Synthetic Amino Acid Mixtures Research Articles

Sinorhizobium meliloti Chemotaxis to Multiple Amino Acids Is Mediated by the Chemoreceptor McpU.

The legume symbiont Sinorhizobium meliloti is chemoattracted to compounds exuded by germinating seeds of its host alfalfa. This response is mainly mediated by the S. meliloti chemoreceptor McpU. McpU also has a prominent contribution in sensing a synthetic amino acid (aa) mixture mimicking the amounts and composition observed in seed exudate. Here, we used the hydrogel capillary assay to quantify chemotactic responses of S. meliloti to individual aa exuded by germinating alfalfa seeds and to define the role of McpU in this behavior. S. meliloti exhibited positive chemotaxis responses to all proteinogenic aa, except for aspartate, and to citrulline, cystine, gamma-aminobutyric acid, and ornithine. Wild-type responses were diverse in intensity, while a strain lacking mcpU displayed strongly diminished responses. Differential scanning fluorimetry demonstrated interaction of the purified periplasmic region of McpU (McpU-PR) with the aa, except glutamate and aspartate. We additionally tested organic acids and sugars, but there were no significant interactions with the McpU ligand-binding domain, except for citrate. Using ligand displacement, we confirmed the interaction of McpU-PR with aa representing strong and weak attractants. Our results show that S. meliloti McpU is a broad-range aa receptor mediating differential responses to individual attractants, which does not bind negatively charged aa.

Contribution of Individual Chemoreceptors to Sinorhizobium meliloti Chemotaxis Towards Amino Acids of Host and Nonhost Seed Exudates.

Plant seeds and roots exude a spectrum of molecules into the soil that attract bacteria to the spermosphere and rhizosphere, respectively. The alfalfa symbiont Sinorhizobium meliloti utilizes eight chemoreceptors (McpT to McpZ and IcpA) to mediate chemotaxis. Using a modified hydrogel capillary chemotaxis assay that allows data quantification and larger throughput screening, we defined the role of S. meliloti chemoreceptors in sensing its host, Medicago sativa, and a closely related nonhost, Medicago arabica. S. meliloti wild type and most single-deletion strains displayed comparable chemotaxis responses to host or nonhost seed exudate. However, while the mcpZ mutant responded like wild type to M. sativa exudate, its reaction to M. arabica exudate was reduced by 80%. Even though the amino acid (AA) amounts released by both plant species were similar, synthetic AA mixtures that matched exudate profiles contributed differentially to the S. meliloti wild-type response to M. sativa (23%) and M. arabica (37%) exudates, with McpU identified as the most important chemoreceptor for AA. Our results show that S. meliloti is equally attracted to host and nonhost legumes; however, AA play a greater role in attraction to M. arabica than to M. sativa, with McpZ being specifically important in sensing M. arabica.

Single amino acid supplementation in aminoacidopathies: a systematic review

Aminoacidopathies are a group of rare and diverse disorders, caused by the deficiency of an enzyme or transporter involved in amino acid metabolism. For most aminoacidopathies, dietary management is the mainstay of treatment. Such treatment includes severe natural protein restriction, combined with protein substitution with all amino acids except the amino acids prior to the metabolic block and enriched with the amino acid that has become essential by the enzymatic defect. For some aminoacidopathies, supplementation of one or two amino acids, that have not become essential by the enzymatic defect, has been suggested. This so-called single amino acid supplementation can serve different treatment objectives, but evidence is limited. The aim of the present article is to provide a systematic review on the reasons for applications of single amino acid supplementation in aminoacidopathies treated with natural protein restriction and synthetic amino acid mixtures.

Effect of different dietary levels of potato protein concentrate supplemented with feed attractants on growth performance of rainbow trout (Oncorhynchus mykiss)

The potential of potato protein concentrate (PPC) as a replacement for fish meal (FM) protein was evaluated for rainbow trout (Oncorhynchus mykiss). Five diets were formulated by partial and total replacement of FM protein (20%, 40%, 60%, 80% and 100%, designated as PP20, PP40, PP60, PP80, PP100) with PPC (glycoalkaloid 23.2mgkg−1dryweight) and one experimental diet without PPC as control. Blood meal and a synthetic amino acid mixture and betaine were supplemented as feed attractants. Experimental diets were fed twice per day over a period of 56days to triplicate experimental groups until apparent satiation. Within all groups the feed intake was unaffected compared to control (P>0.05). Feed conversion ratio was negatively affected when fish fed to PP60, PP80 and PP100. Above a FM protein replacement of 60% growth performance was reduced (P<0.05). The nutritional status (body composition, blood parameters) of all groups suggests no health impact of PPC at all. Compared to preceding studies, where the dietary incorporation of PPC was restricted to lower levels, the use of feed attractants (80gkg−1 blood meal, 10gkg−1 synthetic amino acids) increased inclusion levels of PPC up to 60% without any negative effects on fish performance. In conclusion, we presented an optimized PPC-based diet for rainbow trout as proxy for alternative aqua feeds and a sustainable development of aquaculture.

Hydrolyzed Casein Influences Intestinal Mucin Gene Expression in the Rat

The effect of hydrolyzed casein (HC) on the expression of three mucin genes (Muc2, Muc3, and Muc4) in the rat intestine was investigated using quantitative real-time polymerase chain reaction. After a 10 day acclimatization period, rats received for 8 days the test diets containing either HC or a synthetic amino acid (SAA) mixture as the sole source of nitrogen or a protein-free (PF) diet (n = 12 per treatment). The addition of HC or the SAA mixture to the diet significantly improved average daily gain, average daily food intake, and gain:feed ratio as compared with the PF diet. Terminal ileal endogenous N flow was significantly higher for the HC-fed rats in comparison with either the SAA or the PF rats (p < or = 0.001). HC supported a significant increase of Muc3 mRNA (277 and 229% of that for diets PF and SAA, respectively; p < or = 0.05) in the small intestinal tissue and Muc4 mRNA (325 and 265% of that for diets PF and SAA, respectively; p < or = 0.05) in the colon. In conclusion, HC enhances ileal endogenous N flow and up-regulates in vivo the expression of some individual mucin genes.

Flow‐injection determination of cysteine in pharmaceuticals based on luminol–persulphate chemiluminescence detection

A flow injection (FI) method is reported for the determination of l-cysteine, based on its enhancement on chemiluminescence (CL) emission of luminol oxidized by sodium persulphate in alkaline solution. The calibration graph was linear over the range 1.0 x 10(-9)-5.0 x 10(-7) mol/L (r(2) = 0.9992), with relative standard deviations (RSDs) in the range 1.1-2.3% (n = 4). The limit of detection (3 sigma blank) was 5.0 x 10(-10) mol/L with a sample throughput of 120/h. The method was applied to pharmaceuticals and the results obtained were in reasonable agreement with the amount labelled. The proposed method was also applied to cysteine in synthetic amino acid mixtures. Calibration graphs of N-acetylcysteine and glutathione over the range 1.0-50 x 10(-8) and 0.5-7.5 x 10(-7) mol/L were also established (r(2) = 0.998 and 0.9986) with RSDs in the range 1.0-2.0% (n = 4), and the limits of detection (3 sigma blank) were 5.0 x 10(-9) and 1.0 x 10(-8) mol/L, respectively.

Energy expenditure in patients with propionic and methylmalonic acidaemias.

Resting energy expenditure (REE) was investigated in 8 children with propionic and methylmalonic acidaemias because a lowered REE has been reported in the literature. We observed a marginally elevated REE and think that adequate caloric intake and the use of a synthetic amino acid mixture are responsible for this.

A Study on the Optimal Amino Acid Pattern at the Proximal Duodenum in Growing Sheep

Nine crossbred castrated lambs fitted with rumen and duodenum cannula and fed a diet of hay and concentrate formulated with ground corn and soybean meal as main ingredients were used to assess the duodenal ideal amino acid pattern. Three synthetic amino acid mixtures with different profile of essential amino acids were duodenally infused in order to get three different amino acid patterns flowing into the duodenum. The mixtures were designed to have similar amino acid profile as rumen microbial protein (Pm), casein (Pc) and modified muscle amino acid (Pmm). Results showed a lower urine nitrogen excretion (p=0.05), a higher nitrogen retention (p=0.04) and bodyweight gain with treatment Pmm. The modified muscle amino acid pattern also promoted a lower ratio of Gly to other amino acids in plasma (Gly/OAA) and a higher RNA and RNA/DNA concentration in the liver of the sheep. Meanwhile, the urea concentration in plasma was reduced and the insulin concentration was increased with Pmm treatment. No differences in glucose and growth hormone concentration in plasma were found among three treatments. All results obtained indicate that the modified muscle amino acid pattern (Lys 100%, Met+Cys 39%, Thr 76%, His 41%, Arg 72%, Leu 158%, Ile 81%, Val 105%, Phe 81% and Trp 13%) was the best for growing sheep.

REGULATORY EFFECTS OF ENVIRONMENTAL CHEMICAL SIGNALS ON SEARCH BEHAVIOR AND FORAGING SUCCESS

To appreciate the mechanisms governing olfactory-mediated behavior, pro- cesses of chemical signal production and transmission in fluid media (air or water) must be understood. With new tools becoming available in analytical chemistry and fluid dy- namics, investigators can now quantitatively address the processes governing chemical signals in field habitats. This study identifies the role of amino acids as signal molecules regulating search behavior and foraging success by estuarine mud snails (Ilyanassa ob- soleta). For the first time, methods are described for measuring chemical signal production, release, and transport in field habitats, over temporal and spatial scales consistent with olfactory information processing. Rates of advection and turbulent mixing were determined, and shear velocities and roughness Reynolds numbers were estimated to characterize bottom boundary layer flows. Nearly instantaneous chemical measurements were made using a computerized microprobe system and conservative tracer to establish the environmental distributions of signal molecules at rates similar to those sampled by olfactory receptor neurons. In addition, we determined the dissolved free amino acid (DFAA) compositions (up to 18 amino acids), concentrations, and effluent release rates for live intact and injured fiddler crabs (Uca pugilator) and hard clams (Mercenaria mercenaria), which are common prey from mud snail habitats. The field site populated by mud snails was found to be more conducive at broadcasting stronger chemical signals over longer distances than most other estuarine and ocean habitats. Live fiddler crabs released amino acids at very low fluxes (0.1 nmol-min-'-g (wet tissue aiass)-V), while live intact clams took up amino acids from seawater. Once injured, hard clams and fiddler crabs released DFAAs at 88 and 6804 nmol-min-1-g-1, respectively. Mud snails were significantly attracted to injured clams and crabs, but not to intact prey, as compared with controls. Synthetic mixtures of amino acids, simulating fluids leaking from injured prey, were also highly attractive. When we tested for effects of amino acid composition, concentration, mean volume flow rate (of chemical input), and flux, in separate experiments, only flux directly correlated with the number of mud snails attracted. The attraction of mud snails is thus more tightly coupled to the physical transport of chemical stimuli than to the molecular properties of specific amino acids.

Growth of methionine-dependent human prostate cancer (PC-3) is inhibited by ethionine combined with methionine starvation.

Methionine (MET) is required for cell metabolism. MET endogenously synthesized from homocysteine (HCY) supports the proliferation of normal cells, but not that of numerous malignant cells, as shown previously. MET starvation should have an anti-tumour effect, and its deleterious effects on the hosts might be prevented by HCY. Anti-tumour effects of MET starvation must be reinforced by ethionine (ETH), a MET analogue. MET dependency of PC-3, a human prostate cancer cell line, was studied in vitro. Proliferation of PC-3 cells, cultivated in MET-free medium, was 29% compared with growth in MET+HCY- medium. Addition of HCY to MET-free medium increased the proliferation rate to 56%. The concentration of ETH required to decrease the PC-3 cell proliferation rate to 50% (IC50) was 0.5 mg ml(-1) in MET-HCY- medium. ETH-induced inhibition was abolished by MET addition and was reinforced by HCY. PC-3 cell cycle was blocked in the S-G2-phase after 30 h culture in the absence of MET; this blockage was not reversed by addition of HCY. ETH at the IC50 in MET-HCY+ medium blocked DNA replication. Apoptotic cells appeared after 30 h incubation in MET-HCY+ medium only when ETH was added. ATP pools were decreased after 15 h of culture in MET-free medium. In vivo, MET starvation was obtained by feeding tumour-bearing mice a diet containing a synthetic amino acid mixture as the protein supply, in which HCY replaced MET. Given to nude mice bearing xenografted PC-3, from day 1 after grafting and for 3 weeks, this diet inhibited tumour growth (34% on day 20, P < 0.007); this effect was potentiated by ETH (200 mg kg(-1) day(-1) i.p.) (56% on day 20, P < 5 x 10(-5)). The differences between the effects of these two treatments were significant (P < 0.017) and optimal on day 20. These data showed that combination of ETH and HCY slowed the proliferation of prostate cancer cells in vitro and in vivo, decreased ATP synthesis and caused cell cycle arrest and apoptosis. Experimental therapy based on cancer cell MET metabolism deficiency could be efficient for treating advanced prostate cancers refractory to current therapies.

Free amino acids exhibit anthozoan "host factor" activity: they induce the release of photosynthate from symbiotic dinoflagellates in vitro.

Reef-building corals and other tropical anthozoans harbor endosymbiotic dinoflagellates. It is now recognized that the dinoflagellates are fundamental to the biology of their hosts, and their carbon and nitrogen metabolisms are linked in important ways. Unlike free living species, growth of symbiotic dinoflagellates is unbalanced and a substantial fraction of the carbon fixed daily by symbiont photosynthesis is released and used by the host for respiration and growth. Release of fixed carbon as low molecular weight compounds by freshly isolated symbiotic dinoflagellates is evoked by a factor (i.e., a chemical agent) present in a homogenate of host tissue. We have identified this "host factor" in the Hawaiian coral Pocillopora damicornis as a set of free amino acids. Synthetic amino acid mixtures, based on the measured free amino acid pools of P. damicornis tissues, not only elicit the selective release of 14C-labeled photosynthetic products from isolated symbiotic dinoflagellates but also enhance total 14CO2 fixation.

In-vitro formation of cocoa-specific aroma precursors: aroma-related peptides generated from cocoa-seed protein by co-operation of an aspartic endoprotease and a carboxypeptidase

Cocoa-specific aroma precursors were obtained when acetone-dry powder prepared from unfermented cocoa seeds was subjected to autolysis at pH 5·2. Hydrophobic free amino acids and hydrophilic peptides were preferentially generated under these conditions. When this mixture of proteolysis products was formulated and roasted in the presence of reducing sugars, cocoa and/or chocolate aroma was detected by two independent panels. Aroma precursors extracted and partially purified from fermented cocoa seeds also consisted predominantly of hydrophilic peptides and hydrophobic free amino acids. No cocoa-specific aroma precursors were obtained when the acetone-dry powder from unfermented cocoa seeds was incubated at pH 3·5. Few free amino acids were released under these conditions, but a large number of hydrophobic peptides were formed. When these hydrophobic peptides were digested with carboxypeptidase A from porcine pancreas, mixtures of hydrophilic peptides and hydrophobic free amino acids were generated, which were shown to contain cocoa-specific aroma precursors. No typical cocoa aroma was, however, obtained when synthetic mixtures of amino acids adapted to the spectrum of free amino acids present in fermented cocoa seeds (or aroma-precursor extracts) were roasted in the presence of reducing sugars. Our findings therefore indicate that the essential cocoa-specific aroma precursors are among the hydrophilic oligopeptides. Ungerminated cocoa seeds contain one predominant endoprotease (an aspartic endoprotease with a pH optimum at pH 3·5) and a carboxypeptidase activity (pH optimum: 5·8). Our findings indicate that the co-operative action of these two enzymes on cocoa-seed protein is required for the generation of the cocoa-specific aroma precursors.

Submerged culture conidial germination and conidiation of the bioherbicideColletotrichum truncatum are influenced by the amino acid composition of the medium

Submerged culture experiments were conducted to determine the optimal nitrogen source for rapidly producing conidia of the bioherbicide,Colletotrichum truncatum. Germination ofC. truncatum conidial inocula in submerged culture occurred most rapidly (>95% in 6 h) in media provided with a complete complement of amino acids. When (NH4)2SO4, urea, or individual amino acids were provided as the sole nitrogen source, conidial germination was less than 20% after 6 h incubation. Conidia production was delayed inC. truncatum cultures grown in media with urea or individual amino acids as nitrogen sources compared to cultures supplied with Casamino acids or complete synthetic amino acid nitrogen sources. The use of methionine, lysine, tryptophan, isoleucine, leucine or cysteine as a sole nitrogen source severely inhibitedC. truncatum conidia production. Media with synthetic amino acid mixtures less these inhibitory amino acids produced significantly higher conidia yields compared to media with amino acid mixtures containing these amino acids. When various amounts of each individual inhibitory amino acid were added to media which contained amino acid mixtures, cysteine and methionine were shown to be most effective in reducing conidiation. An optimal nitrogen source forC. truncatum conidiation in submerged culture should contain a complete mixture of amino acids with low levels of cysteine, methionine, leucine, isoleucine, lysine and tryptophan for rapid conidiation and optimal conidia yield.

Studies on the effect of synthetic amino acid mixtures on bile secretion in the isolated perfused rat liver

Intrahepatic cholestasis may occur in patients receiving parenteral nutrition. Recent work has implicated amino acids in the perfusates in the pathogenesis of this cholestasis. Thus, we examined the effect of currently used amino acid solutions on liver structure and function using the perfused rat liver. Livers from male rats were perfused with Kreb's-Ringer bicarbonate buffer for periods up to 90 min. Two different solutions (Travasol R, Vamin R) were each added to the perfusion medium at amino acid concentrations of 0.05%, to 1.5%. The perfusate was sampled every 15 min for GTP, K + and Na +. Bile flow and bile acid secretion rate were monitored every 15 min. Livers were examined by light and electron microscopy. Infusion of amino acids did not change Na + and K + concentrations in the perfusate but decreased transaminases at all times studied. Bile flow decreased by 20% to 98% in a dose-dependent manner with Vamin R being slightly less cholestatic than Travasol R. Bile acid independent flow (BAIF) of the bile secreted was also reduced. The hepatic ultrastructure following amino acid perfusion was similar to that of livers after buffer alone and indicated absence of cellular damage. This study suggests that the amino acid solutios may induce cholestasis by interfering with the BAIF.

The isolated perfused rat liver: Conceptual and practical considerations

The isolated perfused rat liver: Conceptual and practical considerations

Inhibition of bile flow in the isolated perfused rat liver by a synthetic parenteral amino acid mixture: associated net amino acid fluxes.

To identify a role for amino acids in cholestasis associated with total parenteral nutrition, we measured bile formation by the isolated perfused rat liver in the presence and absence of added amino acids. All livers were infused constantly with sodium [14C]taurocholate (0.28 mumoles per min) for 90 min. At 40 min, a primed-constant infusion of a synthetic L-amino acid mixture (121 + 19.3 mumoles of N per min) was administered for an additional 50 min. Mean bile flow rates during the amino acid infusion were reduced from 15.4 microliter per min per 10 gm liver weight to 10.4 microliter per min per 10 gm (p less than 0.005). There was no significant change during saline infusion of control livers, and there was no significant difference in perfusate osmolalities in the two groups. Although biliary recovery of infused taurocholate was slightly lower in the experimental perfusions than in controls (95.3% vs. 101.7%, p less than 0.05), there was no significant reduction in taurocholate excretion rate during the infusion in either group. Bile flow changes were related to ambient concentrations and net fluxes of individual amino acids in the perfusate. Of the 14 infused amino acids, glycine and arginine achieved levels greater than 3 times greater than reported physiological postprandial portal venous concentrations in the rat, and together constituted about 25% of the 90-min perfusate amino acids (8.3 mM). The highest net hepatic uptake was for glycine (125 mumoles per hr per 10 gm), which was almost 50% of its infusion rate.(ABSTRACT TRUNCATED AT 250 WORDS)

THEORETICAL MODEL TO ESTIMATE RENAL NET ACID EXCRETION

Natural food is originated from cell material with constant pH. Patients fed natural food show renal net acid excretion (NAE) far below maximum. During parenteral nutrition or under synthetic diets high NAE was observed. Therefore, a model was developed to calculate NAE under a synthetic diet from the analytical data of this diet. According to Relman (J.Clin. Invest. 40, 1621, 1961) NAE corresponds to the difference of urinary anions [sulfate (SO4) + organic acids (OA) + chloride (Cl) + phosphate (P)] and urinary cations [sodium (Na) + potassium (K) + calcium (Ca) + magnesium (Mg)]. If urinary excretion of each parameter can be estimated from the data of intake, NAE can be predicted. In steady state conditions the urinary excretion of electrolytes correlates with their intestinal absorption, which can be estimated from intake and absorption rate. Absorption from the gut was estimated in the following way: Na + Cl: 100%; K: 90%; Ca: 0.17 × Ca intake (mg) + 60; Mg: 0.39 × Mg intake (mg) −23; P: 0.642 × P intake (mg) − 11. 75% of the ingested organic sulfur (methionine and cystine) is oxidized to SO4 and excreted in the urine. OA excretion is almost independent from the diet (38 meq/d/1.73m2). The usefulness of this theoretical model to estimate NAE was demonstrated in acid-base studies of children with phenylketonuria receiving two synthetic amino acid mixtures (PAM with a high NAE of 4-6 meq/kg/d, PAM74 with a low NAE of 1 meq/kg/d).

Olfactory responses of yellowfin tuna (Thunnus albacares) to prey odors: Chemical search image

Yellowfin tuna can detect and distinguish between odors of intact prey organisms, responding to odor quality of various prey rinses with different amino acid profiles. Chemical fractions of natural prey odors and synthetic mixtures of amino acids are less effective than whole natural rinses. Assuming uniform dilution, tuna can detect amino constituents of prey odors at about 10−11 M. Intensity of the behavioral response is modified by hunger. Feeding experience with new prey causes a gradual shift to maximum responses to its odor. We propose that one of the functions of the tuna's olfactory system is to form “chemical search images” which focus food search behavior for specific prey. The search image is thought to be modifiable and to be induced by prevailing prey abundance, so as to optimize the predator's feeding efficiency.

On the analysis of phenylthiohydantoin amino acids by high performance liquid chromatography

The key experimental variables and the corresponding separations of synthetic mixtures of phenylthiohydantoin amino acids achieved by high performance liquid chromatography have been abstracted from the literature and are listed in tabular form. From this table it is seen that the large majority of separations reported since 1977 have been performed utilizing stainless steel tubing of large internal diameter (i.d.≥3.9 mm). The reason for the widespread adoption of this particular practice is that a number of earlier studies have indicated that use of small diameter columns leads to significant loss of chromatographic resolution. An experimental study, employing two commercial chomatographic systems and tubings of differing internal diameter (i.d. 4.5 to 1.5 mm), leads to the conclusion that columns of reverse phase support material packed in stainless steel micro-bore tubing (i.d.≤2 mm) can effect separation of phenylthiohydantoin amino acids with similar efficiency as larger diameter columns. The practical advantages of employing micro-bore columns in general are discussed and adoption of micro-bore columns for the routine separation of this particularly important group of amino acid derivatives is recommended.

Plasma amino acid response to single test meals in humans. VI. Determination of available lysine.

The plasma amino acid response to single test meals in young adults was used to determine the amount of physiologically available lysine in heat-treated milk samples. The plasma amino acid responses were evaluated as PAA ratios according to a modified calculation procedure based on the method proposed by Longenecker and Hause. The amount of available lysine was determined by two separate series of single-meal tests. In the first, a relationship was established between the plasma amino acid response (PAA ratio) and the dietary level of lysine (given as synthetic amino acid mixtures). This relationship could be described by linear regression (r = 0.90). This regression line was then used to quantitatively evaluate the lysine PAA ratios obtained from single test meals with heat-treated milk samples. The heat-treatment of the milk samples caused a marked reduction of the amount of physiologically available lysine. The quantitative estimates obtained by the present plasma amino acid technique were in good agreement with determinations of available lysine in the same milk samples based on rat bioassay and chemical analysis.