Concentrations Of L-glutamine Research Articles

Multi-objective based superimposed optimization method for enhancement of l-glutaminase production by Bacillus subtilis RSP-GLU

l-Glutaminase (l-glutamine amidohydrolase, EC 3.5.1.2) is a deamiding enzyme that has current focus on a therapeutic potentials and flavor enhancement agent in food industry. It was noticed that the amidase production was depended on the bacterial growth. However, the enhancement of bacterial growth by other substrates suppress the secretion of l-glutaminase. Governing the growth as well as amidase production is challenging task. In this study a multi-objective based superimposed optimization method was employed to control the biomass as well as to enhance the production of l-glutaminase. Various parameters such as volume of medium, pH, incubation temperature, inoculum level, l-glutamine concentration and rpm were optimized. The interactive influence of these parameters with their effect on enzyme and biomass production was analyzed using fractional factorial central composite design (FFCCD). The coefficient of determination (R2) was calculated as 0.9862 and 0.9513 for l-glutaminase and biomass production respectively. Overlay graphs were plotted by superimposing the contours for the various response surfaces of biomass and enzyme production. Based on superimposing method the cultural conditions obtained was volume of media 85 mL, temperature 37 °C, pH 7.24, inoculum concentration 2.47 mL, l-glutaminase concentration 1.01 g/L and agitation speed of 109 rpm. At these conditions 225 U/mL l-glutaminase and 0.79 g/L biomass were obtained. The current study provides a good description for balanced optimization of fermentation process of multi response or objective systems by using FFCCD method.

Ametryn removal by Metarhizium brunneum: Biodegradation pathway proposal and metabolic background revealed

Ametryn is a representative of a class of s-triazine herbicides absorbed by plant roots and leaves and characterized as a photosynthesis inhibitor. It is still in use in some countries in the farming of pineapples, soybean, corn, cotton, sugar cane or bananas; however, due to the adverse effects of s-triazine herbicides on living organisms use of these pesticides in the European Union has been banned. In the current study, we characterized the biodegradation of ametryn (100 mg L−1) by entomopathogenic fungal cosmopolite Metarhizium brunneum. Ametryn significantly inhibited the growth and glucose uptake in fungal cultures. The concentration of the xenobiotic drops to 87.75 mg L−1 at the end of culturing and the biodegradation process leads to formation of four metabolites: 2-hydroxy atrazine, ethyl hydroxylated ametryn, S-demethylated ametryn and deethylametryn. Inhibited growth is reflected in the metabolomics data, where significant differences in concentrations of L-proline, gamma-aminobutyric acid, L-glutamine, 4-hydroxyproline, L-glutamic acid, ornithine and L-arginine were observed in the presence of the xenobiotic when compared to control cultures. The metabolomics data demonstrated that the presence of ametryn in the fungal culture induced oxidative stress and serious disruptions of the carbon and nitrogen metabolism. Our results provide deeper insights into the microorganism strategy for xenobiotic biodegradation which may result in future enhancements to ametryn removal by the tested strain.

Gut microbiota of mice putatively modifies amino acid metabolism in the host brain.

Recently, it has been found that the gut microbiota influences functions of the host brain by affecting monoamine metabolism. The present study focused on the relationship between the gut microbiota and the brain amino acids. Specific pathogen-free (SPF) and germ-free (GF) mice were used as experimental models. Plasma and brain regions were sampled from mice at 7 and 16 weeks of age, and analysed for free d- and l-amino acids, which are believed to affect many physiological functions. At 7 weeks of age, plasma concentrations of d-aspartic acid (d-Asp), l-alanine (l-Ala), l-glutamine (l-Gln) and taurine were higher in SPF mice than in GF mice, but no differences were found at 16 weeks of age. Similar patterns were observed for the concentrations of l-Asp in striatum, cerebral cortex and hippocampus, and l-arginine (l-Arg), l-Ala and l-valine (l-Val) in striatum. In addition, the concentrations of l-Asp, d-Ala, l-histidine, l-isoleucine (l-Ile), l-leucine (l-Leu), l-phenylalanine and l-Val were significantly higher in plasma of SPF mice when compared with those of GF mice. The concentrations of l-Arg, l-Gln, l-Ile and l-Leu were significantly higher in SPF than in GF mice, but those of d-Asp, d-serine and l-serine were higher in some brain regions of GF mice than in those of SPF mice. In conclusion, the concentration of amino acids in the host brain seems to be dependent on presence of the gut microbiota. Amino acid metabolism in the host brain may be modified by manipulating microbiota communities.

A sensitive single-enzyme assay system using the non-ribosomal peptide synthetase BpsA for measurement of L-glutamine in biological samples

The ability to rapidly, economically and accurately measure L-glutamine concentrations in biological samples is important for many areas of research, medicine or industry, however there is room for improvement on existing methods. We describe here how the enzyme BpsA, a single-module non-ribosomal peptide synthetase able to convert L-glutamine into the blue pigment indigoidine, can be used to accurately measure L-glutamine in biological samples. Although indigoidine has low solubility in aqueous solutions, meaning direct measurements of indigoidine synthesis do not reliably yield linear standard curves, we demonstrate that resolubilisation of the reaction end-products in DMSO overcomes this issue and that spontaneous reduction to colourless leuco-indigoidine occurs too slowly to interfere with assay accuracy. Our protocol is amenable to a 96-well microtitre format and can be used to measure L-glutamine in common bacterial and mammalian culture media, urine, and deproteinated plasma. We show that active BpsA can be prepared in high yield by expressing it in the apo-form to avoid the toxicity of indigoidine to Escherichia coli host cells, then activating it to the holo-form in cell lysates prior to purification; and that BpsA has a lengthy shelf-life, retaining >95% activity when stored at either −20 °C or 4 °C for 24 weeks.

L-glutamine Induces Expression of Listeria monocytogenes Virulence Genes.

The high environmental adaptability of bacteria is contingent upon their ability to sense changes in their surroundings. Bacterial pathogen entry into host poses an abrupt and dramatic environmental change, during which successful pathogens gauge multiple parameters that signal host localization. The facultative human pathogen Listeria monocytogenes flourishes in soil, water and food, and in ~50 different animals, and serves as a model for intracellular infection. L. monocytogenes identifies host entry by sensing both physical (e.g., temperature) and chemical (e.g., metabolite concentrations) factors. We report here that L-glutamine, an abundant nitrogen source in host serum and cells, serves as an environmental indicator and inducer of virulence gene expression. In contrast, ammonia, which is the most abundant nitrogen source in soil and water, fully supports growth, but fails to activate virulence gene transcription. We demonstrate that induction of virulence genes only occurs when the Listerial intracellular concentration of L-glutamine crosses a certain threshold, acting as an on/off switch: off when L-glutamine concentrations are below the threshold, and fully on when the threshold is crossed. To turn on the switch, L-glutamine must be present, and the L-glutamine high affinity ABC transporter, GlnPQ, must be active. Inactivation of GlnPQ led to complete arrest of L-glutamine uptake, reduced type I interferon response in infected macrophages, dramatic reduction in expression of virulence genes, and attenuated virulence in a mouse infection model. These results may explain observations made with other pathogens correlating nitrogen metabolism and virulence, and suggest that gauging of L-glutamine as a means of ascertaining host localization may be a general mechanism.

Optimization of medium composition for in vitro shoot proliferation and growth of date palm cv. Mejhoul.

The effects of major mineral salts, l-glutamine, myo-inositol and carbon source on shoot bud proliferation of date palm (Phoenix dactylifera L.) cv. Mejhoul were evaluated. Different concentrations of ammonium nitrate (NH4NO3; 550, 825 or 1650 mg/L), potassium nitrate (KNO3; 633.3, 950 or 1900 mg/L), calcium chloride dehydrate (CaCl2·2H2O; 147, 220 or 440 mg/L), potassium dihydrogen phosphate (KH2PO4; 57, 85 or 170 mg/L), magnesium sulfate heptahydrate (MgSO4·7H2O; 123, 185 or 370 mg/L), l-glutamine and myo-inositol (0.5, 1, 1.5 and 2 g/L), sucrose, sorbitol, mannitol or commercial granulated sugar (10, 20, 30, 40 or 50 g/L) were tested. The highest number of shoot buds per explant (18.7) occurred on the medium containing 825 mg/L NH4NO3, 1900 mg/L KNO3, 220 mg/L CaCl2·2H2O, 170 mg/L KH2PO4, 370 mg/L MgSO4·7H2O as well as 1 g/L l-glutamine, 2 g/L myo-inositol and 30 g/L sucrose. The results showed that the frequency of hyperhydricity significantly increased in media containing 1650 mg/L NH4NO3. The concentrations of l-glutamine, myo-inositol and carbon source significantly affected the number of shoot buds per explant. However, they had no effect on hyperhydricity, tissue browning and precocious rooting. Shoots of 4.5–6.0 cm in length were isolated and transferred onto hormone-free media for elongation and rooting. After 3 months, the developed plantlets were successfully transplanted in a glasshouse and over 90 % survived acclimatization.

Structural and Dynamic Basis for Low-Affinity, High-Selectivity Binding of L-Glutamine by the Glutamine Riboswitch.

Naturally occurring L-glutamine riboswitches occur in cyanobacteria and marine metagenomes, where they reside upstream of genes involved in nitrogen metabolism. By combining X-ray, NMR, and MD, we characterized an L-glutamine-dependent conformational transition in the Synechococcus elongatus glutamine riboswitch from tuning fork to L-shaped alignment of stem segments. This transition generates an open ligand-binding pocket with L-glutamine selectivity enforced by Mg(2+)-mediated intermolecular interactions. The transition also stabilizes the P1 helix through a long-range "linchpin" Watson-Crick G-C pair-capping interaction, while melting a short helix below P1 potentially capable of modulating downstream readout. NMR data establish that the ligand-free glutamine riboswitch in Mg(2+) solution exists in a slow equilibrium between flexible tuning fork and a minor conformation, similar, but not identical, to the L-shaped bound conformation. We propose that an open ligand-binding pocket combined with a high conformational penalty for forming the ligand-bound state provide mechanisms for reducing binding affinity while retaining high selectivity.

Growth performance, serum biochemical profile, jejunal morphology, and the expression of nutrients transporter genes in deoxynivalenol (DON)- challenged growing pigs.

BackgroundFusarium infection with concurrent production of deoxynivalenol (DON) causes an increasing safety concern with feed worldwide. This study was conducted to determine the effects of varying levels of DON in diets on growth performance, serum biochemical profile, jejunal morphology, and the differential expression of nutrients transporter genes in growing pigs.ResultsA total of twenty-four 60-day-old healthy growing pigs (initial body weight = 16.3 ± 1.5 kg SE) were individually housed and randomly assigned to receive one of four diets containing 0, 3, 6 or 12 mg DON/kg feed for 21 days. Differences were observed between control and the 12 mg/kg DON treatment group with regards to average daily gain (ADG), although the value for average daily feed intake (ADFI) in the 3 mg/kg DON treatment group was slightly higher than that in control (P<0.01). The relative liver weight in the 12 mg/kg DON treatment group was significantly greater than that in the control (P<0.01), but there were no significant differences in other organs. With regard to serum biochemistry, the values of blood urea nitrogen (BUN), alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate amino transferase (AST) in the 3 treatment groups were higher than those in the control, and the serum concentrations of L-valine, glycine, L-serine, and L-glutamine were significantly reduced in the 3 treatment groups, especially in the 12 mg/kg DON group (P<0.01). Serum total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) were markedly decreased after exposure to DON contaminated feeds (P<0.01). The villi height was markedly decreased and the lymphocyte cell numbers markedly increased in the 3 DON contaminated feeds (P<0.01). The mRNA expression levels of excitatory amino acid transporter-3 (EAAC-3), sodium-glucose transporter-1 (SGLT-1), dipeptide transporter-1 (PepT-1), cationic amino acid transporter-1 (CAT-1) and y+L-type amino acid transporter-1 (LAT-1) in control were slightly or markedly higher than those in the 3 DON treatment groups.ConclusionsThese results showed that feeds containing DON cause a wide range of effects in a dose-dependent manner. Such effects includes weight loss, live injury and oxidation stress, and malabsorption of nutrients as a result of selective regulation of nutrient transporter genes such as EAAC-3, SGLT-1, PepT-1, CAT-1 and LAT-1.

Alanyl-glutamine and glutamine plus alanine supplements improve skeletal redox status in trained rats: Involvement of heat shock protein pathways

AimsWe hypothesized that oral l-glutamine supplementations could attenuate muscle damage and oxidative stress, mediated by glutathione (GSH) in high-intensity aerobic exercise by increasing the 70-kDa heat shock proteins (HSP70) and heat shock factor 1 (HSF1). Main methodsAdult male Wistar rats were 8-week trained (60-min/day, 5days/week) on a treadmill. During the last 21days, the animals were supplemented with either l-alanyl-l-glutamine dipeptide (1.5g/kg, DIP) or a solution containing the amino acids l-glutamine (1g/kg) and l-alanine (0.67g/kg) in their free form (GLN+ALA) or water (controls). Key findingsPlasma from both DIP- and GLN+ALA-treated animals showed higher l-glutamine concentrations and reduced ammonium, malondialdehyde, myoglobin and creatine kinase activity. In the soleus and gastrocnemius muscle of both supplemented groups, l-glutamine and GSH contents were increased and GSH disulfide (GSSG) to GSH ratio was attenuated (p<0.001). In the soleus muscle, cytosolic and nuclear HSP70 and HSF1 were increased by DIP supplementation. GLN+ALA group exhibited higher HSP70 (only in the nucleus) and HSF1 (cytosol and nucleus). In the gastrocnemius muscle, both supplementations were able to increase cytosolic HSP70 and cytosolic and nuclear HSF1. SignificanceIn trained rats, oral supplementation with DIP or GLN+ALA solution increased the expression of muscle HSP70, favored muscle l-glutamine/GSH status and improved redox defenses, which attenuate markers of muscle damage, thus improving the beneficial effects of high-intensity exercise training.

Abstract C154: Kinetic analysis of the glutaminase activity of L-asparaginases derived from Erwinia chrysanthemi (Erwinase®) and Eshericia coli (Kidrolase).

Abstract Glutamine is an essential nutrient for cancer cells and increased glutaminolysis is widely recognised as a key feature of the metabolic reprogramming that occurs during malignant transformation. As a consequence of the role glutamine plays in cancer biology, targeting glutamine metabolism is of considerable interest with inhibition of the mitochondrial enzyme glutaminase showing promise in experimental models. An alternative or adjunct to the strategy of inhibiting glutamine metabolism intracellularly is to deplete extracellular pools of glutamine. L-asparaginases are known to possess glutaminase activity and the purpose of this study is to characterise the kinetics of the glutaminase activity of two clinically approved L-asparaginases, Kidrolase (derived from E. coli) and Erwinase (derived from Erwinia chrysanthemi). Glutaminase activity was measured using a two step reaction: initial reduction of glutamine to glutamate by Kidrolase/Erwinase followed by the NAD dependent conversion of glutamate to ammonia by L-glutamate dehydrogenase (GDH). Each reaction consisted of NAD (2mM), K2PO4 (50mM), GDH (3U), Erwinase or Kidrolase (10U), L-glutamine (concentrations ranged from 5mM to 1.25 μM) in a final volume of 1 ml Tris Acetate buffer (50mM, pH8.6). The reaction was started by the addition of Erwinase/Kidrolase and the formation of NADH monitored for 30 seconds at 340nm using a Cary UV/Vis spectrophotometer and a NADH molar extinction coefficient of 6,220 M-1cm-1. The reaction was linear between glutamine concentrations 100 and 1.25 μM. Using 10U in each reaction, Hanes Woolf kinetic plots generated Vmax parameters of 46.29 and 7.57 μM/min for Erwinase and Kidrolase respectively. Km values were 96.05 and 33.06 μM for Erwinase and Kidrolase respectively. Preliminary studies demonstrate that glutamine depletion induced a dose dependent reduction in cell proliferation in HCT116 p53+/+, HCT116 p53-/-, MDA-MD-231, MDA-MB-453, MCF7, TK10, A549 and T47D cell lines in vitro. In conclusion, these studies demonstrate that Erwinase has significant glutaminase activity and is superior to Kidrolase in terms of glutaminase activity. Erwinase is a good candidate for exploring the efficacy of glutamine depletion strategies and further studies are required to determine whether Erwinase can potentiate the activity of other approaches aimed at restricting the glutamine dependence of tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C154. Citation Format: Roger M. Phillips, Mohammed U. Saleem, Adrian Williams, Jon Morgan. Kinetic analysis of the glutaminase activity of L-asparaginases derived from Erwinia chrysanthemi (Erwinase®) and Eshericia coli (Kidrolase). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C154.

Organic nitrogen composition of the tissue culture medium influences Agrobacterium tumefaciens growth and the recovery of transformed Pinus radiata embryonal masses after cocultivation

Repeated attempts to genetically transform Pinus radiata embryonal masses through cocultivation with Agrobacterium tumefaciens on MSG medium were unproductive due to Agrobacterium overgrowth. Timentin at either 200 or 400 mg l−1 was ineffective in inhibiting bacterial growth after cocultivation. In this study, the causes of the abundant bacterial growth were investigated by comparing MSG medium with two other media (mLV and DCR) commonly used in conifer somatic embryogenesis. Statistical analysis of the growth data (optical density and number of cell-forming units) showed that bacterium grew significantly more on MSG than on mLV or DCR during the 48-h cocultivation. This enhanced growth was attributed to the higher concentration of L-glutamine in MSG. Lowering the concentration of L-glutamine in MSG to 0.5 g l−1 resulted in similar growth of Agrobacterium compared with the other two media. MSG was also superior for the growth of radiata pine cells, with a statistically significant difference after 14 d of culture. Hence, to avoid bacterial overgrowth during and after cocultivation, a two-medium protocol was developed in which cocultivation was carried out on mLV, followed by 5 d on mLV with 400 mg l−1 Timentin. Selection for transformed cells and further control of bacterial growth was then performed using MSG with Timentin and Geneticin. By sequential application of these two media, 2,096 cell colonies were selected; of these, 94 were analyzed and 49 were transgenic. These results highlight yet another factor that might be critical for the success of transformation experiments but has not been sufficiently studied until now: the growth dynamics and ability to eliminate A. tumefaciens on various plant tissue culture media.

L-DOPA Suppressed Scratching Behavior and Modified Brain Monoamine/ Amino Acid Concentrations in Mice

The effects of L-DOPA (0, 25, and 50 mg/kg body weight) administration on peripheral pruritogen-induced scratching behavior and on brain monoamine and amino acid metabolism were investigated in mice. Intraperitoneal injection of L-DOPA significantly suppressed the number of scratches caused by subcutaneous injection of compound 48/80 (C48/80) in a dose-dependent manner. In the brain stem, C48/80 increased the norepinephrine turnover rate, but the change in the frequency of scratching was not paralleled by the changes in monoamine levels or by the monoamine turnover rates in the brain. On the other hand, most amino acids in the brain stem showed the highest levels in mice treated with C48/80 without L-DOPA, and these values were clearly decreased by L-DOPA administration. These changes in the concentrations of amino acids may be involved in the regulation of the frequency of scratching induced by L-DOPA. In particular, treatment with L-DOPA and C48/80 markedly decreased the concentrations of GABA, L-glutamine, Lglutamic acid, and glycine in the brain stem. In conclusion, L-DOPA administration may attenuate the frequency of the scratching through changing amino acid metabolism in the brain.

The impact of chronic imipramine treatment on amino acid concentrations in the hippocampus of mice

The relationship between antidepressants and monoamine concentrations in the brain has been well investigated, but few studies have investigated the relationship between antidepressants and amino acid concentrations in the brain. The purpose of the present study was therefore to investigate the effect of the chronic antidepressant imipramine on amino acid and monoamine concentrations in the mouse brain and plasma. Chronic imipramine treatment decreased the concentration of 5-hydroxyindoleaceticacid/5-hydroxytryptamine in the cerebral cortex and increased that of norepinephrine (NE) in the hippocampus. Since these changes were conspicuous effects of the antidepressant, we concluded that imipramine acts on the central nervous system. No change in amino acid concentrations in plasma was induced by chronic imipramine treatment, but several changes were confirmed in the cerebral cortex, the hypothalamus and the hippocampus. Chronic imipramine treatment caused increases in l-methionine, l-tyrosine, and l-lysine in the cerebral cortex, and an increase in l-aspartate in the hypothalamus. Contrary to this, the concentrations of l-aspartate, l-serine, l-asparagine, glycine, l-glutamine, gamma-aminobutyric acid, l-threonine, l-arginine, l-proline, l-valine, and l-methionine in the hippocampus were decreased by chronic imipramine treatment. The present results demonstrate that the metabolism of several amino acids in the brain, but not of those in plasma, was altered by chronic imipramine treatment. The findings in the present study may help to further elucidate the relationship between amino acids and the effects and side effects of antidepressants.

Amino Acid Profiles in Human Tear Fluids Analyzed by High-Performance Liquid Chromatography and Electrospray Ionization Tandem Mass Spectrometry

To identify the 23 amino acid profiles in human tear fluids, and to evaluate whether the ocular disease conditions reflect the amino acid profiles. Laboratory investigation. We evaluated the concentrations and relative composition of 23 amino acids in tear fluids obtained from 31 healthy volunteers using reversed-phase high-performance liquid chromatography and electrospray ionization tandem mass spectrometry, and compared them with those in plasma and aqueous humor. We also evaluated the tear-fluid amino acid profiles from 33 affected subjects. The amino acid profiles of the basal tear and reflex tear were found to be similar, and 4 distinct groups of healthy volunteers (male, female, young, and elderly) showed similar profiles. Absolute concentrations of taurine (Tau) and L-glutamine were significantly dominant in these tear fluids. The relative compositions of Tau, L-glutamic acid, L-arginine (Arg), and citrulline in the tear fluid were significantly higher than those in the plasma and aqueous humor. Analysis of the hierarchical clustering of the amino acid profiles clearly distinguished severe ocular surface diseases from non-ocular surface diseases. The relative compositions of Tau, L-methionine, and Arg decreased in severe ocular surface disease subjects compared with non-ocular surface disease subjects. Tear-fluid amino acid profiles differ from those in plasma and aqueous humor. Steady-state tear-fluid amino acid profiles might reflect ocular-surface homeostasis and the observed changes of amino acids might have a close relation with the disease conditions on the ocular surface.

Effect of Inducers and Physical Parameters on the Production Of L-Asparaginase Using Aspergillus Terreus

An effort has been made to find the best medium and operating conditions for the production of extracellular L-asparaginase by Aspergillus terreus in shake culture fermentation through stepwise optimization strategies like studies on the effect of pH and temperature on L-asparaginase, studies on the enzyme activity in various concentration of L-glutamine and study the growth kinetics for L-glutamine medium. Our study shows that L- glutamine combined with L-asparagine influence the asparaginase production level and physical parameter such as pH and temperature has more contribution in enzyme production. The pH 8 and temperature 35°C found to be good environmental condition the production of extracellular L-asparaginase by Aspergillus terreus.

Glucose promotion of GABA metabolism contributes to the stimulation of insulin secretion in β-cells

We have demonstrated recently that branched-chain α-keto acid stimulation of insulin secretion is dependent on islet GABA (γ-aminobutyric acid) metabolism: GABA transamination to succinic semialdehyde is increased by 2-oxoglutarate, generated in α-keto acid transamination to its corresponding α-amino acid. The present work was aimed at investigating whether glucose also promotes islet GABA metabolism and whether the latter contributes to the stimulation of insulin secretion. Glucose (20 mM) decreased both the content and release of islet GABA. Gabaculine (1 mM), a GABA transaminase inhibitor, partially suppressed the secretory response of rat perifused islets to 20 mM glucose at different L-glutamine concentrations (0, 1 and 10 mM), as well as the glucose-induced decrease in islet GABA. The drug also reduced islet ATP content and the ATP/ADP ratio at 20 mM glucose. Exogenous succinic semialdehyde induced a dose-dependent increase in islet GABA content by reversal of GABA transamination and a biphasic insulin secretion in the absence of glucose. It depolarized isolated β-cells and triggered action potential firing, accompanied by a reduction of membrane currents through ATP-sensitive K(+) channels. The gene expression and enzyme activity of GABA transaminase were severalfold higher than that of 2-oxoglutarate dehydrogenase in islet homogenates. We conclude that, at high glucose concentrations, there is an increased diversion of glucose metabolism from the citric acid cycle into the 'GABA shunt'. Semialdehyde succinic acid is a cell-permeant 'GABA-shunt' metabolite that increases ATP and the ATP/ADP ratio, depolarizes β-cells and stimulates insulin secretion. In summary, an increased islet GABA metabolism may trigger insulin secretion.

FHANT‐AC genes of the ectomycorrhizal fungus Laccaria bicolor are not repressed by l‐glutamine allowing simultaneous utilization of nitrate and organic nitrogen sources

In boreal and temperate forest ectomycorrhizal fungi play a crucial role in nitrogen cycling by assimilating nitrogenous compounds from soil and transferring them to tree hosts. The expression profile of fHANT-AC genes, nitrate transporter (Lbnrt), nitrate reductase (Lbnr) and nitrite reductase (Lbnir), responsible for nitrate utilization in the ectomycorrhizal fungus Laccaria bicolor, was studied on variable N regimens. The three genes were shown to be under a common regulation: repressed in the presence of ammonium while growth on nitrate resulted in high transcripts accumulation. The presence of nitrate was shown not to be indispensable for activation of Laccaria fHANT-AC as also N starvation and growth on urea and l-asparagine resulted in high transcript levels. Equally high expression of Laccaria fHANT-AC genes was detected in mycelia grown on variable concentrations of l-glutamine. This finding shows that in L. bicolor N metabolite repression of fHANT-AC is not signalled via l-glutamine like described in ascomycetes. The expression patterns of Lbnrt and Lbnir were also studied in an Lbnr RNA-silenced Laccaria strain. No differences were observed on the N source regulation or the degree of transcript accumulation of these genes, indicating that the presence of high nitrate reductase activity is not a core regulator of L. bicolor fHANT-AC expression. The simultaneous utilization of nitrate and organic N sources, already suggested by high transcript levels of Laccaria fHANT-AC genes on organic N, was supported by the increase of culture medium pH as a result of nitrate transporter activity. The possible ecological and evolutionary significance of the herein reported high regulatory flexibility of Laccaria nitrate utilization pathway for ectomycorrizal fungi and the ectomycorrhizal symbiosis is discussed.

Oral Glutamine Dipeptide Prevents against Prolonged Hypoglycemia Induced by Detemir Insulin in Rats

The role of glutamine dipeptide (GDP) to prevent against prolonged insulin induced hypoglycemia (IIH) in overnight fasted rats was investigated. The glycemia was measured 0, 2, 4, 8, and 10 h after an intraperitoneal (i.p.) injection (1 U/kg) of Detemir insulin. Because the lowest glycemia was obtained 4 h after insulin administration, the experiments were done at this time. The hypoglycemia obtained 4 h after insulin injection was partially prevented with increasing oral doses of GDP (1.56, 3.12, 6.25, 12.5 mg/kg). The best result was obtained with 12.5 mg/kg. However, from this dose (25.0, 50.0, 100.0 mg/kg), the values of glycemia progressively decreased (p<0.05). The effect of GDP to prevent prolonged IIH was mediated, partly at least, by an intensification of liver gluconeogenesis. Moreover, the increased portal availability of GDP, L-alanine and L-glutamine after GDP administration also contributed to the IIH prevention, since the rate of gluconeogenesis progressively augmented with the infusion of increasing concentrations of these substances. However, after getting the maximal value, the rate of liver gluconeogenesis decreased (p<0.05) if a more elevated concentration of L-alanine or L-glutamine was infused. On the other hand, the liver gluconeogenesis during the infusion of increasing concentrations of GDP was unchanged. Because, GDP did not directly inhibit liver gluconeogenesis, but an inhibition of this metabolic pathway was observed with low ammonia concentrations (from 0.062 mM) it is possible that the ammonia from the catabolism of GDP by extra hepatic tissues could inhibit liver gluconeogenesis. This mechanism could help to explain the lower glycemia obtained with more elevated doses of oral GDP.

Colonic luminal ammonia and portal blood l-glutamine and l-arginine concentrations: a possible link between colon mucosa and liver ureagenesis

The highest ammonia concentration in the body is found in the colon lumen and although there is evidence that this metabolite can be absorbed through the colonic epithelium, there is little information on the capacity of the colonic mucosa to transfer and metabolize this compound. In the present study, we used a model of conscious pig with a canula implanted into the proximal colon to inject endoluminally increasing amounts of ammonium chloride and to measure during 5 h the kinetics of ammonia and amino acid concentration changes in the portal and arterial blood. By injecting as a single dose from 1 to 5 g ammonia into the colonic lumen, a dose-related increase in ammonia concentration in the portal blood was recorded. Ammonia concentration remained unchanged in the arterial blood except for the highest dose tested, i.e. 5 g which thus apparently exceeds the hepatic ureagenesis capacity. By calculating the apparent net ammonia absorption, it was determined that the pig colonic epithelium has the capacity to absorb 4 g ammonia. Ammonia absorption through the colonic epithelium was concomitant with increase of L-glutamine and L-arginine concentrations in the portal blood. This coincided with the expression of both glutamate dehydrogenase and glutamine synthetase in isolated colonic epithelial cells. Since L-glutamine and L-arginine are known to represent activators for liver ureagenesis, we propose that increased portal concentrations of these amino acids following increased ammonia colonic luminal concentration represent a metabolic link between colon mucosa and liver urea biosynthesis.

Embryogenic tissue initiation and somatic embryogenesis in Fraser fir (Abies fraseir [Pursh] Poir.)

Embryogenic suspensor mass (ESM) was established from immature seeds of Fraser fir. The initiation frequency of ESM was dependent on genotype, collection time, medium, and plant growth regulators (PGR) used. The ESM initiation potential was higher with seeds collected in late June (clone 16-273, 4.7%) or early July (clone 16-45, 2.2%) and decreased as the zygotic embryos matured. Excised proembryo stage of zygotic embryos was most appropriate to initiation of ESM. Most of the ESM arose from the seeds that were at the proembryo stage. From the four different culture media we compared, seven ESM lines were obtained: two lines from Murashige and Skoog (MS) medium with 4.4 μM benzyladenine (BA), one from Schenk and Hildebrandt (SH) medium with 4.5 μM thidiazuron (TDZ), and four from SH with 4.4 μM 6-benzyladenine. However, only one ESM line from clone 16-273 (June 24, SH+TDZ) could be proliferated in subsequent culture. Different concentrations of l-glutamine and casein hydrolysate (CH) in the medium were also compared for their effect on ESM proliferation. The highest proliferation rate (1.16-fold) was obtained from SH medium supplemented with 250 mg/L CH and 3.42 mM l-glutamine. In contrast, the lowest rate was noted when 1,000 mg/L CH plus 3.42 mM l-glutamine (0.17-fold) was added to the medium. As for somatic embryo maturation, the highest number of mature precotyledonary (100.1/g−1 FW ESM) or cotyledonary (64.3/g−1 FW ESM) somatic embryos was obtained on a medium containing 20 or 80 μM abscisic acid, 10% polyethyleneglycol, 4% maltose, and 0.3% gellan gum. For germination of the somatic embryos, the cotyledonary somatic embryos derived from maturation medium were transferred on half-strength Litvay medium containing 0.3% gellan gum. The somatic plantlets were recovered from the germination medium and transferred to soils.