Demonstration of Both a Photosynthetic and a Nonphotosynthetic CO(2) Requirement for NH(4) Assimilation in the Green Alga Selenastrum minutum

Nitrogen-limited and nitrogen-sufficient cell cultures of Selenastrum minutum (Naeg.) Collins (Chlorophyta) were used to investigate the dependence of NH₄⁺ assimilation on exogenous CO₂. N-sufficient cells were only able to assimilate NH₄⁺ maximally in the presence of CO₂ and light. Inhibition of photosynthesis with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron also inhibited NH₄⁺ assimilation. These results indicate that NH₄⁺ assimilation by N-sufficient cells exhibited a strict requirement for photosynthetic CO₂ fixation. N-limited cells assimilated NH₄⁺ both in the dark and in the light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, indicating that photosynthetic CO₂ fixation was not required for NH₄⁺ assimilation. Using CO₂ removal techniques reported previously in the literature, we were unable to demonstrate CO₂-dependent NH₄⁺ assimilation in N-limited cells. However, employing more stringent CO₂ removal techniques we were able to show a CO₂ dependence of NH₄⁺ assimilation in both the light and dark, which was independent of photosynthesis. The results indicate two independent CO₂ requirements for NH₄⁺ assimilation. The first is as a substrate for photosynthetic CO₂ fixation, whereas the second is a nonphoto-synthetic requirement, presumably as a substrate for the anaplerotic reaction catalyzed by phosphoenolpyruvate carboxylase.     

Regulation of the high-affinity ammonium transporter (BnAMT1;2) in the leaves of Brassica napus by nitrogen status

Substantial concentrations of NH₄ ⁺ are found in the apoplast of the leaves of Brassica napus. Physiological studies on isolated mesophyll protoplasts with ¹⁵NH₄ ⁺ revealed the presence of a high-affinity ammonium transporter that shared physiological similarity to the high-affinity NH₄ ⁺ transporters in Arabidopsis thaliana (AtAMT1;3). PCR techniques were used to isolate a full-length clone of a B. napus homologue of AMT1 from shoot mRNA which showed 97% similarity to AtAMT1;3. The full-length cDNA when cloned into the yeast expression vector pFL61 was able to complement a yeast mutant unable to grow on media with NH₄ ⁺ as the sole nitrogen source. Regulatory studies with detached leaves revealed a stimulation of both NH₄ ⁺ uptake and expression of mRNA when the leaves were supplied with increasing concentrations of NH₄ ⁺. Withdrawal of NH₄ ⁺ supply for up to 96 h had little effect on mRNA expression or NH₄ ⁺ uptake; however, plants grown continuously at high NH₄ ⁺ levels exhibited decreased mRNA expression. BnAMT1;2mRNA expression was highest when NH₄ ⁺ was supplied directly to the leaf and lowest when either glutamine or glutamate was supplied to the leaves, which directly paralleled chloroplastic glutamine synthetase (GS2) activity in the same leaves. These results provide tentative evidence that BnAMT1;2may be regulated by similar mechanisms to GS2 in leaves.     

Seasonal variation in ammonia compensation point and nitrogen pools in beech leaves (Fagus sylvatica)

Ammonia (NH₃) fluxes between beech leaves (Fagus sylvatica) and the atmosphere were investigated in a 90-year-old forest canopy and related to leaf nitrogen (N) pools and glutamine synthetase (GS) activities. The stomatal ammonia compensation point, ?? _NH3, was measured by both a twig cuvette and bioassay techniques involving measurements of pH and ammonium (NH ₄ ⁺ ) concentration in the leaf apoplastic solution. The ?? _NH3 determined on the basis of the gas exchange measurements followed a seasonal variation with early-season peaks during leaf expansion (9.6 nmol NH₃ mol^?1 air) and late-season peaks during leaf senescence (7.3 nmol NH₃ mol^?1 air). In the mid-season, the ?? _NH3 of mature green leaves was much lower (around 3 nmol NH₃ mol^?1 air) and dropped below the NH₃ concentration in the ambient atmosphere. For comparison, ?? _NH3 obtained by the apoplastic bioassay were 7.0, 3.7 and 6.4 nmol NH₃ mol^?1 air in early-, mid-, and late -season, thus agreeing reasonably well with ?? _NH3 values derived from the gas exchange measurements. Potential NH₃ emission fluxes during early and late season were 1.31 and 0.51 nmol m^?2 leaf surface area s^?1, respectively, while leaves were a sink for NH₃ during mid-season. During leaf establishment and senescence, both apoplastic and bulk tissue NH ₄ ⁺ concentrations were relatively high coinciding with low activities of glutamine synthetase, which is a key enzyme in leaf N metabolism. In conclusion, the exchange of NH₃ between beech leaves and the atmosphere followed a seasonal variation with NH₃ emission peaks being related to N mobilization during early leaf establishment and remobilization during late leaf senescence.     

Hydrogen peroxide is required for abscisic acid-induced NH4+ accumulation in rice leaves

The role of H₂O₂ in abscisic acid (ABA)-induced NH₄⁺ accumulation in rice leaves was investigated. ABA treatment resulted in an accumulation of NH₄⁺ in rice leaves, which was preceded by a decrease in the activity of glutamine synthetase (GS) and an increase in the specific activities of protease and phenylalanine ammonia-lyase (PAL). GS, PAL, and protease seem to be the enzymes responsible for the accumulation of NH₄⁺ in ABA-treated rice leaves. Dimethylthiourea (DMTU), a chemical trap for H₂O₂, was observed to be effective in inhibiting ABA-induced accumulation of NH₄⁺ in rice leaves. Inhibitors of NADPH oxidase, diphenyleneiodonium chloride (DPI) and imidazole (IMD), and nitric oxide donor (N-tert-butyl-α-phenylnitrone, PBN), which have previously been shown to prevent ABA-induced increase in H₂O₂ contents in rice leaves, inhibited ABA-induced increase in the content of NH₄⁺. Similarly, the changes of enzymes responsible for NH₄⁺ accumulation induced by ABA were observed to be inhibited by DMTU, DPI, IMD, and PBN. Exogenous application of H₂O₂ was found to increase NH₄⁺ content, decrease GS activity, and increase protease and PAL-specific activities in rice leaves. Our results suggest that H₂O₂ is involved in ABA-induced NH₄⁺ accumulation in rice leaves.     

A computer-controlled system for studying ammonia exchange, photosynthesis and transpiration of plant canopies growing under controlled environmental conditions

A fully automatic growth chamber system was built in order to study NH₃, exchange and NH₃, compensation points of plant canopies growing under controlled environmental conditions in which atmospheric NH₃, concentrations corresponded to those naturally occurring over terrestrial ecosystems. The system included plant cuvettes with separate root and shoot compartments constructed of coated polycarbonate. This material did not change the spectral composition of photosynthetically active light and had a low adsorption of NH₃, and water vapour. Atmospheric NH₃, concentrations in the inlet of the cuvettes were controlled by mass-flow controllers. Inlet and outlet NH₃, concentrations were measured on-line with a modified chemiluminescent NH₃, monitor. At airflow rates per unit leaf area of about 3 dm³ m^?2 s^?1, the system allowed accurate determinations of NH₃, exchange rates down to about 0.1 nmol NH₃, m^?2 s^?1. The NH₃, compensation points at anthesis for barley cultivars Laevigatum and Golf were 4.2±2.8 and 4.6±2.9nmol mol^?1 of NH₃, in air (SE, n=4), respectively. NH₃, absorption in both cultivars increased linearly with atmospheric NH₃, concentration in the range 0–30 nmol mol^?1 of NH₃, in air. NH₃, absorption was much higher in the light than in the dark, indicating a strong stomatal and/or metabolic control of NH₃, exchange. Photosynthesis and transpiration were not affected by exposure to NH₃, concentrations in the range 0–30nmol mol^?1 for 7d.     

Effect of influent substrate ratio on anammox granular sludge: performance and kinetics

Effect of influent substrate ratio on anammox process was studied in sequencing batch reactor. Operating temperature was fixed at 35 ± 1 °C. Influent pH and hydraulic retention time were 7.5 and 6 h, respectively. When influent NO₂ ^?-N/NH₄ ⁺-N was no more than 2.0, total nitrogen removal rate (TNRR) increased whereas NH₄ ⁺-N removal rate stabilized at 0.32 kg/(m³ d). ΔNO₂ ^?-N/ΔNH₄ ⁺-N increased with enhancing NO₂ ^?-N/NH₄ ⁺-N. When NO₂ ^?-N/NH₄ ⁺-N was 4.5, ΔNO₂ ^?-N/ΔNH₄ ⁺-N was 1.98, which was much higher than theoretical value (1.32). The IC₅₀ of NO₂ ^?-N was 289 mg/L and anammox activity was inhibited at high NO₂ ^?-N/NH₄ ⁺-N ratio. With regard to influent NH₄ ⁺-N/NO₂ ^?-N, the maximum NH₄ ⁺-N removal rate was 0.36 kg/(m³ d), which occurred at the ratio of 4.0. Anammox activity was inhibited when influent NH₄ ⁺-N/NO₂ ^?-N was higher than 5.0. With influent NO₃ ^?-N/NH₄ ⁺-N of 2.5–6.5, NH₄ ⁺-N removal rate and NRR were stabilized at 0.33 and 0.40 kg/(m³ d), respectively. When the ratio was higher than 6.5, nitrogen removal would be worsened. The inhibitory threshold concentration of NO₂ ^?-N was lower than NH₄ ⁺-N and NO₃ ^?-N. Anammox bacteria were more sensitive to NO₂ ^?-N than NH₄ ⁺-N and NO₃ ^?-N. TNRR would be enhanced with increasing nitrogen loading rate, but sludge floatation occurred at high nitrogen loading shock. The Han-Levenspiel could be applied to simulate nitrogen removal resulting from NO₂ ^?-N inhibition.     

Control of Flowering in the Grapevine (Vitis vinifera L.): Formation of Inflorescences in Vitro by Isolated Tendrils

Photosynthesis, primary productivity, N content, and N₂ fixation were determined as a function of applied NH₄⁺ in peas (Pisum sativum L. cv. Alaska) which were inoculated or not inoculated with Rhizobium leguminosarum. Cabon dioxide exchange rate (CER) increased 10-fold, total N content 7-fold, and total dry weight 3-fold in 26-day-old uninoculated plants as applied NH₄⁺ was increased from 0 to 16 millimolar. In inoculated plants of the same age CER and dry weight were maximal at 2 millimolar NH₄⁺, and total N content increased between 0 and 2 millimolar NH₄⁺ but did not change significantly with higher NH₄⁺ applications. Per cent N content of uninoculated plants was significantly lower than that of inoculated plants except at the highest NH₄⁺ concentration (16 millimolar). Symbiotic N₂ fixation by inoculated plants was maximal in peas grown with 2 millimolar NH₄⁺; and apparent relative efficiency of N₂ fixation, calculated from C₂H₂ reduction and H₂ evolution, was maximal in the 2 to 4 millimolar NH₄⁺ concentration range. The capacity to fix N₂ through the Rhizobium-legume symbiosis significantly enhanced the rate and efficiency of photosynthesis and plant N content when NH₄⁺ concentration in the nutrient solution was below 8 millimolar. Above 8 millimolar NH₄⁺ concentration uninoculated plants had greater CER, N content, and dry weight.     

Copper(II) complexation by D-glucosamine. Spectroscopic and potentiometric studies

The Cu(II) complex formation equilibria of D- glucosamine were studied in aqueous solution by potentiometric and spectroscopic (ESR, CD, absorption spectra) techniques. All data agree that two major species are formed in the pH region 6–9 involving two D-glucosamine ligand molecules bound to the cupric ion via NH₂(CuL₂) or NH₂ and O^? (CuH_?2L₂). In the latter case deprotonated hydroxyls were found to be very effective coordination sites for Cu(II) giving rise to chelate complexes. On the contrary, no complex formation was observed for the Cu(II) N-acetyl-D-glucosamine system.     

Kinetics of Virus Inactivation by Ammonia

Ammonia has been shown to be virucidal in sludge and NH₄Cl solutions, although the rates at which viruses are inactivated have not been thoroughly studied. In the present studies, the kinetics of the poliovirus type 1 (strain CHAT) and bacteriophage f2 inactivation were examined in such a way that the effects of OH⁻ and NH₄⁺ could be separated from those of NH₃. Purified virus stocks were placed into solutions of NH₄Cl and control solutions containing an equivalent concentration of NaCl and incubated at 20°C. The percentage of virus surviving was calculated, and the kinetics were evaluated by constructing semilogarithmic plots of data. At all pH values and NH₃ concentrations studied, the kinetics of the inactivation of both viruses were pseudo-first order. OH⁻ had no measurable effect on the viruses, whereas the effects of NH₄⁺ and Na⁺ were similar. A dose-response relationship between NH₃ and the viruses was also found. Bacteriophage f2 was approximately 4.5 times more resistant to the effects of NH₃ than was poliovirus.     

Preparation of membrane vesicles in lithium chloride from cells of Nitrosomonas europaea

The preparation of membrane vesicles in lithium chloride is described which oxidizes NH₄Cl via hydroxylamine to nitrite. The following properties of the vesicles were determined: the uptake of NH₄Cl and O₂ by electrode methods, the production of NH₂OH and NO₂ from NH₄Cl as well as ATPase activity. The stoichiometry of NH₄⁺:O₂:NO₂^? was found to be 1:1.2:0.7 in vesicles compared with 1:1.5:1.0 in either spheroplasts or washed cells. It is shown that the membrane vesicles also contain a Cu and energy-dependent NH₄^? translocase as in spheroplasts and cells.     

Ammonia Production and Assimilation in Glutamate Synthase Mutants of Arabidopsis thaliana

Ammonia production and assimilation¹ were examined in photorespiratory mutants of Arabidopsis thaliana L. lacking ferredoxin-dependent glutamate synthase (Fd-GluS) activity. Although photosynthesis was rapidly inhibited in these mutants in normal air, NH₄⁺ continued to accumulate. The accumulation of NH₄⁺ was also seen after an initial lag of 30 minutes in 2% O₂, 350 microliters per liter of CO₂ and after 90 minutes in 2% O₂, 900 microliters per liter of CO₂. The accumulation of NH₄⁺ in normal air and low O₂ was also associated with an increase in the total pool of amino acid-N and glutamine, and a decrease in the pools of glutamate, aspartate, alanine, and serine. Upon return to dark conditions, or to 21% O₂, 1% CO₂ in the light, the NH₄⁺ which had accumulated in the leaves was reassimilated into amino acids. The addition of methionine sulfoximine (MSO) resulted in higher accumulations of NH₄⁺ in glutamate synthase mutants and prevented the reassimilation of NH₄⁺ upon return to the dark. The addition of MSO also resulted in the accumulation of NH₄⁺ in glutamate synthase mutants in the light and in 21% O₂, 1% CO₂. These results indicate that glutamine synthetase is essential for the reassimilation of photorespiratory NH₄⁺ and for primary N assimilation in the leaves and strongly suggest that glutamate dehydrogenase plays only a minimal role in the assimilation of ammonia. Levels of NADH-dependent glutamate synthase (NADH-GluS) appear to be sufficient to account for the assimilation of NH₄⁺ by a GS/NADH-GluS cycle.     

Effect of soil heterogeneity on gross nitrogen mineralization measured by 15N-pool dilution techniques

Pool dilution techniques, where the soil ammonium pool is labeled with ¹⁵NH₄ ⁺, are commonly used to estimate gross N mineralization rates in soil. To estimate the rates unbiased, it is assumed that the ¹⁵NH₄ ⁺ is distributed homogenously in ambient ¹⁴NH₄ ⁺ pool of the soil. However, completely homogeneous distribution of ¹⁵NH₄ ⁺ may commonly not be feasible. The objective of this paper was to examine the importance of the spatial distribution of ¹⁴NH₄ ⁺ and ¹⁵NH₄ ⁺ on the measured gross N mineralization rate. In a 2-day incubation experiment, gross N mineralization rates were measured in soil, where different distributions of ¹⁴NH₄ ⁺ and ¹⁵NH₄ ⁺ were combined. Generally, distribution of ¹⁵NH₄ ⁺ to 50% of the soil volume did not alter the measured gross mineralization rate however more heterogeneous distribution caused the rate to be underestimated. Certain combinations of ¹⁴NH₄ ⁺ and ¹⁵NH₄ ⁺ distributions caused the rate to be overestimated. Based on the experimental results, we developed a 2-dimensional model array of soil compartments, to estimate the gross N mineralization and gross NH₄ ⁺ consumption rates in local microsites in the soil. If one of the nitrogen-isotopes was more abundant in a compartment with high NH₄ ⁺-concentration, and the other nitrogen-isotope was more abundant in a compartment with low NH₄ ⁺-concentration, the nitrogen-isotopes would have different apparent bioavailability, hence the gross N mineralization rate would be erroneously estimated. On the other hand, in soil where all compartments had low NH₄ ⁺-concentrations, heterogeneous distribution of ¹⁴NH₄ ⁺, ¹⁵NH₄ ⁺ and microbial activity did not influence the measured gross N mineralization rate significantly.     

Switching substrate specificity of AMT/MEP/ Rh proteins

In organisms from all kingdoms of life, ammonia and its conjugated ion ammonium are transported across membranes by proteins of the AMT/Rh family. Efficient and successful growth often depends on sufficient ammonium nutrition. The proteins mediating this transport, the so called Ammonium Transporter (AMT) or Rhesus like (Rh) proteins, share a very similar trimeric overall structure and a high sequence similarity even throughout the kingdoms. Even though structural components of the transport mechanism, like an external substrate recruitment site, an essential twin histidine pore motif, a phenylalanine gate and the hydrophobic pore are strongly conserved and have been analyzed in detail by molecular dynamic simulations and mutational studies, the substrate(s), which pass the central pores of the AMT/Rh subunits, NH₄⁺, NH₃ + H⁺, NH₄⁺ + H⁺ or NH₃, are still a matter of debate for most proteins, including the best characterized AmtB protein from Escherichia coli. The lack of a robust expression system for functional analysis has hampered proof of structural and mutational studies, although the NH₃ transport function for Rh-like proteins is rarely disputed. In plant transporters belonging to the subfamily AMT1, transport is associated with electrical currents, while some plant transporters, notably of the AMT2 type, were suggested to transport NH₃ across the membrane, without associated ionic currents. Here we summarize data in favor of each substrate for the distinct AMT/Rh classes, discuss mutants and how they differ in structure and functionality. A common mechanism with deprotonation and subsequent NH₃ transport through the central subunit pore is suggested.     

Switching substrate specificity of AMT/MEP/ Rh proteins

In organisms from all kingdoms of life, ammonia and its conjugated ion ammonium are transported across membranes by proteins of the AMT/Rh family. Efficient and successful growth often depends on sufficient ammonium nutrition. The proteins mediating this transport, the so called Ammonium Transporter (AMT) or Rhesus like (Rh) proteins, share a very similar trimeric overall structure and a high sequence similarity even throughout the kingdoms. Even though structural components of the transport mechanism, like an external substrate recruitment site, an essential twin histidine pore motif, a phenylalanine gate and the hydrophobic pore are strongly conserved and have been analyzed in detail by molecular dynamic simulations and mutational studies, the substrate(s), which pass the central pores of the AMT/Rh subunits, NH₄⁺, NH₃ + H⁺, NH₄⁺ + H⁺ or NH₃, are still a matter of debate for most proteins, including the best characterized AmtB protein from Escherichia coli. The lack of a robust expression system for functional analysis has hampered proof of structural and mutational studies, although the NH₃ transport function for Rh-like proteins is rarely disputed. In plant transporters belonging to the subfamily AMT1, transport is associated with electrical currents, while some plant transporters, notably of the AMT2 type, were suggested to transport NH₃ across the membrane, without associated ionic currents. Here we summarize data in favor of each substrate for the distinct AMT/Rh classes, discuss mutants and how they differ in structure and functionality. A common mechanism with deprotonation and subsequent NH₃ transport through the central subunit pore is suggested.     

Dysbalance of Astrocyte Calcium under Hyperammonemic Conditions

Increased brain ammonium (NH₄ ⁺/NH₃) plays a central role in the manifestation of hepatic encephalopathy (HE), a complex syndrome associated with neurological and psychiatric alterations, which is primarily a disorder of astrocytes. Here, we analysed the influence of NH₄ ⁺/NH₃ on the calcium concentration of astrocytes in situ and studied the underlying mechanisms of NH₄ ⁺/NH₃-evoked calcium changes, employing fluorescence imaging with Fura-2 in acute tissue slices derived from different regions of the mouse brain. In the hippocampal stratum radiatum, perfusion with 5 mM NH₄ ⁺/NH₃ for 30 minutes caused a transient calcium increase in about 40% of astrocytes lasting about 10 minutes. Furthermore, the vast majority of astrocytes (∼90%) experienced a persistent calcium increase by ∼50 nM. This persistent increase was already evoked at concentrations of 1–2 mM NH₄ ⁺/NH₃, developed within 10–20 minutes and was maintained as long as the NH₄ ⁺/NH₃ was present. Qualitatively similar changes were observed in astrocytes of different neocortical regions as well as in cerebellar Bergmann glia. Inhibition of glutamine synthetase resulted in significantly larger calcium increases in response to NH₄ ⁺/NH₃, indicating that glutamine accumulation was not a primary cause. Calcium increases were not mimicked by changes in intracellular pH. Pharmacological inhibition of voltage-gated sodium channels, sodium-potassium-chloride-cotransporters (NKCC), the reverse mode of sodium/calcium exchange (NCX), AMPA- or mGluR5-receptors did not dampen NH₄ ⁺/NH₃-induced calcium increases. They were, however, significantly reduced by inhibition of NMDA receptors and depletion of intracellular calcium stores. Taken together, our measurements show that sustained exposure to NH₄ ⁺/NH₃ causes a sustained increase in intracellular calcium in astrocytes in situ, which is partly dependent on NMDA receptor activation and on release of calcium from intracellular stores. Our study furthermore suggests that dysbalance of astrocyte calcium homeostasis under hyperammonemic conditions is a widespread phenomenon, which might contribute to the disturbance of neurotransmission during HE.     

The Uptake of NO3?, NO2?, and NH4+ by Intact Wheat (Triticum aestivum) Seedlings : I. Induction and Kinetics of Transport Systems

The inducibility and kinetics of the NO₃⁻, NO₂⁻, and NH₄⁺ transporters in roots of wheat seedlings (Triticum aestivum cv Yercora Rojo) were characterized using precise methods approaching constant analysis of the substrate solutions. A microcomputer-controlled automated high performance liquid chromatography system was used to determine the depletion of each N species (initially at 1 millimolar) from complete nutrient solutions. Uptake rate analyses were performed using computerized curve-fitting techniques. More precise estimates were obtained for the time required for and the extent of the induction of each transporter. Up to 10 and 6 hours, respectively, were required to achieve apparent full induction of the NO₃⁻ and NO₂⁻ transporters. Evidence for substrate inducibility of the NH₄⁺ transporters requiring 5 hours is presented. The transport of NO₃⁻ was mediated by a dual system (or dual phasic), whereas only single systems were found for transport of NO₂⁻ and NH₄⁺. The K_m values for NO₃⁻, NO₂⁻, and NH₄⁺ were, respectively, 0.027, 0.054, and 0.05 millimolar. The K_m for mechanism II of NO₃⁻ transport could not be defined in this study as it exhibited only apparent first order kinetics up to 1 millimolar.     

Variation inRhizobium leguminosarum response to short term application of NH4NO3 to nodulatedPisum sativum L.

Summary This study was conducted to determine the effect of short term application of NH₄NO₃ on nodule function and to determine whether the rhizobial isolate used was a significant factor in this effect. Pea plants were inoculated with 10 differentRhizobium leguminosarum isolates and grown for 3 weeks in N-free medium before addition of 0, 1, 2 or 5 mM NH₄NO₃ for 2 to 7 days. Acetylene reduction and leghemoglobin content decreased with increasing exposure time to NH₄NO₃ and with increasing concentration of NH₄NO₃. NH ₄ ⁺ and NO ₃ ⁻ depletion from the nutrient medium were assayed in plants exposed to 5 mM NH₄NO₃ and mean uptake rates were similar for each ion. There were significant differences among isolates in the rate of decrease of C₂H₂ reduction with increasing NH₄NO₃ concentration (C₂H₂ reduction responsiveness to NH₄NO₃) 4 and 7 days after addition of NH₄NO₃ but no differences after 2 days of exposure to NH₄NO₃. There were significant differences among isolates in NH ₄ ⁺ depletion from the nutrient medium but these differences were not correlated with the differences observed in C₂H₂ reduction. Ranking of the isolates for C₂H₂ reduction responsiveness to NH₄NO₃ applied to plants with nodules was different from that obtained when NH₄NO₃ was applied at seeding. Isolates with varying sensitivity to NH₄NO₃ may be useful tools for determining the mechanisms responsible for inhibition of symbiotic N₂ fixation by combined nitrogen. NRCC paper no. 25863.     

Impaired electron transfer accounts for the photosynthesis inhibition in wheat seedlings (Triticum aestivum L.) subjected to ammonium stress

No single mechanism can provide an adequate explanation for the inhibition of photosynthesis when plants are supplied with ammonium (NH₄⁺) as the sole nitrogen (N) source. We performed a hydroponic experiment using two N sources [5 mM NH₄⁺ and 5 mM nitrate (NO₃^?)] to investigate the effects of NH₄⁺ stress on the photosynthetic capacities of two wheat cultivars (NH₄⁺‐sensitive AK58 and NH₄⁺‐tolerant XM25). NH₄⁺ significantly inhibited the growth and light‐saturated photosynthesis (A_sat) of both cultivars, but the extent of such inhibition was greater in the NH₄⁺‐sensitive AK58. The CO₂ concentration did not limit CO₂ assimilation under NH₄⁺ nutrition; though both stomatal and mesophyll conductance were significantly suppressed. Carboxylation efficiency (CE), light‐saturated potential rate of electron transport (J_max), the quantum efficiency of PSII (Φ_PSII), electron transport rate through PSII [Je(PSII)], and F_v/F_m were significantly reduced by NH₄⁺. As a result, NH₄⁺ nutrition resulted in a significant increase in the production of hydrogen peroxide (H₂O₂) and superoxide anion radicals (O₂^??), but these symptoms were less severe in the NH₄⁺‐tolerant XM25, which had a higher capacity of removing elevated reactive oxygen species (ROS). Thus, NH₄⁺ N sources might decreased electron transport efficiency and increased the production of ROS, exacerbating damage to the electron transport chain, leading to a reduced plant photosynthetic capacity.     

The effect of anions on the reaction catalyzed by lupine-nodule glutamate dehydrogenase

The kinetics of the reductive amination reaction of lupine-nodule glutamate dehydrogenase (l-glutamate:NAD oxidoreductase (deaminating), EC 1.4.1.2) were found to vary with the identity of the ammonium salt which was used as a substrate. Normal Michaelis-Menten kinetics were obtained with (NH₄)₂SO₄ but when NH₄Cl or NH₄-acetate was varied apparent substrate inhibition was observed. Linear double-reciprocal plots were obtained with NH₄Cl and NH₄-acetate, however, if the concentration of Cl^? or acetate was maintained constant by adding KCl or K-acetate. Chloride and acetate were subsequently found to cause linear noncompetitive inhibition with respect to NH₄⁺ and the apparent substrate inhibition by NH₄Cl and NH₄-acetate can be explained as the result varying a substrate and a noncompetitive inhibitor in constant ratio. Other anions were also found to be inhibitors of the glutamate dehydrogenase reaction; I^? caused parabolic noncompetitive inhibition with respect to NH₄⁺ and NO₃^? caused slope-parabolic noncompetitive inhibition with respect to all three substrates of the reductive amination reaction. For the oxidation deamination reaction, Cl^? was a linear competitive inhibitor with respect to both NAD and l-glutamate whereas NO₃^? caused parabolic competitive inhibition with respect to these reactants. To explain the results, it is proposed that anions bind to an allosteric site and cause a change in some of the rate constants of the reaction. Specifically, the results are consistent with anions causing decreases in the rates of association of NADH and 2-oxoglutarate with the enzyme and an increase in the rate of dissociation of NAD.     

Synthesis,in vitro stability,and antiproliferative effect of d‐cysteine modified GnRH‐doxorubicin conjugates

Overexpression of gonadotropin‐releasing hormone (GnRH) receptor in many tumors but not in normal tissues makes it possible to use GnRH analogs as targeting peptides for selective delivery of cytotoxic agents, which may help to enhance the uptake of anticancer drugs by cancer cells and reduce toxicity to normal cells. The GnRH analogs [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH, [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH, and [d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH were conjugated with doxorubicin (Dox), respectively, through N‐succinimidyl‐3‐maleimidopropionate as a linker to afford three new GnRH‐Dox conjugates. The metabolic stability of these conjugates in human serum was determined by RP‐HPLC. The antiproliferative activity of the conjugates was examined in GnRH receptor‐positive MCF‐7 human breast cancer cell line by MTT assay. The three GnRH‐Dox conjugates showed improved metabolic stability in human serum in comparison with AN‐152. The antiproliferative effect of conjugate II ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH‐Dox) on MCF‐7 cells was higher than that of conjugate I ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH‐Dox) and conjugate III ([d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH‐Dox), and the cytotoxicity of conjugate II against GnRH receptor‐negative 3T3 mouse embryo fibroblast cells was decreased in comparison with free Dox. GnRH receptor inhibition test suggested that the antiproliferative activity of conjugate II might be due to the cellular uptake mediated by the targeting binding of [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH to GnRH receptors. Our study indicates that targeting delivery of conjugate II mediated by [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH is a promising strategy for chemotherapy of tumors that overexpress GnRH receptors.