Application of Nitrate Reductase for the Determination of Nitrate in Meat and Fishery Products

A rapid and specific method is described for the determination of nitrate in meat and fishery products.

Nitrate separated from foods by extraction with 1/50Ν sodium hydroxide and ultrafiltration was readily reduced to nitrite by the use of respiratory nitrate reductase (NR) from Escherichia coli K-12. The nitrite so obtained can be determined by the specific diazotation-coupling reaction method.

The use of an enzymatic reaction resulted in quantitative reduction of nitrate, and the method was relatively free of interferences. Recoveries of 10 and 100 ppm of nitrate from 5 samples of meat and fishery products ranged from 92.8 to 97.8% for 10 ppm and 97.8 to 99.4% for 100 ppm with a detection limit of 0.5 ppm.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-arabitol by a newly isolated <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis>

A newly isolated Zygosaccharomyces rouxii NRRL 27,624 produced d-arabitol as the main metabolic product from glucose. In addition, it also produced ethanol and glycerol. The optimal conditions were temperature 30°C, pH 5.0, 350 rpm, and 5% inoculum. The yeast produced 83.4 ± 1.1 g d-arabitol from 175 ± 1.1 g glucose per liter at pH 5.0, 30°C, and 350 rpm in 240 h with a yield of 0.48 g/g glucose. It also produced d-arabitol from fructose, galactose, and mannose. The yeast produced d-arabitol and xylitol from xylose and also from a mixture of xylose and xylulose. Resting yeast cells produced 63.6 ± 1.9 g d-arabitol from 175 ± 1.8 g glucose per liter in 210 h at pH 5.0, 30°C and 350 rpm with a yield of 0.36 g/g glucose. The yeast has potential to be used for production of xylitol from glucose via d-arabitol route. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. department of Agriculture.     

Micellization of conjugated chenodeoxy- and ursodeoxycholates and solubilization of cholesterol into their micelles: comparison with other four conjugated bile salts species

Micelle formations of sodium glyco- and taurochenodeoxycholate (NaGCDC and NaTCDC) and sodium glyco- and tauroursodeoxycholates (NaGUDC and NaTUDC) was studied at 308.2 K for their critical micelle concentrations at various NaCl concentrations by pyrene fluorescence probe, and the degree of counterion binding to micelle was determined using the Corrin-Harkins plots. The degree of counterion binding was found to be 0.37-0.38 for chenodeoxycholate conjugates, while the determination of the degree was quite difficult for ursodeoxycholate conjugates. The change of I1/I3 values on the fluorescence spectrum with the conjugate bile salt concentration suggested two steps for their bile salt aggregation. The first step is a commencement of smaller aggregates, the first cmc, and the second one is a starting of stable aggregates, the second cmc. The aggregation number was determined at 308.2 K and 0.15 M NaCl concentration by static light scattering: 16.3 and 11.9 for sodium NaGCDC and NaTCDC, and 7.9 and 7.1 for NaGUDC and NaTUDC, respectively. The solubilization of cholesterol into the bile salt micelles in the presence of coexisting cholesterol phase and the maximum additive concentration (MAC) of cholesterol was determined against the bile salt concentration. The standard Gibbs energy change for the solubilization was evaluated, where the micelles were regarded as a chemical species. The solubilization was stabilized in the order of NaGUDC approximately = NaTUDC < NaTC < NaGC < NaTCDC < NaGCDC < NaTDC < NaGDC, where the preceding results were taken into the order.     

Physiological stress exacerbates murine colitis by enhancing proinflammatory cytokine expression that is dependent on IL-18

Psychological stress is an environmental factor considered to be a precipitating factor of inflammatory bowel disease. Interleukin (IL)-18 plays a role in stress-induced aggravation in some diseases. The aim of this study was to establish a model of murine colitis exacerbated by psychological stress and to clarify the role of IL-18 in this model. Male C57Bl/6 mice and IL-18(-/-) mice were used for this study. The mice received dextran sulfate sodium (DSS) for induction of colitis. Some mice were exposed to psychological stress using a communication box. Body weight, colonic length, and histological inflammation were measured for assessment of colitis. Tumor necrosis factor (TNF)-α and IL-18 expression in the colon and IL-18 expression in the adrenal gland were analyzed using real-time PCR. The effect of anti-IL-18 antibody was also investigated. Effects of TNF-α and IL-18 on cytokine expressions were studied using the colonic epithelial cell line LS174T. Induction of psychological stress in DSS-treated wild-type mice significantly exacerbated colitis with enhanced expression of proinflammatory cytokines and IL-18. However, induction of psychological stress in DSS-treated IL-18(-/-) mice did not aggravate colitis compared with that in the IL-18(-/-) group given only DSS treatment. Stress-induced aggravation of colitis was ameliorated significantly by anti-IL-18 antibody treatment. IL-18 did not enhance TNF-α-induced expression of intercellular adhesion molecule-1 or IL-8 in LS174T. We established a model of colitis exacerbated by psychological stress. Psychological stress enhanced IL-18 expression and plays a proinflammatory role in stress-induced aggravation of colitis.     

Induction of Manganese Superoxide Dismutase by Cytokines and Lipopolysaccharide in Cultured Mouse Astrocytes

Abstract: To determine whether cytokines or lipopolysaccharide (LPS) are involved in the induction of superoxide dismutase (SOD) in the nervous system, we examined the effects of these substances on the levels of SOD in cultured mouse astrocytes. Treatment of astrocytes with 10² to 10⁴ U/ml tumor necrosis factor-α for 3 days increased the levels of Mn SOD in a dose- and time-dependent manner to as much as six times the level under nontreated conditions. Treatment with 1.0 µg/ml LPS for 3 days elicited a fourfold increase in levels of Mn SOD, and the effect of LPS was also dose dependent. Furthermore, Mn SOD in astrocytes was induced by a 3-day exposure to interleukin-1α at concentrations of 10² or 10³ U/ml. However, these stimuli had no effect on levels of copper-zinc SOD (Cu/Zn SOD) in astrocytes. By contrast, interferon-γ did not change the levels of either Mn or Cu/Zn SOD in the cells. The results indicate that the selective induction of Mn SOD by cytokines and LPS, which has been observed in nonnervous tissues, may also occur in nervous tissues. The induction of Mn SOD may represent a mechanism for protection of cells from oxidative stress.     

Emergence of Proteases in Germinating Cucumber Cotyledons and Their Roles in the Two-Step Degradation of Storage Protein

The degradation of storage protein in germinating cucumber seedswas shown to proceed via two distinct steps. First, severalproteases with acidic isoelectric points (pIs) were involvedin solubilization and partial degradation of 11S globulin. Treatmentof seedlings with cycloheximide inhibited this step and theexpression of these proteases. Thus, the first step appearedto be governed by these proteases, which were synthesized denovo after imbibition. The first step was observed in dark-growncotyledons, but the complete degradation of 11S globulin didnot occur in the absence of illumination. An additional protease,with a pI of 4.5, was induced by illumination, and it was involvedin the further cleavage of the partially degraded products ofIIS globulin. Thus, the complete degradation of the storageprotein proceeded via a two-step process in illuminated germinatingseedlings. Light is needed to induce the second step in thedegradation of 11S globulin that supplies the nitrogen requiredfor development of the photosynthetic apparatus in the greeningcotyledon. (Received November 9, 1995; Accepted January 31, 1996)     

NADH-dependent nitrate reductase from two-row barley roots: Purification, characteristics and comparison with leaf enzyme

NADH-nitrate reductase (EC 1.6.6.1) was purified 800-fold from roots of two-row barley ( Hordeum vulgare L. cv. Daisen-gold) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on TSK-gel (G3000SW). The specific activity of the purified enzyme was 6.2 μmol nitrite produced (mg protein)⁻¹ min⁻¹ at 30°C.
Besides the reduction of nitrate by NADH, the root enzyme, like leaf nitrate reductase, also catalyzed the partial activities NADH-cytochrome c reductase, NADH-ferricyanide reductase, reduced methyl viologen nitrate reductase and FMNH₂-nitrate reductase. Its molecular weight was estimated to be about 200 kDa, which is somewhat smaller than that for the leaf enzyme. A comparison of root and leaf nitrate reductases shows physiologically similar or identical properties with respect to pH optimum, requirements of electron donor, acceptor, and FAD, apparent K_m for nitrate, NADH and FAD, pH tolerance, thermal stability and response to inorganic orthophosphate. Phosphate activated root nitrate reductase at high concentration of nitrate, but was inhibitory at low concentrations, resulting in increases in apparent K_m for nitrate as well as V_max whereas it did not alter the K_m for NADH.     

Isolation and characterization of cDNAs encoding vacuolar H+-pyrophosphatase isoforms from rice (Oryza sativa L.)

The vacuolar H⁺-pyrophosphatase (V-PPase) is an electrogenic H⁺ pump, which was found in the plant vacuolar membrane. Two cDNA clones (OVP1 and OVP2) encoding the V-PPase were isolated from cultured rice (Oryza sativa L.) cells and subsequently sequenced. The sequence analysis has revealed thatOVP1 contains 2316 nucleotides of open reading frame (ORF) and 362 nucleotides of the 3-untranslated region, whereasOVP2 comprises 2304 nucleotides of ORF and 312 nucleotides of the 3-untranslated region. The nucleotide sequences of ORF ofOVP1 andOVP2 are 80.7% identical, and their 5- and 3-untranslated regions have 39.4% and 48.4% identity, respectively. The polypeptides encoded by the ORF ofOVP1 andOVP2 contain 771 and 767 amino acids, respectively, and the sequences of the OVP proteins are very similar to those of other V-PPases, which are shown to have 85–91% homology. Chromosomal mapping by RFLP techniques demonstrates that OVP1 and OVP2 are isoforms encoded by different genes. BothOVP1 andOVP2 are mapped on the same chromosome (chromosome 6) to a distance of ca. 90 cM. Northern analysis indicates that theOVP1 andOVP2 are also expressed in intact rice plants andOVP2 shows higher expression in the calli than the roots and shoots, compared toOVP1. These results show that at least two genes encoding the V-PPases are present in rice genome and their expressions are probably regulated in a different manner.     

Purification and Characterization of Thiol Proteinase as a Nitrate Reductase-Inactivating Factor from Leaves of Hordeum distichum L.

A thiol proteinase was purified 6,400-fold from leaves of Hordeumdistichum L. by a sequence of ammonium sulfate fractionation,gel filtration, ion exchange chromatography, hydrophobic chromatographyand chromatofocusing. This enzyme also had nitrate reductase(NR)-inactivating activity, which was associated with proteolyticactivity in almost constant proportions during the purificationprocedures. Its molecular weight was estimated as 74,000 bygel filtration, and it had an isoelectric point of 4.05 andan apparent K_m of 0.83 mg ml^–1 for azocasein. The respectiveoptimum pH for proteolytic and NR-inactivating activities were6.0 and 7.0. On electrophoresis, the proteinase gave a majorband that coincided with both activities; it also produced afaint band associated with no activity. Our purified thiol proteinase inactivated FMNH₂-NR and MVH-NRas well as NADH-NR, but it had only a slight effect on NADHcytochrome c reductase activity. This enzyme also inactivatedglutamine synthetase. (Received September 16, 1983; Accepted January 26, 1984)     

Amperometric dimethyl sulfoxide sensor using dimethyl sulfoxide reductase from Rhodobacter sphaeroides

An amperometric dimethyl sulfoxide (DMSO) sensor was constructed based on DMSO reductase (DMSO-R). DMSO-R from Rhodobacter sphaeroides f. sp. denitrificans was immobilized by BSA-glutaraldehyde cross-linking at the surface of a glassy carbon electrode. Mediators were added to the sample solution in a free form. Several mediators (methyl viologen (MV), benzyl viologen (BV), neutral red (NR), safranin T (ST), FMN, phenazine methosulfate (PMS)), which can donate electrons to DMSO-R, were examined with the DMSO-R immobilized electrode. Among them MV was selected as a model mediator because of its wide linear response range and fast response time. The response current was effected by the measurement temperature but hardly effected by the pH of the sample solution. The response current was increased with the measurement temperature up to 50 degrees C. A response current was observed at 1 microM DMSO and the response time was 20 s under the optimum conditions. The response was observed for approximately 2 weeks. By the reduction of Schiff base in the cross-linking layer the response range became narrower but most of the response current was retained at 300 microM of DMSO for more than 5 weeks.