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.

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.

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.

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.

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