目的分离珍稀濒危兰科药用铁皮石斛谷氨酸脱羧酶(GAD)基因并进行生物信息学和表达分析。方法采用RT-PCR和RACE技术获基因cDNA全长;利用生物信息学软件分析蛋白理化性质、结构域和三维建模等分子特性;用DNASTAR 6.0和MEGA 4.0分别进行氨基酸多序列比对和进化树分析;借助实时定量PCR检测基因表达。结果分离到DoGAD基因,cDNA全长1795 bp,编码一条由498个氨基酸组成的多肽,分子量55.90 kD,等电点5.32;DoGAD蛋白不含跨膜域或信号肽,具有谷氨酸脱羧酶和磷酸吡哆醛依赖的脱羧酶结构域(17-443、37-381);DoGAD与植物GADs蛋白一致性为69.5%~78.8%,隶属于GADs分子进化树的植物类群;DoGAD转录本在石斛叶和茎中相对表达量较高,分别为根中的5.16和3.92倍。结论成功克隆得到铁皮石斛谷氨酸脱羧酶基因全长,DoGAD的表达特征暗示其可能在铁皮石斛叶和茎中发挥重要的调控作用。

ObjectiveThis study is aimed to isolate and characterize a glutamate decarboxylase (GAD) geneDoGAD fromDendrobium officinale, a rare endangered medicinal orchid species. MethodsRT-PCR and RACE technologies were used for gene isolation. The physiochemical properties, conserved domains and three dimensional structure of the deduced DoGAD protein were determined using a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 6.0 and MEGA 4.0, respectively. Real time quantitative PCR was used for gene expression analysis.Results The full-length cDNA ofDoGAD, with 1795 bp in size, was deduced to encode a 498-aa protein with molecular weight of 55.90 kD and isoelectric point of 5.32. The deduced DoGAD protein, without transmembrane or signal peptide residues, contained glutamate decarboxylase and pyridoxal phosphate dependant decarboxylase domains (17-443, 37-381). DoGAD had high identities (69.5% - 78.8%) with a number

目的：研究幽门螺杆菌（HP）感染的1型糖尿病患者血浆胰岛细胞抗体（ICA）、谷氨酸脱羧酶抗体（GADA）及胰岛素抗体（IAA）阳性率并与HP阴性糖尿病进行比较，同时观察根除HP治疗前后的变化。方法应用ELISA法测定糖尿病抗体，应用奥美拉唑20 mg，2次/天；克拉霉素0.5 g，2次/天；阿莫西林1.0 g，2次/天三联方法根除HP，对治疗前后糖尿病抗体进行比较。结果 HP阳性的1型糖尿病患者ICA阳性率明显高于HP阴性1型糖尿病患者，并有统计学意义。2组间比较IAA及GADA阳性率差异无统计学意义。治疗前后比较IAA、ICA及GADA阳性率相似，差异无统计学意义（P〉0.05）。结论HP感染使糖尿病患者ICA阳性率增加，根除HP治疗对GABA、IAA阳性率无影响。

Objective To investigate the positive rates of islet cell autoantibody (ICA), glutamic acid decarboxylase antibody (GADA) and insulin autoantibody (IAA) in plasma of patients with type 1 diabetes mellitus (T1DM) and Helicobacter pylori (HP) infection versus patients with T1DM but not HP, and then to detect the changes in these antibodies after HP eradication. Methods HP was eradicated using triple therapy with omeprazole (20 mg, bid), clarithromycin (0.5 g, bid) and amoxicillin (1.0 g, bid). Before and after HP eradication, enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of diabetes-related antibodies. Results Compared with patients with T1DM, higher positive rate of ICA was observed in patients with both T1DM and HP infection. Furthermore, the result was statistically signiifcant. In contrast, there were no differences in GADA and IAA between two groups of patients. After HP eradication, the positive rates of ICA, GADA and IAA were not altered sign

目的：探讨临床表现酷似僵人综合征的神经系统副肿瘤综合征患者的诊断学特征。方法回顾性分析1例临床表现为僵人综合征的神经系统副肿瘤综合征患者的临床资料，并复习相关文献。结果患者临床表现为僵直、肌肉痉挛、癫痫，脑脊液检查示细胞数、蛋白正常；抗钾通道相关抗体、抗 N-甲基-D-天冬氨酸受体抗体、抗谷氨酸脱羧酶抗体、单胞病毒抗体正常，肌电图示连续的运动单位活动。颅脑强化磁共振示右蝶骨嵴区脑膜瘤，颈椎磁共振示 C3-4、C4-5、C5-6、C6-7椎间盘轻度后突出；胸部 CT 示纵隔内及双侧锁骨上下窝区多发肿大淋巴结，锁骨上肿大淋巴结病理结果显示转移性小细胞癌。结论临床表现似僵人综合征的患者，如找不到病因，应行全身检查尤其是肺部检查排除神经系统副肿瘤综合征。

Objective To explore the diagnostic features of paraneoplstic syndrome (PNS ) presented with stiff-person syndrome(SPS).Methods One patient with SPS caused by tumor was diagnosed as paraneoplastic neurological syndrome.The relevant literatures were reviewed.Results One patient was mainly presented with rigidity,muscular spasm and epilepsy;the cerebro-spinal fluid (CSF)protein content and CSF cell count were normal.The anti-potassium channel antibody,anti-N-methyl-D-aspartate receptor antibody,anti-glutamate decarboxylase antibody and anti-herpes simplex virus antibody were all negative.The electromyography revealed continuous motor unit activity.The sphenoid ridge meningoma was found on cerebral enhanced MR.Mild cervical disc herniation was presented on cervical spine MR.Chest CT showed multiple swollen lymph nodes in mediastinal and supradavicular and the pathologic result of the supraclavicular lymph node showed metastatic small cell cancer.Conclusions For patients presente

目的探讨糖尿病自身抗体联合检测在糖尿病分型中的应用价值,为正确分型糖尿病提供理论依据。方法选择我院内分泌科收治的126例糖尿病患者,其中1型糖尿病组51例,2型糖尿病组75例,同时选择健康对照组60例,采用免疫印迹试剂法检测胰岛细胞抗体(ICA)、胰岛素自身抗体(IAA)和谷氨酸脱羧酶抗体(GADA)表达情况,计算阳性率,并对结果进行比较分析。结果 ICA、IAA和GADA在1型糖尿病患者中的阳性率分别为35.3%、31.4%和60.8%,显著高于2型糖尿病患者的1.3%、1.3%、2.7%及对照组的1.3%、0.0%、0.0%,均有显著性差异(P0.05)。ICA/IAA/GADA、GADA/ICA、GADA/IAA三种联合检测组合对1型糖尿病诊断的敏感性分别为92.2%、84.3%和82.4%,均显著高于单一抗体检测,均有显著性差异(P0.05)。结论检测血清ICA、IAA和GADA能够为正确鉴别1型糖尿病和2型糖尿病提供重要依据,联合检测能够提高诊断阳性率,避免漏诊,值得临床推广和普及。

Objective To explore the diagnostic value of detection of diabetes antibodies in the classification of dia-betes mellitus and provide a theoretical basis for the correct typing of diabetes mellitus.Methods 126 cases of diabetic patients were hospitalized in the Department of Endocrinology,including 51 cases of type 1 diabetes,75 cases of type 2 diabetes,and 60 healthy controls were detected as control group.Islet cell antibodies (ICA),insulin autoantibody (IAA),glutamic acid decarboxylase antibody (GADA)expression and the positive rate were tested.Results The posi-tive rates of ICA,IAA,GADA in patients with type 1 diabetes were 35.3%,31.4%,60.8%,were significantly higher than 1.3%,1.3%,2.7% and 1.3% in patients with type 2 diabetes (P 82.4%,were significantly higher than those of single detection (P <0.05).Conclusion The detection of serum ICA, IAA,GADA can provide an important basis for the correct identification of type 1 diabetes and type 2 diabetes.Joint de-tection can improve the

为了降低细胞壁和细胞膜的传质阻力对全细胞催化过程的不利影响，采用有机溶剂对表达重组谷氨酸脱羧酶(GAD)的工程菌BL21(DE3)-pET28a-gadB进行了透性化处理。结果表明，菌体GAD表观催化活力的改善程度与有机溶剂的介电系数和疏水性具有较强相关性，低介电常数(0.68)的有机溶剂可以有效提高工程菌的GAD催化活性，其中最有效的透性化试剂为二甲苯(介电常数为2.4，log P为3.1)。当二甲苯的用量为5μL?mg?1(干重细胞)、处理时间5 min时,菌体表观催化活力达到最优值，为7.94 U?mg?1(干重细胞),是处理前表观催化活力的12.4倍。为了充分利用菌体的催化活力，采用海藻酸钙包埋的方法对二甲苯处理过的菌体进行固定化，结果发现固定化的透性化菌体可以较好地保持催化活力，反复催化10次后发现，第10次的GABA产率为第1次GABA产率的72.04%。研究为系统的选择有机溶剂来有效提高GAD工程菌表观催化活性提供了参考，同时制备的固定化的透性化菌体显示了良好工业应用前景。

RecombinantEscherichia coli cells over-expressing glutamate decarboxylase (GAD) (BL21(DE3)-pET28a-gadB)were permeabilized with organic solvents to overcome the permeability barrier of cell envelope against substrate and product diffusion. The results show that the change in apparent GAD activity is correlated with the physicochemical properties of the organic solvents used for permeabilization. Solvents with lower dielectricconstant ( 0.68) are relatively effective in increasing apparent GAD activity of cells. Xylene (dielectricconstant value is 2.4, logP value is 3.1)is the most effective permeabilizer for improving the cell GAD activity, and an optimal GAD activity (7.94 U·mg-1 dry biomass) is obtained by treating the cells using xylene at 5 μL?mg?1 dry biomass for 5 min. The GAD activity of the xylene-permeabilized cells is 12.4 times higher than that of the controls. The xylene-permeabilized cells are immobilized with Ca-alginate gel and the conversion rate is 72.04% af