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L-Glutamate (L-Glu), the major excitatory neurotransmitter in the mammalian Central Nervous System (CNS), is essential to cognitive functions. However, when L-Glu is accumulated in large concentrations at the synaptic cleft, it can induce excitotoxicity that results in secondary damage implicated in many neurological disorders. Current therapies for the treatment of neurological disorders are ineffective and have side effects associated with their use; therefore, there is a need to develop novel treatments. In this regard, previous studies have shown that neuroactive compounds obtained from the venom of the spider Parawixia bistriata have neuroprotective effects in vitro and in vivo. In this sense, this work aimed to evaluate potential neuroprotective effects of fraction RT10, obtained from this spider venom, on primary cultures of neuron and glial cells subjected to glutamate excitotoxicity insults. Methods: Primary cultures of neurons and glia were obtained from the cerebral tissue of 1-day-old postnatal Wistar rats. After 7 days in vitro (DIV), the cultures were incubated with fraction RT10 (0.002; 0.02; 0.2 and 2 µg/µL) or riluzole (100 µM) for 3-hours before application of 5 mM L-Glu. After 12 hours, the resazurin sodium salt (RSS) test was applied to measure metabolic activity and proliferation of living cells, whereas immunocytochemistry for MAP2 was performed to measure neuronal survival. In addition, the cells were immunolabeled with NeuN and GFAP in baseline conditions. Results: In the RSS tests, we observed that pre-incubation with RT10 before the excitotoxic insults from L-Glu resulted in neuroprotection, shown by a 10% reduction in the cell death level. RT10 was more effective than riluzole, which resulted in a cell-death reduction of 5%. Moreover, qualitative analysis of neuronal morphology (by MAP2 staining, expressed as fluorescence intensity (FI), an indirect measure of neuronal survival) indicate that RT10 reduced the toxic effects of L-Glu, as shown by a 38 % increase in MAP2 fluorescence when compared to L-Glu insult. On the other hand, the riluzole treatment resulted in 17% increase of MAP2 fluorescence; therefore, the neuroprotection from RT10 was more efficacious. Conclusion: RT10 fraction exhibits neuroprotective effects against L-Glu excitotoxicity in neuron-glia cultured in vitro.(AU)
Subject(s)
Spider Venoms , Neuroprotection , Neurotransmitter Agents , Excitatory Amino Acid Agonists , Evaluation Studies as TopicABSTRACT
Objective To investigate the protective effect of brain derived neurotrophic factor (BDNF) on Müller cells in the retina of diabetic rats.Methods A total of 54 healthy male SD rats were recruited and randomly divided into control group,diabetic group and BDNF group.Then a diabetic model was established by intraperitoneal injection of streptozotocin in rats of diabetic and BDNF groups.Preparation of BDNF injection was performed using PBS balanced salt solution containing 0.1 g · L 1 BSA.The BDNF group was given BDNF injection,while the control and diabetic group were injected with equal dose of PBS balanced salt solution 4 weeks after successful modeling.And after 8 weeks,the expression of L-glutamate/L-aspartate transporter (GLAST),glutamine synthetase (GS) and synaptophysin (SYN) were detected by immunofluorescence technique and Western blot,and the content of glutamic acid in retina was determined by glutamic acid determination kit.Results Compared with the control group,the expression of GLAST,GS and SYN were significantly decreased in diabetic group,and the content of glutamic acid was increased significantly (all P ＜ 0.01).Compared with the diabetic group,the expression of GLAST,GS and SYN were increased in BDNF group,and the glutamate level was decreased significantly (all P ＜0.01).Conclusion In the early stage of diabetic retinopathy,administration of exogenous BDNF can unregulated the expression of GLAST,SYN and GS and improve the function of Müller cells to protect RGC against damage,suggesting that BDNF has neuroprotective effects on Müller cells in retina of rats with diabetic retinopathy.
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Objective To observe the effects of A2a adenosine receptor antagonist SCH442416 and ZM241385 on the expression of glutamine synthetase(GS) and L-Glutamate/L-Aspartate Transporter(GLAST) in rat retina under chronic ocular hypertension model.Methods Rat chronic ocular hypertension models were induced in the right eye of 12 male Sprague Dawley rats by blocking three episcleral veins,the left eye as control one.Intraocular pressure (IOP) was measured and compared at postoperative 1 week,2 weeks and 3 weeks.54 male chronic ocular hypertension rats were divided into 3 groups randomly,topically applying A2a adenosine receptor antagonist SCH442416,ZM241385 and carrier,respectively,three times a day for three weeks.At three weeks,mRNA and protein expression of GS and GLAST in rat retina were analyzed by RealTime-PCR and Western-blot.Results The average IOP of the modeling eyes at postoperative 1 week,2 weeks and 3 weeks were higher than that of the control eyes (all P ＜ 0.05).The mRNA and protein expression of GS and GLAST in the retina of SCH442416 and ZM241385 groups increased significantly compared to the carrier group (all P ＜ 0.05).However,the differences of mRNA and protein expression of GS and GLAST between SCH442416 and ZM241385 groups was not significant(all P ＞ 0.05).Conclusion Rat chronic ocular hypertension model can be induced by blocking three episceral veins successfully and effectively.A2a adenosine receptor antagonist SCH442416 and ZM241385 increase the expression of GS and GLAST.There seems no difference between the effects of these two drugs.
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Objective:The aim is to establish L-glutamate specific aminotransferase-L-glutamate dehydrogenase coupling 96-well high throughput screening method,which is applied to molecular evolution of aminotransferase WecE from E.coli.Methods:An optical assay for aminotransferase catalytic activity based on aminotransferase-glutamate dehydrogenase coupling system is established by optimization of coupling enzyme loading,signal molecule NADH concentration and coupling time.Mutants library of WecE is obtained by sitedirected saturation mutagenesis.Positive mutants can be screened out through 96-well preliminary screening and flask second screening.Results:The target transamination reaction is coupled with L-glutamate dehydrogenase indicative reaction system which consists of 0.5 U/ml enzyme loading and 0.4 mmol/L NADH.A positive mutant Y321F whose catalytic activity increases 3.4 fold compared to that of wild type is screened out in Tyr 321 saturation mutagenesis library of WecE.Conclusion:An accurate high throughput screening method with weak background interference is established.It offers feasible solution for molecular evolution of L-glutamate specific aminotransferase.
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Immobilization of enzymes is important and widely applied in biocatalysis. Streptomyces platensis gene gox, encoding an extracellular L-glutamate oxidase (Gox), was fused to cellulose binding domain (CBDcex) from Cellulomonas fimi and the recombinant protein Gox-CBD was expressed in Escherichia coli. The fusion protein (Gox-CBD) was immobilized onto microcrystalline cellulose. The preparation conditions, binding capacity, properties and stability of the immobilized enzyme were studied. Under the condition of 4 ℃, for 1 hour, the fusion protein Gox-CBD was able to bind microcrystalline cellulose at a ratio of 9.0 mg of protein per gram of microcrystalline cellulose. Enzymatic properties of free and immobilized L-glutamic oxidase (Gox-CBD) were compared. The specific activity of the immobilized enzyme decreased, but its thermal stability increased a lot compared with that of the free Gox-CBD. After incubation at 60 ℃ for 30 min, 70% of the total activity remained whereas the free recombinant Gox completely lost its activity. The immobilized protein was tightly bound to microcrystalline cellulose at pH below 10 or more than 5 mmol/L NaCl. The fusion protein of Gox-CBD can be specifically immobilized on the microcrystalline cellulose on a single step. Therefore, our findings can provide a novel strategy for protein purification and enzyme immobilization.
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Aims: Even though glutamate is one of the primary endogenous amino acids of the Central Nervous System (CNS) its subunit receptors exist also in non-neuronal tissues outside CNS such as the hematopoietic system. The purpose of this paper is to define the possible in vivo effect of glutamate ionotropic agonist Monosodium l-glutamate (MSG) in the hematopoietic system of Wistar adult rats. Methodology: MSG was administrated intravenously in male Wistar rats of 250-350g weight. Animals treated with MSG (n = 24) were compared to a control group (n = 10). Full blood count with differential, aggregation intensity and bone marrow cellularity were evaluated 12 and 24 hours after drug administration. The results were analyzed by unpaired t-test. Results: MSG showed to affect white blood cell count in a negative way whereas it provoked an increase in Hemoglobin (Hb) and Hematocrit (Hct) levels. Aggregation was only transiently affected using ADP as an agonist and bone marrow counts showed a trend towards normalization. Conclusion: It is concluded that MSG can affect the hematological profile and bone marrow cellular composition of healthy intact rats as well as blood elements functions. Our results suggest a possible role for glutamate receptors on the hematopoietic system’s pathophysiology. Further research is needed in order to better characterize the in vivo effect of glutamate receptors agonists and antagonists on blood elements and bone marrow.
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<p><b>OBJECTIVE</b>In Corynebacterium crenatum, the adjacent D311 and D312 of N-acetyl-L-glutamate kinase (NAGK), as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine, were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for NAGK.</p><p><b>METHODS</b>NAGK enzyme expression was evaluated using a plasmid-based method. Homologous recombination was employed to eliminate the proB.</p><p><b>RESULTS</b>The IC50 and enzyme activity of NAGK M4, in which the D311R and D312R amino acid substitutions were combined with the previously reported E19R and H26E substitutions, were 3.7-fold and 14.6% higher, respectively, than those of the wild-type NAGK. NAGK M4 was successfully introduced into the C. crenatum MT genome without any genetic markers; the L-arginine yield of C. crenatum MT-M4 was 26.2% higher than that of C. crenatum MT. To further improve upon the L-arginine yield, we constructed the mutant C. crenatum MT-M4 proB. The optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine yield. After L-proline was added to the medium at 10 mmol/L, the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation, approximately 70.1% higher than the yield attained using C. crenatum MT.</p><p><b>CONCLUSION</b>Feedback inhibition of L-arginine on NAGK in C. crenatum is clearly alleviated by the M4 mutation of NAGK, and deletion of the proB in C. crenatum from MT to M4 results in a significant increase in arginine production.</p>
Subject(s)
Animals , Arginine , Corynebacterium , Genetics , Metabolism , Escherichia coli , Feedback, Physiological , Gene Deletion , Mutagenesis, Site-Directed , Phosphotransferases (Carboxyl Group Acceptor) , Genetics , Proline , MetabolismABSTRACT
L-lactate is one of main byproducts excreted in to the fermentation medium. To improve L-glutamate production and reduce L-lactate accumulation, L-lactate dehydrogenase-encoding gene ldhA was knocked out from L-glutamate producing strain Corynebacterium glutamicum GDK-9, designated GDK-9ΔldhA. GDK-9ΔldhA produced approximately 10.1% more L-glutamate than the GDK-9, and yielded lower levels of such by-products as α-ketoglutarate, L-lactate and L-alanine. Since dissolved oxygen (DO) is one of main factors affecting L-lactate formation during L-glutamate fermentation, we investigated the effect of ldhA deletion from GDK-9 under different DO conditions. Under both oxygen-deficient and high oxygen conditions, L-glutamate production by GDK-9ΔldhA was not higher than that of the GDK-9. However, under micro-aerobic conditions, GDK-9ΔldhA exhibited 11.61% higher L-glutamate and 58.50% lower L-alanine production than GDK-9. Taken together, it is demonstrated that deletion of ldhA can enhance L-glutamate production and lower the unwanted by-products concentration, especially under micro-aerobic conditions.
Subject(s)
Corynebacterium glutamicum/enzymology , Corynebacterium glutamicum/metabolism , Gene Deletion , Glutamic Acid/metabolism , L-Lactate Dehydrogenase/genetics , Lactic Acid/metabolism , Metabolic Engineering , Corynebacterium glutamicum/genetics , Oxygen/metabolism , Sequence DeletionABSTRACT
Objective To prepare two types of biodegradable modified materials (chitosan and collagen)and evaluate whether the new materials are suitable for tissue engineering cartilage.Methods Collagen and chitosan were both modified by poly-γ-benzyl-L-glutamate-co-glutamine acid (PBLG-co-PGA) with different proportions.The contact angle,degradation rate,tensile strength,cell attachment and cytocompatibility were tested and compared.Results As the PBLG-co-PGA content varied,the degradation rates of PBLG-co-PGA composites became adjustable,the hydrophilicity of PBLG-co-PGA/chitosan was improved,and the tensile strength increased in PBLG-co-PGA/collagen composite.The composites with 30％ PBLG-co-PGA were chosen for cytocompatibility and cell attachment experiments.The rabbit condrocytes grew significantly better on PBLG-co-PGA/chitosan than on other three materials(P＜0.05).Conclusion PBLG can improve the hydrophilicity,tensile strength and regulate the degradation rate of composite materials,and the cytocompatibility of the composites with 30％ of PBLG is good,among which PBLG-co-PGA/chitosan can even promote cell proliferation.It could be a new choice of scaffold for tissue engineering cartilage.
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Post-weaning protein malnutrition is often related to the development of cardiovascular and metabolic diseases in humans, as well to changed content of neurotransmitters in the central nervous system under experimental conditions. The rostral ventrolateral medulla (RVLM) is a bulbar region that contains sympathetic premotor neurons; the excitatory amino acid L-glutamate seems to be the main neurotransmitter at this level. The aim of the present study was to evaluate the possible change in the L-glutamate sensitivity of the RVLM neurons of malnourished animals. Male Fischer rats were divided into two groups: control (n = 15) and malnourished (n = 19). Four days before the experiments, guide cannulas were implanted bilaterally in direction of the RVLM for microinjection of L-glutamate. Twenty-four hours before the experiments, the femoral artery was cannulated for cardiovascular recordings. The results showed that the baseline heart rate increased in malnourished compared to control animals (412.18 ± 16.03 bpm vs. 370.74 ± 9.59 bpm, respectively). Malnourished animals presented a dissimilar concentration-dependent pressor response curve to L-glutamate and an attenuated baroreflex gain. Our results suggest that post-weaning protein restriction affects glutamatergic neurotransmission of the baroreflex at the RVLM level.
Subject(s)
Animals , Male , Rats , Excitatory Amino Acid Antagonists/pharmacology , Glutamic Acid/pharmacology , Malnutrition/physiopathology , Medulla Oblongata/drug effects , Baroreflex/drug effects , Baroreflex/physiology , Blood Pressure/drug effects , Blood Pressure/physiology , Consciousness , Glutamic Acid/administration & dosage , Heart Rate/drug effects , Heart Rate/physiology , Microinjections , Malnutrition/complications , Medulla Oblongata/physiologyABSTRACT
Extracellular single-unit discharge recording technique was used to examine the effects of Ginkgolide B (BN52021) on the discharges of neurons in CAI area of hippocampal slices and to elucidate the mechanisms involved.The results showed that:(1) In response to the application of ginkgolide B (0.1,1,10 βμmol/L; n =43) into the perfusate for 2 rain,the spontaneous discharge rates (SDR) of 42/43 (97.67%) neurons were significantly decreased in a dose-dependent manner; (2) Pretreatment with L-glutamate (L-Glu,0.2mmol/L) led to a marked increase in the SDR of all 10 (100%) neurons in an epileptiform pattern.The increased discharges were suppressed significantly after ginkgolide B (1 μmol/L) was applied into the perfusate for 2 rain; (3) In 8 neurons,perfusion of the selective L-type calcium channel agonist,Bay K 8644 (0.1 μmol/L),induced a significant increase in the discharge rate of 8/8 (100%) neurons.Ginkgolide B (1 μmoL/L) applied into the perfusate inhibited the discharges of 7/8 (87.5%) slices; (4) In 8 neurons,the broad potassium channels blocker,tetraethylammonium (TEA,1 mmol/L) completely blocked the inhibitory effect of ginkgolide B (1 μmol/L).These results suggest that ginkgolide B can inhibit the electrical activity of CAI neurons.The inhibitory effect may be related to the blockade of L-type voltage-activated calcium channel and may be concerned with delayed rectifier potassium channel (KDR),which indicated that ginkgolide B play a protective role on the central neurons.
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OBJECTIVE: Astrocytes secrete various neurotrophic factors which act to support the survival and growth of neurons. Reactive astrocytes express an increased level of neurotrophic factors in response to central nervous system injury. We demonstrate that reactive astrocytes could express neurotrophic factors to promote neuronal rescue and generate functional recovery. METHODS: To investigate the correlation of neurotrophic factor, brain-derived neurotrophic factor(BDNF) and neurotrophin-3(NT-3) to glutamate-induced reactive gliosis, mRNA expression of BDNF and NT-3 were detected by the RT-PCR technique. RESULTS: Exposure of cultured astrocytes to L-glutamate(1, 100, 200 and 500 microM) and scraped astrocytes for 1 day resulted in significant cell damage and we observed mRNA expression of BDNF and NT-3. The maximal expression of BDNF was observed in the control, scraped and L-glutamate treated astrocytes(1 microM). The basal expression of BDNF mRNA in astrocytes treated with L-glutamate(100, 200 and 500 microM) decreased relative to that of control, scraped and L-glutamate treated astrocytes(1 microM). Reactive gliosis, treatment of control astrocytes with glutamate, showed similar pattern for NT-3 mRNA expression. In a word, the basal content of NT-3 mRNA in scrape and L-glutamate(1, 100, 200 and 500 microM) expressed similar to that of control astrocytes. CONCLUSION: This study indicates that the reactive astrocytes also expressed mRNA of BDNF and NT-3 as normal astrocytes.
Subject(s)
Astrocytes , Brain-Derived Neurotrophic Factor , Central Nervous System , Gliosis , Glutamic Acid , Nerve Growth Factors , Neurons , RNA, MessengerABSTRACT
The purpose of the present study is to determine the role of muscarinic cholinergic receptors of posterior hypothalamus in the central blood pressure regulation when respiration is controlled. In anesthetized and artificially ventilated rats, vasodepressor response was evoked by injection of L-glutamate (10 nmol) neuroexcitatory amino acid into the posterior hypothalamic area. The injection of carbachol (0.5 ~ 8 nmol) into the same area induced dose-dependent vasodepressor and bradycardic responses. Pretreatment with atropine (4 nmol) completely blocked the vasodepressor response to carbachol (2 nmol). In contrast, in spontaneously breathing rats, the injection of carbachol (8 nmol) into the posterior hypothalamic area induced the vasopressor and tachycardic responses. These results suyggest that the muscarinic cholinergic receptors in the posterior hypothalamic area primarily play an inhibitory role in the central regulation of blood pressure and heart rate.
Subject(s)
Animals , Rats , Atropine , Blood Pressure , Carbachol , Glutamic Acid , Heart Rate , Heart , Hypothalamus, Posterior , Receptors, Cholinergic , Receptors, Muscarinic , RespirationABSTRACT
OBJECTIVE:To study the inhibitory effect of melatonin(MT)on the increased influx of Ca 2+ induced by L-glutamate in cultured rat neurons.METHODS:Fura-2/AM,a Ca 2+ sensitive fluorescent indicator was used to load the brain cells isolated from new-born rats,and the intracellular dissociative Ca 2+ concentration(〔Ca 2+ 〕i)was measured with fluores?cence-spectrophotometer.RESULTS:The L-glutamate of concentration500?mol/L could markedly promote the influx of Ca 2+ and intracellular free Ca 2+ concentration(P