Purification and Characterization of Glutamate Decarboxylase of Lactobacillus brevis CGMCC 1306 Isolated from Fresh Milk

A Lactobacillus brevis CGMCC 1306 isolated from fresh milk without pasteurization was found to have higher glutamate decarboxylase （GAD） activity. An effective isolation and purification procedure of GAD from a cell-free extract of Lactobacillus brevis was developed, and the procedure included four steps： 30%-90% saturation （NH4）2SO4 fractional precipitation, Q sepharose FF anion-exchange chromatography, sephacryl S-200 gel filtration, and resource Q anion-exchange chromatography. Using this protocol, the purified GAD was demonstrated to possess electrophoretic homogeneity via SDS-PAGE. The purification fold and activity recovery of GAD were 43.78 and 16.95%, respectively. The molecular weight of the purified GAD was estimated to be approximately 62 kDa via SDS-PAGE. The optimum pH and temperature of the purified GAD were 4.4 and 37℃, respectively. The purified GAD had a half-life of 50rain at 45℃ and the Km value of the enzyme from Lineweaver-Burk plot was found to be 8.22.5＇-pyridoxal phosphate （PLP） had little effect on the regulation of its activity.

Activation of dopamine 2 receptors modulates glutamate decarboxylases 65 and 67 during stroke recovery in mice

Backgrounds:Treatment with levodopa enhances recovery of lost neurological functions in preclinical stroke models and patients.Here,we studied whether dopamine signaling modulates GABAergic neurotransmission in parvalbumin-positive interneurons after experimental stroke.Methods:Following block randomization,mice were subjected to experimental stroke induced by photothrombosis(PT).Two days after the insult,mice were treated either with the D1 receptor antagonist by R(+)-SCH-23390(0.1 mg/kg),the selective D1 receptor agonist(R)-(+)-SKF-38393 hydrochloride(1 mg/kg),the D2 receptor agonist R(−)-2,10,11-trihydroxy-Npropyl-noraporphine hydrobromide hydrate(TNPA)(1 mg/kg),the D2 receptor antagonist S-(−)-eticlopride hydrochloride(0.3 mg/kg),or vehicle(saline)by daily intraperitoneal injection for five consecutive days,respectively.Recovery of function was assessed by paw placement and foot fault test before and on Days 2 and 7 after surgery.Results:Mice treated with TNPA showed a statistically significant improvement of recovery compared to all other treatment conditions.Synthesis of gamma-aminobutyric acid(GABA)was quantified by levels of full-length and cleaved glutamate acid decarboxylase 67 and 65(GAD65 and GAD67)in the peri-infarct area and homotypic regions of the contralateral cortex.Compared to the other treatments,TNPA significantly reduced the level of the GAD67 isoform both in the ischemic and contralateral hemispheres.Levels of GAD65 were found significantly higher in the contralateral hemisphere in TNPA-treated mice after PT accompanied by an increase in the 58 kDa-truncated form.Conclusion:Our results point toward reduced GABA synthesis in a D2 receptor-mediated mechanism possibly contributing to counteract functional inhibition after stroke.

High-level production of𝛾γ-aminobutyric acid via efficient co-expression of the key genes of glutamate decarboxylase system in Escherichia coli

Biosynthesis of the functional factor𝛾γ-aminobutyric acid(GABA)in bacteria involves two key proteins an intra-cellular glutamate decarboxylase(GadB)and a membrane-bound antiporter(GadC).Efficient co-expression of suitable GadB and GadC candidates is crucial for improving GABA productivity.In this study,gadBΔC11 of Lacti-plantibacillus plantarum and gadCΔC41 of Escherichia coli were inserted into the designed double promoter(P T7lac and P BAD)expression system.Then,E.coli Lemo21(DE3)was chosen as the host to minimize the toxic effects of GadCΔC41 overexpression.Furthermore,a green and high-efficiency GABA synthesis system using dormant engineered Lemo21(DE3)cells as biocatalysts was developed.The total GABA yield reached 829.08 g/L with a 98.7%conversion ratio within 13 h,when engineered E.coli Lemo21(DE3)cells were concentrated to an OD 600 of 20 and reused for three cycles in a 3 M L-glutamate solution at 37℃,which represented the highest GABA productivity ever reported.Overall,expanding the active pH ranges of GadB and GadC toward physiological pH and employing a tunable expression host for membrane-bound GadC production is a promising strategy for high-level GABA biosynthesis in E.coli.

Developmental changes of glutamate acid decarboxylase 67 in mouse brain after hypoxia ischemia

Objective To study the developmental changes of glutamic acid decarboxylase-67 （ GAD-67, a GABA synthetic enzyme） in normal and hypoxic ischemic （HI） brain. Methods C57/BL6 mice on postnatal day （P） 5, 9, 21 and 60, corresponding developmentally to premature, term, juvenile and adult human brain were investigated by using both Western blot and immunohistochemistry methods either in normal condition or after hypoxic ischemic insult. Results The immunoreactivity of GAD67 was up regulated with brain development and significant difference was seen between mature （P21, P60） and immature （P5, P9） brain. GAD67 immunoreactivity decreased in the ipsilateral hemisphere in all the ages after hypoxia ischemia （HI） insult, but, significant decrease was only seen in the immature brain. Double labeling of GAD67 and cell death marker, TUNEL, in the cortex at 8h post-HI in the P9 mice showed that （15.6±7.0）% TUNEL positive cells were GAD67 positive which was higher than that of P60 mice. Conclusion These data suggest that GABAergic neurons in immature brain were more vulnerable to HI insult than that of mature brain.

On-line near-infrared spectroscopy optimizing and monitoring biotransformation process of γ-aminobutyric acid

Near-infrared spectroscopy （NIRS） with its fast and nondestructive advantages can be qualified for the real-time quantitative analysis. This paper demonstrates that NIRS combined with partial least squares （PLS） regression can be used as a rapid analytical method to simultaneously quantify L-glutamic acid （L- GIu） and γ-aminobutyric acid （GABA） in a biotransformation process and to guide the optimization of production conditions when the merits of NIRS are combined with response surface methodology. The high performance liquid chromatography （HPLC） reference analysis was performed by the o-phthaldialdehyde pre-column derivatization. NIRS measurements of two batches of 141 samples were firstly analyzed by PLS with several spectral pre-processing methods. Compared with those of the HPLC reference analysis, the resulting determination coefficients （R2）, root mean square error of prediction （RMSEP） and residual predictive deviation （RPD） of the external validation for the L-GIu concentration were 99.5%, 1.62 g/L, and 11.3, respectively. For the GABA concentration, R2, RMSEP, and RPD were 99.8%, 4.00 g/L, and 16.4, respectively. This NIRS model was then used to optimize the biotransformation process through a Box- Behnken experimental design. Under the optimal conditions without pH adjustment, 200 gjL L-GIu could be catalyzed by 7148 U/L glutamate decarboxylase （GAD） to GABA, reaching 99% conversion at the fifth hour. NIRS analysis provided timely information on the conversion from L-GIu to GABA. The results suggest that the NIRS model can not only be used for the routine profiling of enzymatic conversion, providing a simple and effective method of monitoring the biotransformation process of GABA, but also be considered to be an optimal tool to guide the optimization of production conditions.

Six-year follow-up of pancreatic β cell function in adults with latent autoimmune diabetes

AIM: To investigate the characteristics of the progression of islet β cell function in Chinese latent autoimmune diabetes in adult (LADA) patients with glutamic acid decarboxylase antibody (GAD-Ab) positivity, and to explore the prognostic factors for β cell function. METHODS: Forty-five LADA patients with GAD-Ab positivity screened from phenotypic type 2 diabetic (T2DM) patients and 45 T2DM patients without GAD-Ab matched as controls were followed-up every 6 mo. Sixteen patients in LADA1 and T2DM1 groups respectively have been followed-up for 6 years, while 29 patients in LADA2 and T2DM2 groups respectively for only 1.5 years. GAD-Ab was determined by radioligand assay, and C-peptides (CP) by radioimmune assay.RESULTS: The percentage of patients whose fasting CP(FCP) decreased more than 50% compared with thebaseline reached to 25.0% at 1.5th year in LADA1 group, and FCP level decreased (395.8±71.5 vs 572.8±72.3 pmol/L, P＜0.05) at 2.5th year and continuously went down to the end of follow-up. No significant changes of the above parameters were found in T2DM1 group. The average decreased percentages of FCP per year in LADA and T2DM patients were 15.8% (4.0-91.0%) and 5.2% (-3.5 to 35.5%, P= 0.000) respectively. The index of GAD-Ab was negatively correlated with the FCP in LADA patients (rs= -0.483, P = 0.000). The decreased percentage of FCP per year in LADA patients were correlated with GAD-Ab index, body mass index (BMI) and age at onset (rs = 0.408, -0.301 and -0.523 respectively, P＜0.05). Moreover, GAD-Ab wasthe only risk factor for predicting βcell failure in LADA patients (B = 1.455, EXP (B) = 4.283, P = 0.023). CONCLUSION: The decreasing rate of islet β cell function in LADA, being highly heterogeneous, is three times that of T2DM patients. The titer of GAD-Ab is an important predictor for the progression of islet β cell function, and age at onset and BMI could also act as the predictors.

Effects of electroacupuncture on pain sensation in a rat model of hyperalgesia with nicotine dependence

Tobacco smoking is considered to be one of the main risk factors in the development of chronic pain.Long-term chronic exposure to nicotine and other forms of tobacco have been shown to be associated with an increased incidence of pain.Studies have shown that acupuncture can help smokers to reduce their desire to smoke,reduce their withdrawal symptoms,and avoid a relapse after treatment.However,little has been reported about the effects of acupuncture on pain sensitivity caused by long-term smoking.Models of hyperalgesia were established in rats exposed to nicotine for 6 weeks.After 6 weeks of continuous nicotine exposure,electroacupuncture at bilateral acupoints Zusanli(ST36)and Taichong(LR3)was performed 20 minutes per day for 6 days at a continuous wave with a frequency of 2 Hz and a stimulus intensity of 1 m A.The results revealed that electroacupuncture treatment increased the mechanical response threshold of hind paw of nicotine-dependent rats with hyperalgesia and up-regulated the protein expression of pain-related factorsμ-opioid receptor,β-endorphin and glutamic acid decarboxylase 65 in the spinal cord and midbrain periaqueductal gray and the protein expression of glutamic acid decarboxylase 67 in the spinal cord.These findings suggest that electroacupuncture treatment has positive analgesic effects on pain sensitivity caused by long-term chronic nicotine exposure.One possible mechanism for the improved analgesia is that electroacupuncture increases the expression of painrelated factors in the spinal cord and midbrain periaqueductal gray.This study was approved by Institutional Animal Care and Use Committee(IACUC)of the University of Miami(#18-167)on December 12,2018.

Effects of antisense glutamic aciddecarboxylase oligodeoxynucleotide on epileptic rats induced by pentylenetetrazol

OBJECTIVE: To 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). METHODS: A 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. RESULTS: Antisense 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. CONCLUSION: The 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.

Construction and functional activity of a recombinant vector expressing rat glutamic acid decarboxylase 65

Objective Glutamic acid decarboxylase 2（GAD65） is a gamma-aminobutyric acid（GABA） synthetase.This study aimed to construct a recombinant lentivirus-rGAD65（rLV-rGAD65） vector containing the cDNA of rat GAD65（rGAD65） and assess its functional activity in vitro and in vivo.Methods cDNA of rGAD65 was amplified by RT-PCR and subcloned into the LV vector,forming the rLV-GFP-rGAD65 plasmid.The recombinant lentivirus particles（rLVrGAD65） were packaged by the LV Helper-Free System and the titer was measured.Primary rat lung fibroblasts were transfected with rLV-rGAD65.The expression of rGAD65 in fibroblasts was detected by immunocytochemistry and western blot and the level of GABA in the medium was assessed by high-performance liquid chromatograph（HPLC）.In vivo,rLV-rGAD65 was injected into the subthalamic nucleus（STN） of Sprague-Dawley rats using stereotaxic methods,and rGAD65 protein levels in the STN were assessed by immunohistochemistry and Western blot,while the GABA concentration in the substantia nigra pars reticulata（SNr） was assayed by HPLC.Results The sequence of rGAD65 cDNA was in accord with that in GenBank.The amino-acid sequence of rGAD65 had no mutations and the titer of rLVrGAD65 reached 6.8 × 108/mL.The efficiency of infection of fibroblasts was 80%,and the concentration of GABA in the medium was（48.14 ± 9.35） nmol/L.In vivo,rGAD65 expression was detected in the STN,and the concentration of GABA in the SNr increased from（5.95 ± 1.09） to（12.44 ± 3.79） nmol/g tissue.Conclusion The recombinant LVGFP-rGAD65 vector was successfully constructed.rLV-rGAD65-infected primary fibroblasts in vitro and the expressed rGAD65 catalyzed the formation of GABA from glutamic acid.In vivo,the concentration of GABA in the SNr was increased after rLV-rGAD65 injection into the STN.

Glutamic acid decarboxylase 65 autoantibody levels discriminate two subtypes of latent autoimmune diabetes in adults

Objective To compare the clinical characteristics between type 2 diabetes mellitus (T2DM) and latent autoimmune diabetes in adults ( LADA) with different titers of glutamic acid decarboxylase autoantibody (GADA) and to define the two distinct subtypes of LADA.Methods Sera of 750 patients with an initial diagnosis of T2DM from central south of China were screened for GADA using a radioligand assay. The distribution and frequency of GADA levels were described. Two hundred and ninety-five patients were divided into the T2DM group (n =233) and the LADA group ( n = 62) to compare the age of onset, body mass index, HbA1c, C-peptide, hypertension, dyslipidemia and chronic diabetic complications. Furthermore, LADA patients with different GADA titers were subdivided to analyze the same indexes as the above.Results The prevalence of LADA (defined as GADA≥0. 05, namely GADA positive) was 9. 7% in the 750 initially diagnosed type 2 diabetic patients. Compared with T2DM, LADA patients were younger at their ages of onset, had lower C-peptide and body mass index, and also had less cases with hypertension and with dyslipidemia. However, only patients with high titer of GADA had poorer beta cell functions and less diabetic complications compared to T2DM and low GADA titer of LADA patients. Patients with low GADA titer were similar to T2DM patients, except that they were prone to develop ketosis more frequently.Conclusions Two clinically distinct subtypes of LADA can be identified by GADA levels in patients initially-diagnosed as type 2 diabetes. Patients with high titer of GADA (GADA≥0. 5) subsequently develope more insulin dependency, which are classified as LADA-type 1; while those with lower GADA titer (0.05≤GADA < 0. 5) and having clinical and metabolic phenotypes of type 2 diabetes are classified as LADA-type 2.

Change of glutamic acid decarboxylase antibody and protein tyrosine phosphatase antibody in Chinese patients with acute-onset type 1 diabetes mellitus

Background Glutamic acid decarboxylase antibody （GADA） and protein tyrosine phosphatase antibody （IA-2A） are two major autoantibodies, which exert important roles in the process of type 1 diabetes mellitus （T1D）. Our study aimed to investigate the changes in positivity and titers of GADA and IA-2A during the course of Chinese acute-onset T1D patients and their relationships with clinical features.

Diagnostic role of antibodies to glutamic acid decarboxylase in latent autoimmune diabetes mellitus in adults

Objective To investigate the diagnostic role of antibodies to glutamic acid decarboxylase (GAD 65 Ab) in latent autoimmune diabetes of adults (LADA) and the frequency of GAD Ab in Chinese patients initially diagnosed as non insulin dependent diabetes mellitus (NIDDM) Methods Forty five control subjects and 195 consecutive inpatients initially classified as NIDDM with ≥35 years of age at onset and nonketotic history for >6 months after diagnosis, were recruited In vitro transcripted and translated recombinant human 35 S GAD 65 was used in radioligand assay of GAD Ab Results The overall prevalence of GAD 65 Ab was 14 8% (29/195) in NIDDM patients and 2 2% (1/45) in control subjects, respectively Of the 29 GAD 65 Ab positive patients, 17 (58 6%) were insulin deficient while 12 (41 4%) were non insulin deficient The prevalence of GAD 65 Ab in NIDDM group with age of <40 years at diabetes onset, ketotic history, body mass index (BMI) <21 kg/m 2, were significantly higher than that of corresponding control diabetic subgroups (2 5, 4 1 and 3 2 times, respectively) The sex, duration, symptoms of polyphagia, polydipsia, polyuria and weight loss at onset of the disease were not related to the prevalence of GAD 65 Ab positivity Conclusions In China, patients initially diagnosed as NIDDM may in many cases suffer from LADA Testing by GAD 65 Ab may be of assistance to identifying LADA at the earliest stage of disease

The role of anti-glutamic acid decarboxylase autoantibodies in mood disorders

Gamma-aminobutyric acid(GABA)possibly plays a causative role in mood disorders.This hypothesis originated with studies on the beneficial effect of valproate in mania and as a mood stabilizer.Since valproate is known for its action in increasing the level of GABA,it was indirectly suggested that decreasing levels of GABA were responsible for mood alterations.To identify factors causing the decreased levels of GABA,studies have concentrated on the activity of the enzyme L-glutamic acid decarboxylase(GAD),which catalyzes the transformation of glutamate to GABA,as a decreasing function of this enzyme induces lower levels of the neurotransmitter.Moreover,a very limited amount of research investigated the possible role of glutamic acid decarboxylase antibodies(GADA)in determining a decreased enzymatic function of GAD.If these findings are confirmed,it will be possible to improve diagnosis and treatment of mood disorders.In addition,if the presence of GADA is associated with a genetic trait,this would allow and facilitate early diagnoses.

Effects of germination and aeration treatment following segmented moisture conditioning on theγ-aminobutyric acid accumulation in germinated brown rice

Brown rice was treated by segmented moisture conditioning to reach the suitable water content and aerated with air for germination.The effects of germination and aeration treatment on theγ-aminobutyric acid(GABA)content in germinated brown rice were studied.The results showed that the germination rate,glutamic acid,glutamate decarboxylase activity and GABA content in germinated brown rice increased significantly and then decreased with the increase of germination and aeration treatment parameters.Correlation analysis also revealed that there was a significant positive correlation between GABA accumulation and glutamic acid content,glutamate decarboxylase activity.These results suggested that the aeration treatment during germination following segmented moisture conditioning could contribute to a high GABA content of germinated brown rice.

The cross-reactivity of I-A^(g7) and I-A^d tetramers

OBJECTIVE: To investigate the cross-reactivity between glutamic acid decarboxylase (GAD)-I-A(g7) and I-A(d) tetramer in diabetes-prone non-obese diabetic (NOD) mice (I-A(g7)) and diabetes-free Balb/c mice (I-A(d)). METHODS: Two GAD peptide I-A(g7) and I-A(d) tetramers were generated and compared for phenotype and function of sorted GAD peptide I-A(g7) and I-A(d) tetramer-positive (tet+) T cells. RESULTS: The cross-reactivity is shown in either tetramer positive percentage or tetramer staining intensity. The NOD and Balb/c derived-tet+ T cells were able to be cross-stained by GAD peptide I-A(g7) and I-A(d) tetramers, and responded to both irradiated NOD and Balb/c splenotyes under stimulation by synthetic and recombinant GAD peptides. CONCLUSION: Although I-A(g7) and I-A(d) are closely related in biochemical and biological aspects, their most notable difference is the presence or absence of a negatively charged residue at position beta57 that links to insulin-dependent diabetes mellitus.

Efect of the Gad system on Actinobacillus succinogenes during acid stress

The glutamate decarboxylase(Gad)system is an important amino acid-dependent acid resistance system commonly found in microorganisms.Actinobacillus succinogenes is one of the best natural producers of succinic acid(SA)but lacks glutamate decarboxylase.This study assessed the efects of Gad system introduction into A.succinogenes.The recombinant strains gadB-SW and gadBC-SW were constructed by heterologous expression of gadB alone,or gadB together with gadC,respectively.After 1.0 and 1.5 h of acid stress at pH 4.6,cell survival of gadBC-SW was greater than gadB-SW.The growth of gadB-SW and gadBC-SW was both afected by the expression of heterologous proteins and byγ-aminobutyric acid,with gadBC-SW growth reduced at a neutral pH.SA production in acidic conditions was evaluated by a shake fask and by 3-L bioreactor fermentation.The results showed gadBC-SW to increase SA production by 8.4%in shake fask compared to the parent strain,SW.For a 3-L bioreactor batch fermentation under acidic environment,the highest conversion rate of sugar to SA was observed for gadBC-SW,reaching 96%.However,SA concentration by gadBC-SW was only 47 g/L and 31 g/L at pH 6.5 and pH 6.0,respectively.In summary,the introduction of heterologous gadB and gadC into A.succinogenes not only improved acid tolerance but also infuenced the synthesis of SA and added a metabolic burden.

Multiple autoimmune antibody limbic encephalitis:a case in a pregnant woman

Autoimmune limbic encephalitis is most commonly associated with antibodies against the N‑methyl‑D‑aspartate receptor(NMDAR),among other neuronal cell surface receptors.Here,a case of a pregnant female with limbic encephalitis in the presence of multiple additional autoimmune antibodies is described.The patient was a 36‑year‑old female who presented with 4 days of confusion,hallucinations,hypersexuality,disinhibition,and pressured speech.The patient’s work‑up detected the presence of anti‑NMDAR antibodies,anti‑glutamic acid decarboxylase antibodies,and a yet uncharacterized neuronal autoantibody.The patient was also found to be pregnant.No evidence of ovarian or other pelvic malignancy was discovered.Symptomatic control was achieved with plasma exchange.

The cross-reactivity of I-Ag7 and I-Ad tetramers

To investigate the cross reactivity between glutamic acid decarboxylase (GAD) I A g7 and I A d tetramer in diabetes prone non obese diabetic (NOD) mice (I A g7 ) and diabetes free Balb/c mice (I A d) Methods Two GAD peptide I A g7 and I A d tetramers were generated and compared for phenotype and function of sorted GAD peptide I A g7 and I A d tetramer positive (tet +) T cells Results The cross reactivity is shown in either tetramer positive percentage or tetramer staining intensity The NOD and Balb/c derived tet + T cells were able to be cross stained by GAD peptide I A g7 and I A d tetramers, and responded to both irradiated NOD and Balb/c splenotyes under stimulation by synthetic and recombinant GAD peptides Conclusion Although I A g7 and I A d are closely related in biochemical and biological aspects, their most notable difference is the presence or absence of a negatively charged residue at position β57 that links to insulin dependent diabetes mellitus