Free Glycine Research Articles

Effect of potassium chloride and calcium chloride induced stress on in vitro cultures of Bacopa monnieri (L.) Pennell and accumulation of medicinally important bacoside A

Growth, osmotic adjustment, antioxidant enzyme defense and principle medicinal component bacoside A was studied in in vitro raised shoots of Bacopa monnieri under different concentrations of KCl and CaCl2 (0, 50, 100, 150 or 200 mM). Significant reduction was observed in shoot number per culture; shoot length, fresh weight, dry weight and tissue water content (TWC) when shoots were exposed to increasing KCl and CaCl2 concentrations (50–200 mM) as compared to control. Minimum damage to the membrane as assessed by malondialdehyde (MDA) content was noticed in control in contrast to sharp increase in KCl and CaCl2 stressed shoots. Higher amounts of free proline, glycine betaine and total soluble sugars (TSS) accumulated in KCl and CaCl2 exposed shoots compared to the controls. Among different concentrations of KCl and CaCl2, increasing concentration of CaCl2 showed more increase in osmolyte accumulation. Na+ content decreased with increasing concentrations of KCl and CaCl2. Accumulation of K+ increased significantly in KCl (50–100 mM) stressed shoots as compared to control, while it decreased in CaCl2 treated shoots indicating that it prevents the uptake of K+ ions. Ca2+ accumulation significantly increased with increasing concentrations of CaCl2 up to 150 mM but decreased at higher concentrations. Shoots treated with KCl and CaCl2 (0–100 mM) showed higher antioxidant enzyme (SOD, CAT, APX and GPX) activities but KCl suppressed the activities at higher concentrations. Accumulation of bacoside A was enhanced with an increase in KCl and CaCl2 concentration up to 100 mM. It appears from the data that accumulation of osmolytes, and elevated activities of antioxidant enzymes play an important role in osmotic adjustment in shoot cultures of Bacopa and the two salts tested have a positive effect on bacoside accumulation.

Zinc–amino acid complexes are more stable than free amino acids in saline and washed soils

Biodegradability of metal–amino acids (AA) complexes has remained undocumented. Such knowledge is necessary to assess role of AA in metal mobility and bioavailability in the soil environments. In this study, biodegradation of free glycine (Gly), glutamine (Gln), arginine (Arg) and histidine (His) was compared with their Zn complexes in a natural saline soil before and after washing. The soil samples were collected from 0 to 15 cm depth of a saline soil before and 7 days after washing. The heavy washing of the saline soils using low salinity irrigation water (EC < 2 dS m−1) is a common practice applied by the farmers in the region before plant seeding. Mineralization of AA, revealed by CO2 release (35–66% in saline soil and 20–42% in washed soil) after 48 h incubation indicating rapid degradation of AA in soil. Complexation with Zn significantly reduced mineralization of AA. In saline soil treated with AA, the cumulative release of CO2 after 48 h incubation varied from 12.4 to 47.5 mg C kg−1, while it was 5.8–37.9 mg C kg−1 in the same soil treated with Zn–AA complexes (ZnAAC). The effect of complexation with Zn on CO2 release was dependent on AA type. Lower C mineralization and thus, higher stability of ZnAAC compared with free AA supports a conclusion that complexation of metal greatly decreases degradability of AA. According to the results, the effectiveness of complexation with proteinaceous AA in metal mobilization in soil has to be given greater consideration than that reported previously.

Free Glycine Accelerates the Autoproteolytic Activation of Human Asparaginase

Human asparaginase 3 (hASNase3), which belongs to the N-terminal nucleophile hydrolase superfamily, is synthesized as a single polypeptide that is devoid of asparaginase activity. Intramolecular autoproteolytic processing releases the amino group of Thr168, a moiety required for catalyzing asparagine hydrolysis. Recombinant hASNase3 purifies as the uncleaved, asparaginase-inactive form and undergoes self-cleavage to the active form at a very slow rate. Here, we show that the free amino acid glycine selectively acts to accelerate hASNase3 cleavage both in vitro and in human cells. Other small amino acids such as alanine, serine, or the substrate asparagine are not capable of promoting autoproteolysis. Crystal structures of hASNase3 in complex with glycine in the uncleaved and cleaved enzyme states reveal the mechanism of glycine-accelerated posttranslational processing and explain why no other amino acid can substitute for glycine.

Growth Medium-Dependent Glycine Incorporation into the Peptidoglycan of Caulobacter crescentus

The peptidoglycan (PG) is a macromolecular component of the bacterial cell wall that maintains the shape and integrity of the cell. The PG of Caulobacter crescentus, unlike that of many other Gram-negative bacteria, has repeatedly been shown to contain significant amounts of glycine. This compositional peculiarity has been deemed an intrinsic characteristic of this species. By performing a comprehensive qualitative and quantitative analysis of the C. crescentus PG by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), we show here that glycine incorporation into the C. crescentus PG depends on the presence of exogenous glycine in the growth medium. High levels of glycine were detected at the fifth position of the peptide side chains of PG isolated from C. crescentus cells grown in the complex laboratory medium PYE or in defined medium (M2G) supplemented with casamino acids or glycine alone. In contrast, glycine incorporation was undetectable when cells were grown in M2G medium lacking glycine. Remarkably, glycine incorporation into C. crescentus peptidoglycan occurred even in the presence of low millimolar to sub-millimolar concentrations of free glycine. High glycine content in the PG had no obvious effects on growth rates, mode of PG incorporation or cell morphology. Hence, the C. crescentus PG is able to retain its physiological functions in cell growth and morphogenesis despite significant alterations in its composition, in what we deem to be unprecedented plasticity.

Structures of Apo and Product-Bound Human l-Asparaginase: Insights into the Mechanism of Autoproteolysis and Substrate Hydrolysis

Asparaginases catalyze the hydrolysis of the amino acid asparagine to aspartate and ammonia. Bacterial asparaginases are used in cancer chemotherapy to deplete asparagine from the blood, because several hematological malignancies depend on extracellular asparagine for growth. To avoid the immune response against the bacterial enzymes, it would be beneficial to replace them with human asparaginases. However, unlike the bacterial asparaginases, the human enzymes have a millimolar K(m) value for asparagine, making them inefficient in depleting the amino acid from blood. To facilitate the development of human variants suitable for therapeutic use, we determined the structure of human l-asparaginase (hASNase3). This asparaginase is an N-terminal nucleophile (Ntn) family member that requires autocleavage between Gly167 and Thr168 to become catalytically competent. For most Ntn hydrolases, this autoproteolytic activation occurs efficiently. In contrast, hASNas3 is relatively stable in its uncleaved state, and this allowed us to observe the structure of the enzyme prior to cleavage. To determine the structure of the cleaved state, we exploited our discovery that the free amino acid glycine promotes complete cleavage of hASNase3. Both enzyme states were elucidated in the absence and presence of the product aspartate. Together, these structures provide insight into the conformational changes required for cleavage and the precise enzyme-substrate interactions. The new understanding of hASNase3 will serve to guide the design of variants that possess a decreased K(m) value for asparagine, making the human enzyme a suitable replacement for the bacterial asparaginases in cancer therapy.

Synaptic and Extrasynaptic NMDA Receptors Are Gated by Different Endogenous Coagonists

N-methyl-d-aspartate receptors (NMDARs) are located in neuronal cell membranes at synaptic and extrasynaptic locations, where they are believed to mediate distinct physiological and pathological processes. Activation of NMDARs requires glutamate and a coagonist whose nature and impact on NMDAR physiology remain elusive. We report that synaptic and extrasynaptic NMDARs are gated by different endogenous coagonists, d-serine and glycine, respectively. The regionalized availability of the coagonists matches the preferential affinity of synaptic NMDARs for d-serine and extrasynaptic NMDARs for glycine. Furthermore, glycine and d-serine inhibit NMDAR surface trafficking in a subunit-dependent manner, which is likely to influence NMDARs subcellular location. Taking advantage of this coagonist segregation, we demonstrate that long-term potentiation and NMDA-induced neurotoxicity rely on synaptic NMDARs only. Conversely, long-term depression requires both synaptic and extrasynaptic receptors. Our observations provide key insights into the operating mode of NMDARs, emphasizing functional distinctions between synaptic and extrasynaptic NMDARs in brain physiology.

The effect of dipeptide, Lys-Gly, supplemented diets on digestive tract histology in juvenile yellow perch (Perca flavescens)

The aim of the present study was to evaluate the effects of three formulated diets: wheat-gluten-protein-based diets supplemented with Lys-Gly dipeptide (LG) or free lysine and glycine (FL), a control diet without lysine supplementation (C) and commercial starter Bio Oregon (BO) for on the growth and digestive system morphology of yellow perch. After 48 days of experimental feeding, fish fed LG diet showed the highest body mass. Fish fed LG diet showed the highest number of gastrin/cholecystokinin positive cells and the lowest number of CD3-positive cells. The brush border of anterior intestine was the most PepT1 immunopositive in fish fed LG diet, the weakest in fish fed C diet. The largest hepatocytes were observed in fish fed BO, while the smallest in those fed FL diet, the difference being statistically significant. Relative hepatocyte cytoplasm volume occupied by lipids was higher in fish fed BO and FL compared to those fed C and LG. Number of proliferating cell nuclear antigen-positive hepatocyte nuclei did not significantly differ among experimental groups. These results indicate that wheat-gluten-protein-based diets supplemented with dipeptide Lys-Gly (LG) were appropriate for yellow perch.

Experimental thermodynamics of free glycine conformations: the first Raman experiment after twenty years of calculations

Low-frequency, gas-phase vibrational (Raman) spectroscopy was used in conjunction with a jet-cooled technique and ab initio calculations to study the intrinsic thermodynamic properties of the free (gas-phase) amino acid--glycine (Gly, H(2)NCHRCOOH). The first experimental evaluation of the enthalpy differences between the Gly conformations in the vapor phase is presented. The enthalpy values were determined to be 0.33 ± 0.05 and 1.15 ± 0.07 kcal mol(-1) for the ccc and gtt rotamers, respectively; the corresponding relative entropy values were -2.86 ± 0.12 and -0.12 ± 0.16 cal mol(-1) K(-1), respectively. It was proven that the low-frequency Raman and infrared spectroscopy is capable of estimating intrinsic thermodynamic parameters of protein building blocks, such as intermolecular hydrogen bonds (ccc conformer) and rotation around one of the bonds (N-C, gtt conformer). The inaccuracy of the RRHO approximation to Gly conformers was experimentally confirmed. Benchmark data for quantum theory and molecular dynamics were provided.

Abstract 18444: Effects of Cardiac Resynchronization Therapy on Transcardiac Metabolomic Profile and Substrate Utilization

Introduction: Cardiac remodeling in heart failure (HF) is associated with conduction abnormalities and dyssynchrony which results in poorly coordinated contractions precipitating metabolic heterogeneity and energetic inefficiency. Cardiac resynchronization therapy (CRT) improves HF symptoms, ventricular function and overall survival. The aim of this study was to determine the changes in metabolomic profiles and substrate utilization after CRT. Methods: 18 patients with advanced HF and 7 healthy controls were included in this study. Blood samples were collected from peripheral vein, femoral artery (FA) and coronary sinus (CS) before and 10-min after CRT. Metabolomic profiling of plasma samples were performed using GC/MS and 1 H NMR spectroscopy. Partial Least Squares Discriminant Analysis (PLS-DA) score plots and regression coefficients were used to examine metabolomic profiles. Results: HF patients have altered plasma metabolomic profile (Fig. A) with distinct panel of metabolites including free fatty acids (FFA), glycerol, 2-hydroxybutyrate, glycine, succinate and uric acid important in group classification compared to controls. Increased release of fumarate and glycerol-3-phosphate from myocardium in HF indicate mitochondrial and substrate shuttle dysfunction. Immediately after CRT, transcardiac metabolomic profile of plasma from CS shifted to a different state with increased myocardial uptake and metabolism of FFA and decreased release of creatinine indicating improved energetic conditions (Fig. B). Conclusion: Metabolomic profiling indicates that CRT improves myocardial substrate metabolism and utilization in patients with advanced HF.

Chemical and Textural Properties in Commercial Fermented Soybean Curds of Sufu

A survey aiming to find out the chemical and textural properties of commercial fermented soy bean curd called sufu was conducted. Sixteen brands of plain sufu produced in the Northern or the Southern part of China were collected and examined for their crude protein, crude fat, texture profiles, free amino acids, and free fatty acid contents. Twenty-one free amino acids were extracted and derivatized using a commercial kit followed by separation and analyzed by the gas chromatography-mass spectrometry (GC-MS). Similarly, ten free fatty acids were extracted using alumina, eluted, separated and analyzed. The content ranges of crude fat and protein were 22～36% and 31～38%, respectively. In texture profile analysis, ranges of the texture parameters were 131～493 g (hardness), 0.4～0.5 (cohesiveness), -137 to -50 gs (adhesiveness), 0.6～1 (springiness), 47～220 g (gumminess) and 32～177 g (chewiness). Twenty-one different free amino acids, especially alanine, glycine, α-aminobutyric acid, valine, leucine, allo-isoleucine, aspartic acid, glutamic acid and lysine in large amount, as well as ten fatty acids in total, notably linoleic acid (9-octadecanoic acid), oleic acid (9,12-octadecadienoic acid), linolenic acid (9,12,15-octadecadienoic acid), hexadecanoic acid and octadecanoic acid were found. This information provides important quality reference ranges for product developers and manufacturers to optimize and produce the plain sufu.

Metabolic and Genomic Response to Dietary Isocaloric Protein Restriction in the Rat

We have examined hepatic, genomic, and metabolic responses to dietary protein restriction in the non-pregnant Sprague-Dawley rat. Animals were pair-fed either a 6 or 24% casein-based diet for 7-10 days. At the end of the dietary period, a microarray analysis of the liver was performed, followed by validation of the genes of interest. The rates of appearance of phenylalanine, methionine, serine, and glucose and the contribution of pyruvate to serine and glucose were quantified using tracer methods. Plasma and tissue amino acid levels, enzyme activities, and metabolic intermediates were measured. Protein restriction resulted in significant differential expression of a number of genes involved in cell cycle, cell differentiation, transport, transcription, and metabolic processes. RT-PCR showed that the expression of genes involved in serine biosynthesis and fatty acid oxidation was higher, and those involved in fatty acid synthesis and urea synthesis were lower in the liver of protein-restricted animals. Free serine and glycine levels were higher and taurine levels lower in all tissues examined. Tracer isotope studies showed an ∼50% increase in serine de novo synthesis. Pyruvate was the primary (∼90%) source of serine in both groups. Transmethylation of methionine was significantly higher in the protein-restricted group. This was associated with a higher S-adenosylmethionine/S-adenosylhomocysteine ratio and lower cystathione β-synthase and cystathionine γ-lyase activity. Dietary isocaloric protein restriction results in profound changes in hepatic one-carbon metabolism within a short period. These may be related to high methylation demands placed on the organism and caused by possible changes in cellular osmolarity as a result of the efflux of the intracellular taurine.

Effects of various diet formulations (experimental and commercial) on the morphology of the liver and intestine of rainbow trout (Oncorhynchus mykiss) juveniles

The aim of the present study was to evaluate the effects of three formulated diets: wheat gluten protein-based diets supplemented with Lys–Gly dipeptide (PP) or free lisine and glycine (AA), and a control diet without lysine supplementation (CON), two commercial starters: Aller Futura (AF), Aglo Norse (AN) and frozen Chironomidae larvae (CH) for on the growth and digestive system development of rainbow trout. The experiment lasted 28 days and survival at the end of the experiment was the highest in the group fed AF (87.9±4.2%) and the lowest in the group fed the CON diet (74.0±4.3%). Fish fed the CON diet showed a significantly lower crude protein content, and the AA group had a significantly higher lipid level (P≤0.05) in body carcass compared with the other groups. The highest density of intestinal goblet cells (P≤0.05) was observed in fish fed the CON diet, while the lowest density was seen in the distal intestine in the AF group. The hepatocyte cytoplasm of all fish stored more glycogen than lipids. These results indicate that wheat gluten protein-based diets supplemented with dipeptide Lys–Gly (PP) or free lysine and glycine (AA) supported growth, protein efficiency rate and intestine epithelium homeostasis. On the other hand, fish fed CON (lysine-deficient diet) showed lower survival and growth rate, and disturbances of intestine epithelium homeostasis, probably as a result of lysine deficiency.

Reduction of palladium(II) monoglycinate complexes at rotating disc palladium electrode in acid media

Reduction of palladium(II) glycinate complexes in strongly acid 0.5 M NaClO4 solutions (pH 0.6 and 1.0) with variable palladium(II) complex and free glycine concentration was studied by the taking of cyclic voltammograms at palladium rotating disc electrode. It is shown that it was a chelate monoglycinate palladium(II) complex that was present in all studied solutions and underwent the reduction. The diffusion coefficient of the chelate monoglycinate palladium(II) complex D = (6.5 ± 0.5) × 10−6 cm2/s was determined from the limiting diffusion current of the complex reduction. The monoglycinate palladium(II) complex reduction occurred in the double-layer segment of the palladium charging curve; it was not complicated by hydrogen adsorption at electrodes. The palladium(II) complex reduction half-wave potential was determined (E 1/2 = ∼0.300 to 0.330 V (SCE)). It is shown that the decreasing of the number of ligands coordinated by palladium via nitrogen atom facilitates the complex reduction process. In particular, the reduction potentials of palladium(II) complexes with different ligand number at palladium electrode shifted markedly toward negative potentials in the series: Pdgly+ < Pd(gly)2 < Pd(gly) 4 2− .

The effect of plant protein-based diet supplemented with dipeptide or free amino acids on digestive tract morphology and PepT1 and PepT2 expressions in common carp ( Cyprinus carpio L.)

Common carp ( Cyprinus carpio) of average body mass 0.07 ± 0.02 g were fed three formulated diets: wheat gluten protein-based diet supplemented with Lys–Gly dipeptide (PP), wheat gluten protein-based diet supplemented with free lysine and glycine (AA), and a wheat gluten protein-based control diet without lysine supplementation (CON), frozen zooplankton (Z) (restricted diet), and a commercial starter food Aglo Norse (AN). After 4 weeks of experimental feeding, fish fed AN diet showed the highest body mass and length. Significantly lower mass occurred in groups fed PP, AA, CON, and Z. Fish fed CON diet showed the lowest intestinal folds and the highest number of mucous cells. Fish fed PP diet showed a significantly higher number of gastrin/cholecystokinin (CCK) positive cells. The diameter of lipid vacuoles in hepatocyte cytoplasm of fish fed formulated diets (PP, AA and CON) was significantly higher than in fish fed zooplankton (Z) and the commercial diet (AN). Hepatocytes of fish fed AA and CON showed a higher nucleus proliferation rate than in the other experimental groups. The quantitative analysis of the number of proliferating cell nuclear antigen (PCNA) and caspase-3(rabbit polyclonal antibody CPP-32)-positive cells showed that the highest proliferation rate was accompanied by the high apoptosis in the intestine of fish fed AA and CON. After 4 weeks of experimental feeding the highest relative expression of PepT1 gene was observed in fish fed PP diet, while the lowest expression occurred in fish fed CON. Feeding carp plant protein-based diet supplemented with Lys–Gly dipeptide (PP) had a beneficial influence on fish growth and metabolism in the digestive tract as compared to fish fed control diet without lysine supplementation (CON).

Oral and integumental uptake of free exogenous glycine by the Japanese spiny lobsterPanulirus japonicusphyllosoma larvae

The possibility of direct integumental absorption of the amino acid glycine from a solution in seawater was investigated in 250-260 day old (16.9-50.0 mg wet mass) phyllosoma larvae of the Japanese spiny lobster Panulirus japonicus Von Siebold 1824. The uptake of the amino acid was assessed by autoradiography and liquid scintillation counting (LSC) of larvae incubated with [2-(3)H]glycine and the net uptake was estimated by a time course high-performance liquid chromatography (HPLC) analysis of the concentration of glycine in the incubation medium. Autoradiography revealed the presence of labelled glycine in the cuticle, epidermis and internal tissues (digestive system, muscle, haemocytes) within 30 min of the onset of incubation. Absorption through the integument was confirmed by autoradiography and LSC as glycine uptake was observed even in larvae whose mouths were artificially sealed with cyanoacrylate bond prior to incubation. Scanning electron microscopic examination of the body surface revealed no bacterial population that could have mediated the uptake. HPLC revealed a consistent net uptake (0.29-0.39 micromol g(-1) body mass h(-1)) of glycine in larvae incubated in 6 micromol l(-1) glycine and high individual variation (e.g. absorption or release) in larvae incubated at higher concentrations (30 and 60 micromol l(-1)). Thus, the results of this study provide clear confirmation that, in addition to the known mode of oral feeding on macroscopic food masses, P. japonicus phyllosoma larvae are also able to absorb nutrients directly from the surrounding medium.

Comparative evaluation of hydro-, chemo- , and hormonal-priming methods for imparting salt and PEG stress tolerance in Indian mustard (Brassica juncea L.)

The present study was aimed to evaluate the effect of different seed priming methods to enhance the sodium chloride (NaCl) and polyethylene glycol-8000 (PEG-8000) stress tolerance in Indian mustard (Brassica juncea L.). Seeds subjected to different priming treatments such as water (hydro-priming), calcium chloride (CaCl2) (chemo-priming), and abscisic acid (ABA) (hormonal-priming) showed increased rate of germination as compared to non-primed seeds. The primed and non-primed seeds were grown for 15 days and then the seedlings were independently subjected to iso-osmotic salt (150 mM NaCl) or PEG-8000 (20%) stress. The different biochemical responses were studied 10 days after treatment. Under NaCl and PEG stress, the dry weight and total chlorophyll content were higher in primed sets as compared to non-primed treatment which was also evident by the phenotype of the seedlings. In general, the higher activities of superoxide dismutase and glutathione reductase resulted in lower oxidative damage, in terms of malondialdehyde content, under NaCl and PEG stress in hydro-primed set as compared to non-primed, ABA-, and CaCl2-primed treatments. Besides, the level of total phenolics and accumulation of osmolytes such as free proline, glycine betaine, and total soluble sugars was also lower in hydro-primed set as compared to other primed and non-primed treatments. The study thus suggests the use of hydro-priming as a simple and cost-effective strategy to alleviate the NaCl and PEG induced stress in B. juncea.

Regeneration of plantlets of guava (Psidium guajava L.) from somatic embryos developed under salt-stress condition

The present study demonstrates the regeneration of plantlets of guava (Psidium guajava L.) from somatic embryos developed under salt-stress conditions. With increasing concentrations of NaCl in induction medium (MS + 4.52 μM 2,4-d + 5% sucrose) from 0 to 200 mM, the number of somatic embryos per responsive explant decreased. Somatic embryos induced on 0–100 mM NaCl containing medium developed into torpedo stages, whereas, the development of somatic embryos that differentiated on 150 and 200 mM NaCl-supplemented medium was arrested prior to torpedo stage and did not undergo maturation phase. Somatic embryos that developed on NaCl-containing medium, showed better germination in the presence of NaCl as compared with those developed on medium without NaCl. The effect of increasing salt-stress was also investigated on plant growth, chlorophyll and carotenoids, Na+ and K+, and proline and glycine betaine accumulation in in vitro grown plantlets. The level of Na+ in leaves increased with increasing concentrations of NaCl in the medium. Accumulation of free proline and glycine betaine in leaves significantly increased with increasing salinity. The results suggest that accumulation of proline and glycine betaine may be important for osmotic adjustment in guava under salinity stress.

Failure of carnitine in improving hepatic nitrogen content in alcoholic and non-alcoholic malnourished rats

AIMS:To investigate the effect of carnitine supplementation on alcoholic malnourished rats' hepatic nitrogen content.METHODS:Malnourished rats, on 50% protein‐calorie restriction with free access to water (malnutrition group) and malnourished rats under the same conditions with free access to a 20% alcohol/water solution (alcohol group) were studied. After the undernourishment period (4 weeks with or without alcohol), both groups were randomly divided into two subgroups, one of them nutritionally recovered for 28 days with free access to a normal diet and water (recovery groups) and the other re‐fed with free access to diet and water plus carnitine (0.1 g/g body weight/day by gavage) (carnitine groups). No alcohol intake was allowed during the recovery period.RESULTS:The results showed: i) no difference between the alcohol/no alcohol groups, with or without carnitine, regarding body weight gain, diet consumption, urinary nitrogen excretion, plasma free fatty acids, lysine, methionine, and glycine. ii) Liver nitrogen content was highest in the carnitine recovery non‐alcoholic group (from 1.7 to 3.3 g/100 g, P<0.05) and lowest in alcoholic animals (about 1.5 g/100g). iii) Hepatic fat content (∼10 g/100 g, P>.05) was highest in the alcoholic animals.CONCLUSION:Carnitine supplementation did not induce better nutritional recovery.

Betaines and Dimethylsulfoniopropionate as Major Osmolytes in Cnidaria with Endosymbiotic Dinoflagellates

Most marine invertebrates and algae are osmoconformers whose cells accumulate organic osmolytes that provide half or more of cellular osmotic pressure. These solutes are primarily free amino acids and glycine betaine in most invertebrates and small carbohydrates and dimethylsulfoniopropionate (DMSP) in many algae. Corals with endosymbiotic dinoflagellates (Symbiodinium spp.) have been reported to obtain from the symbionts potential organic osmolytes such as glycerol, amino acids, and DMSP. However, corals and their endosymbionts have not been fully analyzed for osmolytes. We quantified small carbohydrates, free amino acids, methylamines, and DMSP in tissues of the corals Fungia scutaria, Pocillopora damicornis, Pocillopora meandrina, Montipora capitata, Porites compressa, and Porites lobata (all with symbionts) plus Tubastrea aurea (asymbiotic) from Kaneohe Bay, Oahu (Hawaii). Glycine betaine, at 33-69 mmol/kg wet mass, was found to constitute 90% or more of the measured organic solutes in all except the Porites species. Those were dominated by proline betaine and dimethyltaurine. DMSP was found at 0.5-3 mmol/kg in all species with endosymbionts. Freshly isolated Symbiodinium from Fungia, P. damicornis, and P. compressa were also analyzed. DMSP and glycine betaine dominated in the first two; Porites endosymbionts had DMSP, proline betaine, and dimethyltaurine. In all specimens, glycerol and glucose were detected by high-performance liquid chromatography only at 0-1 mmol/kg wet mass. An enzymatic assay for glycerol plus glycerol 3-phosphate and dihydroxyacetone phosphate yielded 1-10 mmol/kg. Cassiopeia andromeda (upside-down jelly; Scyphozoan) and Aiptasia puchella (solitary anemone; Anthozoan) were also analyzed; both have endosymbiotic dinoflagellates. In both, glycine betaine, taurine, and DMSP were the dominant osmolytes. In summary, methylated osmolytes dominate in many Cnidaria; in those with algal symbionts, host and symbiont have similar methylated amino acids, as do congeners. However, little glycerol was present as an osmolyte and was probably metabolized before it could accumulate.

The freshwater Amazonian stingray, Potamotrygon motoro, up-regulates glutamine synthetase activity and protein abundance, and accumulates glutamine when exposed to brackish (15‰) water

This study aimed to examine whether the stenohaline freshwater stingray, Potamotrygon motoro, which lacks a functional ornithine-urea cycle, would up-regulate glutamine synthetase (GS) activity and protein abundance, and accumulate glutamine during a progressive transfer from freshwater to brackish (15 per thousand) water with daily feeding. Our results revealed that, similar to other freshwater teleosts, P. motoro performed hyperosmotic regulation, with very low urea concentrations in plasma and tissues, in freshwater. In 15 per thousand water, it was non-ureotelic and non-ureoosmotic, acting mainly as an osmoconformer with its plasma osmolality, [Na+] and [Cl-] comparable to those of the external medium. There were significant increases in the content of several free amino acids (FAAs), including glutamate, glutamine and glycine, in muscle and liver, but not in plasma, indicating that FAAs could contribute in part to cell volume regulation. Furthermore, exposure of P. motoro to 15 per thousand water led to up-regulation of GS activity and protein abundance in both liver and muscle. Thus, our results indicate for the first time that, despite the inability to synthesize urea and the lack of functional carbamoyl phosphate synthetase III (CPS III) which uses glutamine as a substrate, P. motoro retained the capacity to up-regulate the activity and protein expression of GS in response to salinity stress. Potamotrygon motoro was not nitrogen (N) limited when exposed to 15 per thousand water with feeding, and there were no significant changes in the amination and deamination activities of hepatic glutamate dehydrogenase. In contrast, P. motoro became N limited when exposed to 10 per thousand water with fasting and could not survive well in 15 per thousand water without food.