Free Asn Research Articles

Reducing Acrylamide Formation Potential by Targeting Free Asparagine Accumulation in Seeds.

Acrylamide is a probable carcinogen in humans and is formed when reducing sugars react with free asparagine (Asn) during thermal processing of food. Although breeding for low reducing sugars worked well in potatoes, it is less successful in cereals. However, reducing free Asn in cereals has great potential for reducing acrylamide formation, despite the role that Asn plays in nitrogen transport and amino acid biosynthesis. In this perspective, we summarize the efforts aimed at reducing free Asn in cereal grains and discuss the potentials and challenges associated with targeting this essential amino acid, especially in a seed-specific manner.

Reducing α-Synuclein in Human CSF; An Evaluation of Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of MEDI1341, an α-Synuclein-specific antibody, in Healthy Volunteers and Parkinson’s Disease Patients (P1-11.007)

<h3>Objective:</h3> To evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of MEDI1341 (TAK-341) in single (SAD; NCT03272165) and multiple ascending dose (MAD; NCT04449484) studies undertaken in healthy volunteers (HVs) and Parkinson’s disease patients, respectively. <h3>Background:</h3> MEDI1341 is a human IgG1λ monoclonal antibody, engineered for selective, high-affinity binding of α-synuclein (ASN) and for reduced effector function. MEDI1341 recognises both monomeric and aggregated forms of human ASN. In rats and cynomolgus monkeys, MEDI1341 decreases free ASN in brain interstitial fluid and cerebrospinal fluid (CSF). In a mouse model, MEDI1341 attenuates ASN axonal propagation and accumulation in the brain. <h3>Design/Methods:</h3> HVs aged 18–80 years were eligible for the SAD study. Subjects eligible for the MAD study were aged 40–85 years with a diagnosis of mild-moderate Parkinson’s disease according to the UK Parkinson’s Disease Society Brain Bank criteria and stage 1–3 on the Hoehn and Yahr scale. In the SAD study, N=49 subjects received placebo or MEDI1341 administered intravenously (IV) across six dose levels. In the MAD study, two dose levels were evaluated in Parkinson’s patients (N=25), administered IV every 4 weeks for 3 infusions. <h3>Results:</h3> Administration of MEDI1341 or placebo to HVs or Parkinson’s patients was not associated with any medically important changes in safety parameters across all doses tested. Dose-dependent changes in serum concentrations for MEDI1341 were observed across cohorts. CSF concentrations demonstrated central nervous system penetration of MEDI1341. PD data showed dose-dependent suppression of free ASN in CSF, consistent with the results seen in preclinical species. <h3>Conclusions:</h3> MEDI1341 was generally well tolerated following IV administration to HVs or Parkinson’s patients after single or repeat doses, respectively. The PK and PD data provide evidence of dose-dependent and selective ASN target engagement by MEDI1341 in the central compartment. Full study design and data from the Phase 1 studies will be presented. <b>Disclosure:</b> Dr. Shering has received personal compensation for serving as an employee of AstraZeneca. An immediate family member of Dr. Shering has received personal compensation for serving as an employee of eResearch Technology Inc. Dr. Shering has stock in AstraZeneca. Dr. Shering has received intellectual property interests from a discovery or technology relating to health care. Michael Pomfret has nothing to disclose. Dr. Kubiak has received personal compensation for serving as an employee of AstraZeneca. Dr. Kubiak has stock in AstraZeneca. Mrs. Pouliquen has received personal compensation for serving as an employee of AstraZeneca. Mrs. Pouliquen has received personal compensation for serving as an employee of GSK. Mrs. Pouliquen has stock in GSK. Mrs. Pouliquen has stock in AstraZeneca. Wei Yin has received personal compensation for serving as an employee of Takeda. Wei Yin has stock in Takeda. Arthur Simen has received personal compensation for serving as an employee of Takeda. Dr. Ratti has received personal compensation for serving as an employee of Takeda. Dr. Ratti has received personal compensation for serving as an employee of Biogen. Dr. Ratti has stock in Takeda. Dr. Ratti has stock in Biogen. Dr. Perkinton has stock in AstraZeneca. Dr. Tan has received personal compensation for serving as an employee of AstaZeneca. An immediate family member of Dr. Tan has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for SOBI. Dr. Tan has stock in AstraZeneca. Dr. Chessell has received personal compensation in the range of $100,000-$499,999 for serving as a Employee with AstraZeneca. Dr. Ostenfeld has received personal compensation for serving as an employee of AstraZeneca. Dr. Ostenfeld has stock in AstraZeneca. Dr. Ostenfeld has received intellectual property interests from a discovery or technology relating to health care.

Direct Pathway For Bacillus anthracis tRNA Asparaginylation

Protein synthesis is essential for life and requires the correct pairing of amino acids to their cognate transfer tRNA (aminoacylation). To date, only two routes exist to attach asparagine (Asn), to it cognate tRNAAsn: the direct and the indirect pathways. The direct path uses asparaginyl‐tRNA synthetase to attach Asn to tRNAAsn when free Asn is present. The Asn is synthesized by asparagine synthetase A. The second pathway involves synthesizing Asn on the tRNA itself. The causative agent for anthrax, Bacillus anthracis, appears to encode both routes for Asn‐tRNAAsn formation. We would like to understand why B. anthraciscodes for both routes. This semester we have focused on the direct pathway. We report the successful purification of the asparagine synthetase A and asparaginyl‐tRNA synthetase along with activity measurements of asparagine synthetase A in order to characterize the direct pathway under different physiologically relevant conditions.Support or Funding InformationNational Science Foundation (MCB‐1615770)

Impact of Row Distance and Seed Density on Grain Yield, Quality Traits, and Free Asparagine of Organically Grown Wheat

Organic farming faces challenges providing sufficient nutrient supply as manure and crop rotations are often the major nutrient inputs. Larger row distances and fewer seed densities can support nitrogen availability by giving more space to the single plant. As free asparagine (Asn) the main precursor of acrylamide (AA) in plants is closely related to nitrogen uptake and storage, the question arose whether free Asn will be affected by row distance and seed densities in organic farming. This study investigated the effect of row distance and seed density on yield, yield components, baking quality, and free Asn in organic farming. A two-year field trial was carried out including two winter wheat cultivars, two row distances, and two seed densities. Year and cultivar highly influenced all traits. The impact of both treatments was mainly caused by interaction. Nevertheless, enlarged row distances raised baking quality, while free Asn was changed to a minor extent. Thus, we recommend larger row distances for raising baking quality without increasing free Asn. Seed density is of minor relevance. The close relation found between free Asn and grains per spike (R2 = 0.72) indicates that smaller grains contain more Asn than bigger grains. This opens new insights into Asn synthesis during grain development and offers a potential prediction of Asn amounts.

Acrylamide-Formation Potential of Cereals: What Role Does the Agronomic Management System Play?

As bakery products contribute considerably to the daily intake of the carcinogen acting substance acrylamide (AA), the aim of this study was to evaluate the impact of the management system (conventional vs. organic farming) on AA precursor levels of free asparagine (Asn) across different cultivars of the cereal species, namely winter wheat (Triticum aestivum), winter spelt (Triticum aestivum ssp. spelta) and winter rye (Secale cereale) with simultaneous consideration of gained grain yields and flour qualities. For this purpose, orthogonal field trials were established at two sites in Southwest Germany over two growing seasons (2006–2007 and 2007–2008). The results indicated a significant impact of the management system on free Asn contents in white flour. Across all species, free Asn contents in the white flour was 26% lower under organic compared to conventional farming. The impact of the management system on individual cultivars was obvious with a maximum reduction in free Asn contents of 50% in wheat cultivars if organically produced (e.g., for cultivars Ludwig, Privileg, Capo). For spelt, a significant impact of the management system was only found in 2008 with a reduction in free Asn of up to 25% if organically produced. Across both cropping systems, cultivar Franckenkorn reached the lowest levels of free Asn. For rye, a significant impact of the management system was observed only in 2007 with 33% higher Asn amounts in the conventional management system. Independent of the cropping system, rye reached the highest levels of free Asn followed by wheat and spelt. Depending on species, there was also an impact of the two systems on crude protein. The organically cropped wheat had a significantly lower level, but this was not observed for spelt and for rye only in 2007. The possible reason for the low free Asn content in the organically produced wheat flour could partially be the lower crude protein amount. Furthermore, the results indicated that lower AA contents in bakery products can be achieved by proper selection of species (e.g., 66% lower if rye is replaced by wheat) and cultivars. With an appropriate choice of the cultivar, a reduction of up to 65% was possible within wheat, along with a reduction of 44% within spelt and 12.5% within rye. In summary, the results indicated that organically produced wheat especially offers the opportunity to significantly lower the AA potential of bread and bread rolls by the choice of raw materials low in free Asn.

Evaluation of Asparagine Concentration as an Indicator of the Acrylamide Formation in Cereals Grown under Organic Farming Conditions

This study investigated the impact of organically grown cereals on the level of free asparagine (Asn) with simultaneous consideration of grain yields and flour qualities over three growing seasons in Germany. Additionally, the relation of free Asn and acrylamide (AA) was investigated. By including free Asn results of a second trial site, heritability of the trait free Asn was calculated. Free Asn was significantly influenced by species and within species by cultivars. Rye showed the highest free Asn amount, followed by einkorn, emmer, wheat, and spelt. Replacing rye with spelt would reduce free Asn by 85%. Cultivars differed in free Asn by up to 67% (wheat), 55% (spelt), and 33% (rye). Year significantly influenced free Asn levels. Heritability was high for wheat and spelt concerning locations, but regarding years, heritability was low for wheat but high for spelt and rye. For organically grown cereals, the relation between free Asn and AA formation has never been investigated. Across species and years, a correlation of R2 = 0.69 *** was found. Thus, free Asn can serve as an indicator for AA formation. In conclusion, the level of free Asn can be highly influenced by proper selection of species and cultivars.

Impact of Nitrogen and Sulfur Supply on the Potential of Acrylamide Formation in Organically and Conventionally Grown Winter Wheat

In a two-year field trial, the effect of nitrogen (N) and sulfur (S) fertilization was investigated on grain yield, grain quality parameters, formation of acrylamide (AA), and the precursor free asparagine (Asn) in organically and conventionally produced winter wheat cultivars. In both production systems, different types, amounts, and temporal distributions of N were tested. The the effect of S fertilizer types and amounts on free Asn was only tested in the conventional farming system. Within both cropping systems, grain yield and baking quality were significantly influenced by N treatment while the effect on free Asn was only minor. Especially within the organic farming system, increasing N fertilization levels did not increase free Asn significantly. A slight trend of increasing free Asn levels with an intensified N supply was observed, especially in the presence of crude protein contents of 14% or higher. However, only N amounts of 180 kg N ha−1 or higher increased the probability of high free Asn contents considerably, while N supply below that amount led to free Asn values similar to the unfertilized controls. The results indicated that good baking quality can be achieved without significantly increasing free Asn levels. In addition, cultivars affected the levels of free Asn significantly. Compared to cv. Bussard and Naturastar, cv. Capo exhibited the lowest AA formation potential at an N supply of 180 kg N ha−1 while simultaneously reaching a crude protein content &gt; 15% (conventional) and &gt; 12% (organic). Thus, it seems that cultivars differ in their ability to store and incorporate free Asn into proteins. Over all trials, a relation of free Asn and AA was shown by R2 = 0.77, while a relation of free Asn and protein was only R2 = 0.36. Thus, lowering free Asn by adjusting N treatments should not necessarily affect baking quality. S fertilization within conventional farming did not change the free Asn amount or crude protein significantly, probably due to the fact that soil was not sulfate-deficient. In summary, it was evident that free Asn amounts in wheat varied widely both within cultivars and between cropping systems. In order to clearly unravel genotypic differences and their interaction with environmental factors and especially N fertilization, further research is needed.

Reduction in Dietary Acrylamide Exposure-Impact of Potatoes with Low Acrylamide Potential.

Acrylamide forms primarily from a reaction between reducing sugars (e.g., glucose and fructose) and an amino acid (asparagine, Asn) formed naturally in foods, including potatoes. This reaction occurs when carbohydrate-rich foods are heated at temperatures above 120 °C. Multiple potato varieties were transformed with potato genomic DNA that results in down-regulation of the expression of the asparagine synthetase-1 gene (Asn1), significantly reducing synthesis of free Asn, and consequently lowering the potential to form acrylamide during cooking. These potatoes with low acrylamide potential (LAP) were tested in agronomic trials, and processed into French fries and potato chips. Decreased levels of acrylamide were measured in these cooked food products when derived from LAP potatoes compared with those derived from conventional potatoes. These reductions can be directly attributed to reduction in Asn levels in the LAP potatoes. The corresponding average reduction in exposure to acrylamide from French fry and potato chip consumption is estimated to be 65%, which would amount to approximately a 25% reduction in overall dietary exposure. Considering that children consume nearly three times more acrylamide than adults on a per kg body weight basis, they would experience the most impact from the reduced acrylamide associated with LAP potatoes. The potential public health impacts, in context of dietary acrylamide exposure reduction, are discussed in this study.

Discriminating Nature of the B. anthracis Aspartyl‐tRNA Synthetases

Protein synthesis is essential for life and requires the correct pairing of amino acids to their cognate transfer RNA (tRNA). Two routes exist to attach asparagine (Asn), to tRNAAsn: the direct and the indirect pathways. The direct path uses asparaginyl-tRNA synthetase to attach Asn to tRNAAsn when free Asn is present. The second pathway involves two steps in which a non-discriminating AspRS attaches aspartate (Asp) to tRNAAsn. The Asp-tRNAAsn is then amidated to Asn by GatCAB. The causative agent for anthrax, Bacillus anthracis, appears to encode both routes for Asn-tRNAAsn formation along with multiple Asn synthetic pathways and two AspRS enzymes. We demonstrate the B. anthracis AspRS acquired by horizontal gene transfer from archaea can attach Asp to tRNAAsn consistent with the organism synthesizing Asn on tRNAAsn using the indirect pathway. The acquisition of a second AspRS may enable B. anthracis to directly couple Asn biosynthesis with its use in translation. This work was supported by Skidmore College and the National Science Foundation (MCB-1244326).

Dual Routes for S. aureus Asparaginyl‐tRNA Formation

Two routes have evolved for attaching the amino acid asparagine (Asn) to its correct transfer RNA (tRNAAsn), an essential step in protein synthesis. The direct pathway involves aminoacylation of tRNAAsn by asparaginyl‐tRNA synthetase (AsnRS) when free Asn is available. In the indirect pathway, a non‐discriminating aspartyl‐tRNA snthetase (ND‐AspRS) attaches Asp to tRNAAsn, and the tRNA‐bound Asp is then amidated to Asn by GatCAB. Bioinformatics suggests the human pathogen Staphylococccus aureus may encode the indirect pathway in addition to the direct one. We set out to determine if, S. aureus does in fact encode an ND‐AspRS and GatCAB for tRNA‐dependent Asn synthesis. Using the E. coli JF448 system, we demonstrate that the concerted activities of S. aureus AspRS and GatCAB can form Asn on tRNA. In addition, using the E. coli TrpA34 system, we are verifying the S. aureus AspRS enzyme is non‐discriminating supported by in vitro studies with the enzyme. We speculate encoding dual routes for Asn‐tRNAAsn formation enables S. aureus to take advantage of situations when Asn is plentiful.

Impact of different nitrogen fertilizers and an additional sulfur supply on grain yield, quality, and the potential of acrylamide formation in winter wheat

AbstractThe amino acid asparagine (Asn) plays a key role in acrylamide (AA) formation in strongly heated cereal foodstuffs. The influence of different nitrogen (N) fertilizers (calcium ammonium nitrate, CAN; urea ammonium sulfate solution, UAS, applied according to the CULTAN method; urea; urea ammonium nitrate, UAN; ammonium nitrate sulfate containing the nitrification inhibitor 3,4‐dimethyl pyrazole phosphate, Entec 26®; and a combination of liquid manure and CAN) at a nitrogen level of 180 kg N ha–1 and an additional sulfur (S) supply on grain yield, quality, Asn concentration, and the potential of AA formation of winter wheat were studied in a 2‐year field experiment. Grain yields varied between 61 und 104 dt ha–1 dry matter depending on cultivar (cv), fertilization, and year. Quality demands concerning crude protein concentration and sedimentation value were reached when CAN, CAN+S, urea, or a combination of liquid manure and CAN were applied. Asparagine concentrations in flours varied from 2.6 to 13.6 mg per 100 g flour dry matter depending on cultivar, fertilization, and year. In both years, a close nonlinear correlation between crude protein concentration and the concentration of free Asn with r²2004 = 0.93 and r²2005 = 0.94 was observed. Nitrogen fertilizers leading to high crude protein concentrations caused significantly increased Asn concentrations. In both years, a correlation between the concentration of free Asn and the potential of AA formation with r²2004 = 0.72 and r²2005 = 0.84 was found. The application of S (CAN compared to CAN+S) had no beneficial effect on the Asn concentration and the potential of AA formation, most likely because S concentration in grains was sufficient even without additional S supply.

Impact of nitrogen amount and timing on the potential of acrylamide formation in winter wheat ( Triticum aestivum L.)

Acrylamide (AA), a potential human carcinogen, is formed in strongly heated carbohydrate-rich food as a part of the Maillard reaction. The amino acid asparagine (Asn) and reducing sugars are considered to be the main precursors for AA formation. In a 2-year field trial the impact of nitrogen (N) amount and timing on the content of AA precursors and the potential of AA formation in different winter wheat cultivars (cv.) were studied in association with respective grain yields and parameters of baking quality. Depending on year, cultivar and nitrogen treatment Asn contents ranged between 4 and 18 mg 100 g −1 flour dry-matter (DM). Nitrogen treatments affecting crude protein contents in flours above 13% caused a considerable increase in free Asn. Nitrogen amounts of 220 kg N ha −1 increased the contents of free Asn by between 130% and 270% depending on year and cultivar compared to the untreated controls. A close linear correlation between the content of free Asn and the potential of AA formation (2004: R 2 = 0.89, 2005: R 2 = 0.83) could be observed, whereas no correlation could be found between reducing sugars and the potential of AA formation, pointing to the importance of free Asn as the limiting and thus determining factor for the AA formation potential in wheat flours. To reach high crude protein contents and good sedimentation values demanded for breadstuffs, nitrogen amounts of at least 180 kg N ha −1 were necessary. Nitrogen fertilization measures resulting in high crude protein contents above 13% enhanced the potential of AA formation by increasing the content of free Asn in flours. As long as demands from traders and producers for flour with high crude protein contents are not revised, lowering Asn contents and thus the potential for AA formation by application of N amounts below 180 kg N ha −1 and abandoning the late application of N do not appear to be successful ways to reduce the risk of AA formation in breadstuffs.

Biochemical and morphological developments are partially impaired in intestinal mucosa from growing pigs fed reduced-protein diets supplemented with crystalline amino acids1

The objective of this study was to determine if a reduction in dietary CP, with partial replacement of the intact protein with crystalline AA (CAA), would alter growth, morphology, and free or peptide-bound AA concentrations of intestinal mucosa in growing pigs. Twenty-four barrows (37.0 +/- 1.5 kg of BW) were fed 1 of 4 diets for 24 d: 16.1% CP with no CAA, or 12.8, 10.1, or 7.8% CP (analyzed values, as-fed) containing CAA. As CP decreased, CAA were gradually increased to meet requirements on a true ileal digestible basis. Pigs were euthanized 2 h postmeal on d 24, and mucosal samples from duodenum, jejunum, and ileum were collected. Reducing dietary CP decreased ADG, G:F, and final weight (linear, P < 0.05). With reduced dietary CP, mucosal protein concentration decreased in the jejunum (quadratic, P < 0.05) and tended to decrease in the ileum (linear, P = 0.062). Reduction of the dietary CP concentration from 16.1 to 7.8% tended to decrease the crypt depth (linear, P < 0.10) and decreased villus width (linear, P < 0.05) in duodenum and jejunum mucosa but did not reduce villus height or villus surface area in any regions of the small intestine. In the duodenum, a reduction in dietary CP increased free Lys, Met, and Thr (linear, P < 0.05) and peptide-bound Lys and Thr (quadratic, P < 0.10). In the jejunum, reducing CP decreased free Cys (linear P < 0.05) and tended to decrease free Asn and His (linear, P < 0.10) and peptide-bound His (quadratic, P = 0.061) and Ile, Leu, and Val (linear, P < 0.10). In the ileum, reducing CP decreased free Asn, Ser, Tyr, Arg, His, Phe (linear, P < 0.05), and Leu (linear, P = 0.054) and peptide-bound Gly and Ser (linear, P < 0.05) and tended to decrease peptide-bound Ile, Leu, Phe, Val (linear, P < 0.10), and Lys (linear P < 0.05). In conclusion, reduced-CP diets supplemented with CAA lead to a reduction in growth performance, associated with biochemical and morphological modifications of the intestinal mucosa.

Free amino acid profiles suggest a possible role for asparagine in the control of storage-product accumulation in developing seeds of low- and high-protein soybean lines

Several approaches were taken to examine the role of N-assimilate supply in the control of soybean (Glycine max) seed composition. In the first study, developing seeds were grown in vitro with D-[U-14C]sucrose (Suc) and different concentrations of Gln. Light stimulated carbon flux into oil and protein, and was required to sustain Suc uptake and anabolic processes under conditions of elevated nitrogen supply. High Gln supply resulted in higher transcript levels of beta-conglycinin and oleosin. In the second study, analyses of soluble amino acid pools in two genetically related lines, NC103 and NC106 (low- and high-seed protein, respectively) showed that, in the light, NC106 accumulated higher levels of Asn and several other amino acids in developing cotyledons compared with NC103, whereas at the seed coat and apoplast levels both lines were similar. In the dark, NC103 accumulated Gln, Arg, and its precursors, suggesting a reduced availability of organic acids required for amino acid interconversions, while NC106 maintained higher levels of the pyruvate-derived amino acids Val, Leu, and Ile. Comparing NC103 and NC106, differences in seed composition were reflected in steady-state transcript levels of storage proteins and the lipogenic enzyme multi-subunit acetyl CoA carboxylase. In the third study, a positive correlation (P < or = 0.05) between free Asn in developing cotyledons and seed protein content at maturity was confirmed in a comparison of five unrelated field-grown cultivars. The findings support the hypothesis that high seed-protein content in soybean is determined by the capacity of the embryo to take up nitrogen sources and to synthesize storage proteins. Asn levels are probably tightly regulated in the embryo of high-protein lines, and may act as a metabolic signal of seed nitrogen status.

Correlation of ASN2 gene expression with ammonium metabolism in Arabidopsis.

In Arabidopsis, asparagine (Asn) synthetase is encoded by a small gene family (ASN1, ASN2, and ASN3). It has been shown that ASN1 and ASN2 exhibit reciprocal gene expression patterns toward light and metabolites. Moreover, changes in total free Asn levels parallel the expression of ASN1, but not ASN2. In this study, we show that ASN2 expression correlates with ammonium metabolism. We demonstrate that the light induction of ASN2 is ammonium dependent. The addition and removal of ammonium exerted fast and reciprocal effects on the levels of ASN2 mRNA, specifically under light-grown conditions. NaCl and cold stress increased cellular free ammonium and ASN2 mRNA levels in a coordinated manner, suggesting that the effects of stress on ASN2 expression may be mediated via accumulation of ammonium. The correlation between ASN2 and cellular ammonium metabolism was further demonstrated by analysis of ASN2 transgenic plants. When plants were grown on Murashige and Skoog medium containing 50 mm ammonium, ASN2 overexpressors accumulated less endogenous ammonium compared with the wild-type Colombia-0 and ASN2 underexpressors. When plants were subjected to high-light irradiance, ammonium levels built up. Under such conditions, ASN2 underexpressors accumulated more endogenous ammonium than the wild-type Colombia-0 and ASN2 overexpressors. These results support the notion that ASN2 is closely correlated to ammonium metabolism in higher plants.

Overexpression of the ASN1 gene enhances nitrogen status in seeds of Arabidopsis.

In wild-type Arabidopsis, levels of ASN1 mRNA and asparagine (Asn) are tightly regulated by environmental factors and metabolites. Because Asn serves as an important nitrogen storage and transport compound used to allocate nitrogen resources between source and sink organs, we tested whether overexpression of the major expressed gene for Asn synthetase, ASN1, would lead to changes in nitrogen status in the ultimate storage organ for metabolites-seeds. Transgenic Arabidopsis constitutively overexpressing ASN1 under the cauliflower mosaic virus 35S promoter were constructed (35S-ASN1). In seeds of the 35S-ASN1 lines, three observations support the notion that the nitrogen status was enhanced: (a) elevations of soluble seed protein contents, (b) elevations of total protein contents from acid-hydrolyzed seeds, and (c) higher tolerance of young seedlings when grown on nitrogen-limiting media. Besides quantitative differences, changes in the relative composition of the seed amino acid were also observed. The change in seed nitrogen status was accompanied by an increase of total free amino acids (mainly Asn) allocated to flowers and developing siliques. In 35S-ASN1 lines, sink tissues such as flowers and developing siliques exhibit a higher level of free Asn than source tissues such as leaves and stems, despite significantly higher levels of ASN1 mRNA observed in the source tissues. This was at least partially due to an enhanced transport of Asn from source to sink via the phloem, as demonstrated by the increased levels of Asn in phloem exudates of the 35S-ASN1 plants.

Insight into the mechanism of asparaginase-induced depletion of antithrombin III in treatment of childhood acute lymphoblastic leukemia

Asparaginase (ASNase) is a widely used and successful agent against childhood acute lymphoblastic leukemia (ALL). Asparaginase cleaves asparagine (Asn) to give aspartic acid and ammonia, thereby depleting free Asn in the blood. However, treatment with ASNase has been implicated in significant reduction of plasma levels of the coagulation serine protease inhibitor (serpin) antithrombin III (AT3), predisposing patients to thromboembolic complications. Our investigation was designed to delineate the biochemical mechanism of AT3 depletion that can occur in the plasma of ALL patients undergoing ASNase therapy. SDS-PAGE showed no cleavage of purified AT3 following treatment with ASNase. Furthermore, purified AT3 treated with ASNase demonstrated no decrease in inhibitory activity. Human plasma and whole blood treated with approximate therapeutic concentrations of ASNase showed no loss of AT3 activity as detected by a plasma-based factor Xa inhibition assay. Treatment of a confluent monolayer of HepG2 (hepatocarcinoma) cells with ASNase showed no gross loss in AT3 message levels detected by rtPCR. However, a decrease of cell viability was observed in cultures treated with ASNase. Interestingly, medium from HepG2 cells treated with ASNase showed a marked decrease in secretion of AT3 and another serpin, heparin cofactor II. Collectively, these data show that ASNase has no direct effect on AT3 in blood or plasma, but that ASNase may affect plasma levels of AT3 by interfering with translation and/or secretion of the protein in liver cells.

The feedback mechanism of nitrate inhibition of nitrogenase activity in soybean may involve asparagine and/or products of its metabolism

A feedback mechanism which involves sensing of change in phloem N concentration has been proposed to control nodulation and dinitrogen fixation in the presence of external combined N. Whether this control is in response to a change in total N or in some specific signal compound(s) is not known. In the present study we reevaluated the hypothesis that control of nodulation and N2 fixation involves sensing of change in tissue N composition and attempted to identify potential signal molecule(s) involved. Two soybean (Glycine max [L.] Merr.) genotypes (Williams 82 and NOD1‐3) differing in nodule number and tolerance to nitrate were germinated in sand trays. Seven‐day‐old seedlings were inoculated with a solution of Bradyrhizobium japonicum and grown for 28 days in growth chambers, using a hydroponic system with limited N supply to promote nodulation. Half of 28‐day‐old plants were treated with 15 mM NO3−, then control and treated plants were sampled at the onset of nitrogenase inhibition (24 h following NO3−, treatment) for evaluation of nitrogenase activity and tissue concentration of total N and of each individual free amino acid. Phenylisothiocyanate‐(PITC) amino acid derivatives were separated and quantified using HPLC. The decline in nitrogenase activity following the short‐term nitrate treatment was associated with a dramatic asparagine concentration increase in the shoot and an increase in nodule aspartate and glutamate in both genotypes. Asparagine concentration in the shoot increased 35 times from a barely detectable level of 95 to 3 327 nmol g−1 fresh weight in Williams 82, and more than tripled from 509 to 1 753 nmol g−1 fresh weight in NOD1‐3. Increase in levels of free Asn and in total free amino acids in the shoot following the short‐term nitrate treatment was more pronounced in Williams 82 than in its partially nitrate‐tolerant mutant NOD1‐3. These results indicate that the feedback control of nodule activity may involve sensing changes in shoot asparagine levels and/or products of its metabolism (aspartate and glutamate) in the nodule. These results also indicate that partial‐nitrate tolerance of nodulation in the hypernodulated NOD1‐3 mutant is associated with a lesser change in tissue N following nitrate treatment.