A new covalent peroxidase conjugation method using bis(sulfosuccinimidyl) suberate as cross-linking reagent in a two-step procedure

A new method has been developed to prepare horseradish peroxidase (HRP) conjugates using bis(sulfosuccinimidyl)suberate (BS3) as cross-linking reagent in a two-step procedure. The enzyme is reacted with BS3, introducing active ester molecules into the enzyme itself, and it is then used directly to label amino-containing compounds without further treatment. The proteins involved in the conjugation undergo minimal modifications. The reaction conditions are evaluated, as well as studies on the conservation of the biological activities of the conjugated proteins and the stability of the conjugates in time. These conjugates are found to be significantly improved compared with similar products prepared by conventional methods.     

Effects of In Vitro Ethanol and Fetal Ethanol Exposure on Glutathione Stimulation of N-Methyl-D-Aspartate Receptor Function

The present studies investigated the effects of glutathione (GSH; γ-glutamylcysteinylglycine) and its oxidized form (GSSG) on neuronal N -methyl-D-aspartate (NMDA) receptor activation in both acute and chronic preparations of ethanol exposure. It was demonstrated using fura-2-loaded dissociated brain cells from newborn rat pups that both GSH and GSSG (0–4 mM) produced concentration-dependent increases in intracellular calcium similar to those produced by NMDA and other agonists of the NMDA receptor. GSH-stimulated calcium entry was not inhibited by low intoxicating concentrations of ethanol, which contrasts with ethanol's typical inhibitory effect on NMDA-stimulated receptor activation. Behavioral studies in adult rats demonstrated that ethanol-induced sleep times were significantly decreased when 10 μl of GSSG (20 mM) were administered intracere-broventricularly approximately 5 min before an intraperitoneal injection of 20% (w/v) ethanol (3 g/kg). These findings suggest that the less potent effect of ethanol on GSH-stimulated calcium entry as well as the reduction in ethanol-induced sleep times may be related to the presence of glycine in the peptide. The glycine found in GSH may activate the glycine site and block or reduce ethanol's action on this site. It appears that although GSH may play an important role in the activation of the NMDA receptor, this action does not involve a process that is sensitive to acute ethanol exposure. In contrast, when rat pups were chronically exposed to ethanol via prenatal exposure before the fura-2 preparation, increases in NMDA- and GSH-stimulated calcium entry were significantly decreased relative to those in pair-fed and ad libitum-fed controls. Thus, chronic in utero exposure to ethanol may alter the NMDA-receptor complex, such that calcium entry mediated by NMDA or GSH activation is significantly reduced.     

GSTA1基因多态性对环磷酰胺辅助化疗的乳腺癌患者预后的影响

目的 探讨GSTA1基因多态性与接受CTX类药物辅助化疗乳腺癌患者预后的关系.方法 对采用CTX类药物为主的方案进行辅助化疗的137例确诊乳腺癌患者(浸润性导管癌患者124例,浸润性小叶癌患者5例,其他类型癌症患者8例),用PCR-LDR检测其GSTA1基因多态性,Kaplan-Meier法绘制生存曲线,log-rank法比较不同基因型组间生存差异,并用Cox比例风险回归模型进行预后影响因素的多因素分析.结果 137例乳腺癌患者中,GSTA1*A/*A、*A/*B和-B/*B基因型频率分别为67.2%(92/137)、31.4%(43/137)和1.5%(2/137).乳腺癌患者的GSTA1基因型频率在按年龄、临床分期、淋巴结转移、雌、孕激素受体状态等临床病理特征分组的分布差异均无统计学意义(x2值分别为0.722、1.967、3.303、0.226、0.709,P均＞0.05);经Fisher精确概率分析,肿瘤大小、病理组织类型、病理分级的差异均无统计学意义(P均＞0.05).携带GSTA1-A/*A基因型和*A/*B或*B/-B基因型的乳腺癌患者复发率分别为47.8%(44/92)和31.1%(14/45),死亡率分别为22.8%(21/92)和17.8%(8/45).Kaplan-Meier生存曲线和log-rank分析显示,携带GSTA1*A/*B+*B/*B基因型乳腺癌患者的无复发生存率和总生存率均高于携带GSTA1*A/*A基因型患者,且差异均有统计学意义(x2值分别为18.723、7.352,P均＜0.01).经Cox多因素分析,GSTA1基因多态性是影响乳腺癌患者无复发生存[OR=0.222,95%CI:0.108～0.458,P＜0.01]和总生存[OR=0.362,95%CI:0.145～0.902,P＜0.05]的独立因素.结论 GSTA1基因多态性是乳腺癌患者CTX药物辅助化疗无复发生存和总生存的预测标志.

Abstract：

Objective To investigate the association between the genetic polymorphisms in GSTA1 and the clinical outcome of breast cancer patients treated with cyclophosphamide-based adjuvant chemotherapy. Methods A total of 137 breast cancer patients receiving cyclophosphamide-based adjuvant chemotherapy were recruited ( 124 cases with infiltrative ductal carcinoma, 5 cases with infiltrative lobular carcinoma and 8 cases with other histological types). PCR-LDR method was used to detect the genotypes of GSTA1. Survival curves were generated by the Kaplan-Meier method, and verified by the log-rank test. Cox proportional hazards regression analysis was used to estimate the prognostic factors in multivariate analysis. Results Of the 137 breast cancer patients, the genotypic frequencies of the GSTA1 * A/* A,* A/* B and * B/* B were 67.2% ( 92/137 ), 31.4% ( 43/137 ) and 1.5% ( 2/137 ), respectively. No significant differences were found between the genotypic frequencies and groups categorization according to age, stage, lymph node metastasis, ER or PR status (x2 = 0. 722,1. 967, 3. 303, 0. 226 and 0. 709, all P ＞0. 05 ) ;through Fisher exact test, also no significant differences were found between the genotypic frequencies and group categorization according to tumor size, histological types and grading ( all P ＞ 0. 05 ) . The recurrence rates in patients with GSTA1 * A/* A and * A/* B or * B/* B genotypes were 47. 8% (44/92) and 31.1% ( 14/45 ), respectively, and the mortality rates were 22. 8% ( 21/92 ) and 17. 8% ( 8/45 ),respectively. Patients with GSTA1 * A/* B and * B/* B genotypes were significantly associated with reduced hazard of relapse (x2 =18.723, P＜0. 01)and mortality (x2 =7.352, P＜0.01), compared to cases with the common * A/* A genotypes, according to Kaplan-Meier survival analysis and log-rank test. Moreover,Cox multivariate analysis showed that GSTA1 polymorphisms appeared to be an independent risk factor for recurrence-free survival ( OR =0. 222, 95% CI:0. 108-0. 458, P ＜0. 01 ) and overall survival ( OR =0. 362,95% CI:0. 145-0. 902, P ＜ 0. 05 ). Conclusion These data indicate that GSTA1 polymorphism may be a potential prognostic factor for recurrence-free survival and overall survival in breast cancer patients treated with cyclophosphamide-based adjuvant chemotherapy.     

基于纳米四氧化三铁的电化学生物传感器研究

目的 构筑基于纳米四氧化三铁(Fe3O4)的电化学生物传感器,建立过氧化氢(H2O2)和模拟酶的电化学测定方法.方法 利用纳米Fe3O4模拟过氧化物酶催化3,3',5,5'-四甲基联苯胺(TMB)-H2O2的反应,采用传统的三电极体系,应用电流-时间曲线法检测酶催化产物.结果 在最佳实验条件下,电流强度与H2O2浓度在...     

复方甘草酸苷联合还原型谷胱甘肽治疗乙醇性肝病60例疗效观察

目的:观察复方甘草酸苷联合还原型谷胱甘肽治疗乙醇性肝病的临床疗效。方法:将60例酒精性肝病患者随机分成复方甘草酸苷、还原型谷胱甘肽、还原型谷胱甘肽和复方甘草酸苷联合组三组,每组各20例,疗程均为20 d。复方甘草酸苷组给予复方甘草酸苷注射液120 mg/d;还原型谷胱甘肽组给予还原型谷胱甘肽1.8 g/d;联合组给予复方甘草酸苷针120 mg/d和还原型谷胱甘肽针1.8 g/d。结果:治疗后,三组临床症状均有改善。联合组临床症状改善均优于复方甘草酸苷组和还原型谷胱甘肽组。结论:复方甘草酸苷联合还原型谷胱甘肽比两药单用对乙醇性肝病的临床治疗作用更加明显。     

低硒高镉对大鼠免疫功能及过氧化作用影响的研究

用克山病病区粮或加硒和加镉的病区粮喂养Ｓ．Ｄ大鼠，１～２个月后取其组织进行分析。结果显示：用病区粮饲养大鼠，可使大鼠免疫器官的发育发生障碍，低硒和高镉对大鼠组织的损伤呈现相加作用，加硒可拮抗镉的部分毒性，组织中脂质过氧化物水平与循环免疫复合物的含量呈显著正相关，与还原型谷胱甘肽含量呈显著负相关。说明克山病的致病因素是复杂的，低硒是主要的致病因素，高镉则加重其危害程度，并可能涉及自身免疫病理过程。     

Effect of cerulein hyperstimulation on the paracellular barrier of rat exocrine pancreas

Cerulein-induced pancreatitis causes a rapid increase in pancreatic enzyme levels in serum and decreases in pancreatic duct secretion and interstitial edema. One mechanism to explain these early events is disruption of the actin tight junction paracellular seal of acinar and intralobular pancreatic duct cells. To examine the paracellular barrier of the proximal exocrine pancreas, rats were hyperstimulated with 5.0 μg · kg⁻¹ · h⁻¹ of cerulein. Actin was visualized with rhodamine phalloidin and by electron microscopy and tight junctions were visualized with antibodies to the tight-junction protein ZO-1. Paracellular permeability was measured by movement of horseradish peroxidase from interstitium into duct or acinar lumens. In controls, linear actin and ZO-1 staining occurred along the apical membrane of intralobular duct cells and extended to the apical pole of acinar cells. Hyperstimulation caused progressive disruption of the linear staining of f-actin and ZO-1. Actin disruption in duct cells was confirmed by electron microscopy. Horseradish peroxidase entered intralobular ducts and acinar lumens of hyperstimulated animals more frequently than those of controls. The structure and function of the paracellular barrier of acinar and intralobular pancreatic duct cells are disrupted early during cerulein pancreatitis and may contribute to early clinical features.     

Differences in the glutathione system of cultured aortic smooth muscle cells from young and aged rats

We studied the relation between the glutathione (GSH) system and cell proliferation in a model of smooth muscle cells (SMC) derived from the thoracic aorta of 4–6-week-old (young) and 15-month-old (aged) rats. SMC from aged rats showed greater levels of total non-protein thiol compounds (T-SH), increased glutathione transferase (GST) and increased glutathione reductase (GSSG-Red) activities compared with cells from young rats. These changes were associated with an increased proliferation rate of SMC from aged rats. To evaluate the role of GSH on cell proliferation better, a specific inhibitor of gamma-glutamyl-cystein synthetase, -buthionine-SR-sulphoximine (BSO) was used. BSO showed a dose-dependent inhibition of cell growth, with an IC₅₀ of 10⁻⁴ M, after 48–72 h of incubation. Removal of BSO restored cell growth, further suggesting a link between GSH levels and vascular cell proliferation. The inhibitory effect of BSO was about two times greater on SMC from young than on SMC from aged rats. BSO showed 56% inhibition on the proliferation of SMC from young rats and 32% inhibition on SMC from aged rats (10⁻⁴ M, 72 h of incubation). A parallel reduction of GSH levels of 38% and 19% for SMC from young and aged rats, respectively, was observed, suggesting that age-related factors may influence the involvement of GSH system in cell proliferation.     

硒对鸡胚脑发育的影响

目的观察补硒对正常鸡胚脑谷胱甘肽过氧化物酶(GPx)、Ⅱ型脱碘酶(IDⅡ)活性和生长相关蛋白(GAP-43)表达的影响,探讨硒对正常动物脑发育的作用。方法对孵育8 d的鸡胚分别注射不同剂量的MSeC(含0、1、5、10、20、40 μg硒),孵育至第20天时处死。测定脑组织中硒、GPx活性、IDⅡ活性,检测GAP-43蛋白的表达。结果对正常鸡胚补硒,可提高脑硒水平、脑GPx活性及GAP-43的表达,补硒量与脑硒、脑GPx活性显著相关(P< 0.01),而与脑IDⅡ活性无相关性。结论MSeC可有效提高鸡胚脑组织硒水平、脑GPx活性和GAP-43的表达,对脑IDⅡ活性没有影响。对正常鸡胚适量补硒可能会促进神经系统的生长和发育。