SENSITIVE NITRATE DETERMINATION IN WATER AND MEAT SAMPLES BY AMPEROMETRIC BIOSENSOR

This study describes a novel biosensor method for specific determination of nitrate in food and water samples by using nitrate reductase (NR) (EC 1.9.6.1) biosensor based on the detection of oxidation peak current of redox mediator, methyl viologen, related to nitrate concentration. The method was shown to be selective and sensitive to determine the nitrate levels of water samples and processed meat samples. Immobilization procedure and also working conditions of the biosensor were optimized. Dynamic range attained with this method was established as (5.0–90.0 × 10^?9 M) for nitrate concentration with a 10 s response time. Limit of detection (LOD) and quantification (LOQ) of the biosensor were calculated as 2.2 × 10^?9 M and 5.79 × 10^?9 M, respectively. Reproducibility experiments was established on repetitive measurements by using a freshly prepared biosensor for avoiding the memory effect. The RSD was calculated as 1.22% at a nitrate concentration of 4.7 × 10^?8 M (n = 7).     

NITRATE REDUCTASE MUTANTS IN EUDORINA ELEGANS (CHLOROPHYCEAE)1

Three nitrate reductase mutants were independently isolated and characterized in the colonial alga, Eudorina elegans Ehrenberg. nar-1 is a leaky mutant, deficient in the production of nitrate reductase. nar-2 and nar-3 both lack the ability to produce nitrate reductase. However, nar-2 grows and nar-3 does not grow when hypoxanthine is the sole nitrogen source. The specific activity of the next enzyme, in the pathway, nitrite reductase is increased in nar-3 when compared to wild-type, nar-1 and nar-2.     

COMPARISONS OF NITRATE UPTAKE, STORAGE, AND REDUCTION IN MARINE DIATOMS AND FLAGELLATES

Diatoms, but not flagellates, have been shown to increase rates of nitrogen release after a shift from a low growth irradiance to a much higher experimental irradiance. We compared NO₃ ^? uptake kinetics, internal inorganic nitrogen storage, and the temperature dependence of the NO₃ ^? reduction enzymes, nitrate (NR) and nitrite reductase (NiR), in nitrogen‐replete cultures of 3 diatoms (Chaetoceros sp., Skeletonema costatum, Thalassiosira weissflogii) and 3 flagellates (Dunaliella tertiolecta, Pavlova lutheri, Prorocentrum minimum) to provide insight into the differences in nitrogen release patterns observed between these species. At NO₃ ^? concentrations <40 μmol‐N·L ^{? 1}, all the diatom species and the dinoflagellate P. minimum exhibited saturating kinetics, whereas the other flagellates, D. tertiolecta and P. lutheri, did not saturate, leading to very high estimated K _s values. Above ～60 μmol‐N·L ^{? 1}, NO₃ ^? uptake rates of all species tested continued to increase in a linear fashion. Rates of NO₃ ^? uptake at 40 μmol‐N·L ^{? 1}, normalized to cellular nitrogen, carbon, cell number, and surface area, were generally greater for diatoms than flagellates. Diatoms stored significant amounts of NO₃ ^? internally, whereas the flagellate species stored significant amounts of NH₄ ⁺ . Half‐saturation concentrations for NR and NiR were similar between all species, but diatoms had significantly lower temperature optima for NR and NiR than did the flagellates tested in most cases. Relative to calculated biosynthetic demands, diatoms were found to have greater NO₃ ^? uptake and NO₃ ^? reduction rates than flagellates. This enhanced capacity for NO₃ ^? uptake and reduction along with the lower optimum temperature for enzyme activity could explain differences in nitrogen release patterns between diatoms and flagellates after an increase in irradiance.     

重庆市蔬菜硝酸盐、亚硝酸盐含量及其与环境的关系

在重庆市的主要蔬菜中,硝酸盐含量依次为根菜类＞叶菜类＞葱蒜类＞瓜类＞豆类＞茄果类,前３类的硝酸盐含量超标,但各种蔬菜的亚硝酸盐含量一般较低,未超过卫生标准。就同一种蔬菜的不同样品而言,硝酸盐和亚硝酸盐含量的差异很大,说明品种或环境条件可显著影响其含量。重庆市多雾少光,冬春低温,土壤含氮量和钠高子含量较高,有效钾含量较低可能是导致蔬菜大量积累硝酸盐的环境因素。蔬菜不同器官硝酸盐和亚硝酸盐含量各异,根＞茎＞叶柄＞叶片。     

VARIABILITY IN NITRATE UPTAKE KINETICS IN THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

Short-term (1–9 min) nitrate uptake kinetics were measured in Thalassiosira pseudonana (Hust.) Hasle & Heimdal grown in nitrate-limited, ammonium-limited, and nitrate-sufficient continuous cultures. For all cultures, maximal nitrate uptake rates did not develop until approximately 3 min after nitrate addition; thereafter, nitrate uptake rates remained constant or declined slightly. The K_s and V_max for the nitrate-limited cultures were higher at any growth rate than those for the ammonium-limited or nitrate-sufficient cultures. Thus, much higher nitrate concentrations would be required to saturate nitrate uptake in nitrate-limited Thalassiosira pseudonana than is usually considered necessary. The lack of data for other species grown under a range of environmental conditions makes it difficult to generalize about the effect of preconditioning on nitrate uptake kinetics.     

RELATIONSHIPS BETWEEN NITRATE REDUCTASE ACTIVITY AND RATES OF GROWTH AND NITRATE INCORPORATION UNDER STEADY-STATE LIGHT OR NITRATE LIMITATION IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

Although activity of the enzyme nitrate reductase (NR) can potentially be used to predict the rate of nitrate incorporation in field assemblages of marine phytoplankton, application of this index has met with little success because the relationship between the two rates is not well established under steady-state conditions. To provide a basis for using NR activity measurements, the relationships among NR activity, growth rate, cell composition, and nitrate incorporation rate were examined in cultures of Thalassiosira pseudonana (Hustedt)Hasle and Heimdal, growing a) under steady-state light limitation, b) during transitions between low and high irradiance (15 or 90 μmol quanta.m^?2.s^?1), and c) under steady-state nitrate limitation. Using a modified assay for NR involving additions of bovine serum albumin to stabilize enzyme activity, NR activity in light-limited cultures was positively and quantitatively related to calculated rates of nitrate incorporation, even in cultures that were apparently starved of selenium. During transitions in irradiance, growth rates acclimated to new conditions within 1 day; through the transition, the relationship between NR activity and nitrate incorporation rate remained quantitative. In nitrate-limited chemostat cultures, NR activity was positively correlated with growth rate and with nitrate incorporation rates, but the relationship was not quantitative. NR activity exceeded nitrate incorporation rates at lower growth rates (<25% of nutrient-replete growth rates), but chemostats operating at such low dilution rates may not represent ecologically relevant conditions for marine diatoms. The strong relationship between NR activity and nitrate incorporation provides support for the idea that NR is rate-limiting for nitrate incorporation or is closely coupled to the rate-limiting step. In an effort to determine a suitable variable for scaling NR activity, relationships between different cell components and growth rate were examined. These relationships differed depending on the limiting factor. For example, under light limitation, cell volume and cell carbon content increased significantly with increased growth rate, while under nitrate limitation cell volume and carbon content decreased as growth rates increased. Despite the differences found between cell composition and growth rate under light and nitrate limitation, the relationships between NR activity scaled to different compositional variables and growth rate did not differ between the limitations. In field situations where cell numbers are not easily determined, scaling NR activity to particulate nitrogen content may be the best alternative. These results establish a strong basis for pursuing NR activity measurements as indices of nitrate incorporation in the field.     

VARIABILITY OF NITRATE UPTAKE CAPACITY IN MACROCYSTIS PYRIFERA (LAMINARIALES,PHAEOPHYTA) WITH NITRATE AND LIGHT AVAILABILITY1

The nitrate uptake capacity of mature blade tissue of the giant kelp, Macrocystis pyrifera (L.) C. Ag., was examined as a function of the availability of light and nitrate. Time course measurements indicated that nitrate uptake rate, as measured by the incorporation of ¹⁵N, was significantly increased by N starvation. The response was linear over the first hour of exposure regardless of the N status of the tissue indicating that surge uptake was not responsible for the increase. The Michaelis-Menten parameters V_max and K_s, however, were not significantly changed by either growth nitrate concentration or growth irradiance as a result of high variability among blades. Similarly, the initial slope (α) of the nitrate uptake kinetics curves was unaffected. Concentration of photosynthetic pigments increased in response to increased nitrate availability but not to increased growth irradiance. Time course and pigment data demonstrated that mature blade tissue responds to increased N availability by decreasing its capacity to take up nitrate and by increasing its investment in photosynthetic pigments, perhaps for N storage or enhanced light-harvesting capabilities and the increase in reducing power available for N assimilation. This study provides evidence for a dynamic regulatory system that responds to changes in nitrate availability in an integrated manner.     

NITRATE UPTAKE IN MARINE PHYTOPLANKTON: (NITRATE,CHILORIDE)-ACTIVATED ADENOSINE TRIPHOSPHATASE FROM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1,2

A membrane-bound (nitrate, chloride)-activated AT Pase was found in the neritie diatom, Skeletonema costatum (Grev.) Cleve. The enzyme is suggested to translocate NO₃⁻ across the plasmalemma, against a concentration gradient. The K_m of the enzyme with respect to NO₃⁻ is ca. 0.9 × 10⁻⁶ M, and is in close agreement with the reported K₈ for NO₃⁻ uptake by the whole cell.     

INTRASPECIFIC COMPARISONS OF NITRATE UPTAKE IN THREE MARINE DIATOMS1

Short-term (within 5 min) and long-term (≤2 h) rates of nitrate uptake were determined for the marine diatoms, Nitschiella longissima (Cleve), Skeletonema costatum (Greville) Cleve and Asterionella japonica (Cleve). Pigment levels, cell carbon, nitrogen and cell volume were also determined for cells in the logarithmic and stationary phases of growth. For each species, one clone isolated from oligotrophic coastal water and one clone isolated from eutrophic coastal water were compared. Long-term NO₃^? uptake typically followed saturation kinetics describable by the Michaelis-Menten expression. Under experimental conditions, half-saturation constants ranged from 0.6 to 2.2 μM NO₃^?. Generally, the oligotrophic clones had lower Ks and V_max (on a per cell basis) than their eutrophic counterparts, though this was only statistically significant in one pair of clones. Eutrophic and oligotrophic clones also differed in their short-term response to nutrient addition; oligotrophic clones showed greatest rate of uptake at the lowest nitrate addition while uptake by eutrophic clones increased with increasing nitrate concentration. However, all clones had very similar V_max values expressed on a dry weight basis. Under N-starvation, cellular C and pigment levels (and N to a lesser extent) generally declined more in eutrophic than in oligotrophic clones. While the differences between inshore and offshore clones were not great, the results are consistent with the hypothesis that eutrophic waters support algae which grow faster and are less conservative biochemically than cells in oligotrophic waters.     

CHARACTERIZATION OF DIATOM (BACILLARIOPHYCEAE) NITRATE REDUCTASE GENES AND THEIR DETECTION IN MARINE PHYTOPLANKTON COMMUNITIES1

The complete assimilatory nitrate reductase (NR) gene from the pennate diatom Phaeodactylum triconutum Bohlin was sequenced from cDNA and compared with NR sequences from fungi, green algae, vascular plants, and the recently sequenced genome of the centric diatom Thalassiosira pseudonana Hasle and Heimdal CCMP1335. In all the major eukaryotic nitrate reductase (Euk‐NR) functional domains, diatom NR gene sequences are generally 50%–60% identical to plant and alga sequences at the amino acid level. In the less conserved N‐terminal, hinge 1, and hinge 2 regions, homology to other NR sequences is weak, generally<30%. Two PCR primer sets capable of amplifying Euk‐NR from plants, algae, and diatoms were designed. One primer set was used to amplify a 750‐base pair (bp) NR fragment from the cDNA of five additional diatom strains. The PCR amplicon spans part of the well‐conserved dimer interface region, the more variable hinge 1 region, and part of the conserved cytochrome b heme binding region. The second primer set, targeted to the dimer region, was used to amplify an approximately 400‐bp fragment of the NR gene from DNA samples collected in Monterey Bay, California and in central New Jersey inner continental shelf (LEO‐15 site) waters. Only diatom‐like NR sequences were recovered from Monterey Bay samples, whereas LEO‐15 samples yielded NR sequences from a range of photosynthetic eukaryotes. The prospect of using DNA‐ and RNA‐based methods to target the NR genes of diatoms specifically is a promising approach for future physiological and ecological experiments.     

水稻叶片硝酸还原酶mRNA的体外翻译

水稻叶片总RNA经Oligo(dT)—Cellulose柱层析分离Poly(A)~+RNA。在往层析中采用变温洗脱的方法,可减少rRNA的混杂,提高mRNA的纯度和体外翻译活力。它对麦胚无细胞系统蛋白质合成的促进可稳定在10倍,有时高达20多倍。NR—mRNA的体外翻译产物可用以NR为载体的火箭免疫电泳方法进行鉴定,其量可用火箭峰内的放射强度来表示。     

NITRATE AND PHOSPHATE UPTAKE INTERACTIONS IN A MARINE PRYMNESIOPHYTE1

The short- and long-term uptake of nitrate and phosphate ions, and their interactions, were studied as functions of the preconditioning of Pavlova lutheri (Droop) Green. Populations were preconditioned in continuous culture at a variety of growth rates and N:P supply ratios. The maximum uptake rates cell^?1 for nitrate and phosphate were of similar magnitudes, in spite of the forty-fold smaller requirement for phosphorus. Short-term phosphate uptake was independent of the nitrate concentration, but the short-term nitrate uptake rate was reduced in the presence of phosphate. The severity of inhibition of nitrate uptake by phosphate was positively correlated with the preconditioning N:P supply ratio and the preconditioning growth rate. In response to large additions of nutrients, P. lutheri was able to increase its phosphorus content sixty-fold, but was only able to take up enough nitrate to double its nitrogen content. The high rate of phosphate uptake relative to its requirement, the inhibition of nitrate uptake by phosphate, and the large capacity for phosphorus storage relative to its requirement, all of which were observed even under N limitation, may imply that even where nitrogen is limiting there can be interspecific competition for available phosphate.     

NITRATE UPTAKE KINETICS AND CLONAL VARIABILITY IN THE NERITIC DIATOM BIDDULPHIA AURITA1

Nitrate uptake experiments were conducted at 18 and 25 C on subtropical and temperate isolates of the neritic diatom Biddulphia aurita (Lyngb.) Breb. & Godey. Two clones (STX-88 and B1) were grown in a 12:12 LD cycle in NO₃-limited continuous cultures used for uptake experiments. The half-saturation constant (K_s) and the maximum NO₃- uptake rate (V_m) were measured under conditions in which external NO₃- concentration controlled uptake rate. The kinetic constants for both clones were higher at the higher temperature. The subtropical clone (STX-88) had lower kinetic values than temperate clone (B1) at both temperatures. The differences appear to be of adaptive significance since STX-88 has a NO₃- uptake system, described by its lower K_s value, which minimizes the double stress effects of high temperature-low NO₃- concentration conditions characteristic of its habitat. Cytoplasmic enzyme electrophoretic analyses were conducted on the clones. Differences in banding patterns and mobilities showed evidence of allelic substitution. The results suggest genetic variation at the intraspecific level.     

硫和6-苄氨基腺嘌呤对水稻幼苗硝酸还原酶活性的影响

缺硫培养6天的水稻幼苗,其叶片和根中的硝酸还原酶(NR)活性明显下降。用1pPm 的6-苄氨基腺嘌呤(6-BA)处理培养了10天的水稻幼苗根系,24小时后缺硫培养的水稻幼苗叶片和根系的 NR 活性升高,加硫培养的水稻幼苗叶片和根中的 NR 活性下降。用~(35)S示踪发现,6-BA 可降低加硫幼苗对~(35)S 的吸收和转化,但促进缺硫幼苗对~(35)S 的转化。     

光呼吸代谢物乙醇酸、乙醛酸和草酸对烟草叶片硝酸还原的影响

乙醇酸、乙醛酸和草酸能明显促进烟草(Nicotiana rustica)叶片在黑暗中的硝酸还原,光呼吸抑制剂a-羟基吡啶甲烷磺酸能消除前二者的促进作用而不能完全消除草酸的作用。草酸+NAD~+能显著促进离体的硝酸还原。烟叶提取液加入草酸和NAD~+后生成NADH和CO_2认为活体内由乙醛酸氧化生成的草酸是经脱氢生成NADH供硝酸还原之用。未能证明在烟叶内存在乙醇酸脱氨酶,因此排除由乙醇酸直接脱氢以还原硝酸的可能。     

PHASED OSCILLATIONS IN CELL NUMBERS AND NITRATE IN BATCH CULTURES OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE)1

Alexandrium tamarense (M. Lebour) Balech strains isolated in spring 2007 from a single bloom in Thau lagoon have been grown in nonaxenic artificial media. For three strains showing large oscillations in biomass (crashes followed by recoveries) on a scale of several days, a significant relationship was observed between changes in cell densities (as in vivo fluorescence) and changes in nitrate concentrations. Increases in cell densities were accompanied by decreases in nitrate, while decreases in cell densities corresponded to increases in nitrate, presumably due to nitrification. Net increases in nitrate could reach up to 15 μmol N · L^?1 · d^?1 indicating a very active nitrifying archaeal/bacterial population. However, following population crashes, algal cells can recover and attain biomass levels similar to those reached during the first growth phase. This finding indicates that those archaea/bacteria do not compete for nutrients or do not hamper algal growth under those conditions. In contrast to diatoms, dinoflagellates such as A. tamarense do not excrete/exude dissolved organic matter, thus preventing excessive bacterial growth. This mechanism could help explain the recovery of this species in the presence of bacteria.