both the throphogenic and tropholytic zones. The samples were filtered throught GF/C whatman filters presoaked in distilled water during 24 h. The PNRA ranged between 0 and 5.80 mg NO[2]-1[-1]-h[-1]. The majority of the relationships between the PNRA and several abiotic and biotic parameters corresponded to trends reported in the literature. However, PNRA was negatively and highly significantly correlated to nitrate concentrations [r = 0,485 for 28 d.f., p <0.01]. This finding contradicts the generally accepted idea about the inductive character of the NRA enzyme system. This discrepancy may be explained by the interpretation of FINLAY [1985], about the important role the ciliates [i.e. Loxodes which is a dominant member of the ciliate community during our investigations] play in utilization of nitrates during summer in the aerobic/anaerobic interface of the lake