Implications of missing efflux sites on convective ventilation and amino acid metabolism in Phragmites australis

Three stands of Phragmites australis (Cav.) Trin. ex Steudel were investigated regarding the relationship between the number of efflux culms and convective ventilation efficiency affecting the hypoxic status of roots and rhizomes. The lack of old (efflux) culms after mowing the preceding winter caused a significantly higher counterpressure within the rhizome, thereby diminishing air flushing rate, i.e. oxygen supply, of rhizomes. The levels of alanine and c-aminobutyric acid in basal culm internodes increased significantly. Both amino acids indicate the hypoxic status of the root and rhizome metabolism of P. australis . Amino acid patterns of the basal culm internodes are discussed with respect to the maintenance of aerobic root metabolism and nutrient availability.     

Analysis of energetic amino acid metabolism in Acyrthosiphon pisum: A multidimensional approach to amino acid metabolism in aphids

Aphids are highly specialized insects that feed on the phloem-sap of plants, the amino acid composition of which is very unbalanced. Amino acid metabolism is thus crucial in aphids, and we describe a novel investigation method based on the use of ¹⁴C-labeled amino acids added in an artificial diet. A metabolism cage for aphids was constructed, allowing for the collection and analysis of the radioactivity incorporated into the aphid body, expired as CO₂, and rejected in the honeydew and exuviae. This method was applied to the study of the metabolism of eight energetic amino acids (aspartate, glutamate, glutamine, glycine, serine, alanine, proline, and threonine) in the pea aphid, Acyrthosiphon pisum. All these amino acids except threonine were subject to substantial catabolism as measured by high ¹⁴CO₂ production. The highest turnover was displayed by aspartate, with 60% of its carbons expired as CO₂. For the first time in an aphid, we directly demonstrated the synthesis of three essential amino acids (threonine, isoleucine, and lysine) from carbons of common amino acids. The synthesis of these three compounds was only observed from amino acids that were previously converted into glutamate. This conversion was important for aspartate, and lower for alanine and proline. To explain the quantitative results of interconversion between amino acids, we propose a compartmentation model with the intervention of bacterial endosymbiotes for the synthesis of essential amino acids and with glutamate as the only amino acid supplied by the insect to the symbiotes. Moreover, proline exhibited partial conversion into arginine, and it is suggested that proline is probably indirectly involved in excretory nitrogen metabolism. © 1995 Wiley-Liss, Inc.     

褪黑素抑制低氧引起大鼠大脑皮层氨基酸递质释放

为研究褪黑素对低氧引起大鼠大脑皮层脑片氨基酸释放变化的影响,利用反相高效液相色谱结合荧光检测法,测定了孵育液中氨基酸类神经递质的含量。低氧条件为通入９１．６％Ｎ２和８．４％Ｏ２的混合气体。低氧３０ｍｉｎ时,大鼠大脑皮层脑片孵育中,氨基酸类神经递质天冬氨酸、谷氨酸、谷氨酰胺、甘氨酸、牛磺酸和γ－氨基丁酸的含量显著增加,其含量分别是正常氧组的２４０．４％,３３４．３％,２００．６％,２１０．４％,１６     

Effects of dissolved oxygen levels and the role of extra- and intracellular amino acid concentrations upon the metabolism of mammalian cell lines during batch and continuous cultures

The effects of dissolved oxygen and the concentration of essential amino acids upon the metabolism of two mammalian cell lines (rCHO producing human active (t-PA) and a mouse-mouse hybridoma) were investigated in batch, chemostat, and perfusion cultures. Intracellular amino acid concentrations were measured for both cell lines during repeated batch cultures and the K_S-values for the essential amino acids were calculated using Monod equations via computer simulation. The K_S-values were in the range of 10 mmol L⁻¹ and the pool of most intracellular amino acids remained constant at about 10–100 fold higher in concentration than in the medium. No significant differences were observed between the hybridoma and CHO cell. The specific nutrient uptake rates corresponded with the cell specific growth rate and the effects of reduced dissolved oxygen concentrations only became evident when the DO dropped below 5% of air saturation (critical concentration below 1%). Nevertheless, a correlation between nutrient concentration and specific oxygen uptake was detected. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nitrogen and carbohydrate pools of two rhizome types of Phragmites australis (Cav.) Trin. ex Steudel

Carbohydrate and total nitrogen contents as well as free amino acid (FAA) concentrations were determined in distinct types of rhizomes of two genetically homogeneous stands of reed (Phragmites australis (Cav.) Trin. ex Steudel) differing in morphology, productivity and nutrient supply in order to evaluate the storage capacity of vertical rhizomes and expansion rhizomes. The expansion rhizomes possess significantly higher amounts of FAA and of total nitrogen but similar carbohydrate concentrations in comparison to the vertical rhizomes. However, no significant differences were found for total nitrogen, FAA and total carbohydrates between both investigated stands indicating a comparable storage capacity of rhizomes independent of nutrient availability. Only the composition of the FAA pool varied in the alanine/asparagine ratio probably influenced by the oxygen supply of the rhizome/root system.     

Protein restriction and branched‐chain amino acid restriction promote geroprotective shifts in metabolism

The proportion of humans suffering from age‐related diseases is increasing around the world, and creative solutions are needed to promote healthy longevity. Recent work has clearly shown that a calorie is not just a calorie—and that low protein diets are associated with reduced mortality in humans and promote metabolic health and extended lifespan in rodents. Many of the benefits of protein restriction on metabolism and aging are the result of decreased consumption of the three branched‐chain amino acids (BCAAs), leucine, isoleucine, and valine. Here, we discuss the emerging evidence that BCAAs are critical modulators of healthy metabolism and longevity in rodents and humans, as well as the physiological and molecular mechanisms that may drive the benefits of BCAA restriction. Our results illustrate that protein quality—the specific composition of dietary protein—may be a previously unappreciated driver of metabolic dysfunction and that reducing dietary BCAAs may be a promising new approach to delay and prevent diseases of aging.     

Kinetic resolution of amino acid esters catalyzed by lipases

Racemic amino acids were resolved by lipase via hydrolysis of their esters. Lipases (Pseudomonas lipase from Amano PS, Rhizopus lipase from Serva, and porcine pancrease lipase from Sigma) could selectively hydrolyze the L-amino acid esters in aqueous solution with high reactivities and selectivities. The effect of the structural changes in the ester moiety on the stereoselectivity of the lipases was also investigated using D ,L -homophenylalanine as a model. Procedures were developed for the resolution of natural and unnatural amino acids. © 1996 Wiley-Liss, Inc.     

Aromatic amino acid metabolism during organogenesis in rice callus cultures

The activity during root and shoot initiation of key enzymes involved in aromatic amino acid metabolism was examined in rice ( Oryza sativa L. cv. Bala) callus cultures. Increased activities of the enzymes quinate:NAD⁺ oxidoreductase (EC 1.1.1.24), shikimate kinase (EC 2.7.1.71), chorismate mutase (EC 5.4.99.5), anthranilate synthase (EC 4.1.3.27) and tryptophan synthetase (EC 4.2.1.20) were noticed in organ-forming callus compared to proliferating callus of rice, especially prior to the visible manifestation of form. These results suggest a correlation between organogenesis and the aromatic amino acid pathway.     

Branched-chain amino acid metabolism controls membrane phospholipid structure in Staphylococcus aureus

Branched-chain amino acids (primarily isoleucine) are important regulators of virulence and are converted to precursor molecules used to initiate fatty acid synthesis in Staphylococcus aureus. Defining how bacteria control their membrane phospholipid composition is key to understanding their adaptation to different environments. Here, we used mass tracing experiments to show that extracellular isoleucine is preferentially metabolized by the branched-chain ketoacid dehydrogenase complex, in contrast to valine, which is not efficiently converted to isobutyryl-CoA. This selectivity creates a ratio of anteiso:iso C₅-CoAs that matches the anteiso:iso ratio in membrane phospholipids, indicating indiscriminate utilization of these precursors by the initiation condensing enzyme FabH. Lipidomics analysis showed that removal of isoleucine and leucine from the medium led to the replacement of phospholipid molecular species containing anteiso/iso 17- and 19-carbon fatty acids with 18- and 20-carbon straight-chain fatty acids. This compositional change is driven by an increase in the acetyl-CoA:C₅-CoA ratio, enhancing the utilization of acetyl-CoA by FabH. The acyl carrier protein (ACP) pool normally consists of odd carbon acyl-ACP intermediates, but when branched-chain amino acids are absent from the environment, there was a large increase in even carbon acyl-ACP pathway intermediates. The high substrate selectivity of PlsC ensures that, in the presence or the absence of extracellular Ile/Leu, the 2-position is occupied by a branched-chain 15-carbon fatty acid. These metabolomic measurements show how the metabolism of isoleucine and leucine, rather than the selectivity of FabH, control the structure of membrane phospholipids.     

Effects of l-leucine in ovo feeding on thermotolerance,growth and amino acid metabolism under heat stress in broilers

Recently, we found that in ovo feeding of l-leucine (l-Leu) afforded thermotolerance, stimulated lipid metabolism and modified amino acid metabolism in male broiler chicks. However, the effects of in ovo feeding of l-Leu on thermoregulation and growth performance until marketing age of broilers are still unknown. In this study, we investigated the effects of in ovo feeding of l-Leu on body weight (BW) gain under control thermoneutral temperature or chronic heat stress. We measured changes of body temperature and food intake, organ weight, as well as amino acid metabolism and plasma metabolites under acute and chronic heat stress in broilers. A total of 168 fertilized Chunky broiler eggs were randomly divided into 2 treatment groups in experiments. The eggs were in ovo fed with l-Leu (34.5 µmol/500 µl per egg) or sterile water (500 µl/egg) during incubation. After hatching, male broilers were selected and assigned seven to nine replicates (one bird/replicate) in each group for heat challenge experiments. Broilers (29- or 30-day-old) were exposed to acute heat stress (30 ± 1°C) for 120 min or a chronic heat cyclic and continued heat stress (over 30 ± 1°C; ages, 15 to 44 days). In ovo feeding of l-Leu caused a significant suppression of enhanced body temperature without affecting food intake, plasma triacylglycerol, non-esterified fatty acids, ketone bodies, glucose, lactic acid or thyroid hormones under acute heat stress. Daily body temperature was significantly increased by l-Leu in ovo feeding under chronic heat stress. Interestingly, in ovo feeding of l-Leu caused a significantly higher daily BW gain compared with that of the control group under chronic heat stress. Moreover, some essential amino acids, including Leu and isoleucine, were significantly increased in the liver and decreased in the plasma by l-Leu in ovo feeding under acute heat stress. These results suggested that l-Leu in ovo feeding afforded thermotolerance to broilers under acute heat stress mainly through changing amino acid metabolism until marketing age.     

Recognition of specific patterns of amino acid inhibition of growth in higher plants, uncomplicated by glutamine-reversible `general amino acid inhibition''

The complexity of the regulatory mechanisms that govern amino acid biosynthesis, particularly in multibranched pathways, frequently results in sensitivity to growth inhibition by exogenous amino acids. Usually the inhibition caused by a given amino acid(s) is relieved by another amino acid(s), thus indicating the cause of inhibition to be a specific interference with endogenous formation of the latter amino acid(s). We recently summarized the evidence that Nicotiana silvestris (and probably most higher plants), in suspension culture, exhibits a separate phenomenon of amino acid mediated growth inhibition called general amino acid inhibition. Every amino acid provokes general amino acid inhibition except for

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-glutamine. In fact,

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-glutamine completely overcomes general amino acid inhibition. We have now demonstrated that specific amino acid inhibition can be recognized and characterized at the level of growth inhibition without interference caused by general amino acid inhibition by the simple provision of exogenous

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-glutamine. Several examples of specific amino acid inhibition of growth were demonstrated in N. silvestris. In one case,

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-threonine inhibits growth partially in the presence of

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-glutamine. The residual amino acid inhibition was overcome by the additional presence of

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-lysine and

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-methionine, indicating that exogenous

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-threonine specifically inhibits the biosynthesis of both

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-lysine and

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-methionine. As a second example, the

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-valine-mediated inhibition of growth that persisted in the presence of

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-glutamine was overcome by

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-isoleucine, indicating that exogenous

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-valine inhibits

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-isoleucine biosynthesis. The use of amino acid analogs as experimental tools for biochemical-genetic studies in higher plants is also complicated by general amino acid inhibition. Conditions were demonstrated under which p-fluorophenylalanine and m-fluorotyrosine could be used as specific antimetabolites of

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-phenylalanine and

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-tyrosine biosynthesis without interference from general amino acid inhibition. We thus present a rigorous basis for recognition of specific relationships between metabolic branches that can guide detailed enzymological analyses.     

Modulation of phosphoinositide metabolism in rat brain slices by excitatory amino acids,arachidonic acid,and GABA

In rat brain slices the synthesis of [³H]phosphoinositides and the production of [³H]inositol monophosphate (IP₁) induced by norepinephrine (NE) were inhibited by glutamate. Calcium concentrations were varied to test if these inhibitory effects of glutamate were mediated by a calcium-dependent process. Although reducing calcium or addition of the calcium antagonist verpamil reduced the inhibitory effects of glutamate, these results were equivocal because reduced calcium directly decreased agonist-induced [³H]phosphoinositide synthesis. The inhibitory effects of glutamate were mimicked by quisqualate in a dose-dependent manner, but none of a variety of excitatory amino acid receptor antagonists modified the inhibition caused by quisqualate. It is suggested that glutamate activates a quisqualate-sensitive receptor (for which an antagonist is not available) and causes inhibition of phosphoinositide hydrolysis mediated in part by a direct or indirect inhibitory effect of calcium on phosphoinositide synthesis. Modulatory effects of arachidonic acid were examined because glutamate and calcium can activate phospholipase A₂. Arachidonic acid caused a rapid and dose-dependent inhibition of [³H]phosphoinositide synthesis and of NE-stimulated [³H]IP₁ production. A similar inhibition of the response to carbachol also occurred. The inhibition caused by arachidonic acid was unchanged by addition of inhibitors of cyclooxygenase or lipoxygenase. Activation of phospholipase A₂ with melittin caused inhibitory effects similar to those of arachidonic acid. Inhibitors of phospholipase A₂ were found to impair phosphoinositide metabolism, likely due to their lack of specificity for phospholipase A₂. Further studies were carried out in slices that were prelabelled with [³H]inositol in an attempt to separate modulatory effects on [³H]phosphoinositide synthesis and agonist-stimulated [³H]IP₁ production. Several excitatory amino acid agonists inhibited NE-stimulated [³H]IP₁ production. This inhibitory inter-action could be due to impaired synthesis of [³H]phosphoinositides because, even though the slices were prelabeled, addition of unlabelled inositol reduced NE-stimulated [³H]IP₁ production, indicating that continuous regeneration of [³H]phosphoinositides is required. In contrast to the inhibitory effects of the excitatory amino acids, gamma-aminobutyric acid (GABA) enhanced the response to NE in cortical and hippocampal slices. GABA also enhanced the response to carbachol in hippocampal and striatal slices and to ibotenic acid in hippocampal slices. Baclofen potentiated the response to NE similarly to the effect of GABA and baclofen partially blocked the inhibitory effect of arachidonic acid but did not alter that of quisqualate.Abbreviations AMPA -amino-3-hydroxy-5-methyl-4-isoxazolepropionic - acid AP₄ dl-2-amino-4-phosphonobutyric acid - BPB bromphenacyl bromide - BSA bovine serum albumin - CNQX 6-cyano-7-nitroquinoxaline-2,3-dione - DFMO -difluoromethylornithine - DIDS diisothiocyanotostilbene-2,2-disulfonic acid - EGTA ethyleneglycol-bis-N - N, N N-tetraacetic acid - GABA -aminobutyric acid - GDEE glutamate diethyl ether - -GG -glutamylglycine - IP₁ inositol monophosphate - IP₂ inositol bisphosphate - IP₃ inositol trisphosphate - NDGA nordihydroguaiaretic acid - NE norepinephrine - NMDA N-methyl-d-aspartate     

Standardised ileal crude protein and amino acid digestibilities in protein supplements for piglets

Standardised ileal digestibilities (SID) of crude protein and amino acids (AA) originating from 24 different feed ingredients, including 11 feed ingredients produced from soybeans, seven by-products of starch processing, four whey products and two fish meals, were determined in piglets by means of the difference method. For the indispensable AA, the highest SID values were obtained in three out of four whey proteins (SID ≥90% for most indispensable AA), one out of two fish meals (SID ≥86%), soy protein concentrate, hydrolysed soy protein isolate (SID ≥86% for most indispensable AA), and by-products of starch processing (SID ≥84% for most indispensable AA). The lowest SID values were obtained in extruded soybeans and microbially fermented soy protein (SID ≤78% for most indispensable AA), whereas the SID values for high-protein soybean meal were intermediate (SID 80–89% for indispensable AA except for Thr). The SID values in the three enzymatically fermented soy proteins (SID 80–94% for most indispensable AA) were similar to those in high-protein soybean meal and soy protein concentrate. The results of the present study indicate that SID values of AA in feed ingredients for piglets differ considerably from those reported for grower-finisher pigs, thus there may be a need for separate feed tables for piglets.     

Colloidal and dissolved organic matter in lake water: Carbohydrate and amino acid composition,and ability to support bacterial growth

Bacterial utilization of dissolved organic matter (DOM) was studied in water from a humic and a clearwater oligotrophic lake. Indigenous bacteria were inoculated into either 0.2 m natural filtered lake water, or lake water enriched fivefold with colloidal DOM >100 kD but below 0.2 m. Consumption of DOM was followed from changes in concentrations of total dissolved organic carbon (DOC), dissolved combined and free carbohydrates and amino acids (DCCHO and DFCHO, and DCAA and DFAA, respectively) and by uptake of monosaccharide and amino acid radioisotopes. DCCHO and DCAA made up 8% (humic lake) to 33–44% (clear-water lake) of the natural DOC pools, while DFCHO and DFAA contributed at most 1.7% to the DOC pools. Addition of >100 kD DOM increased the DOC concentrations by 50% (clearwater lake) to 92% (humic lake), but it only resulted in a higher bacterial production (by 63%) in the humic lake. During the incubations 13 to 37% of the DOC was assimilated by the bacteria, at estimated growth efficiencies of 4–8%. Despite the measured reduction of DOC, statistically significant changes of specific organic compounds, especially of DCCHO and DCAA, generally did not occur. Probably the presence of high molecular weight DOC interfered with the applied analytical procedures. Addition of radiotracers indicated, however, that DFAA sustained 17–58% and 29–100% of the bacterial carbon and nitrogen requirements, respectively, and that glucose met 1–3% of the bacterial carbon requirements. Thus, our experiments indicate that radiotracers, rather than measurements of concentration changes, should be used in studies of bacterial utilization of DOC in freshwaters with a high content of humic or high molecular weight organic matter.     

Carbohydrate and amino acid metabolism during batch culture of a human lymphoblastoid cell line,BTSN6

This work presents data on the carbohydrate and amino acid metabolism of a lymphoblastoid cell line producing an IgG1 antibody. In static culture, it was observed that lactate levels were significantly lowered when the cells were cultured on galactose as a carbon source. The use of carbohydrate substitution may be useful in lowering lactate levels, if it is established that this component is toxic to the cells. In addition, carbohydrate substitution may be used to modify glycosylation patterns and hence pharmacokinetic properties of glycoproteins.The amino acids glutamine and tryptophan were shown to be limiting in batch culture on this medium (DR, a 1:1 mixture of DMEM and RPMI, with 4mM glutamine). Amino acids produced included alanine, proline and glutamate. Serine was consumed to exhaustion, which was followed by a depletion of extracellular glycine. Amino acid metabolism, specific antibody productivity and specific growth rate were shown to be functions of the inoculation density in stirred flask culture. The results have implications for the design of media for both low and high density antibody manufacture by these cell lines.     

A study on the estimation of sulfur-containing amino acid metabolism by the determination of urinary sulfate and taurine

Summary.  Sulfate and taurine are major end products of sulfur-containing amino acid metabolism in mammals including humans, and they are excreted in urine. Average excretions (μmol/mg of creatinine) in the morning urine of 58 female college students were: total (free plus ester) sulfate (a), 12.53 ± 3.85; free sulfate, 11.57 ± 3.69; taurine, 0.78 ± 0.53. Ratio of total sulfate and taurine was 10 : 0.6. Regression lines obtained by plotting total sulfate, free sulfate, or total sulfate plus taurine against urea have shown that the former excretions are significantly correlated with urea excretion. Excretion of total sulfate at zero point of urea excretion (b) was 5.30, which corresponded to 42.3% of average excretion (12.53) and was assumed to be derived from dietary sulfate. The difference 7.23 (a − b) seemed to be derived from sulfur-containing amino acids. It was pointed out that the difference of average sulfate excretion and sulfate excretion at zero urea excretion, namely a − b, was appropriate for the metabolic index of sulfur-containing amino acids of the group examined. As free sulfate constituted 92.3% of total sulfate, excretion of ester sulfate was at a constant level, and that of taurine was not significantly correlated with urea excretion, the value of free sulfate corresponding to the value a − b of total sulfate mentioned above seemed to be a reliable and convenient index in the assessment of sulfur-containing amino acid metabolism. Received December 3, 2001 Accepted January 2, 2002 Published online August 30, 2002 Authors' address: Dr. Toshihiko Ubuka, Department of Clinical Nutrition, Kawasaki University of Medical Welfare, Kurashiki Okayama, 701-0193 Japan, E-mail: ubukatos@mw.kawasaki-m.ac.jp     

Nature of amino acid substitutions in homologous proteins during evolution

Replacement substitutions of mitochondrial cytochrome $\text{c}$ and α- and β-chains of haemoglobin have been studied by considering the structural similarity among amino acid residues at the secondary and tertiary structural levels. Secondary structural similarity explains ～70% while tertiary structural similarity explains ～50% of observed replacements for most of the cases. These structural similarities could not account for all the replacement substitutions. The study was extended to consider the composition of codons, and the chemical nature and polarity of the replacing and replaced residues. These also could not individually account for all the affected replacements. In general, no property of amino acid residues is conserved for substitutions occurring at any single position during evolution of proteins.     

Porcine pancreatic lipase-catalized enantioselective hydrolysis of N-protected amino acid methyl-esters

Summary A preparative-scale enantioselective hydrolysis of racemic methyl esters of several N-protected amino acid has been carried out by using crude porcine pancreatic lipase (Triacylglycerol lipase, EC 3.1.1.3) PPL as a hydrolytic enzyme. In all cases 50% of the racemic methyl ester was hydrolysed to the N-protected L-amino acid with high yield and high optical purity.Hydrolysis rates were very close related not only to the amino acid structure but also to the steric and/or electronic nature of the ester and N-protecting groups. Thus, the very convenient ester methyl group can be enantioselectively hydrolysed with PPL when N-protecting group is a carbonyl derivative, as it is the usual benzoyl group.     

Genetic screening of Lactobacillus sakei and Lactobacillus curvatus strains for their peptidolytic system and amino acid metabolism, and comparison of their volatilomes in a model system

A total of 51 Lactobacillus sakei and 28 Lactobacillus curvatus strains from different origins were screened for their potential to produce biogenic amines (BAs), and for their diversity of peptidolytic systems and specific aminotransferases (AraT, BcaT) that initiate amino acid conversion to volatiles relevant for aroma formation in meat products. The profiles of volatiles formed (volatilomes) were analysed in the headspace of fermentations by solid phase microextraction followed by GC-MS analysis. Tyramine-forming potential was detected only within L. curvatus and was strain-dependent. Histamine decarboxylase (HDC) activity could only be detected in one L. sakei strain, previously described as histidine decarboxylase positive (HDC⁺). Peptide transporters and peptidases were nearly ubiquitous in L. sakei and only a few strains lacked single peptidases. In L. curvatus, differences were detected in the occurrence of peptidase genes detected with PCR primers derived from L. sakei. All strains lacked known aminotransferases specific for branched-chain amino acids (BCAAs) and aromatic amino acids (ACAAs). Although L. sakei is suggested as a genetically very heterogenous species, and relatedness between L. curvatus and L. sakei at the genomic level is rather low, they appeared to be nearly uniform in the genes forming the peptidolytic system. The volatilomes of L. sakei and L. curvatus strains were qualitatively nearly identical. However, slight differences in the formation of single volatile compounds and the interaction with staphylococci may impact upon sausage fermentation which occurs over a period of many weeks. Among the compounds expected to contribute to the aroma were dimethyldisulphide, 3-methyl-1-butanol, acetic acid, 1-butanol and butanoic acid.