GABA Accumulation Research Articles

Stress-induced changes in the free amino acid composition of two wheat cultivars with difference in drought resistance

Developmental changes in the levels of free amino acids of flag leaves in drought-tolerant (Triticum aestivum L. cv. Zagros) and drought-sensitive (cv. Marvdasht) wheat genotypes was investigated under controlled water deficit condition during grain filling. Cultivars were grown in pots and treated with either well-watered (control) or drought-stress (stress) from anthesis to maturity. Total soluble amino nitrogen in the leaves of two cultivars rose as relative water content (RWC) declined, the peak concentrations were attained early in the drought period (seven days after anthesis), when the Zagros and Marvdasht leaf RWC approached 80 and 70%, respectively. However, irrespective of water deficit, the total amino acids concentration declined with time. Interestingly, Zagros and Marvdasht did not accumulate the same amino acids. Zagros accumulated large amounts of proline and -aminobutyrate, whereas Marvdasht preferentially accumulated asparagine and arginine. It is concluded that; (i) modifications in the amino acid metabolism during dehydration stress were different depending on leaf development and (ii) proline and -aminobutyrate (Gaba) accumulation in Zagros leaves were essential for the acquisition of desiccation tolerance. On the contrary, the accumulation of large amounts of asparagine and arginine that serves as nitrogen and carbon reservoirs, in the Marvdasht during dehydration may be as a result of restriction of transport efficiency of storage material that led to low yield in drought sensitive cultivar.

489 Endogenous H2S Produced in Prevertebral Sympathetic Ganglia Predominantly by Cse in Neurons and Glia Cells Modulates Central Cholinergic Synaptic Input

Hydrogen sulfide(H2S), mainly produced endogenously by two enzymes, cystathionine γlyase(CSE) and cystathionine β-synthase(CBS), is a messenger molecule in the central nervous system, in the GI tract and in sympathetic prevertebral ganglia. Our previous study found, in mouse superior mesenteric ganglion(SMG), that exogenous H2S acts selectively on splanchnic nerve terminals to potentiate fast excitatory post synaptic potentials (F-EPSPs). In this study, we investigated the distribution of CSE and CBS and the role of endogenously produced H2S on central sympathetic input. Methods: The SMG, splanchnic nerve trunks and colonic nerve trunk were dissected from adult SJL/J mice. Immunostaining was performed with antibodies for CSE and CBS and for vesicular acetylcholine transporter(VAChT) to identify cholinergic nerve terminals. Microelectrodes were used for intracellular recordings. Results: CSE immunoreactivity(IR) was found in neurons and glia cells and did not co-localize with VAChT-IR. CBS-IR was found only in glia cells. To determine whether the F-EPSPs could be potentiated by endogenously released H2S, we tested the effect of inhibiting H2S breakdown with stigmatellin, a specific sulfide quinone reductase inhibitor, on F-EPSPs. Stigmatellin(1μM) significantly(P<0.05) increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(21.0±4.8mV and 441±107ms●mV with stigmatellin vs. 18.4±5.3mV and 338±108ms●mV before stigmatellin, n=4) but had no significant effect on F-EPSPs evoked by colonic nerve stimulation. In contrast, the CSE inhibitor, dl-propargylglycine(PAG, 1mM), significantly(P<0.05) decreased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.7±3.9mV and 824±288ms●mV with PAG vs. 27.0±4.1mV and 1230±414ms●mV before PAG, n=6), but had no effect on F-EPSPs evoked by colonic nerve stimulation. Surprisingly, the CBS inhibitor, aminooxyacetic acid(AOAA, 0.5mM), increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.6±12.1mV and 651±455ms●mV with AOAA vs. 20.2±10.9mV and 438±318ms●mV before AOAA, n=4, P<0.05) and increased the amplitude and the area of F-EPSPs evoked by colonic nerve stimulation(36.2±8.2mV and 1609±468ms●mV with AOAA vs. 28.9±8.1mV and 1084±402ms●mV before AOAA, n=6, P<0.05). Bicuculline(20μM), a specific GABA-A receptor inhibitor, blocked the potentiating effect of AOAA suggesting that the AOAA induced potentiation of F-EPSPs was due to the inhibition of GABA transaminase-induced accumulation of GABA. Conclusion: We conclude that endogenously produced H2S in the mouse SMG modulates fast cholinergic synaptic input in a pathwayspecific manner. H2S modulates fast cholinergic synaptic input from central splanchnic nerve terminals but has no affect on input from peripheral cholinergic input. (Funded by NIH DK17238)

490 Studies of the Psychophysiological Markers and the Brain Processing of Nausea in Healthy Humans Using a Novel Virtual Reality Video

Hydrogen sulfide(H2S), mainly produced endogenously by two enzymes, cystathionine γlyase(CSE) and cystathionine β-synthase(CBS), is a messenger molecule in the central nervous system, in the GI tract and in sympathetic prevertebral ganglia. Our previous study found, in mouse superior mesenteric ganglion(SMG), that exogenous H2S acts selectively on splanchnic nerve terminals to potentiate fast excitatory post synaptic potentials (F-EPSPs). In this study, we investigated the distribution of CSE and CBS and the role of endogenously produced H2S on central sympathetic input. Methods: The SMG, splanchnic nerve trunks and colonic nerve trunk were dissected from adult SJL/J mice. Immunostaining was performed with antibodies for CSE and CBS and for vesicular acetylcholine transporter(VAChT) to identify cholinergic nerve terminals. Microelectrodes were used for intracellular recordings. Results: CSE immunoreactivity(IR) was found in neurons and glia cells and did not co-localize with VAChT-IR. CBS-IR was found only in glia cells. To determine whether the F-EPSPs could be potentiated by endogenously released H2S, we tested the effect of inhibiting H2S breakdown with stigmatellin, a specific sulfide quinone reductase inhibitor, on F-EPSPs. Stigmatellin(1μM) significantly(P<0.05) increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(21.0±4.8mV and 441±107ms●mV with stigmatellin vs. 18.4±5.3mV and 338±108ms●mV before stigmatellin, n=4) but had no significant effect on F-EPSPs evoked by colonic nerve stimulation. In contrast, the CSE inhibitor, dl-propargylglycine(PAG, 1mM), significantly(P<0.05) decreased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.7±3.9mV and 824±288ms●mV with PAG vs. 27.0±4.1mV and 1230±414ms●mV before PAG, n=6), but had no effect on F-EPSPs evoked by colonic nerve stimulation. Surprisingly, the CBS inhibitor, aminooxyacetic acid(AOAA, 0.5mM), increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.6±12.1mV and 651±455ms●mV with AOAA vs. 20.2±10.9mV and 438±318ms●mV before AOAA, n=4, P<0.05) and increased the amplitude and the area of F-EPSPs evoked by colonic nerve stimulation(36.2±8.2mV and 1609±468ms●mV with AOAA vs. 28.9±8.1mV and 1084±402ms●mV before AOAA, n=6, P<0.05). Bicuculline(20μM), a specific GABA-A receptor inhibitor, blocked the potentiating effect of AOAA suggesting that the AOAA induced potentiation of F-EPSPs was due to the inhibition of GABA transaminase-induced accumulation of GABA. Conclusion: We conclude that endogenously produced H2S in the mouse SMG modulates fast cholinergic synaptic input in a pathwayspecific manner. H2S modulates fast cholinergic synaptic input from central splanchnic nerve terminals but has no affect on input from peripheral cholinergic input. (Funded by NIH DK17238)

Accumulation of γ-aminobutyric acid in germinated soybean (Glycine max L.) in relation to glutamate decarboxylase and diamine oxidase activity induced by additives under hypoxia

High levels of γ-aminobutyric acid (GABA) accumulate in plant tissues under various stresses and exogenous additives. The purpose of this research is to provide an effective finding that can prove a rapid accumulation of GABA in germinated soybean (Glycine max L.) in response to different additives under hypoxia. Hypoxia-induced GABA accumulation in soybean embryo resulted in part from polyamine oxidation. Response to different concentration of glutamate (Glu), pyridoxal phosphate, arginine, CuCl2, NaCl, and CaCl2, a significant difference including GABA accumulation, changes of Glutamate decarboxylase (GAD), and Diamine oxidase activity (DAO) activity in germinated soybean under hypoxia occurred (p < 0.05) and the maximum accumulation of GABA were 4.07, 3.02, 3.50, 3.26, 4.00, and 3.30 g kg−1 DW respectively, which were significantly higher than those germinated soybean under normal culture (CK) and hypoxia culture (CK0) (p < 0.05). The GAD and DAO have different distributions in cotyledon and embryo of germinated soybean, and the enzyme activity mainly located in embryo of germinated soybean. Germinated soybean is a good resource of GABA-rich food. Different additives have significant effects on GABA production, among which Glu and NaCl are ideal material for GABA accumulation.

Accumulation Mechanism of γ-Aminobutyric Acid in Tomatoes (Solanum lycopersicum L.) under Low O2 with and without CO2

The storage of ripe tomatoes in low-O(2) conditions with and without CO(2) promotes γ-aminobutyric acid (GABA) accumulation. The activities of glutamate decarboxylase (GAD) and α-ketoglutarate-dependent GABA transaminase (GABA-TK) were higher and lower, respectively, following storage under hypoxic (2.4 or 3.5% O(2)) or adjusted aerobic (11% O(2)) conditions compared to the activities in air for 7 days at 25 °C. GAD activity was consistent with the expression level of mRNA for GAD. The GABA concentration in tomatoes stored under hypoxic conditions and adjusted aerobic conditions was 60-90% higher than that when they are stored in air on the same day. These results demonstrate that upregulation of GAD activity and downregulation of GABA-TK activity cause GABA accumulation in tomatoes stored under low-O(2) conditions. Meanwhile, the effect of CO(2) on GABA accumulation is probably minimal.

Homeostatic Strengthening of Inhibitory Synapses Is Mediated by the Accumulation of GABAAReceptors

Mechanisms of homeostatic plasticity scale synaptic strength according to changes in overall activity to maintain stability in neuronal network function. This study investigated mechanisms of GABAergic homeostatic plasticity. Cultured neurons exposed to depolarizing conditions reacted with an increased firing rate (high activity, HA) that normalized to control levels after 48 h of treatment. HA-treated hippocampal neurons displayed an attenuated response to further changes in depolarization, and the firing rate in HA-treated neurons increased above normalized levels when inhibition was partially reduced back to the level of control neurons. The amplitude and frequency of mIPSCs in hippocampal neurons increased after 48 h of HA, and increases in the size of GABA(A) receptor γ2 subunit clusters and presynaptic GAD-65 puncta were observed. Investigation of the time course of inhibitory homeostasis suggested that accumulation of GABA(A) receptors preceded presynaptic increases in GAD-65 puncta size. Interestingly, the size of GABA(A) receptor γ2 subunit clusters that colocalized with GAD-65 were larger at 12 h, coinciding in time with the increase found in mIPSC amplitude. The rate of internalization of GABA(A) receptors, a process involved in regulating the surface expression of inhibitory receptors, was slower in HA-treated neurons. These data also suggest that increased receptor expression was consolidated with presynaptic changes. HA induced an increase in postsynaptic GABA(A) receptors through a decrease in the rate of internalization, leading to larger synaptically localized receptor clusters that increased GABAergic synaptic strength and contributed to the homeostatic stabilization of neuronal firing rate.

Climbing fiber activity reduces 14-3-3-θ regulated GABA A receptor phosphorylation in cerebellar Purkinje cells

Cerebellar adaptive plasticity regulates posture and movement in response to changing conditions of sensory stimulation. Study of adaptive plasticity of cerebellar circuitry in vitro confines experimental interest to mechanisms with a time scale of minutes. However, cerebellar plasticity, measured behaviorally or electrophysiologically in vivo, occurs over a time scale of tens of minutes and hours. Here we investigate how optokinetically-evoked increases in climbing fiber activity influence expression of key subcellular signaling proteins that regulate the accumulation of GABA A receptors (GABA ARs) in the cytoplasm of Purkinje cells and their insertion into the plasma membrane. We used long-term horizontal optokinetic stimulation (HOKS) to activate climbing fibers that project to the flocculus of mice. Although long-term increases in climbing fiber activity in vivo do not alter the expression of any of the subunits of GABA ARs expressed by Purkinje cells, they do influence other subcellular events such as transcription and interaction of signaling proteins. Specifically, increased climbing fiber activity evoked decreased expression of 14-3-3-θ, reduced serine phosphorylation of GABA Ag 2, and reduced the interaction of 14-3-3-θ with protein kinase C-γ (PKC-γ). Knockdown of 14-3-3-θ in vivo reduced the serine phosphorylation of GABA Aγ 2. Conversely, treatment of cerebellar lysates with phorbol 12-myristate-13-acetate (PMA), a PKC activator, increased serine phosphorylation of GABA Aγ 2. Knockdown of 14-3-3-θ or PKC-γ in N2a cells in vitro reduced serine phosphorylation of GABA Aγ 2 and reduced its cell-surface expression. We interpret these data to mean that a prolonged increase in climbing fiber activity decreases the cell-surface expression of GABA ARs in Purkinje cells and thereby reduces their sensitivity to GABAergic inhibition. This provides a homeostatic mechanism by which Purkinje cells become less sensitive to stellate cell inhibition also evoked by climbing fiber activity.

Effects of valproate on glutamate metabolism in rat brain slices: A 13C NMR study

Sodium valproate is a drug widely used for the treatment of epilepsy and mood disorders. We studied the effect of valproate on cerebral energy metabolism by incubating rat brain slices with 5 mM [3-(13)C]glutamate in the absence and the presence of 1 mM valproate. Substrate removal and product formation were measured by enzymatic and carbon 13 NMR methods. Fluxes through the enzymatic steps involved were calculated with an original mathematical model. We demonstrate that, in the presence of valproate, glutamate consumption and aspartate accumulation and labeling were inhibited, whereas GABA accumulation and labeling were increased. Consistent with these observations, this drug inhibited the unidirectional flux from glutamate to α-ketoglutarate and fluxes through several enzymes (gamma aminobutyric acid aminotransferase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, malic enzyme, pyruvate dehydrogenase, pyruvate carboxylase and citrate synthase). By contrast, glutamic acid decarboxylase flux was increased. With 2 mM glutamate+1 mM valproate and with 5 mM glutamate+2 mM valproate, GABA and aspartate labelings were similarly altered. On the basis of the effects of valproate, it is concluded that our cellular model and our cellular metabolomic approach appear suitable to study the beneficial and adverse interactions of neurotropic compounds with the cerebral metabolic pathways.

Malting revisited: Germination of barley (Hordeum vulgare L.) is inhibited by both oxygen deficiency and high carbon dioxide concentrations

During malting, barley (Hordeum vulgare L.) seeds are germinated to promote the mobilisation of storage compounds. Germination is strongly influenced by O2 and CO2; however, any distinction between the particular effects is missing. Since, in this study, the ambient O2 concentration was maintained when high CO2 concentrations were applied, for the first time the impacts of CO2 and of O2 deficiency could be distinguished unambiguously.Germination was inhibited by both O2 deficiency and high CO2 (80%) concentrations, documented by the lack of any growth of coleoptiles and any increase of α-amylase and β-glucanase activity. In contrast, the related impacts of O2 starvation and high CO2 on fermentation differ strongly, demonstrated by quite different patterns of ethanol emission. Additionally, the stress metabolism – monitored by the means of GABA accumulation – was also differently impacted. The elucidation of the underlying, so far unknown, mechanisms will provide novel opportunities to improve malting.

Brain-derived Neurotrophic Factor (BDNF) Enhances GABA Transport by Modulating the Trafficking of GABA Transporter-1 (GAT-1) from the Plasma Membrane of Rat Cortical Astrocytes

The γ-aminobutyric acid (GABA) transporters (GATs) are located in the plasma membrane of neurons and astrocytes and are responsible for termination of GABAergic transmission. It has previously been shown that brain derived neurotrophic factor (BDNF) modulates GAT-1-mediated GABA transport in nerve terminals and neuronal cultures. We now report that BDNF enhances GAT-1-mediated GABA transport in cultured astrocytes, an effect mostly due to an increase in the V(max) kinetic constant. This action involves the truncated form of the TrkB receptor (TrkB-t) coupled to a non-classic PLC-γ/PKC-δ and ERK/MAPK pathway and requires active adenosine A(2A) receptors. Transport through GAT-3 is not affected by BDNF. To elucidate if BDNF affects trafficking of GAT-1 in astrocytes, we generated and infected astrocytes with a functional mutant of the rat GAT-1 (rGAT-1) in which the hemagglutinin (HA) epitope was incorporated into the second extracellular loop. An increase in plasma membrane of HA-rGAT-1 as well as of rGAT-1 was observed when both HA-GAT-1-transduced astrocytes and rGAT-1-overexpressing astrocytes were treated with BDNF. The effect of BDNF results from inhibition of dynamin/clathrin-dependent constitutive internalization of GAT-1 rather than from facilitation of the monensin-sensitive recycling of GAT-1 molecules back to the plasma membrane. We therefore conclude that BDNF enhances the time span of GAT-1 molecules at the plasma membrane of astrocytes. BDNF may thus play an active role in the clearance of GABA from synaptic and extrasynaptic sites and in this way influence neuronal excitability.

Factors Influencing Diamine Oxidase Activity and γ-Aminobutyric Acid Content of Fava Bean (Vicia faba L.) during Germination

Factors (germination time, spectra, temperature, pH, and chemical inhibitors) influencing diamine oxidase (DAO, EC 1.4.3.6) activity and γ-aminobutyric acid (GABA) content of fava bean (Vicia faba L.) during germination were investigated in this study. DAO activity significantly increased in germinating seeds but varied with different organs. The enzyme activity was higher in shoot than that in cotyledon, hypocotyl, and radicle. When seeds were germinated in the dark, DAO activity was 2.35-, 2.00-, 2.36-, 4.40-, and 1.67-fold of that under white, red, blue, green, and yellow spectra, respectively. The optimum germination temperature and pH value for increasing DAO activity were 30 °C and 3.0, respectively. The DAO activity was inhibited significantly by aminoguanidine and sodium ethylenediamine tetracetate, while it was activated by CuCl(2) and CaCl(2). Germinating at an appropriate temperature and pH, 30% of GABA formation was supplied by DAO. Calcium was related to the regulation of DAO activity and GABA accumulation.

The Dominant Glutamic Acid Metabolic Flux to Produce γ‐Amino Butyric Acid over Proline in Nicotiana tabacum Leaves under Water Stress Relates to its Significant Role in Antioxidant Activity

γ-Amino butyric acid (GABA) and proline play a crucial role in protecting plants during various environmental stresses. Their synthesis is from the common precursor glutamic acid, which is catalyzed by glutamate decarboxylase and Δ(1) -pyrroline-5-carboxylate synthetase respectively. However, the dominant pathway under water stress has not yet been established. To explore this, excised tobacco leaves were used to simulate a water-stress condition. The results showed GABA content was much higher than that of proline in leaves under water-deficit and non-water-deficit conditions. Specifically, the amount of GABA significantly increased compared to proline under continuous water loss for 16 h, indicating that GABA biosynthesis is the dominant pathway from glutamic acid metabolism under these conditions. Quantitative reverse transcription polymerase chain reaction and protein Western gel-blot analysis further confirmed this. To explore the function of GABA accumulation, a system producing superoxide anion (O(2) (-) ), peroxide hydrogen (H(2) O(2) ), and singlet oxygen ((1) O(2) ) was employed to investigate the scavenging role on free-radical production. The results demonstrated that the scavenging ability of GABA for O(2) (-) , H(2) O(2) , and (1) O(2) was significantly higher than that of proline. This indicated that GABA acts as an effective osmolyte to reduce the production of reactive oxygen species under water stress.

Differential Regulation of the Postsynaptic Clustering of γ-Aminobutyric Acid Type A (GABAA) Receptors by Collybistin Isoforms

Collybistin promotes submembrane clustering of gephyrin and is essential for the postsynaptic localization of gephyrin and γ-aminobutyric acid type A (GABA(A)) receptors at GABAergic synapses in hippocampus and amygdala. Four collybistin isoforms are expressed in brain neurons; CB2 and CB3 differ in the C terminus and occur with and without the Src homology 3 (SH3) domain. We have found that in transfected hippocampal neurons, all collybistin isoforms (CB2(SH3+), CB2(SH3-), CB3(SH3+), and CB3(SH3-)) target to and concentrate at GABAergic postsynapses. Moreover, in non-transfected neurons, collybistin concentrates at GABAergic synapses. Hippocampal neurons co-transfected with CB2(SH3-) and gephyrin developed very large postsynaptic gephyrin and GABA(A) receptor clusters (superclusters). This effect was accompanied by a significant increase in the amplitude of miniature inhibitory postsynaptic currents. Co-transfection with CB2(SH3+) and gephyrin induced the formation of many (supernumerary) non-synaptic clusters. Transfection with gephyrin alone did not affect cluster number or size, but gephyrin potentiated the clustering effect of CB2(SH3-) or CB2(SH3+). Co-transfection with CB2(SH3-) or CB2(SH3+) and gephyrin did not affect the density of presynaptic GABAergic terminals contacting the transfected cells, indicating that collybistin is not synaptogenic. Nevertheless, the synaptic superclusters induced by CB2(SH3-) and gephyrin were accompanied by enlarged presynaptic GABAergic terminals. The enhanced clustering of gephyrin and GABA(A) receptors induced by collybistin isoforms was not accompanied by enhanced clustering of neuroligin 2. Moreover, during the development of GABAergic synapses, the clustering of gephyrin and GABA(A) receptors preceded the clustering of neuroligin 2. We propose a model in which the SH3- isoforms play a major role in the postsynaptic accumulation of GABA(A) receptors and in GABAergic synaptic strength.

Unraveling the role of fermentation in the mode of action of acetolactate synthase inhibitors by metabolic profiling

Herbicides that inhibit branched chain amino acid biosynthesis induce aerobic fermentation. The role of fermentation in the mode of action of these herbicides is not known, nor is the importance of this physiological response in the growth inhibition and the lethality caused by them. Metabolic profiling was used to compare the effects of the herbicide imazethapyr (IM) on pea plants with two other treatments that also induce fermentation: hypoxia and the exogenous supply pyruvate for seven days. While hypoxic roots did not show internal anoxia, feeding pyruvate or applying IM to the roots led to internal anoxia, probably related to the respiratory burst detected. The three treatments induced ethanol fermentation, but fermentation induced following herbicide treatment was earlier than that following pyruvate supply and was not associated with a decrease in the energy status. No striking changes were detected in the metabolic profiling of hypoxic roots, indicating that metabolism was only slightly impaired. Feeding pyruvate resulted in marked succinate accumulation and a general amino acid accumulation. IM-treated roots showed a general accumulation of glycolytic metabolites upstream of pyruvate, a decrease in some TCA intermediates and an increase in the free amino acid pool sizes. All treatments caused GABA and putrescine accumulation. Our results indicate that IM supply impairs carbon/nitrogen metabolism and this impaired metabolism is likely to be related to the growth arrest detected. As growth is arrested, carbohydrates and glycolytic intermediates accumulate and energy becomes more available.

GABA Accumulation Causes Cell Elongation Defects and a Decrease in Expression of Genes Encoding Secreted and Cell Wall-Related Proteins in Arabidopsis thaliana

GABA (γ-aminobutyric acid), a non-protein amino acid, is a signaling factor in many organisms. In plants, GABA is known to accumulate under a variety of stresses. However, the consequence of GABA accumulation, especially in vegetative tissues, remains poorly understood. Moreover, gene expression changes as a consequence of GABA accumulation in plants are largely unknown. The pop2 mutant, which is defective in GABA catabolism and accumulates GABA, is a good model to examine the effects of GABA accumulation on plant development. Here, we show that the pop2 mutants have pollen tube elongation defects in the transmitting tract of pistils. Additionally, we observed growth inhibition of primary root and dark-grown hypocotyl, at least in part due to cell elongation defects, upon exposure to exogenous GABA. Microarray analysis of pop2-1 seedlings grown in GABA-supplemented medium revealed that 60% of genes whose expression decreased encode secreted proteins. Besides, functional classification of genes with decreased expression in the pop2-1 mutant showed that cell wall-related genes were significantly enriched in the microarray data set, consistent with the cell elongation defects observed in pop2 mutants. Our study identifies cell elongation defects caused by GABA accumulation in both reproductive and vegetative tissues. Additionally, our results show that genes that encode secreted and cell wall-related proteins may mediate some of the effects of GABA accumulation. The potential function of GABA as a growth control factor under stressful conditions is discussed.

Isolation, characterization, and utilization of γ-aminobutyric acid (GABA)-producing lactic acid bacteria from Myanmar fishery products fermented with boiled rice

γ-Aminobutyric acid (GABA)-producing lactic acid bacteria (LAB) were isolated from four types of Myanmar traditional fermented fishery products with boiled rice. All of them belonged to the genus Lactobacillus, and comparison of the effects of these representatives on GABA accumulation in fermented fishery products with boiled rice revealed that Lactobacillus farciminis D323 is the most effective strain as a starter culture. These results may contribute to the development of traditional fermented fishery products with functional properties. In addition, this study is the first to show in detail the distribution of GABA-producing LAB in Southeast Asian fermented fishery products.

Effects of soaking and aeration treatment on γ-aminobutyric acid accumulation in germinated soybean (Glycine max L.)

Effects of soaking temperature, soaking time, opportunity of aeration treatment, culture temperature, pH value, and air flow rate on γ-aminobutyric acid (GABA) accumulation during germination of soybean (Glycine max (L.) Merr.) were investigated in this study. The objective of this study was to optimize the culture conditions of GABA production in germinated soybeans. Results showed that soaking at 30 °C for 4 h was found to be the most effective soaking ways that made soybean seeds have sufficient moisture, satisfactory germination, and GABA accumulation. The suitable stress opportunity was dark culture for 2 days with distilled water and then hypoxia stress in aerated culture medium for 2 days in a dark incubator at 30 °C. Under these conditions, the maximum GABA content (2.24 mg/g DW) was 3.5 times higher than the initial sample of aeration treatment (0 h) and 12.5 times higher than the raw material. Correlation analysis also revealed that GABA accumulation was significantly correlated to the corresponding physiochemical indexes (p < 0.01). Box-Behnken experimental analysis showed that the optimal condition with aeration treatment for GABA accumulation in germinated soybean was at a temperature of 30.5 °C, a pH value of 4.1, and an air flow rate of 0.9 L/min; the predicted highest GABA yield was 2.60 mg/g DW, which was 15.2 times higher than raw seeds. Analysis of variance and confirmatory trials for the regression model suggested that the model can quite exactly predict GABA accumulation in soybean during germination.

Effect of Exogenous γ-Aminobutyric Acid Treatment on Proline Accumulation and Chilling Injury in Peach Fruit after Long-Term Cold Storage

The effect of exogenous γ-aminobutyric acid (GABA) on chilling injury of peach fruit was investigated. Freshly harvested peaches were treated with 1, 5, or 10 mM GABA at 20 °C for 10 min and then stored at 1 °C for up to 5 weeks. The results showed that all of the GABA treatments could reduce chilling injury of peach fruit with 5 mM being the most effective concentration. GABA treatment significantly enhanced the accumulation of endogenous GABA and proline, which resulted from the increased activities of glutamate decarboxylase, Δ(1)-pyrroline-5-carboxylate synthetase, and ornithine δ-aminotransferase and decreased proline dehydrogenase activity. Our results revealed that GABA treatment may be a useful technique to alleviate chilling injury in cold-stored peach fruit, and the reduction in chilling by GABA may be due to the induction of endogenous GABA and proline accumulation. These data are the first evidence that exogenous GABA induced chilling tolerance in postharvest horticultural products.

Retinal nerve fibre layer attenuation: clinical indicator for vigabatrin toxicity

To investigate whether persistent visual field defects among patients exposed once to the antiepileptic drug vigabatrin (VGB) were associated with peripapillary retinal nerve fibre layer thickness (RNFLT) attenuation. Nine individuals with partial epilepsy and VGB-attributed visual field loss (group 1; 18 eyes) and seven age- and gender-matched individuals with epilepsy and no previous VGB exposure (group 2; 14 eyes) were included in the study. Full-field 120 point screening perimetry out to 60 degrees from central fixation using the Humphrey Field Analyzer was performed. RNFLT was quantified by optical coherence tomography (OCT) using Fast RNFLT protocol, Stratus OCT (3.0) after pupillary dilation. The results from the right eye are presented in this article. Among the patients with VGB-attributed visual field loss, five patients had only peripheral field defect (group 1a) and the remaining four had advanced field defects both in the periphery and within 30° from central fixation (group 1b). None of the patients in the control group had manifest visual field loss. The mean RNFLT among the patients with VGB-attributed visual field loss was significantly attenuated compared to the controls [mean total RNFLT: group 1: 75.6 ± 12.7 μm, group 2: 103.5 ± 9.7 μm, mean difference 27.9 μm, (CI 15.9-39.9; p < 0.001)]. RNFLT values classified as borderline according to normative database (Stratus OCT) occurred more frequently among individuals with VGB-attributed visual field loss than in controls (frequency in group 1: 6/9; group 2: 0/7, p = 0.011). The nasal, superior and inferior quadrants of RNFLT in individuals with VGB-attributed visual field loss were significantly attenuated, while no difference was detected in temporal quadrants compared to controls. Both individuals with peripheral and those with advanced visual field losses in the VGB group had attenuated mean total RNFLT compared to controls (p = 0.006, p = 0.002, respectively). Occurrence of borderline classification of total RNFLT ≤5th percentile was more frequent among individuals with advanced visual field loss than among controls (p = 0.048). Persistent visual field loss attributed to VGB is associated with reduced peripapillary RNFLT and was detected both among patients with advanced and among patients with only peripheral visual field defects. Measurements of RNFLT with OCT might be considered as a diagnostic supplement in the follow-up of patients exposed to vigabatrin.

ACCUMULATION OF γ-AMINOBUTYRIC ACID IN APPLE, STRAWBERRY AND TOMATO FRUIT IN RESPONSE TO POSTHARVEST TREATMENTS

γ-Aminobutyric acid (GABA) concentrations have been investigated in apple and strawberry fruit exposed to elevated CO 2 levels, and in tomato fruit of S. pennellii introgression lines in cultivated tomato (S. lycopersicum) stored at 3°C. In apple fruit, external CO 2 injury was much greater in fruit exposed to 2.5 and 5% CO 2 than at 1% CO 2 , and injury was enhanced by 1-methylcyclopropene (1-MCP) and inhibited by diphenylamine (DPA) treatment before exposure to 5% CO 2 . GABA accumulated in fruit skin to a greater extent when exposed to 2.5 and 5% CO 2 than to 1% CO 2 , while 1-MCP treated fruit had enhanced GABA accumulation and DPA treated fruit less GABA accumulation compared with 5% CO 2 alone. Strawberries contained undetectable amounts of GABA at harvest, but its concentration increased markedly during storage in 20% CO 2 . Tomato fruits of L. pennellii introgression lines stored at 3°C for 3 weeks showed elevated GABA concentrations, and the % increase over GABA concentrations at harvest was greater in fruit of introgression lines with higher susceptibility to chilling injury. Overall, the results suggest that GABA accumulations may be related to postharvest stresses in fruit, but further research is required to understand the relationships.