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Hydrogen Recycling by Rhizobium leguminosarum Isolates and Growth and Nitrogen Contents of Pea Plants (Pisum sativum L.).

The ability to recycle H(2) evolved by nitrogenase is thought to be of importance in increasing the efficiency of N(2) fixation and to be a factor in increasing plant yield in symbiotic systems. To determine whether this ability is a significant factor in the Rhizobium leguminosarum-Pisum sativum L. system, plants were inoculated with R. leguminosarum isolates which differed in their ability to oxidize H(2) and in their relative efficiency of N(2) fixation. These plants were grown at three levels of irradiance and harvested after 3, 4, and 5 weeks of growth for determination of C(2)H(2) reduction, H(2) evolution and uptake, plant dry weight, and N content. Plants inoculated with uptake hydrogenase-positive (Hup) isolates did not exhibit higher dry weight or N content than those inoculated with Hup isolates under any of the growth conditions studied. The efficiency of the nitrogenase system of Hup isolates increased at a low irradiance, a factor which may allow them to compete successfully with Hup isolates. In some HupR. leguminosarum isolates, H(2) oxidation is coupled to ATP formation, whereas in others, it is not. There were no differences in plant dry weight and N content in plants inoculated with the two types and grown for 5 weeks at three irradiance levels. The addition of H(2) to Hup nodules whose supply of photosynthate had been removed by stem excision did not increase C(2)H(2) reduction in either coupled or uncoupled types.

Journal Article↗

Use of precision agriculture technology to investigate spatial variability in nitrogen yields in cut grassland.

Spatial variability in N uptake and utilisation by swards within uniformly managed field units could be responsible for a significant proportion of the NH3, N2O, NO3- and NOx (NO and NO2) 'pollutants' generated by agriculture and released to the environment. An investigation was commenced, therefore, to quantify, map and explain the spatial variability in sward N yield in a 'large' silage field and to assess the potential for managing this variability using some of the latest precision agriculture technology. Sward dry matter (DM) and N yields were predicted from the results of plant tissue analyses using mathematical models. Sward N yields were found to vary greatly across the field seemingly because of differences in net soil N mineralisation, but the pattern of variability appeared to remain constant with time. Conventional soil analysis of a range of soil chemical and physical properties, however, failed to explain this variability. It was concluded that the N-yield distribution map might be used in place of soil analysis as the basis for varying the rates of N application to different parts of the field with the twin objectives of maximising fertiliser use efficiency and minimising N emissions to air and water.

Agriculture↗

Effects of graded amounts of duodenal infusions of methionine on the mammary uptake of major milk precursors in dairy cows.

Arteriovenous differences in plasma and rate of blood flow were measured across the udder of four lactating dairy cows to examine the effects of increased supplies of a limiting AA on nutrient balance to the mammary gland. Treatment consisted of graded amounts of duodenal infusions of DL-Met (0, 8, 16, and 32 g/d) over 4-d periods according to a 4 x 4 Latin square design. Cows were fed a diet formulated to meet protein and energy requirements and based on 70:30 corn silage:concentrate ratio and dehydrated alfalfa (1.5 kg/d of DM). The basal diet met 70% of Met requirements. Yields of milk, fat, and protein and milk fat content were unaffected by infusions of Met, but milk protein content tended to increase by 5% to a maximum at the third treatment. Surprisingly, the rate of mammary blood flow declined in response to infusions of Met. The mammary uptake remained constant, nevertheless, for oxygen, energy-yielding nutrients, and nonessential AA. In contrast, the quantity of essential AA taken up by the udder decreased. As a result, milk protein yield seemed sustained by an increased efficiency of the mammary gland. In addition, in response to the Met infusions, the arterial supply of Met nearly increased 3-fold, and the mammary uptake of Met was similar across all treatments. The uptake of Met by the udder seems to be more related to the quantity of Met required for milk protein synthesis than to its concentration in the artery.

Animals↗

Monoclonal antibodies to a nitroxide lipid hapten.

The isolation and characterization of a hybridoma cell line producing a monoclonal IgG1 antibody against a spin-label nitroxide group is described. The antibody recognizes a synthetic hapten containing linked dinitrophenyl and 2,2,6,6-tetramethylpiperidinyl-1-oxy groups, having an affinity of 3.6 +/- 1.0 . 10(6) M-1 for the soluble hapten at 25 degrees. The antibody binds to phospholipid vesicles containing 2 mol% of spin label-derivitized lipid (lipid hapten) with an affinity of 1.5 +/- 0.2 . 10(8) M-1. This monoclonal IgG1 mediates the binding of hapten-bearing lipid vesicles to mouse macrophage RAW264 cells bearing Fc receptors. The cellular responses to this binding are similar to those observed previously using polyclonal rabbit anti-hapten IgG. As with the heterogeneous antibodies, the monoclonal IgG1 is more efficient in mediating cellular uptake when the vesicles are in the "fluid' physical state (dimyristoylphosphatidylcholine at 37 degrees C) compared to "solid' (dipalmitoylphosphatidylcholine at 37 degrees C). Despite the enhanced binding of "fluid' phospholipid vesicles to cells, only the "solid' vesicles triggered a significant respiratory burst in Raw264 macrophages.

Animals↗

Brain metabolism of branched-chain amino acids.

The synthesis of brain glutamate requires an amino group donor that is efficiently transported into the brain and that is readily transaminated. The branched-chain amino acids (BCAA), particularly leucine, play this important role. The uptake of leucine across the blood-brain barrier is faster than any other amino acid. Studies with 15N-labelled branched-chain amino acids indicate that at least one third of the amino groups of brain glutamate are derived from the BCAA; leucine alone probably donates at least 25%. Transamination occurs in large measure in astrocytes, which articulate closely with the brain capillaries across which amino acids must past as they are transported from the blood. After using BCAA nitrogen for glutamate synthesis, the astrocytes may release the branched-chain ketoacid, e.g., alpha-ketoisocaproate, to the extracellular fluid, from which it can be taken up in a neuronal compartment and there converted back to leucine. This process, which consumes an equimolar amount of glutamic acid, may provide a mechanism for the "buffering" of brain glutamate if levels of this excitatory (and potentially toxic) neurotransmitter become elevated. Leucine so formed in neurons is released to the extracellular fluid and transported back to the astrocytes, thereby completing a "leucine-glutamate cycle."

Amino Acids, Branched-Chain↗

Utilization of 2-aminoethylarsonic acid in Pseudomonas aeruginosa.

This paper describes the metabolism, transport and growth inhibition effects of 2-aminoethylarsonic acid (AEA) and 3-aminopropylarsonic acid (APrA). The former compound supported growth of Pseudomonas aeruginosa, as sole nitrogen source. The two arsonates inhibited the growth of this bacterium when 2-aminoethylphosphonic acid (AEP) but not alanine or NH4Cl, was supplied as the only other nitrogen source. The analogy between AEA and the natural compound AEP led us to examine the in vitro and in vivo interaction of AEA with the enzymes of AEP metabolism. The uptake system for AEP (Km 6 microM) was found to be competitively inhibited by AEA and APrA (Ki 18 microM for each). AEP-aminotransferase was found to act on AEA with a Km of 4 mM (3.85 mM for AEP). Alanine and 2-arsonoacetaldehyde was generated concomitantly, in a stoichiometric reaction. In vivo, AEA was catabolized by the AEP-aminotransferase since it was able to first induce this enzyme, then to be an efficient substrate. The lower growth observed may have been due to the slowness with which the permease and the aminotransferase were induced, and hence to a poor supply of alanine by transamination.

Aminoethylphosphonic Acid↗

Relationship of low lysine and high arginine concentrations to efficient ethanolic fermentation of wheat mash.

Very high gravity wheat mashes containing 20 or more grams of carbohydrates per 100 mL were fermented completely by Saccharomyces cerevisiae, even though these mashes contained low amounts of assimilable nitrogen. Supplementation of wheat mashes with various amino acids or with yeast extract, urea, or ammonium sulfate reduced the fermentation time. However, lysine or glycine added as single supplements, inhibited yeast growth and fermentation. With lysine, yeast growth was severely inhibited, and a loss of cell viability as high as 80% was seen. Partial or complete reversal of lysine-induced inhibition was achieved by the addition of a number of nitrogen sources. All nitrogen sources that relieved lysine-induced inhibition of yeast growth also promoted uptake of lysine and restored cell viability to the level observed in the control. They also increased the rate of fermentation. Experiments with minimal media showed that for lysine to be inhibitory to yeast growth, assimilable nitrogen in the medium must be in growth-limiting concentrations or totally absent. In the presence of excess nitrogen, lysine stimulated yeast growth and fermentation. Results indicate that supplementing wheat mash with other nitrogen sources increases the rate of fermentation not only by providing extra nitrogen but also by reducing or eliminating the inhibitory effect of lysine on yeast growth.

Arginine↗

Milk production and nitrogen utilization in response to postruminal infusion of sodium caseinate in lactating cows.

Five lactating, rumen-fistulated Holstein cows were used to obtain additional information concerning the effects of postruminal infusion of sodium caseinate on milk production and amino acid utilization. A 7-day continuous abomasal infusion of approximately 450 g/day of sodium caseinate was preceded and followed by 7-day infusions of an isonitrogenous-isocaloric solution of glucose, monosodium glutamate, and urea. Total collections of milk, urine, and feces were obtained during the last 5 days of each infusion period. On the last day of each period, arterial and mammary venous blood samples were obtained for analysis of plasma free amino acids. During infusion of sodium caseinate, milk production, milk protein (N times 6.38) production, and efficiency of nitrogen utilization for milk crude protein production were increased. Arterial plasma concentrations of free histidine, isoleucine, phenylalanine, valine, and total essential amino acids were elevated above control levels during infusion of sodium caseinate, while ornithine and tryrosine were decreased. Calculation of the relative concentration of essential amino acids in arterial plasma and in milk protein indicated that methionine and lysine were least abundant in plasma relative to their requirement for milk protein synthesis. A high precentage extraction from arterial plasma by the mammary gland also suggested that methionine and lysine may have been the essential amino acids in most critical supply for milk protein synthesis. Calculation of uptake to output ratios of individual plasma amino acids by the mammary gland suggested that significant quantities of extracted arginine, isoleucine, leucine, threonine, and valine were utilized in pathways other than direct incorporation into milk protein.

Amino Acids↗

Carbon and nitrogen assimilation in relation to yield: mechanisms are the key to understanding production systems.

Improved understanding of crop production systems in relation to N-supply has come from a knowledge of basic plant biochemistry and physiology. Gene expression leads to protein synthesis and the formation of metabolic systems; the ensuing metabolism determines the capacity for growth, development and yield production. This constitutes the genetic potential. These processes set the requirements for the supply of resources. The interactions between carbon dioxide (CO(2)) and nitrate () assimilation and their dynamics are of key importance for crop production. In particular, an adequate supply of, its assimilation to amino acids (for which photosynthesized carbon compounds are required) and their availability for protein synthesis, are essential for metabolism. An adequate supply of stimulates leaf growth and photosynthesis, the former via cell growth and division, the latter by larger contents of components of the light reactions, and those of CO(2) assimilation and related processes. If the supply of resources exceeds the demand set by the genetic potential then production is maximal, but if it is less then potential is not reached; matching resources to potential is the aim of agriculture. However, the connection between metabolism and yield is poorly quantified. Biochemical characteristics and simulation models must be better used and combined to improve fertilizer-N application, efficiency of N-use, and yields. Increasing N-uptake at inadequate N-supply by increasing rooting volume and density is feasible, increasing affinity is less so. It would increase biomass and N/C ratio. With adequate N, at full genetic potential, more C-assimilation per unit N would increase biomass, but energy would be limiting at full canopy. Increasing C-assimilation per unit N would increase biomass but decrease N/C at both large and small N-supply. Increasing production of all biochemical components would increase biomass and demand for N, and maintain N/C ratio. Changing C- or N-assimilation requires modifications to many processes to effect improvements in the whole system; genetic engineering/molecular biological alterations to single steps in the central metabolism are unlikely to achieve this, because targets are unclear, and also because of the complex interactions between processes and environment. Achievement of the long-term objectives of improving crop N-use and yield with fewer inputs and less pollution, by agronomy, breeding or genetic engineering, requires a better understanding of the whole system, from genes via metabolism to yield.

Agriculture↗

The influence of phosphorus deficiency on growth and nitrogen fixation of white clover plants.

The effects of P deficiency on growth, N(2)-fixation and photosynthesis in white clover (Trifolium repens L.) plants were investigated using three contrasting relative addition rates of P, or following abrupt withdrawal of the P supply. Responses to a constant below-optimum P supply rate consisted of a decline in N(2)-fixation per unit root weight and a small reduction in the efficiency with which electrons were allocated to the reduction of N(2) in nodules. Abrupt removal of P arrested nodule growth and caused a substantial decline in nitrogenase activity per unit root weight, but not per unit nodule mass. Similarly, the rate of photosynthesis per unit leaf area was unaffected by abrupt P removal, whereas CO(2) acquisition for the plant as a whole decreased due to a decline in total leaf area, leaf area per unit leaf weight and utilization of incoming radiation. These changes followed the decline in tissue P concentrations. The ratio between CO(2)-fixation and N(2)-fixation was maintained under short-term P deprivation but increased under long-term low P supply, indicating a regulatory inhibition of nodule activity following morphological and growth adjustments. It is concluded that N(2)-fixation did not limit the growth of clover plants experiencing P deficiency. A low P status induced changes in the relative growth of roots, nodules and shoots rather than changes in N and/or C uptake rates per unit mass or area of these organs.

Carbon Dioxide↗

Opposite fluxes of glutamine and alanine in the splanchnic area are an efficient mechanism for nitrogen sparing in rats.

Glutamine release by the liver constitutes a process of nitrogen salvage through the recycling of a part of the nitrogen, which prevents irreversible nitrogen losses as urea. The aim of this work was to study the nitrogen cycling in the splanchnic bed under different nutritional conditions: fed state, postabsorptive state (16 h food deprivation) or prolonged starvation (24 or 40 h). Rats were adapted to a 15% casein diet for 15 d and then sampled. The digestive, hepatic and splanchnic balances of glucose, lactate, ketone bodies, urea and amino acids were determined. There was a net release of lactate and alanine by the digestive tract, due to the high rate of glycolysis and glutaminolysis. During prolonged starvation, ketone bodies became major energy fuel for the intestine. In fed rats, there was a net uptake of most amino acids by the liver, except for glutamine and glutamate. Urea, glutamine and glutamate released represented 33, 24 and 6% of total nitrogen taken up by the liver, respectively. In postabsorptive rats, compared with fed rats, there was a significant reduction of ureagenesis, and glutamine became the major form of nitrogen released by the liver. In fact, nitrogen cycling in the form of glutamine or glutamate in the liver may be interpreted as a nitrogen salvage process, rather than as an acid-base control process. In the splanchnic area, in parallel with a highly active cycling of glucose as lactate, there exists a nitrogen cycling involving opposite fluxes of glutamine and alanine.

Alanine↗

Oxygen binding and NO scavenging properties of truncated hemoglobin, HbN, of Mycobacterium smegmatis.

Unraveling of microbial genome data has indicated that two distantly related truncated hemoglobins (trHbs), HbN and HbO, might occur in many species of slow-growing pathogenic mycobacteria. Involvement of HbN in bacterial defense against NO toxicity and nitrosative stress has been proposed. A gene, encoding a putative HbN homolog with conserved features of typical trHbs, has been identified within the genome sequence of fast-growing mycobacterium, Mycobacterium smegmatis. Sequence analysis of M. smegmatis HbN indicated that it is relatively smaller in size and lacks N-terminal pre-A region, carrying 12-residue polar sequence motif that is present in HbN of M. tuberculosis. HbN encoding gene of M. smegmatis was expressed in E. coli as a 12.8kD homodimeric heme protein that binds oxygen reversibly with high affinity (P50 approximately 0.081 mm Hg) and autooxidizes faster than M. tuberculosis HbN. The circular dichroism spectra indicate that HbN of M. smegmatis and M. tuberculosis are structurally similar. Interestingly, an hmp mutant of E. coli, unable to metabolize nitric oxide, exhibited very low NO uptake activity in the presence of M. smegmatis HbN as compared to HbN of M. tuberculosis. On the basis of cellular heme content, specific nitric oxide dioxygenase (NOD) activity of M. smegmatis HbN was nearly one-third of that from M. tuberculosis. Additionally, the hmp mutant of E. coli, carrying M. smegmatis HbN, exhibited nearly 10-fold lower cell survival under nitrosative stress and nitrite derived reactive nitrogen species as compared to the isogenic strain harboring HbN of M. tuberculosis. Taken together, these results suggest that NO metabolizing activity and protection provided by M. smegmatis HbN against toxicity of NO and reactive nitrogen is significantly lower than HbN of M. tuberculosis. The lower efficiency of M. smegmatis HbN for NO detoxification as compared to M. tuberculosis HbN might be related to different level of NO exposure and nitrosative stress faced by these mycobacteria during their cellular metabolism.

Amino Acid Motifs↗

In-stream uptake dampens effects of major forest disturbance on watershed nitrogen export.

Between January 4 and 10, 1998, a severe ice storm impacted large areas of northern New York, New England, and eastern Canada. This storm struck the Hubbard Brook Experimental Forest in New Hampshire on January 7-8, 1998, and caused extensive forest crown damage (>30%) in a narrow elevation band (600-740 m) across the south-facing experimental watersheds. Stream water has been collected and chemically analyzed since 1963 in six experimental watersheds at Hubbard Brook Experimental Forest; thus, we were able to examine the effect of this severe natural disturbance on watershed nutrient export and changes in instream nitrate (NO3-) processing. The ice storm caused large increases in watershed export of NO3- for 2 years after the disturbance, but our examination of in-stream processing suggests that NO3- losses would have been much more dramatic had there not been an increase in in-stream, nitrogen-processing efficiency after the ice storm. The canopy damage that resulted from the ice storm led to increased light availability and large inputs of woody debris to the stream. We suspect that increases in algal production and storage and processing of terrestrial litter account for the increase in inorganic nitrogen processing in these streams. Our results indicate that, without in-stream processing, export of NO3- from the damaged watersheds would have been 80-140% higher than was observed. These results point to an intriguing negative feedback mechanism whereby the same disturbance that causes watershed NO3- loss may simultaneously lead to increased in-stream retention and transformation.

Ecosystem↗

A feasibility study of a Salix viminalis gravel hydroponic system to renovate primary settled wastewater.

A Salix viminalis/gravel system based on hydroponics was developed for wastewater renovation in order to avoid the problems of soil damage and pollution associated with long-term application of wastewater to soil. For such a system to work the mineral elements applied must match closely the requirements of the tree species. To examine this the growth and nutrient uptake of S. viminalis in wastewater was compared with that in Long Ashton nutrient solution (1/4 strength). S. viminalis grew more slowly in wastewater than in Long Ashton solution, but exhibited no obvious deficiency or toxicity symptoms. Since industrial wastewaters often contain metals, the extent to which copper might inhibit wastewater treatment in this system was also examined. S. viminalis was grown in wastewater amended with 10 and 100 ppm copper. Trees were unaffected by wastewater with 10 ppm copper when compared to trees grown in wastewater alone. Wastewater containing with 100 ppm copper was too toxic for the trees to thrive and wastewater treatment was reduced. Treatment efficiencies for unamended wastewater were 57.7% for nitrogen, 90.6% for phosphorus and 24.9% for potassium. These efficiencies are much greater than those quoted for a Salix/soil system, and thus Salix/gravel systems may have potential for wastewater treatment in environmentally sensitive areas or situations.

Biodegradation, Environmental↗

Leaching and crop uptake of nitrogen from nitrogen-15-labeled green manures and ammonium nitrate.

Green manures can be used as an N source for agricultural crops as a substitute for inorganic N fertilizers. The effects of using green manures on leaching and uptake of N by spring barley (Hordeum vulgare L.) were evaluated in a 2-yr lysimeter study. Ryegrass (Lolium perenne L.) and red clover (Trifolium pratense L.) labeled with (15)N were applied in May of the first year at 160 kg total N ha(-1). Simultaneously, (15)NH(4)(15)NO(3) was applied at 80 kg N ha(-1) to additional lysimeters and others were left without N additions (control). During the second year, all lysimeters, except the control, received 80 kg N ha(-1) as unlabeled NH(4)NO(3). The cumulative, average loads of total N leached during the two years were: 37 (control), 62 (NH(4)NO(3)), 50 (ryegrass manure), and 73 (red clover manure) kg ha(-1). The differences among the treatments were not significant (P > 0.05), but the control had significantly smaller (P < 0.05) leaching loads than the treatments. About 24% of ryegrass- and red clover-derived N and 43% of NH(4)NO(3) were removed through spring barley grain and stover during the two growing seasons. Thus, the N use efficiency in barley was substantially larger when grown with inorganic N fertilizer than when grown with green manure. Viewed in combination with the tendency for larger N leaching loads under red clover manure, claims about water quality benefits of legume-based green manures should be evaluated with regard to the timing of N release and demand for N by the plant.

Agriculture↗

The effect of supplementary protein on in vivo metabolism of the mammary gland in lactating dairy cows.

Four lactating cows equipped with rumen and duodenal cannulas were fed a diet of grass silage and concentrates containing either 12.4 or 17.2% CP (DM basis) in a change-over design. Additional protein was supplied as white fish meal. Fish meal did not affect molar proportions of VFA in the rumen, but duodenal NAN supply was increased .69 g/g of N in supplementary feed. In Experiment 2, three lactating dairy cows that had been prepared with catheters across the mammary gland were fed the same diets using a switchback design. Blood samples were taken to determine changes in metabolite flux to the mammary gland. In both experiments, milk production and protein yield were nonsignificantly increased by addition of fish meal. Milk urea output was increased from 3.18 to 4.74 g/d by fish meal supplementation, reflecting increased arterial concentrations of urea. Concentrations of glucose, VFA, and BHBA in blood showed no substantial changes because of dietary supplementation of fish meal. Supply of essential AA increased 26% with fish meal supplementation, mammary uptake increased 34%, but milk protein output only increased 5%. The low efficiency of conversion of supplementary protein to milk protein appears to be related to the inability of the gland to utilize the additional AA.

Amino Acids↗

Identification of a high affinity NH4+ transporter from plants.

Despite the important role of the ammonium ion in metabolism, i.e. as a form of nitrogen that is taken up from the soil by microorganisms and plants, little is known at the molecular level about its transport across biomembranes. Biphasic uptake kinetics have been observed in roots of several plant species. To study such transport processes, a mutant yeast strain that is deficient in two NH4+ uptake systems was used to identify a plant NH4+ transporter. Expression of an Arabidopsis cDNA in the yeast mutant complemented the uptake deficiency. The cDNA AMT1 contains an open reading frame of 501 amino acids and encodes a highly hydrophobic protein with 9-12 putative membrane spanning regions. Direct uptake measurements show that mutant yeast cells expressing the protein are able to take up [14C]methylamine. Methylamine uptake can be efficiently competed by NH4+ but not by K+. The methylamine uptake is optimal at pH 7 with a Km of 65 microM and a Ki for NH4+ of approximately 10 microM, is energy-dependent and can be inhibited by protonophores. The plant protein is highly related to an NH4+ transporter from yeast (Marini et al., accompanying manuscript). Sequence homologies to genes of bacterial and animal origin indicate that this type of transporter is conserved over a broad range of organisms. Taken together, the data provide strong evidence that a gene for the plant high affinity NH4+ uptake has been identified.

Amino Acid Sequence↗

An investigation of 11C-methane, 13N-nitrous oxide and 11C-acetylene as regional cerebral blood flow agents.

Radioisotopes of xenon or krypton are generally used as the inert diffusible indicators in regional cerebral blood flow (rCBF) studies. These gases have disadvantages when introduced into the body inhalation because of their low solubility in body tissues. The use of 11C-methane, 13N-nitrous oxide and 11C-acetylene as inhalation rCBF agents has been investigated. These gases span a wide range of solubilities: methane is four times less soluble than xenon in body tissues while nitrous oxide and acetylene are respectively 3 and 6 times more soluble. Cerebral blood flow measurements were performed on normal volunteers using the three labelled gases as well as 133Xe. All tracers were administered by inhalation and the cerebral clearance of the gases was monitored by coincidence detection. Uptake in the body was found to be dependent on solubility. The computed blood flow values found using the various indicators were in excellent agreement and were within the normal cerebral blood flow range. The high solubilities of acetylene and nitrous oxide result in a more efficient transfer of tracer from lung to brain than the conventionally used agents. The positron emission is also advantageous for tomographic rCBF measurements.

Acetylene↗