Nitrite In Medium Research Articles

Comparison of inhibition of glucose-stimulated insulin secretion in rat islets of Langerhans by Streptozotocin and methyl and ethyl nitrosoureas and methanesulphonates: Lack of correlation with nitric oxide-releasing or O 6-alkylating ability

We have studied inhibition of glucose-stimulated insulin secretion in islets of Langerhans isolated from adult Sprague-Dawley rats and treated with different alkylating agents. Streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-ethyl-N-nitrosourea (ENU) all released nitric oxide, as demonstrated by an increase in medium nitrite and cellular cyclic GMP. Methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), which do not possess a nitroso group, did not show evidence of nitric oxide release. All five compounds, however, decreased glucose-stimulated insulin release, suggesting that nitric oxide release was not necessary for the inhibition of secretion. Lack of involvement of nitric oxide was further suggested by the failure of oxyhaemoglobin to reverse STZ and MNU inhibition of insulin secretion. Since ENU was at least as effective as MNU in inhibiting insulin secretion, it appears that alkylation of DNA at the O 6 position of guanine may not be involved in this process.

Nitric oxide and interleukin-8 as inflammatory components of cystic fibrosis

We examined the production of reactive nitrogen intermediates in the tracheo-bronchial tree of patients with cystic fibrosis (CF). Examination of the soluble phase of sputa from 17 CF patients revealed the presence of high levels of NO2-/NO3- assayed by the Greiss reaction. We also examined the presence of the chemotactic cytokine interleukin-8 (IL-8) in these samples so as to assess another important inflammatory marker; high levels of IL-8 were present in the sputa of cystic fibrosis subjects. The elevated nitrite was not produced by the presence of Pseudomonas bacteria in the sputa, inasmuch as bacteria in culture released undetectable amounts of nitrite in culture media. Neutrophils from the sputa of CF patients with disease exacerbation released higher amounts of nitrite and IL-8. Neutrophils from the sputa were also shown to spontaneously release substantial amounts of nitrite in the supernatants, and this release was partly blocked by the antagonist NG-mono-methyl-L-arginine (L-NMMA). Blood neutrophils were shown to release nitrite only in response to challenge with CF-associated strains of Pseudomonas, and not exposure to cytokines. There was no significant differences in nitrite release between normal and CF blood polymorphonuclear leucocytes (PMNs). A study of upper airway epithelial cell lines showed that these cells released low amounts of nitrite after infection with CF-associated strains of Pseudomonas but not after cytokine exposure. Epithelial cell lines with CF or normal phenotypes were shown to release similar quantities of nitrite, upon stimulation with Pseudomonas. These data demonstrate that elevated levels of reactive nitrogen intermediates and IL-8 are produced in the tracheo-bronchial tree of subjects with CF. Levels of IL-8 and nitrite were higher in the secretions of CF subjects with disease exacerbation. The involvement of nitric oxide and other reactive nitrogen intermediates produced by neutrophils and other cells in the tissue damaging processes in CF deserves further investigation.

Production of nitrite by primary rat astrocytes in response to pneumococci

Recent studies using a rat model of pneumococcal meningitis have shown that nitric oxide synthase (NOS) inhibitors greatly attenuated microvascular changes and brain edema formation. The site of NO production during bacterial meningitis is unknown. In this study we tested whether primary astrocyte cultures from neonatal rat cortex can be induced to release NO upon stimulation with pneumococci. NO production was assessed by measuring nitrite in the cell culture supernatant using the Griess reaction. Stimulation with heat-killed unencapsulated pneumococci (HKP) increased nitrite concentrations in astrocyte culture supernatants in a dose-dependent fashion. Administration of AT-nitro-L-arginine (L-NA), aminoguanidine, L-canavanine, cycloheximide, and dexamethasone prevented the increase in nitrite concentrations. Addition of L-arginine, but not of o-arginine, partially reversed the inhibitory effect of L-NA. Administration of SOD increased nitrite accumulation. Moreover, at 72 h after stimulation with heat-killed pneumococci (10 7 cfu/ml) astrocytes showed an inducible NOS-like immunoreactivity. Accumulation of nitrite was also observed when rat cerebellar neurons and microglia were stimulated with HKP, whereas there was only a slight increase of nitrite in media of rat C6 glioma cells, but no increase of nitrite when the human glioblastoma cell line LN-229 was stimulated with HKP. There was a stronger increase in nitrite levels when astrocytes from Lewis rats were used compared to that from Wistar rats. In conclusion, our study indicates that astrocytes, neurons and microglia are inducible for NO production upon stimulation with pneumococci.

Five nitrate assimilation-related loci are clustered in Chlamydomonas reinhardtii.

Three overlapping clones covering a Chlamydomonas reinhardtii genomic region of about 32 kb appear to contain five genes potentially involved in nitrate assimilation in addition to the nitrate reductase structural locus nit-1. These new loci produced transcripts of 2.8, 2.2, 1.8 and 1.7 kb in nitrate-induced wild-type cells that, like the 3.4 kb transcript of nit-1, were undetectable in cells grown in ammonium. In addition, in a mutant defective at the regulatory locus, nit-2 for nitrate assimilation, which does not express the nit-1 gene transcript, accumulation of the four other transcripts was also blocked. They have been named nar (nitrate assimilation related) genes. The nar-1 and nar-2 loci are transcribed in the same orientation as nit-1. The nar-3 and nar-4 loci are transcribed divergently from nit-1. DNA and RNA sequences from both nar-3 and nar-4 cross-hybridized with each other indicating that they share similar sequences. Four nitrate assimilation-deficient mutants (C2, D2, F6 and G1) were characterized. These mutants lack nar transcripts and have major deletions and/or rearrangements in the nar gene cluster. In contrast to other nitrate reductase-deficient mutants and to wild type, deletion mutants and the regulatory mutant nit-2 were incapable of accumulating intracellular nitrate. Two of the mutants in which expression of all the nar loci did not occur, C2 and D2, grew in nitrite medium and showed wild-type levels of both nitrite uptake and nitrite reductase activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Inverse spectrophotometric detection in flow injection analysis: determination of nitrite using cerium(IV) as the chromophore

The application of inverse spectrophotometric detection in flow injection is introduced and its potential illustrated by a possible application to the determination of nitrite by its reaction with cerium(IV).A single manifold was employed with two reagent concentrations, namely 10 and 20 g l–1, of cerium(IV) ammonium sulphate in 0.5 M sulphuric acid. The working ranges are 10–200 and 200–1000 mg l–1, respectively, for a ca. 30-µl sample injection and the sampling rate is between 60 and 75 injections per hour. The 200–1000 mg l–1 system was applied to the determination of nitrite in culture media and the results were compared with those given by a manual diazotisation method.The same manifold with a smaller sample loop was also employed to demonstrate the extension of the technique to provide chemical back-off. A working range of approximately 2500 mg l–1 of nitrite can be covered anywhere between 1000 and 9000 mg l–1 simply by selection of a suitable reagent concentration.

Inhibitor Evaluation with Immobilized Nitrobacter agilis Cells

Nitrobacter agilis was entrapped in calcium alginate beads and used as a floating bed supplied with a continuous flow of nitrite medium. Complete nitrite oxidation was achieved within 30 h, and the system could be maintained for at least 210 h. The immobilized Nitrobacter system was subjected to sulfur oxyanions, acidity, and metal ions. Thiosulfate and tetrathionate (up to 20 mM each) did not inhibit the nitrite oxidation activity. A low pH of 4.2 resulted in the complete cessation of nitrite oxidation, and the activity was not restored upon increasing the pH to 7. Nitrite oxidation by N. agilis was sensitive to 10 mM each Ni and Al but insensitive to 10 mM MoO(4).

Culture and nitrite uptake in immobilized Scenedesmus obliquus

The green alga Scenedesmus obliquus was immobilized in Ca-alginate beads. The cell growth after immobilization was studied by cell counting. The nitrite uptake was not affected by immobilization, except that a longer lag phase was observed in immobilized cells than in free ones. That result could be due to a barrier effect of the matrix against nitrite diffusion inside the beads. The treatment of cells by glycerol prior to their immobilization in a batch reactor induced an increase of nitrite uptake by the cells. This effect disappeared after a few runs. The glycerol effect on specific rates seemed also to decrease when the number of immobilized cells increased. This decrease can be related to the decrease of light efficiency as well as substrate accessibility when a high cell concentration was used. Several alternating runs of Tris-HCl buffer containing nitrite growth medium depleted in combined nitrogen were tested. Cellular growth occurred inside the beads up to a maximum followed by a decrease of cell number in the beads.

Assimilation of Inorganic Nitrogen Compounds by Nitrobacter agilis

Nitrobacter agilis, a chemolithotrophic bacterium, utilizes ammonia as well as nitrite as a nitrogen source for growth. The growth yields were increased about twofold by growing the bacterium in a nitrite medium supplemented with 2 mm-ammonium chloride. Higher concentrations of ammonium chloride, however, competitively inhibited nitrite oxidation and growth of the bacterium. Washed cells readily incorporated 15NH4 +, 15NH2OH, 15NO2 − and 15NO3 − respectively (in decreasing order) into cell nitrogen. Enzyme activities in cell extracts of ammonia-supplemented cultures, compared to those without ammonia, were greater for glutamate dehydrogenase (EC 1.4.1.2) and similar for glutamine synthetase (EC 6.3.1.2), whereas glutamate synthase (EC 1.4.1.14) was barely detectable. Inhibitors of glutamine synthetase (l-methionine dl-sulphoximine) and glutamate synthase (azaserine) did not affect the incorporation of 15NH4 − and 15NO2 − into cell nitrogen of washed cells. The results indicate that glutamate dehydrogenase is a key enzyme in N. agilis for the assimilation of either nitrite or ammonia. Glutamine synthetase, which was also active in cell extracts, is probably required for the production of glutamine. Glutamate synthase, however, does not appear to be an important enzyme for ammonia assimilation.

Oxygen-18 isotope-induced shifts in nitrogen-15 nuclear magnetic resonance spectroscopy. 1. Isotope shift in nitrite and its application to the study of oxygen exchange of sodium [15N,18O2]nitrite

Substitution of /sup 18/O for /sup 16/O results in an upfield shift in the /sup 15/N nuclear magnetic resonance signal of the /sup 15/N-enriched nitrite and nitrate ions. The magnitude of the shift in the /sup 15/N NMR signal depends on the number of /sup 18/O atoms covalently bonded to the nitrogen atom. In nitrite the shift is 0.138 ppM//sup 18/O. This relatively large isotope shift permits the direct study by nuclear magnetic resonance spectroscopy of oxygen-exchange reactions involving nitrogen species. Acid-catalyzed medium nitrite(oxygen)-water exchange was followed in a continuous assay mode to demonstrate the ease and applicability of the isotope shift to the study of nitrogen(oxygen)-water exchange reactions. It was found that at pH 6.26 and 28/sup 0/C a sequential mode of exchange of the oxygen atoms is observed. This observation agreed with a mechanism postulated earlier on the basis of indirect evidence.

Anaerobic transfer of antibiotic resistance from Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic pathogen that often initiates infections from a reservoir in the intestinal tract, may donate or acquire antibiotic resistance in an anaerobic environment. Only by including nitrate and nitrite in media could antibiotic-resistant and -sensitive strains of P. aeruginosa be cultured in a glove box isolator. These anaerobically grown cells remained sensitive to lytic phage isolated from sewage. After incubation with a phage lysate derived from P. aeruginosa 1822, anaerobic transfer of antibiotic resistance to recipients P. aeruginosa PS8EtBr and PS8EtBrR occurred at frequencies of 6.2 x 10(-9) and 5.0 x 10(-8) cells per plaque-forming unit, respectively. In experiments performed outside the isolator, transfer frequencies to PS8EtBr and PS8EtBrR were higher, 1.3 x 10(-7) and 6.5 x 10(-8) cells per plaque-forming unit, respectively. When P. aeruginosa 1822 was incubated aerobically with Escherichia coli B in medium containing nitrate and nitrite, the maximum concentration of carbenicillin-resistant E. coli B reached 25% of the total E. coli B population. This percentage declined to 0.01% of the total E. coli B population when anaerobically grown P. aeruginosa 1822 and E. coli B were combined and incubated in the glove box isolator. The highest concentration of the recipient population converted to antibiotic resistance occurred after 24 h of aerobic incubation, when an initially high donor/recipient ratio (>15) of cells was mixed. These data indicate that transfer of antibiotic resistance either by transduction between Pseudomonas spp. or by conjugation between Pseudomonas sp. and E. coli occurs under strict anaerobic conditions, although at lower frequencies than under aerobic conditions.

Growth and Pigments of Nitrosoguanidine Treated and Untreated Cells of the Blue-green Alga Anacystis nidulans in Different Nitrogen Sources

Summary NTG-treated cells of the blue-green alga Anacystis nidulans grew well in media supplemented with sodium nitrate and ammonium chloride but not in sodium nitrite in which case a yellowing of the cultures was also noticed. Absorption spectra of acetone extracts showed greater amounts of carotenoids and lesser amount of chlorophyll for the yellow cultures in nitrite medium while the reverse was true for cultures growing in media containing either nitrate or ammonium. No change of this kind occurred in untreated cell suspensions in the presence of different inorganic nitrogen sources supplied to them.

Inhibition of spores of Clostridium spp. by sodium nitrite.

Summary. Sodium nitrite heated in a laboratory medium was more inhibitory to spores of Clostridium spp. than nitrite added as a filter‐sterilized solution to the same medium. Most spores remained refractile after inhibition for >3 months and some proved viable when inoculated into fresh nitrite‐free medium. The inhibitory activity of heated nitrite medium was not stable indefinitely, growth sometimes occurred on re‐inoculation with vegetative cells.

NITROGEN METABOLISM OF AQUATIC ORGANISMS. II. THE ASSIMILATION OF NITRATE, NITRITE, AND AMMONIA BYBIDDULPHIA AURITA1

SUMMARYBiddulphia aurita, a centric diatom, can grow on either nitrate, nitrite, or ammonia as its sole nitrogen, source.Cells remove ammonium nitrogen from the medium 2.3–2.4 times faster than either nitrate or nitrite nitrogen and, when grown for 24 hr in the ammonium medium, contain higher levels of non‐protein nitrogen than cells grown in the nitrate or nitrite medium for the same period of time.The nitrogenous compounds in the nonprotein nitrogen fraction from cells grown in the nitrate, nitrite, or ammonium medium contain the same level of soluble‐free amino nitrogen, combined amino nitrogen, and ammonium nitrogen. The high level of soluble nonprotein nitrogen in the medium of the cells grown in the ammonium medium is due to soluble amide nitrogen which represents 18% of the total soluble nitrogen present in these cells, whereas it represents only 2% in cells from the nitrite medium, and its level is negligible in cells from the nitrate medium.Cells grown in the nitrate medium have both nitrate‐ and nitrite‐reductase activity. Cells grown in the nitrite medium have only nitrite‐reductase activity in significant levels, while cells grown in the ammonium medium lack both enzymes.

Ion-exchange equilibrium distribution studies of some metal ions in nitrite media

Ion-exchange equilibrium distribution of some metal ions has been studied in nitrite media, with cation- as well as anion-exchangers. The distribution coefficients ( K D) show the utility of a new nitrite medium which is a complexing medium for most transition metal ions giving anionic complexes in aqueous solution. Similar studies have been performed in aqueous ethanol. Increase in the concentration of ethanol led to increased complex formation and changed K D values.

The regulation of activity of the enzymes involved in the assimilation of nitrate by higher plants

1. Possible mechanisms regulating the activities of three enzymes involved in nitrate assimilation, nitrate reductase, nitrite reductase and glutamate dehydrogenase, were studied in radish cotyledons. 2. Nitrate-reductase and nitrite-reductase activities are low in nitrogen-deficient cotyledons, and are induced by their substrates. 3. Glutamate dehydrogenase is present regardless of the nitrogen status, and the enzyme can be increased only slightly by long-term growth on ammonia. 4. Although nitrate is the best inducer of nitrate reductase, lower levels of induction are also obtained with nitrite and ammonia. The experiments did not distinguish between direct or indirect induction by these two molecules. 5. Nitrite reductase is induced by nitrite and only indirectly by nitrate. 6. The induction of both nitrate reductase and nitrite reductase is prevented by the inhibitors actinomycin D, puromycin and cycloheximide, indicating a requirement for the synthesis of RNA and protein. 7. The decay of nitrate reductase, determined after inhibition of protein synthesis, is slower than the synthesis of the enzyme. Nitrite reductase is much more stable than nitrate reductase. 8. The synthesis of nitrate reductase is not repressed by ammonia, but is repressed by growth on a nitrite medium. 9. There is no inhibition of nitrate reductase, nitrite reductase or glutamate dehydrogenase by the normal end products of assimilation, but cyanate is a fairly specific inhibitor of nitrate reductase.

Salicylate degradation test for differentiation of Mycobacterium fortuitum from other mycobacteria.

SUMMARY: Most isolates of Mycobacterium fortuitum decomposed salicylate and stained black the Sauton agar medium containing salicylate (0.5 or 1 mg./ml.); and all isolates capable of decomposing salicylate were identified as M. fortuitum. Other species, including rapidly growing mycobacteria, did not decompose salicylate. Most but not all of the salicylate-decomposing isolates showed also PAS degradation and a positive 3-day arylsulphatase test. The salicylate-decomposing isolates utilized nitrite as sole nitrogen source and about one-half of them grew on a nitrite medium within 7 days. These reactions, salicylate degradation, PAS degradation, utilization of nitrite, and 3-day arylsulphatase activity, are shown to be useful for identifying M. fortuitum. The 3-day arylsulphatase test showed some exceptional cases; therefore, a positive reaction in this test was not sufficient to identify M. fortuitum. Positive reactions in the above four tests seemed to mean that an isolate is certainly M. fortuitum.

Stimulation of Nitrobacter agilis by Biotin

Krulwich, Terry A. (Goucher College, Baltimore, Md.), and Helen B. Funk. Stimulation of Nitrobacter agilis by biotin. J. Bacteriol. 90:729-733. 1965.-Addition of biotin to nitrite-mineral medium greatly stimulated the autotrophic growth of four strains of Nitrobacter agilis. Comparisons of cultures of the organisms grown in parallel at 30 C in nitrite medium and in the medium supplemented with 150 mmug of biotin per ml showed that the vitamin promoted: (i) 2- to 4-fold greater rates of utilization of nitrite, and (ii) 100- to 1,000-fold greater populations of cells per milliliter. Avidin specifically inhibited the biotin stimulation of nitrite utilization at an avidin-biotin ratio of 133:1. Incubation of the four strains of N. agilis at 37 C imposed a requirement for biotin that could be met by daily addition of 150 mmug of the vitamin per ml of medium. The stimulatory effects of the vitamin at 30 C suggest that in N. agilis the synthesis of biotin is rate-limiting for growth.

Electrolytic investigations : New electrolytic method for determining silver and separating silver from copper

A new electrolytic method is proposed for determining silver and separating silver from copper. A nitric acid-sodium nitrite medium is used. The deposits are bright and adherent, large and small amounts of silver may be accurately determined, and neither the amperage nor the voltage is critical.