γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGAD^{S24R/D88R/Y309K} was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and Lpgad^{S24R/D88R/Y309K} genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD₆₀₀) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.
Glutamate Decarboxylase/genetics* ; Lactobacillus plantarum/genetics* ; Catalysis ; gamma-Aminobutyric Acid ; Hydrogen-Ion Concentration ; Glutamic Acid

OBJECTIVE: To construct and identify 2 secreted human GAD65 fragment DNA vaccines. METHODS: The GAD(190-315), GAD(490-570) cDNA and hIL-2 signal peptide cDNA were linked through overlapping PCR, respectively. The fusion gene was cloned into eukaryotic expression vector pBudCE4.1. After the DNA vaccine being determined to contain the correct target nucleotide sequence, the expression of fusion proteins was detected by Western blot. RESULTS: The nucleotide sequence of the cloned gene was the same as the reported sequence, and their open reading fragment was correct. The products of these DNA vaccines were expressed and secreted in eukaryotic cell using Western blot. CONCLUSION The pBudCE4.1/SGAD(190-315) and pBudCE4.1/SGAD(490-570) secreted human GAD65 fragment DNA vaccines were successfully constructed, which is a foundation for immune prevention of type 1 diabetes.
Base Sequence ; Cloning, Molecular ; DNA, Complementary ; Genetic Vectors ; Glutamate Decarboxylase ; classification ; genetics ; Humans ; Open Reading Frames ; Peptide Fragments ; genetics ; Recombinant Fusion Proteins ; genetics ; Vaccines, DNA ; biosynthesis ; genetics

In order to enhance gamma-aminobutyric acid production from L-glutamate efficiently, we amplified the key enzyme glutamate decarboxylase (GAD) encoding gene lpgad from the strain Lactobacillus plantarum GB 01-21 which was obtained by way of multi-mutagenesis and overexpressed it in E. coli BL21. Then we purified GAD by Ni-NTA affinity chromatography and characterized the enzyme to optimize the conditions of the whole-cell transformation. The results showed that the recombinant E. coli BL21 (pET-28a-lpgad) produced 8.53 U/mg GAD, which was increased by 3.24 fold compared with the GAD activity in L. plantarum. The optimum pH and temperature of the enzyme were pH 4.8 and 37 degrees C, respectively. At the same time, we found that Ca2+ and Mg2+ could increase the activity significantly. Based on this, we investigated gamma-aminobutyric acid transformation in 5 L fermentor under the optimum transformation conditions. Accordingly, the yield of gamma-aminobutyric acid was 204.5 g/L at 24 h when the 600 g L-glutamate was added and the mole conversion rate had reached 97.92%. The production of gamma-aminobutyric acid was improved by 42.5% compared with that under the unoptimized transformation conditions. This paved a way for the gamma-aminobutyric acid construction of the industrial applications.
Cloning, Molecular ; Escherichia coli ; enzymology ; genetics ; metabolism ; Glutamate Decarboxylase ; biosynthesis ; genetics ; Glutamic Acid ; metabolism ; Lactobacillus plantarum ; enzymology ; genetics ; Recombination, Genetic ; gamma-Aminobutyric Acid ; biosynthesis

BACKGROUNDThe treatment of spinal cord injury is still a challenge. This study aimed at evaluating the therapeutical effectiveness of neurons derived form mesenchymal stem cells (MSCs) for spinal cord injury.

METHODSIn this study, rhesus MSCs were isolated and induced by cryptotanshinone in vitro and then a process of RT-PCR was used to detect the expression of glutamic acid decarboxylase (GAD) gene. The induced MSCs were tagged with Hoechst 33342 and injected into the injury site of rhesus spinal cord made by the modified Allen method. Following that, behavior analysis was made after 1 week, 1 month, 2 months and 3 months. After 3 months, true blue chloride retrograde tracing study was also used to evaluate the re-establishment of axons pathway and the hematoxylin-eosin (HE) staining and immunohistochemistry were performed after the animals had been killed.

RESULTSIn this study, the expression of mRNA of GAD gene could be found in the induced MSCs but not in primitive MSCs and immunohistochemistry could also confirm that rhesus MSCs could be induced and differentiated into neurons. Behavior analysis showed that the experimental animals restored the function of spinal cord up to grade 2-3 of Tarlov classification. Retrograde tracing study showed that true blue chollide could be found in the rostral thoracic spinal cords, red nucleus and sensory-motor cortex.

CONCLUSIONSThese results suggest that the transplantation is safe and effective.

Animals ; Bone Marrow Cells ; cytology ; Glutamate Decarboxylase ; genetics ; Isoenzymes ; genetics ; Macaca mulatta ; Male ; Mesenchymal Stromal Cells ; cytology ; Neurons ; transplantation ; RNA, Messenger ; analysis ; Spinal Cord Injuries ; pathology ; therapy

As an important material in pharmaceutical and chemical industry, β-alanine was mainly produced by chemical methods. L-aspartate-α-decarboxylase could catalyze the α-decarboxylation from L-aspartate to β-alanine. Determinations for specific activities of PanDs from Escherichia coli, Corynebacterium glutamicum and Bacillus subtilis were performed in this study (0.98 U/mg, 7.52 U/mg and 8.4 U/mg respectively). The optimal temperature and pH of PanDs from C. glutamicum and B. subtilis were 65 degrees C, pH 6.5 and 60 degrees C, pH 6.5 respectively. According to our research, PanD from B. subtilis could be more appropriate for industrial application because of the higher activity and thermostability when compared to PanDs from E. coli and C. glutamicum which had been the most studied. We also analyzed and discussed the special post-translation self-cleavage phenomenon and the mechanism based inactivation.
Bacillus subtilis ; enzymology ; Bacterial Proteins ; genetics ; metabolism ; Corynebacterium glutamicum ; enzymology ; Escherichia coli ; enzymology ; Glutamate Decarboxylase ; genetics ; metabolism ; Industrial Microbiology ; Temperature ; beta-Alanine

OBJECTIVETo investigate the effects of antisense glutamic acid decarboxylase (GAD(67)) oligodeoxynucleo-tide (ODN) on behavior, seizure threshold and EEG of hippocampus in the epileptic rats induced by pentylenetetrazol (PTZ).

METHODSA model of chronic epilepsy in rats was established by PTZ. The inhibition of GAD(67) mRNA expression in hippocampus was selectively induced by antisense oligodeoxynucleotide of GAD(67). The effect of antisense GAD(67) ODN on behavior, seizure threshold and EEG recording of kindled rats was examined.

RESULTSAntisense GAD(67) ODN could inhibit the expression of GAD(67) mRNA and the concentration of GABA. It also could significantly shorten the latencies of seizure and increase the level of seizure and the frequency of epileptiform discharges.

CONCLUSIONThe gene of GAD(67) may be an anti-seizure gene, which might inhibit epileptiform discharge. The treatment of epilepsy by GAD(67) gene will have a bright future.

Animals ; Electroencephalography ; Epilepsy ; chemically induced ; physiopathology ; Glutamate Decarboxylase ; genetics ; pharmacology ; Hippocampus ; physiopathology ; Isoenzymes ; genetics ; pharmacology ; Kindling, Neurologic ; Male ; Oligonucleotides, Antisense ; pharmacology ; Pentylenetetrazole ; Rats ; gamma-Aminobutyric Acid ; analysis

Vectors used to carry foreign genes play an important role in gene therapy, among which, the adeno-associated virus (AAV) has many advantages, such as nonpathogenicity, low immunogenicity, stable and long-term expression and multiple-tissue-type infection, etc. These advantages have made AAV one of the most potential vectors in gene therapy, and widely used in many clinical researches, for example, Parkinson's disease. This paper introduces the biological characteristics of AAV and the latest research progress of AAV carrying neurotrophic factor, dopamine synthesis related enzymes and glutamic acid decarboxylase gene in the gene therapy of Parkinson's disease.
Animals ; Aromatic-L-Amino-Acid Decarboxylases ; genetics ; Dependovirus ; genetics ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; Glutamate Decarboxylase ; genetics ; Humans ; Nerve Growth Factors ; genetics ; Neurturin ; genetics ; Parkinson Disease ; therapy

BACKGROUND: Type 1 diabetes mellitus is frequently associated with other autoimmune diseases. The occurrence of common features of autoimmune diseases and the coassociation of multiple autoimmune diseases in the same individual or family supports the notion that there may be common genetic factors. METHODS: To investigate potential clustering of autoimmune thyroid disease (ATD) among type 1 diabetes patients and the contribution of common susceptibility genes to this, HLA DR/DQ alleles as well as antithyroid autoantibodies were measured in 115 Korean patients with type 1 diabetes and their 96 first-degree family members. RESULTS: Twenty-five percent of the patients had ATD, whereas 3 of 36 (8%) age-matched normal controls had ATD (RR = 3.7, p < 0.05). Twenty-six of ninty-six (27%) type 1 diabetes family members had ATD. No differences in the distribution of HLA alleles/haplotypes and genotypes between the patients with and without ATD were found. CONCLUSION: From this finding, we could assess that individuals with type 1 diabetes and their relatives frequently develop ATD, perhaps due to common susceptibility genes that are shared among first degree relatives.
Adult ; Alleles ; Autoantibodies/blood ; Autoimmune Diseases/epidemiology* ; Child ; Child, Preschool ; Diabetes Mellitus, Insulin-Dependent/genetics ; Diabetes Mellitus, Insulin-Dependent/complications* ; Female ; Glutamate Decarboxylase/immunology ; HLA-DQ Antigens/genetics ; HLA-DR Antigens/genetics ; Human ; Male ; Prevalence ; Thyroid Diseases/epidemiology*

OBJECTIVETo investigate the expressions of gamma aminobutyric acid transporter 1 (GAT-1) and glutamate decarboxylase 65 (GAD65) mRNA in different brain regions at brain propofol uptake equilibrium in dogs.

METHODSEighteen 12- to 18-month-old healthy hybrid dogs were randomized equally into control group (group C), low dose group (group L), and high dose group (group H). In groups L and H, anesthesia was administered by intravenous injection of 5.5 and 7.0 mg/kg propofol followed by propofol infusion at a constant rate of 55 and 70 mg·kg(-1)·h(-1) for 50 min, respectively. Blood samples were taken from the internal carotid artery and jugular vein to measure plasma propofol concentrations, and the brain tissues of the hypothalamus, sub thalamus, dorsal thalamus, hippocampus, pons, parietal lobe and frontal lobe were examined for GAT-1 and GAD65 mRNA expressions using quantitative real-time PCR.

RESULTSIn groups L and H, propofol infusion at a constant rate for 50 min resulted in comparable plasma propofol concentrations between the internal carotid artery and jugular vein (P>0.05), but the concentrations differed significantly between the two groups (P<0.01). GAT-1 mRNA levels in the hypothalamus and hippocampus were significantly higher in groups L and H than in group C (P<0.05 and P<0.01), but comparable between the former two groups. The variations of GAT-1 mRNA levels between the hypothalamus and hippocampus were similar in both group L [(61.26∓7.17)% and (79.34∓39.95)%, P>0.05] and group H [(74.64∓19.63)% and (97.12∓32.31)%, P>0.05]. GAT-1 mRNA levels in other brain regions showed no significant difference among the 3 groups. GAD65 mRNA levels were similar between group L and group H, but both significantly higher than that in group C (P<0.01). GAD65 mRNA in other brain regions had no significant difference among the 3 groups.

CONCLUSIONGAT-1 mRNA in the hypothalamus and hippocampus and GAD65 mRNA in the dorsal thalamus are upregulated when propofol uptake reaches an equilibrium in the brain of dogs.

Animals ; Brain ; drug effects ; metabolism ; Dogs ; GABA Plasma Membrane Transport Proteins ; genetics ; metabolism ; Glutamate Decarboxylase ; genetics ; metabolism ; Hippocampus ; drug effects ; metabolism ; Hypothalamus ; drug effects ; metabolism ; Propofol ; pharmacology ; RNA, Messenger ; genetics ; Thalamus ; drug effects ; metabolism

OBJECTIVETo investigate the distribution of GAD67 and the co-localization with bNOS in the main olfactory bulb of GAD67-GFP knock-in mouse.

METHODSPolymerase chain reaction was applied to identify the genotype of GAD67-GFP knock-in mouse, the animals were sacrificed and frozen sections of olfactory bulb were prepared. The Nissl-staining was performed to show an framework of the neuron in the olfactory bulb. The distribution of GAD67 and co-localization with bNOS were detected by immunofluorescence technique.

RESULTSThe proportion of GAD67-positive cells among DAPI-positive cells were (42.98 ± 0.92)% in glomerular layer, (23.64 ± 0.84)% in mitral cell layer and (77.75 ± 0.84)% in granule cell layer; the bNOS-positive cells mainly existed in glomerular layer and mitral cell layer, very few in granule cell layer. No co-localization of GAD67 and bNOS in granule cell layer and mitral cell layer was found, but there was dispersed distribution in glomerular layer.

CONCLUSIONGAD67-positive neurons mainly appear in glomerular layer and granule cell layer, and the bNOS is mostly expressed in glomerular layer and mitral cell layer; while the co-localization of GAD67 and bNOS only occurs in glomerular layer of olfactory bulb.

Animals ; Gene Knock-In Techniques ; Glutamate Decarboxylase ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Mice, Transgenic ; Neurons ; metabolism ; Nitric Oxide Synthase Type I ; metabolism ; Olfactory Bulb ; metabolism ; Tissue Distribution