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Because previous work showed that in the newborn brain, but not in the adult brain, glutamate decarboxylase (GAD) is notably susceptible to heat, we have studied the possible involvement of GAD inhibition in febrile convulsions and the related changes in gamma-aminobutyric acid (GABA) content. Rats of different ages were subjected to hyperthermia, and GAD activity was determined in brain homogenates by measuring the release of 14CO2 from labeled glutamate and by measuring the formation of GABA. The latter method gave considerably lower values than the former in the youngest rats, and was considered more reliable. With this method, we found a 37-48% inhibition of GAD activity in rat pups 2-5 days old, which showed febrile seizures at progressively higher body temperatures, whereas in 10- and 15-day-old animals, which did not show convulsions, GAD activity was not affected by hyperthermia. Whole-brain GABA levels, however, did not change at any age. In contrast to GAD, choline acetyltransferase and lactic dehydrogenase activities were not altered by hyperthermia at any of the ages studied. These results suggest that a decreased efficiency of the inhibitory neurotransmission mediated by GABA, consequent to the inhibition of GAD activity, may be a factor related to febrile convulsions.

A total of 50 lactic acid bacteria (LAB) isolates from Iranian traditional dairy products (Motal and Lighvan cheeses, and artisanal yogurt) were screened for gamma-aminobutyric acid (GABA) production. Firstly, a rapid colorimetric test was performed to evaluate the glutamate decarboxylase (GAD) activity among the LAB isolates examined. Thin layer chromatography (TLC) was then performed on selected strains to identify isolates with high/moderate GABA producing capacity, and a GABase micro-titer plate assay was employed to quantify GABA. Finally, two Lactococcus (Lac.) lactis strains were selected for GABA production optimization via Response Surface Methodology (RSM) following Central Composite Design (CCD). Forty-one out of the 50 isolates showed GAD activity according to the colorimetric assay. Eight isolates displayed strong GAD activity, while nine showed no activity; low to moderate GAD activity was scored for all other isolates. GABA production was confirmed by TLC in all isolates with high GAD activity and in four selected among isoaltes with moderate activity. Among the Lactococcus strains tested, Lac. lactis 311 and Lac. lactis 491 were the strongest GABA producers with amounts of 3.3 and 1.26 mM, respectively. These two strains were subjected to GABA production optimization applying RSM and CCD on three key variables: Monosodium glutamate concentration (MSG) (between 25 and 150 mM), incubation temperature (between 25 and 37 °C), and pH (between 4.0 and 5.0). Optimal conditions for GABA production by Lac. lactis 311 and Lac. lactis 491 of temperature, pH and MSG concentration were, respectively, 35.4 and 30 °C, pH 4.5 and 4.6, and MSG concentration of 89 and 147.4 mM, respectively. Under the above conditions, the amount of GABA produced by Lac. lactis 311 and Lac. lactis 491 was 0.395 and 0.179 mg/mL, respectively. These strains and the optimal culture conditions determined in this study could be used for the biotechnological production of GABA or applied in food fermentations for the development of naturally GABA-enriched foods.

BackgroundSalt stress is a serious abiotic stress that caused crop growth inhibition and yield decline. Previous studies have reported on the the synthesis of gamma-aminobutyric acid (GABA) and its relationship with plant resistance under various abiotic stress. However, the relationship between exogenous GABA alleviating plant salt stress damage and ion flux, amino acid synthesis, and key enzyme expression remains largely unclear. We investigated plant growth, Na+ transportation and accumulation, reactive oxygen species (ROS) metabolism and evaluated the effect of GABA on amino acids, especially SlGADs gene expression and the endogenous GABA content of tomato (Solanum lycopersicum L.) seedlings treated with or without 5 mmol·L− 1 GABA under 175 mmol·L− 1 NaCl stress.ResultsExogenous application of GABA significantly reduced the salt damage index and increased plant height, chlorophyll content and the dry and fresh weights of tomato plants exposed to NaCl stress. GABA significantly reduced Na+ accumulation in leaves and roots by preventing Na+ influx in roots and transportation to leaves. The transcriptional expression of SlGAD1–3 genes were induced by NaCl stress especially with GABA application. Among them, SlGAD1 expression was the most sensitive and contributed the most to the increase in glutamate decarboxylase (GAD) activity induced by NaCl and GABA application; Exogenous GABA increased GAD activity and amino acid contents in tomato leaves compared with the levels under NaCl stress alone, especially the levels of endogenous GABA, proline, glutamate and eight other amino acids. These results indicated that SlGADs transcriptional expression played an important role in tomato plant resistance to NaCl stress with GABA application by enhancing GAD activity and amino acid contents. GABA significantly alleviated the active oxygen-related injury of leaves under NaCl stress by increasing the activities of antioxidant enzymes and decreasing the contents of active oxygen species and malondialdehyde.ConclusionExogenous GABA had a positive effect on the resistance of tomato seedlings to salt stress, which was closely associated with reducing Na+ flux from root to leaves, increasing amino acid content and strengthening antioxidant metabolism. Endogenous GABA content was induced by salt and exogenous GABA at both the transcriptional and metabolic levels.

An enzymatic microassay method for glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was improved to yield a high sensitivity and a low blank. The 20-microns thick freeze-dried sections (0.2-1.5 micrograms dry weight) were prepared from the oviduct and ovary of rat. The analysis of these microsamples by the improved method showed that, contrary to the previous observations, the rat ovary is devoid of GAD activity and contains a trace amount of GABA. Both are present abundantly in the oviduct. In the oviduct mucosa, significant GAD activity was found in the estrous phase, whereas the activity was nearly null during other phases of the estrous cycle. GABA concentration in the oviduct mucosa was 10-fold higher than in the cerebral cortex; its variation during the estrous cycle was not remarkable. In the muscle layer of oviduct, GAD activity had a low peak in the estrous phase and GABA concentration was almost constant during the estrous cycle. The denervation experiment showed that GAD is present in the nerve terminals innervating the oviduct.

Glutamate decarboxylase (GAD) activity was estimated in various areas of the brain in 21 control and 26 parkinsonian subjects matched for age, postmortem delay and premortem state. Retrospective analysis of clinical data was used to define a premortem severity index (PMSI), scaled from 0 to 6, based upon a semiquantitative estimation of the duration of anoxia (0-3) and hypovolaemia (0-3). A significant correlation was found between GAD activity and PMSI in most regions of the brain. In the prefrontal cortex and caudate nucleus, GAD activity was not correlated with age, postmortem delay, sepsis, being bedridden, or with cachexia. Dosage and duration of drug treatment did not influence striatal or cortical GAD levels. In Parkinson's disease, GAD activity did not differ from controls in many brain areas except in the caudate nucleus, hippocampus and the frontal and occipital cortex. No difference in striatal and cortical GAD activity was observed when 10 control and 9 parkinsonian brains were selected for an optimal premortem state which approximated to sudden death (PMSI less than or equal to 2). GAD activity in the caudate nucleus and prefrontal cortex was not significantly influenced by the duration of L-DOPA treatment or withdrawal, disease duration, or severity of intellectual deterioration. Although the number of samples in certain brain areas was too small to allow a definitive conclusion, these results make it doubtful that GABAergic neurons are damaged in this disease.

Region-specific changes in the immunoreactivity of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide receptors (VPAC2, and PAC1 receptor) in the aged rat brains

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system and provides beneficial effects for human and other animals health. To accumulate GABA, samples from two different naked oat cultivars, Baiyan II and Bayou I, were steeped and germinated in an incubator. The content of GABA and glutamic acid as well as the activity of the glutamate decarboxylase (GAD) in oats during steeping and germination were investigated with an amino acid automatic analyzer. Compared with raw groats, an increase in GABA content of oat groats during steeping and germination was continuously observed for two oat cultivars. The activity of GAD increased greatly at the end of steeping and the second stage of germination for Baiyan II and Bayou I, respectively. Glutamic acid content of treated oat groats was significantly lower than that in raw groats until the later period of germination. GABA was correlated (p<0.01) significantly and positively with the glutamic acid rather than GAD activity in the current study. The results indicates that steeping and germination process under highly controlled conditions can effectively accumulate the GABA in oat groats for Baiyan II and Bayou I, which would greatly facilitate production of nutraceuticals or food ingredients that enable consumers to gain greater access to the health benefits of oats. However, more assays need to be further performed with more oat cultivars.

Distribution of GABA in post-mortem brain tissue from control, psychotic and Huntington's chorea subjects

Age-related oxidative inactivation of Na +, K +-ATPase in rat brain crude synaptosomes

Glutamic acid decarboxylase activity associated with cerebral blood vessels appears to be part of a specific cerebrovascular system involving gamma-aminobutyric acid. This activity was characterized kinetically and pharmacologically and compared with that in brain and several nonneuronal tissues. Formation of gamma-aminobutyric acid from [14C]glutamate was measured in a soluble extract of pia-arachnoid blood vessels isolated from bovine brain. The vascular activity was like brain glutamate decarboxylase in that it required pyridoxal phosphate, was completely inhibited by aminooxyacetic acid, and had a similar affinity for glutamate. Cerebrovascular decarboxylase activity differed, however, from brain decarboxylase in that it was less sensitive to sulfhydryl reagents, was stimulated by 3-mercaptopropionic and cysteic acids, and was competitively inhibited by cysteine sulfinic acid. The glutamate decarboxylase activity of the cerebral vessels was similar to that in renal cortex and mesenteric blood vessels in its responses to sulfhydryl reagents and 3-mercaptopropionic acid. These findings are consistent with previous suggestions of a nonneuronal form of the enzyme and offer the possibility that synthesis of gamma-aminobutyric acid in cerebral blood vessels can be manipulated independently from that in neuronal tissue.

Effects of components in culture medium on glutamate decarboxylase activity and γ-aminobutyric acid accumulation in foxtail millet (Setaria italica L.) during germination

In previous work, our research group isolated and bred a γ-aminobutyric acid (GABA)-producing microorganism, Lactobacillus brevis CGMCC 1306, from fresh unpasteurized milk and found that the strain possessed high glutamate decarboxylase (GAD) activity. However, the cells of the strain grew poorly during submerged fermentation, with the highest dry cell weight only 2.78 g/L. To enhance the efficiency of GABA biosynthesis and achieve GAD production in high cell-density fermentations, the gad gene from L. brevis CGMCC 1306 was cloned using degenerate PCR. Nucleotide sequencing analysis showed that the cloned gene comprised 1407 bp and encoded a 468-amino acid protein. The gad gene with a hexa-His tag was inserted into pET-28a(+) and expressed in Escherichia coli BL21. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the recombinant protein product had a molecular weight of approximately 53 kDa, which corresponded to the predicted size of the deduced protein (53.47 kDa). High-performance liquid chromatography analysis showed that the purified recombinant protein was capable of catalyzing α-decarboxylation of L-sodium glutamate into GABA, which confirmed that the protein was derived from a gad gene. GAD activity was the highest at pH 4.8 and 48°C. Using Lineweaver–Burk plots, the K m and V max of the recombinant enzyme were 10.26 mM and 8.86 U/mg, respectively. Its activity was not dependent on the addition of pyridoxal 5'-phosphate. These results strongly suggest that cloning of the gad gene is not only of interest to researchers in terms of affecting GAD production by protein engineering strategies, but that it may increase GAD production by high cell-density fermentation and enhance the efficiency of GABA biosynthesis.

Glutamate is an essential amino acid in both the energy and biosynthetic processes in plant cells. The aim of this work was to study changes in glutamate metabolism upon irradiation of maize (Zea mays L.) leaves with light of different spectral compositions, as well as to identify mechanisms regulating the work of enzymes involved in the studied process. A study was conducted of light-induced changes in glutamate metabolism in maize leaves, mediated by redirecting the glutamate flow to the γ-aminobutyric acid (GABA) shunt. Glutamate dehydrogenase (GDH) was more active in darkness, and the irradiation by red light inhibited the expression of both the Gdh1 and Gdh2 genes. EGTA and ruthenium red abolished the effects of light, indicating the participation of Ca2+ ions in phytochrome signal transduction. Contrary to GDH, glutamate decarboxylase (GAD) activity was moderately higher in the light, stimulated by red light, while far-red light reversed the effect. The effect of light on Gad expression was more pronounced than on GAD activity. Irradiation by red light also resulted in the increase in activity of GABA transaminase (GTA), which was abolished by far-red light. The third enzyme of the GABA shunt, succinic semialdehyde dehydrogenase (SSADH), was also activated by light. The effect of light on the expression of Ssadh1, but not on Ssadh2, was phytochrome-dependent. It is concluded that irradiation by light shifts glutamate metabolism from GDH to GAD with the activation of GABA transaminase and SSADH. This suggests that the GABA pathway plays a role in the maintenance of the tricarboxylic acid cycle in the light via bypassing its reactions when the 2-oxoglutarate dehydrogenase complex is inhibited and the cycle switches to the open mode.

Only in the last decade has consideration been given to glial cells in the aging brain, and the role of glial cells in the neuronal aging process is far from being understood. Morphological changes in number and volume of astroglia cells have been reported in various brain areas in the aging rat (Geinisman et al., 1978; Landfield et al., 1977; Lindsey et al., 1979). The phenomenon of “gliosis” with aging has been interpreted to reflect a compensatory response of astrocytes to their microenvironment and specifically to neuronal loss. On the other hand, the decline in synaptic function with aging (see review by Vernadakis, 1985) may partially be attributed to the lack of responsiveness of glial cells to synaptic plasticity including “reactive synaptogenesis,” a phenomenon observed in neuronal regeneration (Cotman and Nadler, 1978; Cotman and Scheff, 1979). Studies using the hippocampus as a model system to compare reactive synaptogenesis in the mature and aged rat brain have shown that reactive synaptogenesis is slower in the aged animals. Reactive synaptogenesis observed after partial denervation in brain-lesion studies appears to depend upon trophic signals provided by hormones and other growth-regulating factors, and the regulatory role of glial cells, i.e., the ability of glial cells to prepare the neuropil for the growth of new synapses. It is likely that, in the aging brain, glial cells may not supply the required growth factors for new synapse formation. Thus the proposal has been put forth that glial cells change with aging and may ultimately contribute to neuronal aging through changes in the microenvironment.

Haloperidol, sulpiride, domperidone and apomorphine, drugs which influence dopamine (DA) receptors and in turn prolactin (PRL) secretion have been shown to induce parallel changes in medial basal hypothalamic (MBH) glutamate decarboxylase (GAD) activity and serum PRL levels. The possibility that PRL may be involved in the effects of the drugs on MBH GAD activity is suggested in view of the evidence that hypophysectomy completely prevents drug-induced MBH GAD activity changes and that hyperprolactinemia by anterior pituitary homograft results in a significant, although small, change in the enzymatic activity.