湖南长沙及湘西泡状带绦虫分离株线粒体cox1基因的克隆及序列分析

目的研究湖南省长沙及湘西泡状带绦虫分离株线粒体细胞色素C氧化酶第1亚基（cox1）基因部分序列（pcox1）的遗传变异情况,并用peox1序列重构泡状带绦虫与其他带科绦虫的种群遗传关系。方法应用聚合酶链反应（PCR）扩增泡状带绦虫虫株的pcox1,应用Clusta1X1．81程序对序列进行比对。然后用Phylip3．67程序MP法和Mage4．0程序NJ法绘制种系发育树,并用Puzzle5．2程序构建最大似然树;同时利用DNAstar5．0中的Megalign程序进行同源性分析,并与GenBank^TM中已知泡状带绦虫相应基因序列进行比较分析。结果所获得的pcox1序列长度一致,均为343bp,与GenBank^TM公布的带科绦虫序列进行比较分析表明,湖南长沙分离株1和湘西分离株5与已知泡状带绦虫相应基因的相似性分别为99．4％和99．7％,与其它带科绦虫的相似性均小于90．0％;湖南长沙1和湘西分离株5与已知泡状带绦虫相应基因遗传距离分别为0．006和0．003,与其他带科绦虫的遗传距离均大于0．100。种系发育树表明,2个分离株与已知泡状带绦虫位于同一分枝,Bootstrap值在97％以上。结论由于泡状带绦虫pcox1序列种内相对保守,种问差异较大,故可作为种间遗传变异研究的标记,研究结果为进一步研究泡状带绦虫的群体遗传结构奠定了基础。     

MTO1 Mutations are Associated with Hypertrophic Cardiomyopathy and Lactic Acidosis and Cause Respiratory Chain Deficiency in Humans and Yeast

We report three families presenting with hypertrophic cardiomyopathy, lactic acidosis, and multiple defects of mitochondrial respiratory chain (MRC) activities. By direct sequencing of the candidate gene MTO1, encoding the mitochondrial‐tRNA modifier 1, or whole exome sequencing analysis, we identified novel missense mutations. All MTO1 mutations were predicted to be deleterious on MTO1 function. Their pathogenic role was experimentally validated in a recombinant yeast model, by assessing oxidative growth, respiratory activity, mitochondrial protein synthesis, and complex IV activity. In one case, we also demonstrated that expression of wt MTO1 could rescue the respiratory defect in mutant fibroblasts. The severity of the yeast respiratory phenotypes partly correlated with the different clinical presentations observed in MTO1 mutant patients, although the clinical outcome was highly variable in patients with the same mutation and seemed also to depend on timely start of pharmacological treatment, centered on the control of lactic acidosis by dichloroacetate. Our results indicate that MTO1 mutations are commonly associated with a presentation of hypertrophic cardiomyopathy, lactic acidosis, and MRC deficiency, and that ad hoc recombinant yeast models represent a useful system to test the pathogenic potential of uncommon variants, and provide insight into their effects on the expression of a biochemical phenotype.     

Decreased Cytochrome c Oxidase Subunit VIIa in Aged Rat Heart Mitochondria: Immunocytochemistry

Aging decreases oxidative phosphorylation through cytochrome oxidase (COX) in cardiac interfibrillar mitochondria (IFM) in 24‐month old (aged) rats compared to 6‐month old adult Fischer 344 rats, whereas subsarcolemmal mitochondria (SSM) located beneath the plasma membrane remain unaffected. Immunoelectron microscopy (IEM) reveals in aged rats a 25% reduction in cardiac COX subunit VIIa in cardiac IFM, but not in SSM. In contrast, the content of subunit IV remains unchanged in both SSM and IFM, irrespective of age. These subunits are localized mainly on cristae membranes. In contrast, semi‐quantitative immunoblotting, which detects denatured protein, indicates that the content of COX VIIa is similar in IFM and SSM from both aged and adult hearts. IEM provides a sensitive method for precise localizing and quantifying specific mitochondrial proteins. The lack of immunoreaction of COX VIIa subunit by IEM in aged IFM is not explained by a reduction in protein, but rather by a masking phenomenon or by an in situ change in protein structure affecting COX activity. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Novel Mutations in SCO1 as a Cause of Fatal Infantile Encephalopathy and Lactic Acidosis

Isolated cytochrome c oxidase (COX) deficiency is a common cause of mitochondrial disease, yet its genetic basis remains unresolved in many patients. Here, we identified novel compound heterozygous mutations in SCO1 (p.M294V, p.Val93*) in one such patient with fatal encephalopathy. The patient lacked the severe hepatopathy (p.P174L) or hypertrophic cardiomyopathy (p.G132S) observed in previously reported SCO1 cases, so we investigated whether allele‐specific defects in SCO1 function might underlie the genotype–phenotype relationships. Fibroblasts expressing p.M294V had a relatively modest decrease in COX activity compared with those expressing p.P174L, whereas both SCO1 lines had marked copper deficiencies. Overexpression of known pathogenic variants in SCO1 fibroblasts showed that p.G132S exacerbated the COX deficiency, whereas COX activity was partially or fully restored by p.P174L and p.M294V, respectively. These data suggest that the clinical phenotypes in SCO1 patients might reflect the residual capacity of the pathogenic alleles to perform one or both functions of SCO1.     

Mutation in mitochondrial complex IV subunit COX5A causes pulmonary arterial hypertension,lactic acidemia,and failure to thrive

COX5A is a nuclear‐encoded subunit of mitochondrial respiratory chain complex IV (cytochrome c oxidase). We present patients with a homozygous pathogenic variant in the COX5A gene. Clinical details of two affected siblings suffering from early‐onset pulmonary arterial hypertension, lactic acidemia, failure to thrive, and isolated complex IV deficiency are presented. We show that the variant lies within the evolutionarily conserved COX5A/COX4 interface domain, suggesting that it alters the interaction between these two subunits during complex IV biogenesis. In patient skin fibroblasts, the enzymatic activity and protein levels of complex IV and several of its subunits are reduced. Lentiviral complementation rescues complex IV deficiency. The monomeric COX1 assembly intermediate accumulates demonstrating a function of COX5A in complex IV biogenesis. A potential therapeutic lead is demonstrated by showing that copper supplementation leads to partial rescue of complex IV deficiency in patient fibroblasts.     

基于靶基因文库的高通量测序平台建立肥厚型心肌病患者致病基因突变检测方法

目的 基于靶基因文库,应用下一代半导体高通量测序平台,建立快速、 准确的肥厚型心肌病(hypertrophic cardiomyopathy,HCM)常见致病基因突变检测方法 ,有利于HCM患者的早期预防及临床分子诊断.方法 选择国内外公认的与HCM致病相关的常见基因(MYH7、MYBPC3、TNNT2、TNNI3、A...     

Effects of Transient and Prolonged Flashing Light Stimulation on the Cytochrome Oxidase Module System in Layer IV of the Primary Visual Cortex of Kittens

Cytochrome oxidase spots in layer IV of field 17 of the primary visual cortex were studied in kittens aged 33, 49, and 93 days, stimulated with a light flashing at a frequency of 15 Hz. The kittens of one group received stimulation from the moment of eye opening until euthanasia (prolonged stimulation); other groups received stimulation for eight days starting from ages 26, 42, or 85 days (transient stimulation), again until euthanasia. Both types of stimulation were found not to alter the geometrical characteristics of cytochrome oxidase spots, but led to significant increases in the contrast of spots located in the splenial gyrus. Increases in spot contrast in the lateral gyrus occurred only after prolonged stimulation to age 93 days or after transient stimulation from age 26 days to age 33 days. Thus, stimulation of kittens of different ages with a light flashing at a frequency of 15 Hz led to structural-metabolic changes in the primary visual cortex. These changes were apparent to different extents in areas of the cortex responsible for central and peripheral vision. This may be explained, firstly, by the predominant activation of the Y conducting channel of the visual system and, secondly, by the increase in dominance of the contralateral input to the primary visual cortex. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 94, No. 5, pp. 557–565, May, 2008.     

Clinical hematological and biochemical parameters in Swiss,BALB/c,C57BL/6 and B6D2F1 Mus musculus

BackgroundAnimal models are widely used in scientific research in order to obtain information from a whole organism under a specific set of experimental conditions. Various lineages of mice have been used to investigate diseases and new therapeutic strategies, and, consequently, hematological and biochemical tests in these laboratory animals are essential to validate scientific studies. Our study seeks to establish reference values for hematological and biochemical parameters of four lineages of mice.MethodsWe evaluated the hematological and biochemical profiles of 20 males and 20 females from the lineages Swiss (heterogeneous), BALB/c and C57BL/6 (isogenic), and B6D2F1 (hybrid), totaling 160 mice. Analysis were standardized using the systems pocH‐100iV Diff™ for 19 hematological parameters and VITROS^® 350 for 12 biochemical parameters.ResultsResults are shown as means and standard deviation, grouped by lineage and genre. Comparing the values obtained in this study with the values from previous studies, some variations were detected, which could be explained by differences in methodologies or individual variability.ConclusionThus our study shows that knowledge and disclosure of the values of physiological parameters of laboratory animals is necessary, and emphasises the importance of considering variations influenced by gender, lineage and genotype in the choice of the best experimental model.     

Expression and clinical significance of cytochrome c oxidase subunit IV in colorectal cancer patients

Introduction

Previous studies have demonstrated that the expression of cytochrome c oxidase (COX) subunits encoded by mitochondrial DNA is elevated in colorectal cancer (CRC). However, the expression of nuclear DNA-encoded COX IV and its clinical significance have not yet been investigated in CRC.

Material and methods

We examined COX IV expression in paired CRC samples (cancer and pericancerous tissues) by quantitative real-time polymerase chain reaction (qPCR), Western blot and immunohistochemical staining and analyzed its clinical significance.

Results

qPCR and Western blot analyses showed that COX IV expression was significantly elevated at both the mRNA (p = 0.05) and protein levels in CRC tissue samples when compared with those in paired pericancerous tissues. Immunohistochemistry also revealed that COX IV expression was significantly increased in CRC tissues (p < 0.001). Association analyses showed that there was no significant association between COX IV expression and clinical parameters of CRC patients except for gender (p = 0.017). Moreover, we did not find any association between COX IV expression and overall survival or recurrence-free survival of CRC patients. Further analysis showed no significant relationship between the expression of COX IV and proliferating cell nuclear antigen (PCNA), a marker of cell proliferation.

Conclusions

Our findings suggest that elevated COX IV expression may play an important role in colorectal carcinogenesis, but not in progression, which warrants further investigation in future studies.     

Mutations in the SURF1 gene associated with Leigh syndrome and cytochrome C oxidase deficiency

Cytochrome c oxidase (COX) deficiency is one of the major causes of Leigh Syndrome (LS), a fatal encephalopathy of infancy or childhood, characterized by symmetrical lesions in the basal ganglia and brainstem. Mutations in the nuclear genes encoding COX subunits have not been found in patients with LS and COX deficiency, but mutations have been identified in SURF1. SURF1 encodes a factor involved in COX biogenesis. To date, 30 different mutations have been reported in 40 unrelated patients. We aim to provide an overview of all known mutations in SURF1, and to propose a common nomenclature. Twelve of the mutations were insertion/deletion mutations in exons 1, 4, 6, 8, and 9; 10 were missense/nonsense mutations in exons 2, 4, 5, 7, and 8; and eight were detected at splicing sites in introns 3 to 7. The most frequent mutation was 312_321del 311_312insAT which was found in 12 patients out of 40. Twenty mutations have been described only once. We also list all polymorphisms discovered to date.     

Mutations in the glucose-6-phosphatase-alpha (G6PC) gene that cause type Ia glycogen storage disease

Glucose-6-phosphatase-alpha (G6PC) is a key enzyme in glucose homeostasis that catalyzes the hydrolysis of glucose-6-phosphate to glucose and phosphate in the terminal step of gluconeogenesis and glycogenolysis. Mutations in the G6PC gene, located on chromosome 17q21, result in glycogen storage disease type Ia (GSD-Ia), an autosomal recessive metabolic disorder. GSD-Ia patients manifest a disturbed glucose homeostasis, characterized by fasting hypoglycemia, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, lactic acidemia, and growth retardation. G6PC is a highly hydrophobic glycoprotein, anchored in the membrane of the endoplasmic reticulum with the active center facing into the lumen. To date, 54 missense, 10 nonsense, 17 insertion/deletion, and three splicing mutations in the G6PC gene have been identified in more than 550 patients. Of these, 50 missense, two nonsense, and two insertion/deletion mutations have been functionally characterized for their effects on enzymatic activity and stability. While GSD-Ia is not more prevalent in any ethnic group, mutations unique to Caucasian, Oriental, and Jewish populations have been described. Despite this, GSD-Ia patients exhibit phenotypic heterogeneity and a stringent genotype-phenotype relationship does not exist.     

扩张型心肌病IL－6及IL－1活性的初步研究

应用ＭＴＴ比色法及ＣｏｎＡ活化小鼠胸腺细胞增殖法检测了３０例扩张型心肌病（ＤＣＭ）患者及２０例正常人（ＮＣ）的血清白细胞介素６（ＩＬ－６）、白细胞介素１（ＩＬ－１）的活性，结果发现ＤＣＭ患者ＩＬ－６及ＩＬ－１活性均明显高于ＮＣ组（Ｐ＜０．００１，Ｐ＜０．０１）。提示：ＩＬ－６及ＩＬ－１功能紊乱参与了ＤＣＭ的病理过程。     

Biallelic variants in COX4I1 associated with a novel phenotype resembling Leigh syndrome with developmental regression,intellectual disability,and seizures

Autosomal recessive COX4I1 deficiency has been previously reported in a single individual with a homozygous pathogenic variant in COX4I1, who presented with short stature, poor weight gain, dysmorphic features, and features of Fanconi anemia. COX4I1 encodes subunit 4, isoform 1 of cytochrome c oxidase. Cytochrome c oxidase is a respiratory chain enzyme that plays an important role in mitochondrial electron transport and reduces molecular oxygen to water leading to the formation of ATP. Defective production of cytochrome c oxidase leads to a variable phenotypic spectrum ranging from isolated myopathy to Leigh syndrome. Here, we describe two siblings, born to consanguineous parents, who presented with encephalopathy, developmental regression, hypotonia, pathognomonic brain imaging findings resembling Leigh‐syndrome, and a novel homozygous variant on COX4I1, expanding the known clinical phenotype associated with pathogenic variants in COX4I1.     

Morphofunctional characteristic of mast cells in BALB/c and C57Bl/6 mice during cold exposure

Mast cells of the mesentery and subcutaneous tissue in BALB/c and C57Bl/6 mice were studied after single and repeated cold exposure (−20°C, 3 min). Immediate adaptive reactions of mast cells in BALB/c and C57Bl/6 mice did not differ after single cold exposure and were manifested in increased degranulation. Repeated cold exposure of BALB/c mice was followed by an adaptive reaction, which included an increase in the count of mast cells in subcutaneous tissue and normalization of the degranulation index. In C57Bl/6 mice the count of mast cells in subcutaneous tissue decreased, while the degranulation index remained high. These changes reflect the disadaptive response of mast cells to repeated cold exposure. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 138, No. 8, pp. 207–209, August, 2004     

Morphofunctional Characteristic of Mast Cells in BALB/c and C57Bl/6 Mice during Cold Exposure

Mast cells of the mesentery and subcutaneous tissue in BALB/c and C57Bl/6 mice were studied after single and repeated cold exposure (-20 degrees C, 3 min). Immediate adaptive reactions of mast cells in BALB/c and C57Bl/6 mice did not differ after single cold exposure and were manifested in increased degranulation. Repeated cold exposure of BALB/c mice was followed by an adaptive reaction, which included an increase in the count of mast cells in subcutaneous tissue and normalization of the degranulation index. In C57Bl/6 mice the count of mast cells in subcutaneous tissue decreased, while the degranulation index remained high. These changes reflect the disadaptive response of mast cells to repeated cold exposure.     

Comparison of focally induced epileptiform activities in C57BL/6 and BALB/c mice by using in vivo EEG recording

Epilepsy characterized by repeated seizures is influenced by genetic factors. Seizure response of inbred mouse strains changes depending on the variety of stimuli including chemical (e.g., pentylenetetrazole, nicotine, cocaine, NMDA, kainate), physical (e.g., auditory) or electrical. In this study, we compared the susceptibilities of C57BL/6 and BALB/c mice strains to penicillin induced epileptiform activity (a focally induced, experimental epilepsy model), by analyzing the spike onset latency, spike amplitude and spike frequency. The power spectrums of baseline EEGs were also investigated. We found no alterations of spike onset latencies between the C57 and BALB mice. However, spike amplitudes and spike frequencies were found to be higher in BALB mice than C57 mice. With regard to EEG power spectrum, absolute power of investigated bands was not different between the two strains. Interestingly, the relative power of all investigated bands differed significantly between two strains. The relative power of delta and theta was lower whereas relative power of alpha, beta and gamma was higher in C57 mice compared to BALB mice. In conclusion, our findings showed that BALB mice are more sensitive to penicillin induced epileptiform activity when compared to C57 strain.     

牛MBP诱导BALB/c和C57BL/6小鼠实验性变态性脑脊髓炎样反应的研究

为了进一步研究实验性变态反应性脑脊髓炎(EAE)动物模型,采用从新鲜牛脊髓中提取的MBP免疫诱导非敏感品系BALB/c小鼠和C57BL/6小鼠,通过对实验动物的症状、中枢神经系统的病理改变及细胞因子水平变化的检测,较成功地建立了C57BL/6、BALB/c小鼠的EAE模型,实验方法稳定可靠。     

Comparative Characterization of Cytokine Production by Concanavalin A-Activated Splenocytes from BALB/c and C57Bl/6 Mice after Cold Exposure

The level of cytokines produced by ConA activated splenocytes was studied in male BALB/c and C57Bl/6 mice after single and repeated cold exposure (–20°C, 3 min). Single cold exposure significantly decreased IL-2, -3, -4, -5, -10, -12, IFN- production in BALB/c mice and decreased IL-2 content and increased TNF- level in C57Bl/6 mice. Repeated cold exposure normalized the content of IL-2, -4, -10, -12, and IFN- in BALB/c mice, which reflects the development of adaptive immune reactions. In C57Bl/6 mice IL-2, -3, -5, -10, -12, and IFN- production remained significantly decreased, which attested to dysadaptive processes.__________Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 139, No. 2, pp. 188–190, February, 2005     

The role of interleukine-12 in protection induced by CpG ODN against Listeria monocytogenes in BALB/c and C57BL/6

Synthetic oligodeoxynucleotides containing CpG motifs have several immune effects such as cytokine production in normal mice. In this study, we demonstrated the protective effect of CpG ODN against Listeria monocytogenes in BALB/c and C57BL/6. With a single dose of 40 μg/mouse of CpG ODN 48 h before bacterial challenge, protection was achieved in both strains of mice based on survival rates compared with controls. Serum IL-12 from each mouse was measured by using enzyme-linked immunosorbent assay (ELISA), at day 0 (48 h after CpG treatment) and at days 5, 11, and 15 after bacterial challenge. It was shown that serum IL-12 was only elevated at day 0 in BALB/c mice. However, for C57BL/6 mice, IL-12 was elevated at days 0, 5, and 11. These data support the hypothesis that CpG DNA motifs activate protective innate immune defenses.