β‐Sitosterol Enhances Cellular Glutathione Redox Cycling by Reactive Oxygen Species Generated From Mitochondrial Respiration: Protection Against Oxidant Injury in H9c2 Cells and Rat Hearts

Herba Cistanches (Cistanche deserticola Y. C. Ma) is a ‘Yang‐invigorating’ tonic herb in Chinese medicine. Preliminary chemical analysis indicated that β‐sitosterol (BS) is one of the chemical constituents in an active fraction of Herba Cistanches. To investigate whether BS is an active ingredient of Herba Cistanches, the effects of BS on H9c2 cells and rat hearts were examined. The results indicated that BS stimulated the mitochondrial ATP generation capacity in H9c2 cells, which was associated with the increased production of mitochondrial reactive oxygen species. BS also stimulated mitochondrial state 3 and state 4 respiration, with the resultant decrease in coupling efficiency. BS produced an up‐regulation of cellular glutathione redox cycling and protected against hypoxia/reoxygenation‐induced apoptosis in H9c2 cells. However, the protective effect of BS against myocardial ischemia/reperfusion injury was seen in female but not male rats ex vivo. The cardioprotection afforded by BS was likely mediated by an up‐regulation of mitochondrial glutathione redox cycling in female rat hearts. In conclusion, the ensemble of results suggests that BS is an active ingredient of Herba Cistanches. The gender‐dependent effect of BS on myocardial protection will further be investigated. Copyright © 2013 John Wiley & Sons, Ltd.     

喉癌细胞线粒体去极化和凋亡研究

目的线粒体膜电位的缺失是线粒体凋亡途径的早期事件。我们用线粒体膜电位去极化剂(valinomycin)处理3个喉癌细胞系,研究它们的凋亡反应和细胞色素C释放的能力。方法 3个喉癌细胞系和对照细胞系HL-60经过valinomycin处理后,用细胞膜电位探针JC-1染色和凋亡特异性探针annexinV染色,用流式细胞仪分别测量它们的线粒体膜电位和凋亡率。Western印记用来检测细胞浆内的细胞色素C的释放。结果在经过24小时的处理后所有细胞均显示完全的线粒体去极化,只有对照细胞HL-60出现凋亡和细胞色素C释放,而喉癌细胞系没有出现。结论喉癌细胞系表现出线粒体膜电位去极化诱导的凋亡途径的缺陷,线粒体膜电位去极化而没有细胞色素C的释放。线粒体凋亡途径的缺陷是喉癌的一个特点。     

New strain typing method with Sporothrix schenckii using mitochondrial DNA and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique

The complete sequences of mitochondrial DNA (mtDNA) from two strains of different genotypes, American Type Culture Collection 10268 of mtDNA type 1 and KMU2025 of mtDNA type 4, were determined. These are circular molecules, 27 125 and 26 095 bp in length, respectively. The greatest difference between the two strains was found in the region encompassed by atp9 and cox2 genes, which was amplified with polymerase chain reaction (PCR) and used for preliminary restriction fragment length polymorphism (RFLP) analysis. Eight isolates of five mtDNA types were used and RFLP patterns obtained with the restriction enzyme AseI showed that this method seems to have greater discrimination power than the other PCR-RFLP typing method using internal transcribed spacer regions of nuclear DNA.     

LACTATE STRESS TESTING IN SPORADIC AMYOTROPHIC LATERAL SCLEROSIS

Mitochondrial dysfunction is frequently observed in ALS. Mitochondrial dysfunction may result in increased serum lactate at rest or low levels of exercise, being used for diagnostic purposes. The study investigated if resting-lactate-determination is superior to lactate-stress-testing (LST) in demonstrating mitochondrial dysfunction in ALS. Included were 15 ALS patients, 4 women, 11 men, aged 37–72. Severity of the disease was assessed by the Norris-score. The control group comprised 66 healthy subjects, 40 women, 26 men, aged 36–76. Serum lactate was determined before, three times during, and once after a constant workload with 30 W on a bicycle ergometer. According to the EIEscorial criteria 8 patients had definite, 4 probable, 3 possible ALS. Resting lactate was increased in 2 patients, 1 with definite and 1 with possible ALS. The LST was abnormal in 5 patients with definite, 1 with probable and 1 with possible ALS. The mean Norris-score was 67.8 in patients with abnormal LST and 74.6 in patients with normal LST. In conclusion, the LST is more suitable than resting-lactate-determination in demonstrating mitochondrial dysfunction in ALS. The LST suggests mitochondrial dysfunction in half of the ALS patients. Mitochondrial dysfunction in ALS is related to the clinical severity of the disease.     

Value of dynamic 31P magnetic resonance spectroscopy technique in in vivo assessment of the skeletal muscle mitochondrial function in type 2 diabetes

Background  Phosphorous magnetic resonance spectroscopy (³¹P-MRS) has been successfully applied to study intracellular membrane compounds and high-energy phosphate metabolism. This study aimed to evaluate the capability of dynamic ³¹P-MRS for assessing energy metabolism and mitochondrial function in skeletal muscle from type 2 diabetic patients.

Methods  Dynamic ³¹P-MRS was performed on 22 patients with type 2 diabetes and 26 healthy volunteers. Spectra were acquired from quadriceps muscle while subjects were in a state of rest, at exercise and during recovery. The peak areas of inorganic phosphate (Pi), phosphocreatine (PCr), and adenosine triphosphate (ATP) were measured. The concentration of adenosine diphosphate (ADP) and the intracellular pH value were calculated from the biochemistry reaction equilibrium. The time constant and recovery rates of Pi, PCr, and ADP were analyzed using exponential curve fitting.

Results  As compared to healthy controls, type 2 diabetes patients had significantly lower skeletal muscle concentrations of Pi, PCr and β-ATP, and higher levels of ADP and Pi/PCr. During exercise, diabetics experienced a significant Pi peak increase and PCr peak decrease, and once the exercise was completed both Pi and PCr peaks returned to resting levels. Quantitatively, the mean recovery rates of Pi and PCr in diabetes patients were (10.74±1.26) mmol/s and (4.74±2.36) mmol/s, respectively, which was significantly higher than in controls.

Conclusions  Non-invasive quantitative ³¹P-MRS is able to detect energy metabolism inefficiency and mitochondrial function impairment in skeletal muscle of type 2 diabetics.

     

A Study on the D-loop Region of Mitochondrial DNA （mtDNA） Mutation in Cervical Carcinomas

Objective Background-study on genesis and development of tumor is mainly concentrated on gene mutation in nucleus. In recent years, however, the role of mitochondrial DNA （mtDNA） mutation in tumor genesis has been given more and more attention, which is the only extra-nucleus DNA in cells of higher animals. Carcinoma of the uterine cervix is a common tumor in gynecology, but there are few reports of mtDNA mutation in this area. The focus of this study was to investigate the mtDNA mutation in tumor tissues of cervical carcinomas and their relationship to tumorigenesis and tumor development. Methods The D-loop region of 24 cervical carcinomas together with the adjacent normal tissues were amplified by PCR and sequenced. Results Among the 24 cervical carcinomas, 30 mutations in 9 patients＇ specimen were identified with the mutations rate of 37.5 96 （9/24）. There were 8 microsatellite instabilities among the mutations and 13 new polymorphisms which were not reported previously in the Genbank. Conclusions The D-loop region of mitochondrial DNA is a highly polymorphoric and mutable region and the mutation rate is relatively high in patients with cervical carcinomas.     

The biology,function, and applications of exosomes in cancer

Exosomes are cell-derived nanovesicles with diameters from 30 to 150 nm, released upon fusion of multivesicular bodies with the cell surface. They can transport nucleic acids, proteins, and lipids for intercellular communication and activate signaling pathways in target cells. In cancers, exosomes may participate in growth and metastasis of tumors by regulating the immune response, blocking the epithelial–mesenchymal transition, and promoting angiogenesis. They are also involved in the development of resistance to chemotherapeutic drugs. Exosomes in liquid biopsies can be used as non-invasive biomarkers for early detection and diagnosis of cancers. Because of their amphipathic structure, exosomes are natural drug delivery vehicles for cancer therapy.     

线粒体基因组D-loop区基因多态性与年龄及IVF-ET结局相关性

目的:研究线粒体基因组D-loop区基因的变异位点与年龄及IVF-ET或ICSI结局的相关性。方法:采用聚合酶链反应(polymerase chain reaction,PCR)和基因片段直接测序方法测定检测167例行IVF-ET或ICSI治疗患者的外周血线粒体基因组D-loop区nt.128～599区域基因变异情况,比较不同年龄组(A组:<35岁,B组:≥35岁)变异位点、年龄及IVF-ET或ICSI结局之间的关系。结果:共有20个变异位点,其中5个为新发现的变异位点。B组的变异位点数(3.8±1.8)显著多于A组的2.6±1.3,其中nt.332C→T在A组发生率显著低于B组(2.2%vs12.2%,P=0.012),且变异位点数量与年龄间存在线性相关,相关系数为0.333。nt.332C→T、nt.249delA和nt.309del/inC三者的变异组年龄显著高于非变异组,nt.309del/inC变异组的累积胚胎平均评分显著低于非变异组(12.9±0.5vs15.5±0.7,P=0.013),nt.249 delA变异组的受精率及累积胚胎平均评分显著低于非变异组(66.86±27.28%vs75.63±21.42%,P=0.046)(12.0±0.7vs14.7±0.5,P=0.013),变异位点数量与受精率及累积胚胎平均评分间存在线性相关。结论:线粒体基因组D-loop区变异位点发生率与年龄相关,且影响IVF-ET/ICSI的受精率及胚胎质量。     

急性胰腺炎大鼠腺泡细胞XIAP和Smac蛋白的表达

目的：探讨急性胰腺炎（AP）大鼠腺泡细胞中X连锁凋亡抑制蛋白（XIAP）及半胱天冬酶caspase的第二个线粒体激活因子/低等电点的IAP直接结合蛋白（Smac/DIABLO）的表达及其与AP严重程度的关系。方法：通过胆胰管逆行注射不同浓度的牛磺胆酸钠,制备大鼠急性水肿型胰腺炎（AEP）和急性坏死型胰腺炎（ANP）模型,同时制备假手术组为对照组。分别在模型制作3、6和12h取各组大鼠胰腺组织,对其进行病理学评分,并测定血清淀粉酶;应用免疫组织化学和蛋白免疫印迹法检测大鼠胰腺组织中XIAP和Smac蛋白的表达水平。结果：与对照组比较,AEP和ANP组大鼠胰腺组织在各个时间点均有不同程度的病理损害,且血清淀粉酶明显升高（P〈0.01）,其中ANP组升高程度尤为明显（P〈0.01）。免疫组织化学染色显示,XIAP和Smac蛋白在大鼠正常胰腺组织腺泡细胞中均呈弱阳性表达,ANP组XIAP的表达随模型制作后时间的延长而增强,12h时比对照组和AEP组明显增强,AEP组Smac蛋白的表达随时间延长而增强,12h时比ANP组及对照组增强。Westernblot结果显示造模后各时间点对照组XIAP和Smac蛋白表达比较差异无统计学意义（P〉0.05）,3hAEP组XIAP表达较ANP组及对照组明显增强（P〈0.01）,6、12h时ANP组XIAP表达比AEP组和对照组明显增强（P〈0.01）。3h时各组Smac表达差异无统计学意义（P〉0.05）,6、12hAEP组Smac蛋白表达比ANP组和对照组明显增加（P〈0.01）。结论：AP病情轻重程度与XIAP及Smac蛋白表达有关。病情轻者XIAP表达无显著变化,Smac蛋白表达逐渐升高;病情重者XIAP表达逐渐升高,Smac蛋白表达则先升后降。XIAP和Smac蛋白的表达变化在AP的发病中具有一定作用。