Sequential elevation of autoantibodies to thyroglobulin and glutamic acid decarboxylase in type 1 diabetes

We have previously reported the high levels of glutamic acid decarboxylase 65 autoantibodies(GAD65A)in patients with type 1 diabetes and autoimmune thyroid disease.Here we describe a 32-year-old Japanese female with a thirteen-year history of type 1 diabetes whose levels of GAD65A were elevated just after the emergence of anti-thyroid autoimmunity.At 19 years of age,she developed diabetic ketoacidosis and was diagnosed with type 1 diabetes.She had GAD65A,insulinoma-associated antigen-2 autoantibodies(IA-2A),and zinc transporter-8 autoantibodies(ZnT8A),but was negative for antibodies to thyroid peroxidase(TPOAb)and thyroglobulin(TGAb)at disease onset.ZnT8A and IA-2A turned negative 2-3 years after the onset,whereas GAD65A were persistently positive at lower level(approximately 40 U/mL).However,just after the emergence of TGAb at disease duration of 12.5 years,GAD65A levels were reelevated up to5717 U/mL in the absence of ZnT8A and IA-2A.Her thyroid function was normal and TPOAb were consistently negative.She has a HLA-DRB1*03:01/*04:01-DQB1*02:01/*03:02 genotype.Persistent positivity for GAD65A might be associated with increased risk to develop anti-thyroid autoimmunity.

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.

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

Autoantibodies related to ataxia and other central nervous system manifestations of gluten enteropathy

Gluten ataxia and other central nervous system disorders could be linked to gluten enteropathy and related autoantibodies.In this narrative review,we focus on the various neuro-logical manifestations in patients with gluten sensitivity/celiac disease,immunological and autoimmune mechanisms of ataxia in connection to gluten sensitivity and the autoantibodies that could be used as a biomarker for diagnosing and following.We focused on the anti-gliadin antibodies,antibodies to different isoforms of tissue transglutaminase(TG)(anti-TG2,3,and 6 antibodies),anti-glycine receptor antibodies,anti-glutamine acid decarboxylase antibodies,anti-deamidated gliadin peptides antibodies,etc.Most studies found a higher prevalence of these antibodies in patients with gluten sensitivity and neurological dysfunction,presented as different neurological disorders.We also discuss the role of a gluten-free diet on the clinical improvement of patients and also on imaging of these disorders.

RAPID DETERMINATION OF L-GLUTAMIC ACID WITH AN ENZYME REACTOR OF L-GLUTAMIC DECARBOXYLASE IMMOBILIZED ON ION EXCHANGE RESIN

The preparation and characterization of an immobilized L-glutamic decarboxylase (GDC) were studied. This work is to develop a sensitive method for the determination of L-glutamate using a new biosensor, which consists of an enzyme column reactor of GDC immobilized on a novel ion exchange resin (carboxymethyl-copolymer of allyl dextran and N.N?methylene-bisacrylamide CM-CADB) and ion analyzer coupled with a CO2 electrode. The conditions for the enzyme immobilization were optimized by the parameters: buffer composition and concentration, adsorption equilibration time, amount of enzyme, temperature, ionic strength and pH. The properties of the immobilized enzyme on CM-CADB were studied by investigating the initial rate of the enzyme reaction, the effect of various parameters on the immobilized GDC activity and its stability. An immobilized GDC enzyme column reactor matched with a flow injection system-ion analyzer coupled with CO2 electrode-data collection system made up the original form of the apparatus of biosensor for determining of L-glutamate acid. The limit of detection is 1.0×10-5 M. The linearity response is in the range of 5×10 -2-5×10 -5 M . The equation of linear regression of the calibration curve is y= 43.3x + 181.6 (y is the milli-volt of electrical potential response, x is the logarithm of the concentration of the substrate of L-glutamate acid). The correlation coefficient equals 0.99. The coefficient of variation equals 2.7%.

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.

基于稻谷加温加湿技术的γ-氨基丁酸富化

目的:提高糙米品质。方法:以γ-氨基丁酸(GABA)含量和谷氨酸脱羧酶活性作为指标,研究富化条件对GABA含量的影响;基于最优参数,通过分析富化过程中籽粒各部位GABA含量,揭示其迁移规律;最后分析该法对糙米品质的影响,并与浸泡法对比。结果:原料水分含量14.5%,富化温度65℃,最终水分含量20%,富化时间5 h时,GABA含量最高。富化过程中,GABA主要在糠层生成,在生成的同时向内部迁移,各碾减率的糙米GABA含量均在5 h达到峰值,但迁移率在4 h后趋于稳定,胚乳部分的GABA迁移率最高可达87.1%。与浸泡法相比,加温加湿法富化的糙米偏黄,爆腰率较低,米饭更柔软。结论:加温加湿法优于浸泡法,可用于制备高GABA含量的糙米和精米。

微生物法合成γ-氨基丁酸的研究进展

γ-氨基丁酸(γ-aminobutyric acid,GABA)是一种四碳非蛋白质氨基酸,在食品、农业、医药、化工等领域具有广阔的应用前景。微生物法生产GABA因其温和、可持续发展等优势越来越受到人们的关注。因此,为了得到环保、便捷且效率更高的GABA生产方式,以满足食品、制药和畜牧领域对添加剂的严格要求,本文系统介绍了GABA的生产方法、生物体中的合成途径及微生物法生产GABA的研究进展,总结了目前全细胞催化法和微生物从头合成GABA的生产水平。研究者们致力于筛选和优化具有高催化效率和稳定性的酶并通过对微生物的代谢途径进行精细调控,以提高GABA的合成效率,未来的研究需要进一步优化酶和菌株的性能,降低生产成本,并探索更大规模的工业化生产途径。

2型糖尿病患者胰岛细胞自身抗体、抗谷氨酸脱羧酶抗体水平与糖尿病微血管并发症的关系

目的探讨抗胰岛细胞抗体(ICA)、抗胰岛素抗体(IAA)及抗谷氨酸脱羧酶抗体(GADA)与2型糖尿病(T2DM)患者发生糖尿病微血管并发症(DMAP)的关系。方法对2023年11月-2024年3月本院收治的114例T2DM患者临床资料进行回顾性分析,根据是否发生DMAP将患者分别分入单纯T2DM组(n=61)和并发症组(n=53)。检测所有患者入院时IAA、ICA及GADA水平。采用受试者工作特征(ROC)曲线分析IAA、ICA及GADA对DMAP的评估价值,采用多因素logistic回归分析探讨DMAP的影响因素。结果并发症组患者的ICA、IAA、GADA水平均高于单纯T2DM组,差异均有统计学意义(P<0.05)。ICA、IAA及GADA评估T2DM患者发生DMAP的曲线下面积(AUC)分别为0.792、0.736、0.805,联合检测的AUC为0.912。并发症组患者T2DM病程≥8年比例、有饮酒史比例均高于单纯T2DM组患者,差异均有统计学意义(P<0.05)。logistic回归结果显示:T2DM病程≥8年(OR=2.314,95%CI:1.136~4.714)、ICA≥4183.62 RU/mL(OR=4.221,95%CI:1.849~9.633)、IAA≥5587.37 RU/mL(OR=3.449,95%CI:1.654~7.192)、GADA≥3667.92 IU/mL(OR=5.150,95%CI:2.058~12.888)是T2DM患者发生DMAP的危险因素(P<0.05)。结论ICA、IAA及GADA与T2DM患者发生DMAP密切相关,联合检测有助于DMAP的评估。

本氏烟NbGAD1蛋白的原核表达及多克隆抗体制备

γ-氨基丁酸(GABA)作为一种非蛋白质氨基酸,普遍存在于植物体内,在植物生长发育和抗胁迫方面发挥着重要作用。谷氨酸脱羧酶(GAD)是合成GABA的关键蛋白酶,目前关于GAD介导GABA调控植物防御病毒侵染的分子机制尚不清楚。利用RT-PCR克隆本氏烟NbGAD1基因,并与原核表达载体pET-28a连接,构建重组载体pET-28a-NbGAD1,诱导产生重组蛋白,经Ni-NTA柱纯化蛋白后免疫新西兰白兔,最终获得抗血清。结果表明:成功构建原核表达载体pET-28a-NbGAD1,经大肠埃希菌诱导纯化后获得分子量约57 ku的重组蛋白NbGAD1。免疫制备的抗血清经Western blot检测显示,抗血清效价达1∶100000,抗血清稀释2000倍仍能检测到浓度为0.005 mg·mL^(-1)的重组蛋白,且能检测到本氏烟中内源表达的NbGAD1蛋白,说明该抗血清特异性强、灵敏度高,为今后深入探究GAD在植物病毒侵染过程中的作用机制奠定了基础。

谷氨酸脱羧酶的克隆表达与酶学性质

γ-氨基丁酸(γ-aminobutyric acid,GABA)是一种非蛋白天然氨基酸,具有多种生物活性。谷氨酸脱羧酶(glutamate decarboxylase,GAD)能将L-谷氨酸或谷氨酸钠不可逆地催化为γ-氨基丁酸。为探究GAD酶学性质,从植物乳杆菌CPRGJ(Lactobacillus plantarum)中克隆表达GAD,利用镍柱亲和层析对重组GAD进行纯化并探究其酶学性质。结果表明,GAD的酶学性质为最适温度45℃、最适pH5.0、辅酶5-磷酸吡哆醛摩尔浓度250μmol/L,此时有最大酶活力;有机试剂中正丁醇对GAD有强抑制作用,通过Lineweaver Burk方程可得到GAD的米氏常数Km为19.988 mmol/L,Vmax为0.263 mmol/(L·min)。

糖尿病自身免疫抗体联合检测在糖尿病分型中的应用价值

目的分析糖尿病自身免疫抗体联合检测在糖尿病分型中的应用价值。方法将2021年1月—2023年11月于东莞市中医院进行诊治的64例糖尿病患者纳为研究组,其中1型糖尿病(type 1 diabetes mellitus,T1DM)患者22例,2型糖尿病(type 2 diabetes mellitus,T2DM)患者42例。同期体检健康者61例纳为对照组,2组均接受糖尿病自身免疫抗体检测,比较2组的抗体差异性,以及其在糖尿病分型中的价值。结果研究组血清谷氨酸脱羧酶抗体(glutamic acid decarboxylase antibody,GADA)、胰岛细胞抗体(islet cell antibody,ICA)、胰岛素自身抗体(insulin autoantibodies,IAA)以及联合检测阳性率高于对照组,差异有统计学意义(P<0.05);T1DM患者GADA、ICA、IAA以及联合检测阳性率高于T2DM患者,差异有统计学意义(P<0.05);T1DM、T2DM诊断中联合检测的应用价值高于单一抗体检测。结论糖尿病自身免疫抗体联合检测有助于临床更好地鉴别糖尿病分型,为后续治疗提供有价值的指导。

不同茶树品种γ-氨基丁酸(GABA)富集差异分析

以一芽二叶茶鲜叶为试材,采取厌氧/好氧间歇交替富集技术富集茶鲜叶,对富集前后谷氨酸(Glu)含量、谷氨酸脱羧酶(GAD)活性、γ-氨基丁酸(GABA)含量、游离氨基酸(FAA)总量和茶叶色泽、香气差异进行分析,以期为GABA高富集潜力茶树品种的筛选提供参考依据。结果表明:8 h厌氧/好氧间歇交替富集处理显著提高了茶鲜叶GABA含量和FAA总量;不同茶树品种GABA富集潜力、富集前后FAA总量及富集前鲜叶GAD活性和Glu含量均以‘白叶1号’显著最高,其次是‘福鼎大白茶’;茶鲜叶FAA总量、Glu含量和GAD活性可作为筛选GABA高富集潜力茶树品种的参考指标。

谷氨酸脱羧酶1在精神疾病中的研究进展

谷氨酸脱羧酶(glutamic acid decarboxylase, GAD)是γ-氨基丁酸(γ-aminobutyric acid, GABA)的合成酶,主要在脑和胰岛中存在。GABA的异常表达与多种疾病相关,多项研究表明其合成酶GAD1可能参与精神疾病的发病过程,现就GAD1研究的新进展及其可能的致病机制进行综述。

辣木叶γ-氨基丁酸富集方法研究

为了提高辣木叶的γ-氨基丁酸(γ-aminobutyric acid,GABA)含量,通过辣木枝条的导管向叶片细胞补充外源L-谷氨酸(L-glutamic acid,L-Glu),以干叶的GABA含量为评价指标,从辣木叶谷氨酸脱羧酶(glutamate decarboxylase,GAD)的酶学特性、L-Glu溶液pH和浓度、浸吸时间、转化反应时间、转化反应温度等方面对植物导管法富集辣木叶GABA的适宜条件进行研究。结果显示:辣木叶GAD的最适反应温度和pH分别为40℃和6.0,在4~30℃和pH 5.6~6.4相对较稳定;与破碎法(破碎叶片释放GAD转化外源L-Glu合成GABA)相比较,植物导管法(通过植物导管补充外源L-Glu)由于GAD存在细胞的保护而富集效果更佳,植物导管法富集的GABA含量是破碎法的1.71倍;当将鲜辣木枝条切口端插入pH 5.5、50 mmol/L L-Glu溶液于室温放置浸吸1 h,然后摘取辣木叶于35℃保温7 h,辣木叶GABA含量由初始(1.86±0.13)mg/g增加至(5.97±0.36)mg/g,提高了2.21倍。植物导管法富集辣木叶GABA的工艺简单,在富GABA辣木叶产品开发方面具有很好的应用前景。

响应面法优化屎肠球菌T2-2产γ-氨基丁酸的发酵条件

从传统腌制芥菜中筛选获得高产γ-氨基丁酸(γ-aminobutyric acid,GABA)的屎肠球菌T2-2为研究对象,通过单因素实验和响应面法优化菌株发酵条件,提高GABA产量。结果表明:屎肠球菌T2-2产GABA最佳发酵条件为:温度30℃,屎肠球菌T2-2添加量7.50%,L-谷氨酸钠添加量18.93 g/L,pH 6.0,时间1 d,此时菌体生长量为0.338,酶活为3.177μg/mL,GABA产量为1.190 mg/mL,较优化前提高63.01%。本研究为提高乳酸菌产GABA含量提供一定的理论依据。

血清GADA、CypA、SIRT1在糖尿病肾病中的表达及早期诊断价值分析

目的:探究血清谷氨酸脱羧酶抗体(Glutamic acid decarboxylase,GADA)、亲环素A(Cyclophilin A,CypA)、Sirtuin-1(SIRT1)在糖尿病肾病(Diabetic nephropathy,DN)中的表达及早期诊断价值。方法:选取2020年1月至2023年1月期间本院收治的81例DN为研究组,另选取同期81例糖尿病患者为对照组。将DN患者根据肾功能损伤程度进一步分为微量蛋白尿组、大量蛋白尿组、肾功能损伤组。比较两组及不同程度肾损伤患者入院时血清GADA、CypA、SIRT1水平及联合诊断价值。结果:研究组血清GADA、CypA水平高于对照组,SIRT1水平低于对照组(P<0.05);不同程度肾损伤患者血清GADA、CypA水平比较:微量蛋白尿组<大量蛋白尿组<肾功能损伤组,SIRT1水平比较:肾功能损伤组<大量蛋白尿组<微量蛋白尿组(P<0.05);入院时血清GADA、CypA、SIRT1水平联合检测糖尿病肾病的AUC为0.840,最佳敏感度、特异度为88.89%、80.25%。结论:血清GADA、CypA、SIRT1水平与DN病情发展密切相关,可作为早期诊断的辅助指标。