DNA sequence analysis of the apocytochrome b gene of Podospora anserina: a new family of intronic open reading frame

Summary The 5,969 by (base pair) DNA sequence of the apocytochrome b mitochondrial (mt) gene of race A Podospora anserina was located in a 8.5 Kbp region. This gene contained a 2,499 by subgroup IB and a 1,306 by subgroup ID intron as well as a 990 bp subgroup IB intron which is present in race A but not race s. The large subgroup IB intron and the race A specific IB intron both contained potential alternate splice sites which brought their open reading frames into phase with their upstream exon sequences. All three introns were compared with regard to their secondary structures and open reading frames to the other 30 group I introns in Podospora anserina, as well as to other fungal introns. We detected a new family of intronic ORFs comprising seven P. anserina introns, several N. crassa introns, as well as the T4td bacteriophage intron. Sequence similarities to intron-encoded endonucleases were noteworthy. The DNA sequences reported here and in the accompanying paper complete the analysis of race s and race A mitochondrial DNA.     

Prevalence of mitochondrial DNA mutations in childhood/congenital onset non-syndromal sensorineural hearing impairment

Genetic factors are the major causes of childhood hearing impairment. Whereas autosomal recessive mutations account for the majority of prelingual non-syndromic sensorineural hearing impairment (NSSHI), the relative contribution of mitochondrial DNA (mtDNA) mutations to childhood onset NSSHI has not been established.
We screened 202 subjects with congenital/childhood onset NSSHI, consisting of 110 sporadic cases, 75 sib pairs, and 17 families with affected subjects in more than one generation, in order to determine the prevalence of mtDNA mutations associated with NSSHI.
mtDNA mutations were found in three of 10 families (30%) in whom the affected members were related through the maternal lineage. One sporadic case (0.9%) was also found to have a known mtDNA mutation but none was found in the sib pairs.
Although the prevalence of mtDNA mutations was low in the group as a whole (2%), we suggest that screening should be considered in cases of childhood hearing impairment when it is progressive and particularly in families where transmission is compatible with maternal inheritance.


Keywords: mitochondrial DNA; point mutation; hearing impairment     

二氧化锗诱导L6成肌细胞株的MyoD基因表达

目的：探讨成肌细胞的MyoD基因在线粒体肌病发生发展中的作用。方法：采用二氧化锗(GeO2)处理大鼠的成肌细胞系L6，观察细胞形态的变化，利用MTT分析GeO2对成肌细胞的影响，用RT-PCR检测MyoD基因表达。结果：发现GeO2在损伤成肌细胞的同时，能够诱导MyoD基因的表达，表明MyoD基因在线粒体肌病的发生发展中起着重要作用。结论：MyoD基因表达的增强是线粒体肌病中骨骼肌萎缩的一个信号分子，MyoD基因表达有可能成为线粒体肌病检测的参考指标。     

一个遗传性非综合征型耳聋家系的突变分析

目的分析一个遗传性非综合征型耳聋家系的突变，并探讨缝隙连接蛋白beta2(gap junction protein beta 2，GJB2)基因235delC突变是否会加重线粒体A1555G突变导致的非综合征型耳聋症状。方法对一个母系遗传性非综合征型耳聋核心家系72个成员取外周血提取DNA，经聚合酶链反应扩增后，利用Alw26Ⅰ限制性内切酶酶切及直接测序验证，对其线粒体DNA突变进行研究；利用ApaⅠ限制性内切酶酶切及直接测序验证，筛查核心家系中GJB2基因235delC突变情况，并对GJB2基因235delC和线粒体A1555G突变的关系进行研究。结果在27名母系成员中均发现具有线粒体A1555G突变，呈母系遗传；具有耳聋表型的为21人(77．8％)，家族外显率高；所筛查的包括配偶在内的72名个体中，仅3例具有GJB2基因235delC杂合子突变，且均出现在母系成员中，但3例的耳聋表型却不同。结论线粒体A1555G突变是本家系耳聋遗传易感性的基础，在该家系中GJB2基因的235delC杂合子突变未加重线粒体A1555G突变导致的非综合征型耳聋。     

Mutation analysis in 16 patients with mtDNA depletion

Sixteen unrelated Southern European patients with the mitochondrial depletion syndrome (MDS) were analyzed for mutations in the TK2 and DGUOK genes. Three novel mutations were identified in TK2 (R183G, R254X, and 142insG). When we analyzed additional genes involved in the dNTPs pool, such as SLC25A19 (DNC) and NT5M (d-NT2), we did not detect mutations. The current study suggest that scanning the TK2, DGUOK, SLC25A19, and NT5M genes is likely to help about 10% of MDS families in terms of genetic counseling. Also, our findings indicate that genotype-phenotype correlations are not straightforward in MDS.     

线粒体凋亡途径参与表皮膜蛋白1诱导的细胞调亡

目的:探讨人表皮膜蛋白1(hEMP1)诱导细胞凋亡的信号途径.方法:构建包含hEMP1基因编码框的真核表达载体pcDNA3.1( )-EMP1,瞬转HEK293细胞后荧光倒置相差显微镜、流式细胞术检测Caspase-3、Caspase-8、Caspase-9的活力以及线粒体膜电位的变化.结果:荧光倒置相差显微镜、流式细胞术结果显示,过表达EMP1后细胞Caspase-3、Caspasc-9活力显著增强.线粒体膜电位改变增多,而Caspase-8的活力变化不明显.结论:线粒体凋亡途径参与hEMP1诱导的细胞凋亡.     

Localization,structure and polymorphism of two paralogous <Emphasis Type="Italic">Xenopus laevis</Emphasis> mitochondrial malate dehydrogenase genes

Two paralogous mitochondrial malate dehydrogenase 2 (Mdh2) genes of Xenopus laevis have been cloned and sequenced, revealing 95% identity. Fluorescence in-situ hybridization (FISH) combined with tyramide amplification discriminates both genes; Mdh2a was localized into chromosome q3 and Mdh2b into chromosome q8. One kb cDNA probes detect both genes with 85% accuracy. The remaining signals were on the paralogous counterpart. Introns interrupt coding sequences at the same nucleotide as defined for mouse. Restriction polymorphism has been detected in the first intron of Mdh2a, while the individual variability in intron 6 of Mdh2b gene is represented by an insertion of incomplete retrotransposon L1Xl. Rates of nucleotide substitutions indicate that both genes are under similar evolutionary constraints. X. laevis Mdh2 genes can be used as markers for physical mapping and linkage analysis.     

Ultrastructural histochemistry of mesotheliomas and adenocarcinomas in malignant effusions

The results of electron microscopic examination of cytologic specimens from six cases of mesothelioma and 10 cases of metastatic carcinoma of different origins are presented. The formation of cell clusters in malignant effusions from the two neoplasms has been thoroughly investigated: in mesotheliomas, cells had longer, more slender microvilli than in carcinomas and more abundant bundles of intermediate filaments; the central cavity often seen in the clusters frequently contained collagen and showed basement membrane production. The application of periodic acid-silver methenamine (PASM) and phosphotungstic acid (PTA) demonstrated a peculiar ultrastructural difference in cell coat staining in the two tumor types: in mesotheliomas, PTA and PASM were consistently negative along the outer surface of the cell aggregates, while carcinomas displayed a positive reaction either on the outer surface or on both inner and outer surfaces of the clusters. The diagnostic significance of the above-mentioned difference between the two neoplasms will require further investigation in a larger number of cases.     

薏苡附子败酱散对结肠癌细胞HCT116凋亡的影响及机制

目的：探究薏苡附子败酱散对结肠癌细胞HCT116凋亡的影响,并探讨其相关的细胞凋亡机制。方法：不同质量浓度(0.5、1、2、4、6、8、10、12、14、16 g·L^-1 )薏苡附子败酱散干预结肠癌细胞24、48、72 h,细胞增殖与活性检测-8(CCK-8)法检测细胞体外增殖的影响;设空白组、卡培他滨组(1.8 g·L^-1 )和薏苡附子败酱散组(6、10、14 g·L^-1 ),分别处理48 h,采用流式细胞技术检测细胞凋亡率,Hochest 33342荧光染色观察细胞凋亡形态,线粒体红色荧光探针(Mito-Tracker Red CMXRos)分析线粒体膜电位(MMP)变化,蛋白免疫印迹法(Western blot)检测线粒体凋亡途径相关蛋白B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、细胞色素C(Cyt C)、胱天蛋白酶(Caspase)-9、Caspase-3、活化的(cleaved) Caspase-9、cleaved Caspase-3的表达水平,实时荧光定量聚合酶链式反应(Real-time...     

L-Arginine Reduces Nitro-Oxidative Stress in Cultured Cells with Mitochondrial Deficiency

L-Arginine (L-ARG) supplementation has been suggested as a therapeutic option in several diseases, including Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like syndrome (MELAS), arguably the most common mitochondrial disease. It is suggested that L-ARG, a nitric oxide (NO) precursor, can restore NO levels in blood vessels, improving cerebral blood flow. However, NO also participates in mitochondrial processes, such as mitochondrial biogenesis, the regulation of the respiratory chain, and oxidative stress. This study investigated the effects of L-ARG on mitochondrial function, nitric oxide synthesis, and nitro-oxidative stress in cell lines harboring the MELAS mitochondrial DNA (mtDNA) mutation (m.3243A>G). We evaluated mitochondrial enzyme activity, mitochondrial mass, NO concentration, and nitro-oxidative stress. Our results showed that m.3243A>G cells had increased NO levels and protein nitration at basal conditions. Treatment with L-ARG did not affect the mitochondrial function and mass but reduced the intracellular NO concentration and nitrated proteins in m.3243A>G cells. The same treatment led to opposite effects in control cells. In conclusion, we showed that the main effect of L-ARG was on protein nitration. Lowering protein nitration is probably involved in the mechanism related to L-ARG supplementation benefits in MELAS patients.