Molecular cloning, expression, and immobilization of glutamate decarboxylase from Lactobacillus fermentum YS2

Background: GABA (γ-aminobutyric acid) is a four-carbon nonprotein amino acid that has hypotensive, diuretic, and tranquilizing properties. Glutamate decarboxylase (GAD) is the key enzyme to generate GABA. A simple and economical method of preparing and immobilizing GAD would be helpful for GABA production. In this study, the GAD from Lactobacillus fermentum YS2 was expressed under the control of a stress-inducible promoter and was purified and immobilized in a fusion form, and its reusability was investigated. Results: The fusion protein CBM-GAD was expressed in Escherichia coli DH5α carrying pCROCB-gadB, which contained promoter PrpoS, cbm3 (family 3 carbohydrate-binding module from Clostridium thermocellum) coding sequence, the gadB gene from L. fermentum YS2 coding for GAD, and the T7 terminator. After a one-step purification of CBM-GAD using regenerated amorphous cellulose (RAC) as an adsorbent, SDS-PAGE analysis revealed a clear band of 71 kDa; the specific activity of the purified fusion protein CBM-GAD reached 83.6 ± 0.7 U·mg-1. After adsorption onto RAC, the immobilized GAD with CBM3 tag was repeatedly used for GABA synthesis. The protein-binding capacity of RAC was 174 ± 8 mg·g-1. The immobilized CBM-GAD could repeatedly catalyze GABA synthesis, and 8% of the initial activities was retained after 10 uses. We tested the conversion of monosodium glutamate to GABA by the immobilized enzyme; the yield reached 5.15 g/L and the productivity reached 3.09 g/L·h. Conclusions: RAC could be used as an adsorbent in one-step purification and immobilization of CBM-GAD, and the immobilized enzyme could be repeatedly used to catalyze the conversion of glutamate to GABA.

Modeling of glutamic acid production by Lactobacillus plantarum MNZ

Background: L-glutamic acid, the principal excitatory neurotransmitter in the brain and an important intermediate in metabolism acts as a precursor of γ-amino butyric acid (GABA). In the present study, culture condition for enhanced glutamic acid production by Lactobacillus plantarum MNZ was optimized and the influence of such conditions on GABA production was evaluated. Results: Results indicated that glutamic acid increased up to 3-fold (3.35) under the following condition: pH 4.5, temperature 37ºC, 12% (w/v) glucose and 0.7% (w/v) ammonium nitrate; whilst GABA production was enhanced up to 10-fold under the following condition: pH 4.5, temperature 37ºC, 6% (w/v) glucose and 0.7% (w/v) ammonium nitrate. Conclusions: This is the first report for dual biosynthesizing activities of a lactic acid bacterium for the production of glutamic acid and GABA. The results of this study can be further used for developing functional foods rich inglutamic acid and subsequently GABA as a bioactive compound.

Effect of propofol on glutamate and gamma-aminobutyric acid release from rat hippocampal synaptosomes / 华中科技大学学报（医学）（英德文版）

To investigate the effect of propofol on the release of glutamate and gamma-aminobutyric acid (GABA) from rat hippocampal synatosomes, synaptosomes was made from hippocampus and incubated with artificial cerebrospinal fluid (aCSF). With the experiment of Ca(2+)-dependent release of glutamate and GABA, dihydrokainic acid (DHK) and nipectic acid were added into aCSF. For the observation of Ca(2+)-independent release of glutamate and GABA, no DHK, nipectic acid and Ca2+ were added from aCSF. The release of glutamate and GABA were evoked by 20 micromol/L veratridine or 30 mmol/L KCI. The concentration of glutamate and GABA in aCSF was measured by using high-performance liquid chromatography (HPLC). 30, 100 and 300 micromol/L propofol significantly inhibited veratridine-evoked Ca(2+)-dependent release of glutamate and GABA (P 0.05). Veratridine or elevated KCI evoked Ca(2+)-independent release of glutamate and GABA was not affected significantly by propofol (P > 0.05). Propofol could inhibit Ca(2+)-dependent release of glutamate and GABA. However, it has no effect on the Ca(2+)-independent release of glutamate and GABA.

Papel de los aminoácidos excitatorios en la neuropatología / Role of excitatory amino acids in neuropathology

Excitatory amino acids (EAA) became known as neurotransmitters of the central nervous system (CNS) in the last decade. The most studied EAA are glutamate and aspartate. Both are synthesized by the same mechanism as gama-aminobutyric acid. (Fig. 1). Glutamate is widely distributed in the CNS and the spinal cord, being the areas of higher concentration the cerebral cortex, the hypocampus and the cerebellum. There have been identified two type of receptors for glutamate: ionotropic and metabotropic. The former includes three different types: NMDA, AMPA and KA. NMDA receptor is coupled to a Na+, and Ca2+, channel being the second ion the most important one. This receptor has several sites of binding for various substances. Along with the site for N-methyl-D-aspartate, which binds glutamate and/or aspartate, there have been identified a site for the binding of glycine (which is different from the strychnine sensitive one), a site for poliamines such as spermine and spermidine, and a site for the binding of Zn2+ (Table 1). AMPA receptor is associated to a Ca2+ -Na+, channel, being in this case the Na+ the most important ion. There are two metabotropic type receptors: L-AP4 and trans-ACPD. Both are coupled to a G protein and agonists exert their action increasing phospholipase C activity which in turn induces an increment of IP3 and diacyl-glicerol, and a consecutive releasing of Ca2+, from intracellular stores. EAA play a role in some physiological processes. One of them is long-term potentiation (LTP), an electrochemical phenomenon involved in memory consolidation. Antagonists of NMDA and AMPA receptor prevent the development of LTP, and conversely, the agonist of glycine site of NMDA receptor --D-cycloserine -- facilitates memory consolidation. Since 1957, EAA are considered neurotoxic substances and there are many indirect evidences to support this statement. Pathogenesis of neuronal damage elicited by EAA involves the events shown in Fig. 3. Prevention of the cascade of events that provokes neurotoxicity may be achieved by NMDA antagonists, but once it has begun it may be only aborted substracting the Ca2+ from the medium, using nifedipine or blocking AMPA receptor with an antagonist (CNQX). EAA have been shown to play a toxic role in neuronal damage induced by ischemia. Research using various experimental models demonstrated that NMDA receptor antagonists (i.e. MK 801) blocks postischemic damage. Interventions at various levels of the pathogenic cascade shown in Fig. 4 provoke the same results. There is enough evidence to suspect that NMDA and AMPA receptors are altered in epilepsy. NMDA antagonists (i.e. MK801 or AP5) prevent the development of epileptic seizures induced by kindling; CNQX, an AMPA antagonist, blocks the increase in electrical activity induced by K+, in slices of hypocampus; felbamate, an antiepileptic drug, blocks the glycine site (not strychnine sensitive) decreasing NMDA receptor activity. Several neurodegenerative disorders have been associated with exogenous administration or accidental intake of EAA. (i.e. neurolatirism, Guam disease). Similarities between these diseases and lateral amiotrophic sclerosis indicate that in the latter EAA may play a pathogenic role. Finally, the psychotomimetic effect of phencyclidine (an antagonist of NMDA receptor) suggests that in schizophrenia, together with dopaminergic neurotransmission impairment, some dysfunction of glutamate pathways may be present.

Methyl parathion induced alterations in GABAergic system during critical stage of central nervous system development in albino rat pups.

Sublethal doses of methyl parathion (o, o-dimethyl-o-nitrophenyl thiophosphate) injected intraperitoneally to 7th day old developing albino rat pups induced alterations in the inhibitory GABAergic system of CNS. A substantial simulation of the inhibitory system was noticed. A profound increase was found in the level of the inhibitory transmitter, GABA on methyl parathion injection. An increase in the activity levels of the enzymes glutamic acid decarboxylase and 4-aminobutyrate-2-oxoglutarate-amino transferase in the cortex, brain stem and spinal cord of the CNS was found. It is observed that methyl parathion causes potentiation of the inhibitory transmission (GABAergic system) in the wake of inducing suppression of cholinergic system in CNS of developing rat pups.

Effects of plant extract Centella asiatica (Linn.) on cold restraint stress ulcer in rats.

Extract of C. asiatica (Linn.) inhibited significantly gastric ulceration induced by cold and restraint stress (CRS) in Charles-Foster rats, Antiulcer activity of plant extract was compared with famotidine (H2-antagonist) and sodium valproate (anti-epileptic). Plant extract, formotidine and sodium valproate showed a dose dependent reduction of gastric ulceration. Plant extract increased brain GABA level which was also dose dependent. Pretreatment with bicuculline methiodide (specific GABAA-antagonist) at the dose level of 0.5 mg/kg im, reversed the antiulcerogenic activity of both plant extract and sodium valproate. Bicuculline as such did not induce gastric ulceration in normal rat.

Neuropharmacological studies on the venom of Vipera russelli.

Neuropharmacological studies have been conducted on the venom of V. russelli on experimental animals. The venom was found to produce alteration in general behaviour pattern, reduction in spontaneous motility, hypothermia, potentiation of pentobarbitone hypnosis, analgesia, reduction in exploratory behaviour pattern, muscle relaxant action, and suppression of aggressive behaviour. The venom caused a significant increase in brain GABA content in mice. The observations are suggestive of a potent CNS-depressant action of V. russelli venom.

Formation of Y-aminobutyric acid [GABA] from putrescine in the hypothalami of rats and its possible role as a neurotransmitter

Slices prepared from hypothalami removed from male Wistar-derived rats were shown to release 14alpa C alpha [14C GABA] formed from 14C putrescine. The tissue was incubated in Krebs-bicarbonale buffer containing 11 mM glucose. 14C GABA was separated from 14C putrescine by ion-exchange chromatography. In pulse-label experiments highest number of cpm associated with 14C GABA as well as with 14C putrescine were found immediately on removal the latter campound from the incubation medium, thereafter the cpm for both generally kept decreasing was stimulated with 48 M K+, the release of 14C GABA formed 14C putrescine was increased in the but not in the absence of the latter cation, suggesting the possibility that GABA derived front putrescine may have a role as a neurotransmitter. 7-hat the material released was truly 14C GABA was confirmed by performing high voltage paper electrophoresis at a pH of 3.4 and 6.5 with GABA, putrescine and N-monoacetylputrescine as markers