OBJECTIVETo introduce a new method for detecting mitochondrial permeability transition pore (PTP) opening with flow cytometry using the resveratrol-inducing PTP opening model.

METHODSMitochondria were isolated from rat livers and selectively labeled with nonyl acridine orange. The mitochondrial membrane potential was detected using flow cytometry with TMRE (tetramethylrhodamine, ethyl ester) labeling. PTP opening induced by resveratrol was represented by the changes of mitochondrial side-scattering (SSC) detected by flow cytometry.

RESULTSFlow cytometry was capable of defining the purity of the mitochondria isolated. The fluorescence intensities and SSC of the mitochondria were decreased after resveratrol treatment, indicating that resveratrol could induce PTP opening. Ciclosporin A inhibited resveratrol-induced PTP opening.

CONCLUSIONFlow cytometric analysis allows accurate and convenient detection of mitochondrial membrane potential, mitochondrial swelling and PTP opening.

Animals ; Apoptosis ; Flow Cytometry ; Membrane Potential, Mitochondrial ; genetics ; Mitochondria, Liver ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Rats ; Rhodamines

OBJECTIVETo investigate the role of mitofusin-2 gene (mfn2) in apoptosis in human breast carcinoma cell line MCF-7 cells after in vitro transfection.

METHODSpEGFP mfn2 was transfected by sofast in vitro. Expression of GFP was observed by Western blot, and the MCF-7 cell proliferation was measured by MTT and cell counting. Apoptosis in MCF-7 cells was observed in annexin-V/PI and chondrosome transmembrane potential of MCF-7 marked in JC-1 by FCM. The Ultrastructure of cells was observed by transmission electron microscopy.

RESULTSThe stable expression of GFP in MCF-7 cells was confirmed by Western blot. Mfn2 significantly inhibited cell proliferation, revealed by MTT, and decrease chondrosome transmembrane potential. Exogenous mfn2 gene significantly induced apoptosis. The apoptotic rate was increased from 3.6% to 16.0% (P < 0.05). Mfn2 gene induced break down and loss of mitochondrial cristae, and rarefaction of mitochondrial ground substance. Swollen mitochondria intensely aggregated around the cell nuclei.

CONCLUSIONMfn2 can strongly induce apoptosis in MCF-7 cells, which may be associated with decrease of mitochondrial transmembrane potential.

Apoptosis ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Female ; GTP Phosphohydrolases ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Membrane Potential, Mitochondrial ; Membrane Proteins ; genetics ; metabolism ; Mitochondria ; ultrastructure ; Mitochondrial Proteins ; genetics ; metabolism ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVE: To explore the genetic etiology of a Chinese pedigree affected with infantile hepatitis syndrome. METHODS: Genes associated with liver diseases subjected to high-throughput sequencing. Candidate variants were validated by Sanger sequencing of the proband and his parents. The pathogenicity of the variants was analyzed through bioinformatic analysis. RESULTS: High-throughput sequencing revealed that the proband has harbored c.182T>C (p.F61S) and c.293C>T (p.P98L) variants of the MPV17 gene, which were verified by Sanger sequencing to be inherited from his parents. The variant c.182T>C (p.F61S) was unreported previously and predicted to be likely pathogenic by bioinformatic analysis. CONCLUSION The proband was caused by the compound heterozygous variations of MPV17 gene including c.182T>C (p.F61S) and c.293C>T (p.P98L). Discovery of the novel variant has enriched the spectrum of pathogenic variants of the MPV17 gene.
China ; DNA, Mitochondrial/genetics* ; Female ; Genetic Testing ; Humans ; Membrane Proteins/genetics* ; Metabolism, Inborn Errors/genetics* ; Mitochondrial Proteins/genetics* ; Mutation ; Pedigree ; Pregnancy ; Prenatal Diagnosis ; Syndrome

Mitochondrial morphology is dynamically regulated by forming small, fragmented units or interconnected networks, and this is a pivotal process that is used to maintain mitochondrial homeostasis. Although dysregulation of mitochondrial dynamics is related to the pathogenesis of several human diseases, its molecular mechanism is not fully elucidated. In this study, we demonstrate the potential role of miR-27 in the regulation of mitochondrial dynamics. Mitochondrial fission factor (MFF) mRNA is a direct target of miR-27, whose ectopic expression decreases MFF expression through binding to its 3'-untranslated region. Expression of miR-27 results in the elongation of mitochondria as well as an increased mitochondrial membrane potential and mitochondrial ATP level. Our results suggest that miR-27 is a novel regulator affecting morphological mitochondrial changes by targeting MFF.
3' Untranslated Regions ; Cell Line ; Gene Expression Regulation ; Humans ; Membrane Potential, Mitochondrial ; Membrane Proteins/*genetics ; MicroRNAs/*metabolism ; Mitochondria/*genetics/*metabolism ; *Mitochondrial Dynamics ; Mitochondrial Proteins/*genetics ; *Protein Biosynthesis ; RNA, Messenger/genetics/metabolism

Glycerol is the main byproduct in ethanol production by Saccharomyces cerevisiae. In order to improve ethanol yield and the substrate conversion, a cassette about 4.5 kb for gene homologous recombination, gpd2Δ::PGK1(PT)-POS5-HyBR, was constructed and transformed into the haploid strain S. cerevisiae S1 (MATa) to replace the GPD2 gene by POS5 gene. The NADH kinase gene POS5 was successfully over expressed in the recombinant strain S. cerevisiae S3. Comparing with the parent strain, the recombinant strain S. cerevisiae S3 exhibited an 8% increase in ethanol production and a 33.64% decrease in glycerol production in the conical flask fermentation with an initiatory glucose concentration of 150 g/L. Overexpression of NADH kinase gene seems effective in reducing glycerol production and increasing ethanol yield.
Ethanol ; chemistry ; Fermentation ; Glycerol ; chemistry ; Industrial Microbiology ; Mitochondrial Proteins ; genetics ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism

OBJECTIVETo investigate the abundance of metabolic proteins in adult and fetal human livers.

METHODSAdult liver homogenate proteins, fetal liver homogenate proteins, adult liver mitochondrial proteins and fetal liver mitochondrial proteins were obtained from fetal or adult liver tissues and examined using the antibody microarrays containing 19 liver monoclonal mitochondrial antibodies. The protein expression abundances were compared among the 4 protein fractions and the pathways related to protein metabolisms were explored.

RESULTSIn adult liver mitochondria, aldehyde oxidase and carbonyl reductase were up-regulated by 2.6 and 1.7 folds, respectively, whereas corticosteroid 11-beta-dehydrogenase isozyme 1, epoxide hydrolase 1 and fibrinogen beta chain protein were down-regulated by 1.7, 1.9 and 2.2 folds, respectively, compared to those in fetal liver mitochondria. The abundance of epoxide hydrolase 1 and glutathione transferase omega-1 was significantly different between adult and fetal liver homogenate samples.

CONCLUSIONOur results demonstrate a clear difference in the expression profiles of metabolic proteins in the liver between adults and human fetuses to allow a better understanding of the occurrence and development of the metabolic proteins and the identification of markers of liver metabolism.

Adult ; Antibodies ; genetics ; metabolism ; Fetus ; metabolism ; Humans ; Liver ; embryology ; metabolism ; Mitochondria, Liver ; metabolism ; Mitochondrial Proteins ; metabolism ; Protein Array Analysis

OBJECTIVETo observe the effect of survivin antisense oligonucleotides (ASODN) on survivin and Smac gene expression in ovarian cancer SKOV3 cells and explore the role of survivin and Smac genes in ASODN-induced ovarian cancer cell cycle alteration and apoptosis and its molecular mechanisms.

METHODSSurvivin ASODN was introduced via Lipofectamine(TM)2000 into SKOV3 cells, whose growth activity was detected subsequently with MTT assay. The apoptosis index, cell cycle and changes in survivn and Smac protein expression were determined using flow cytometry. The changes in survivn and Smac mRNA expression were detected by RT-PCR.

RESULTSIn comparison with the control cells, cells transfected with difference concentrations of survivin ASODN exhibited significantly inhibited growth, and 48 h after the transfection, the IC(50) was about 600 nmol/L. After a 48-hour transfection of the cells with 600 nmol/L ASODN, the apoptosis index significantly decreased (t=6.3671, P<0.05), and the cell percentage in (1)/G(0) phase increased (t=10.3832, P<0.01), resulting also in significantly down-regulated survivin mRNA and protein expressions (t=3.5031, P<0.05; t=7.8146, P<0.01) and up-regulated Smac mRNA and protein expressions (t= 2.8011, P<0.05; t= 11.3917, P<0.01).

CONCLUSIONASODN against survivin can induce apoptosis of ovarian cancer cell line SKOV3 and results cell growth arrest in G(1)/G(0) phase by up-regulating Smac and down-regulating survivin expression. Survivin and Smac are closely correlated in their action on SKOV3 cell cycle and apoptosis, which is one of the important mechanisms of ovarian cancer development.

Apoptosis ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Inhibitor of Apoptosis Proteins ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Microtubule-Associated Proteins ; genetics ; metabolism ; Mitochondrial Proteins ; genetics ; metabolism ; Oligonucleotides, Antisense ; genetics ; metabolism ; Ovarian Neoplasms ; genetics ; metabolism ; physiopathology

OBJECTIVETo observe spiral ganglion cell (SGC) death pattern caused by cisplatin and investigate pro-apoptotic protein BNIP3 involvement in SGC death.

METHODSCochlear SGC were isolated from the neonatal rats and cultured in vitro. A cochleas insult model were induced by treatment of 100 µmol/L cisplatin. Real-time PCR were used to determine expression of apoptosis related gene in neonatal rat cochlear cultures after cisplatin treatment. Western blotting was used to detect α-spectrin and indirectly determine caspase-3 activity. Double immunohistochemical staining method was performed to indicated the localization and expression of BNIP3 and NF-200.

RESULTSMorphological finding showed that SGC were smaller, and neurofiber were blebbing and broken at treatment of cisplatin for 12 h. NF-200 marker positive cell number decreased. The transcription level of BNIP3 in cisplatin treatment for 3 h, 6 h and 12 h was higher than the control group (P < 0.05). Western blotting results showed that 120 000 of breakdown products of α-spectrin relative gray level were 0.10 ± 0.05 in the control group, 0.49 ± 0.09 and 0.75 ± 0.08 in cisplatin treatment for 6 h and 12 h group. It increased significantly in the group of cisplatin treatment for 6 h and 12 h than the control group (q = 8.63 and 14.61, P < 0.01). When compared between 6 h of cisplatin treatment and 12 h group, significant difference was detected (q = 5.98, P < 0.05). There was weak BNIP3 positive expression in cytoplasm of the control group. However, strongly BNIP3-positive labeled were seen in the nucleus of SGC and cytoplasm of some stromal cells around SGC after cisplatin treatment.

CONCLUSIONSBNIP3 played an important role in cisplatin induced SGC death and followed independent signaling transduction pathway that differ from stromal cells around SGC. It may suggest that BNIP3 enter nucleus to bind DNA and up-regulate apoptotic gene expression to promote cells death.

Animals ; Apoptosis ; drug effects ; Cells, Cultured ; Cisplatin ; pharmacology ; Membrane Proteins ; genetics ; metabolism ; Mitochondrial Proteins ; Proto-Oncogene Proteins ; genetics ; metabolism ; Rats ; Rats, Wistar ; Spiral Ganglion ; cytology ; metabolism

Mesenchymal stem cells (MSCs) are progenitors of connective tissues, which have emerged as important tools for tissue engineering due to their differentiation potential along various cell types. In recent years, accumulating evidence has suggested that the regulation of mitochondria dynamics and function is essential for successful differentiation of MSCs. In this paper, we review and provide an integrated view on the role of mitochondria in MSC differentiation. The mitochondria are maintained at a relatively low activity level in MSCs, and upon induction, mtDNA copy number, protein levels of respiratory enzymes, the oxygen consumption rate, mRNA levels of mitochondrial biogenesis-associated genes, and intracellular ATP content are increased. The regulated level of mitochondrial ROS is found not only to influence differentiation but also to contribute to the direction determination of differentiation. Understanding the roles of mitochondrial dynamics during MSC differentiation will facilitate the optimization of differentiation protocols by adjusting biochemical properties, such as energy production or the redox status of stem cells, and ultimately, benefit the development of new pharmacologic strategies in regenerative medicine.
Adipogenesis ; physiology ; Animals ; Cell Differentiation ; physiology ; Chondrogenesis ; physiology ; Humans ; Mesenchymal Stem Cells ; cytology ; metabolism ; Mitochondria ; genetics ; metabolism ; Mitochondrial Proteins ; genetics ; metabolism ; Osteogenesis ; physiology ; RNA ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; RNA, Mitochondrial ; Reactive Oxygen Species ; metabolism

OBJECTIVETo explore the correlation of the mutation of MTCYB and MTATP6 genes in sperm mitochondria with asthenospermia.

METHODSWe extracted mtDNA from 80 semen samples of asthenospermia and 20 of normal sperm motility, amplified the MTCYB and MTATP6 genes by PCR, and analyzed their mutation by sequencing and BLAST matching.

RESULTSThe deletion of both MTCYB and MTATP6 were detected in 20 of the 80 asthenospermia samples, MTCYB deletion in 16 and MTATP6 deletion in 4, accounting for 20% and 5% respectively. Sequencing and BLAST matching revealed G8887A mutation in the MTATP6 gene in the asthenospermia samples, with a mutation rate of 20%, while no regular mutation was noted in MTCYB. Neither significant deletion nor mutation was observed in any of the 20 samples of normal sperm motility.

CONCLUSIONBoth the deletion and mutation of MTCYB and MTATP6 genes in sperm mitochondria might affect sperm motility in adults.

Adult ; Asthenozoospermia ; genetics ; pathology ; Base Sequence ; Cytochromes b ; genetics ; DNA, Mitochondrial ; genetics ; Humans ; Male ; Mitochondrial Proteins ; genetics ; Mitochondrial Proton-Translocating ATPases ; genetics ; Molecular Sequence Data ; Mutation ; Sequence Homology, Nucleic Acid ; Sperm Count ; Spermatozoa ; metabolism ; pathology