Generation and bioenergetic analysis of cybrids containing mitochondrial DNA from mouse skeletal muscle during aging.

Mitochondrial respiratory chain defects have been associated with various diseases and normal aging, particularly in tissues with high energy demands including skeletal muscle. Muscle-specific mitochondrial DNA (mtDNA) mutations have also been reported to accumulate with aging. Our understanding of the molecular processes mediating altered mitochondrial gene expression to dysfunction associated with mtDNA mutations in muscle would be greatly enhanced by our ability to transfer muscle mtDNA to established cell lines. Here, we report the successful generation of mouse cybrids carrying skeletal muscle mtDNA. Using this novel approach, we performed bioenergetic analysis of cells bearing mtDNA derived from young and old mouse skeletal muscles. A significant decrease in oxidative phosphorylation coupling and regulation capacity has been observed with cybrids carrying mtDNA from skeletal muscle of old mice. Our results also revealed decrease growth capacity and cell viability associated with the mtDNA derived from muscle of old mice. These findings indicate that a decline in mitochondrial function associated with compromised mtDNA quality during aging leads to a decrease in both the capacity and regulation of oxidative phosphorylation.

Nuclear suppression of mitochondrial defects in cells without the ND6 subunit.

Previously, we characterized a mouse cell line, 4A, carrying a mitochondrial DNA mutation in the subunit for respiratory complex I, NADH dehydrogenase, in the ND6 gene. This mutation abolished the complex I assembly and disrupted the respiratory function of complex I. We now report here that a galactose-resistant clone, 4AR, was isolated from the cells carrying the ND6 mutation. 4AR still contained the homoplasmic mutation, and apparently there was no ND6 protein synthesis, whereas the assembly of other complex I subunits into complex I was recovered. Furthermore, the respiratory activity and mitochondrial membrane potential were fully recovered. To investigate the genetic origin of this compensation, the mitochondrial DNA (mtDNA) from 4AR was transferred to a new nuclear background. The transmitochondrial lines failed to grow in galactose medium. We further transferred mtDNA with a nonsense mutation at the ND5 gene to the 4AR nuclear background, and a suppression for mitochondrial deficiency was observed. Our results suggest that change(s) in the expression of a certain nucleus-encoded factor(s) can compensate for the absence of the ND6 or ND5 subunit.

A novel Bi12TiO20/g-C3N4 hybrid catalyst with a bionic granum configuration for enhanced photocatalytic degradation of organic pollutants.

A facile synthesis method was used to produce a novel, multi-layer hybrid carbon nitride with a granum microstromatolite structure (g-C3N4MS). This was combined with Bi12TiO20 (BTO) to produce a catalyst that was useful for decomposing hazardous pollutants. The microstructural investigation of the catalyst showed that the stacked-layer stromatolites of the g-C3N4MS were covered with BTO nanoplates to form the granum-like structures. The coupling between the BTO {310} and the g-C3N4MS {002} facets produced a heterostructure with a large contact area that efficiently separated the photo-generated electrons and holes by a reduction in the CB potential of g-C3N4MS. The photocatalytic performance of this novel catalyst was found to exhibit an optimum efficiency of 97% for the degradation of RhB within 50 min and it had a degradation rate constant that was 11.8 times better than bare BTO and 4.2 times better than g-C3N4MS. Moreover, the synthesized photocatalysts demonstrated good reusability and stability. Based on electron spin resonance results for the novel catalyst, O2- radicals were identified as the main active species in the photocatalytic reaction. A new Z-scheme heterogeneous structure was proposed that reasonably explained the photocatalytic reaction mechanism of the novel catalyst.

Yeast NDI1 improves oxidative phosphorylation capacity and increases protection against oxidative stress and cell death in cells carrying a Leber's hereditary optic neuropathy mutation.

G11778A in the subunit ND4 gene of NADH dehydrogenase complex is the most common primary mutation found in Leber's hereditary optic neuropathy (LHON) patients. The NDI1 gene, which encodes the internal NADH-quinone oxidoreductase in Saccharomyces cerevisiae, was introduced into the nuclear genome of a mitochondrial defective human cell line, Le1.3.1, carrying the G11778A mutation. In transformant cell lines, LeNDI1-1 and -2, total and complex I-dependent respiration were fully restored and largely resistant to complex I inhibitor, rotenone, indicating a dominant role of NDI1 in the transfer of electrons in the host cells. Whereas the original mutant Le1.3.1 cell grows poorly in medium containing galactose, the transformants have a fully restored growth capacity in galactose medium, although the ATP production was not totally recovered. Furthermore, the increased oxidative stress in the cells carrying the G11778A mutation was alleviated in transformants, demonstrated by a decreased reactive oxygen species (ROS) level. Finally, transformants were also shown to be desensitized to induction to apoptosis and also exhibit greater resistance to paraquat-induced cell death. It is concluded that the yeast NDI1 enzyme can improve the oxidative phosphorylation capacity in cells carrying the G11778A mutation and protect the cells from oxidative stress and cell death.

Fluorescence resonance energy transfer of CaF2: Eu2+, Tb3+ applied to dye-sensitized solar cells.

CaF2: Eu2+, Tb3+ introduced into dye-sensitized solar cells (DSSCs) were studied to examine the influence of luminescent materials on photoanodes using a simple method. The emission spectra of CaF2: Eu2+, Tb3+ included the blue light of Eu2+ (4f → 5d) at 430 nm and green emission of Tb3+ (5D4 → 7F5) at 542 nm under the monitoring wavelengths at 398 nm, which matched well the absorption range of the N719 dye in DSSCs. Energy transfer (ET) was verified between Eu2+ and Tb3+ ions and the efficiency of ET found to increase with Tb3+ concentration. Both the fluorescence resonance and luminescence-mediated ETs between phosphor and N719 dye were observed as the main contribution in improving photocurrent and power conversion efficiency (PCE) of these DSSCs. The PCE of DSSCs doped with phosphors was greatly increased by 5.16, to 43.3%, which was comparable to cells made of pure TiO2 photoanodes. Moreover, CaF2: Eu2+, Tb3+ enlarged the surface area of TiO2 photonaodes, which helped the adsorption performance of the TiO2 film.

PGC-1alpha-induced mitochondrial alterations in 3T3 fibroblast cells.

Peroxisome proliferation activator receptor (PPAR) gamma-coactivator 1alpha (PGC-1alpha), a transcription coactivator, functions as a master regulator of a wide array of metabolic and physiological processes and is an essential factor in the process of mitochondrial biogenesis. Transfection of NIH 3T3 fibroblasts with a mouse cDNA for PGC-1alpha led to the induction of markers of mitochondrial biogenesis, that is, mitochondrial transcription factor A (mtTFA), cytochrome c, and mitochondrial DNA (mtDNA). Mitochondrial biogenesis-associated net protein synthesis appears to be accomplished by a reduction in the rate of mitochondrial protein degradation with little or no change in the rate of protein synthesis. Overexpression of PGC-1alpha did not adversely affect cellular proliferation. Cellular ATP levels were increased in the transfected cells and they were more resistant to oxidative stress than the control nontransfected 3T3 cells. This resistance to oxidative stress was manifested by both an improved viability and the maintenance of mitochondrial membrane potential in the transfected cells when exposed to t-butyl hydroperoxide (t-BOOH). It therefore appears that PGC-1alpha overexpression stimulates mitochondrial biogenesis in 3T3 cells making them more resistant to oxidative stressors.

Synergistic anticancer properties of docosahexaenoic acid and 5-fluorouracil through interference with energy metabolism and cell cycle arrest in human gastric cancer cell line AGS cells.

AIM: To explore the synergistic effect of docosahexaenoic acid (DHA)/5-fluorouracil (5-FU) on the human gastric cancer cell line AGS and examine the underlying mechanism. METHODS: AGS cells were cultured and treated with a series of concentrations of DHA and 5-FU alone or in combination for 24 and 48 h. To investigate the synergistic effect of DHA and 5-FU on AGS cells, the inhibition of cell proliferation was determined by MTT assay and cell morphology. Flow cytometric analysis was also used to assess cell cycle distribution, and the expression of mitochondrial electron transfer chain complexes (METCs) I, II and V in AGS cells was further determined by Western blot analysis. RESULTS: DHA and 5-FU alone or in combination could markedly suppress the proliferation of AGS cells in a significant time and dose-dependent manner. DHA markedly strengthened the antiproliferative effect of 5-FU, decreasing the IC50 by 3.56-2.15-fold in an apparent synergy. The morphological changes of the cells were characterized by shrinkage, cell membrane blebbing and decreased adherence. Cell cycle analysis showed a shift of cells into the G0/G1 phase from the S phase following treatment with DHA or 5-FU (G0/G1 phase: 30.04% ± 1.54% vs 49.05% ± 6.41% and 63.39% ± 6.83%, respectively, P < 0.05; S phase: 56.76% ± 3.14% vs 34.75% ± 2.35% and 25.63% ± 2.21%, respectively, P < 0.05). Combination treatment of DHA and 5-FU resulted in a significantly larger shift toward the G0/G1 phase and subsequent reduction in S phase (G0/G1 phase: 69.06% ± 2.63% vs 49.05% ± 6.41% and 63.39% ± 6.83%, respectively, P < 0.05; S phase: 19.80% ± 4.30% vs 34.75% ± 2.35% and 25.63% ± 2.21%, respectively, P < 0.05). This synergy was also reflected in the significant downregulation of the expression of METCs in AGS cells. CONCLUSION: Synergistic anticancer properties of DHA and 5-FU may involve interference with energy production of AGS cells via downregulation of METCs and cell cycle arrest.

Restoration of mitochondrial function in cells with complex I deficiency.

The mammalian mitochondrial NADH dehydrogenase (complex I) is the major entry point for the electron transport chain. It is the largest and most complicated respiratory complex consisting of at least 46 subunits, 7 of which are encoded by mitochondrial DNA (mtDNA). Deficiency in complex I function has been associated with various human diseases including neurodegenerative diseases and the aging process. To explore ways to restore mitochondrial function in complex I-deficient cells, various cell models with mutations in genes encoding subunits for complex I have been established. In this paper, we discuss various approaches to recover mitochondrial activity, the complex I activity in particular, in cultured cells.

[The effect of retinoic acid on induction of osteoporotic model rats and the possible mechanism].

OBJECTIVE: To observe the effect of retinoic acid on induction of osteoporotic model in rats and analyze the mechanism therein involved. METHODS: SD rats were treated with retinoic acid 80 mg/(kg x d) by gastrogavage for 15 days to induce osteoporosis and were killed in batches at 0, 30 and 60 days after drug withdrawal. The levels of Ca, P, BGP, E2, IGF-1, AKP and TRAP in serum were assayed, the collagen and proteoglycan contents of bone and bone mineral density (BMD) were determined, and the morphological changes in cancellous and cortical bone and growth plate cartilage (GPC) of femurs from the experimental rats were observed. RESULTS: After 15 days of induction by retinoic acid, the serum E2 and BGP contents of rats were obviously decreased, the activities of AKP and TRAP were significantly increased, and the levels of BMD were lowered. The masses of spongy bone and cortical bone of femurs from the rats were decreased, and the number of chondrocytes in GPC was reduced. At 30 days, after drug withdrawal, the masses of spongy bone and cortical bone of femurs from the osteoporotic model rats still showed reduction; the activities of AKP in serum were lower than those at 15 days after drug redrawal, but were still higher than those of normal group rats; the chondrocytes in GPC were increased, the serum BGP and Ca contents were increased. At 60 days, after drug withdrawal, only the masses of femoral spongy bone of the osteoporotic model rats continuously showed obvious reduction, the other indices, including BGP, E2, AKP, TRAP and the masses of cortical bone, showed no significant difference between the two groups. CONCLUSION: The short-term effect of retinoic acid on induction of rat's osteoporotic model was noticeable, but the long-term effect was not so good, and the bone loss of spongy bone existed longer and was more obvious than that of cortical bone.

Comparative bioenergetic study of neuronal and muscle mitochondria during aging.

Mitochondrial respiratory chain defects have been associated with various diseases and with normal aging, particularly in tissues with high energy demands, including brain and skeletal muscle. Tissue-specific manifestation of mitochondrial DNA (mtDNA) mutations and mitochondrial dysfunction are hallmarks of mitochondrial diseases although the underlying mechanisms are largely unclear. Previously, we and others have established approaches for transferring mtDNA from muscle and synaptosomes of mice at various ages to cell cultures. In this study, we carried out a comprehensive bioenergetic analysis of cells bearing mitochondria derived from young, middle-aged, and old mouse skeletal muscles and synaptosomes. Significant age-associated alterations in oxidative phosphorylation and regulation during aging were observed in cybrids carrying mitochondria from both skeletal muscle and synaptosomes. Our results also revealed that loss of oxidative phosphorylation capacity may occur at various ages in muscle and brain. These findings indicate the existence of a tissue-specific regulatory mechanism for oxidative phosphorylation.

Pathways related to mitochondrial dysfunction in cartilage of endemic osteoarthritis patients in China.

In this paper, we present the first evidence of differences in the mitochondria-related gene expression profiles of adult articular cartilage derived from patients with Kashin-Beck disease and normal controls. The expression of 705 mitochondria-related genes was analyzed by mitochondria-related gene expression analysis and ingenuity pathways analysis. Mitochondria-related gene expression analysis identified 9 up-regulated genes in Kashin-Beck disease based on their average expression ratio. Three canonical pathways involved in oxidative phosphorylation, apoptosis signaling and pyruvate metabolism were identified, which indicate the involvement of mitochondrial dysfunction in the pathogenesis of Kashin-Beck disease.

An assembled complex IV maintains the stability and activity of complex I in mammalian mitochondria.

In the mammalian mitochondrial electron transfer system, the majority of electrons enter at complex I, go through complexes III and IV, and are finally delivered to oxygen. Previously we generated several mouse cell lines with suppressed expression of the nuclearly encoded subunit 4 of complex IV. This led to a loss of assembly of complex IV and its defective function. Interestingly, we found that the level of assembled complex I and its activity were also significantly reduced, whereas levels and activity of complex III were normal or up-regulated. The structural and functional dependence of complex I on complex IV was verified using a human cell line carrying a nonsense mutation in the mitochondrially encoded complex IV subunit 1 gene. Our work documents that, although there is no direct electron transfer between them, an assembled complex IV helps to maintain complex I in mammalian cells.

Cytochrome c oxidase subunit IV is essential for assembly and respiratory function of the enzyme complex.

Cytochrome c oxidase or complex IV, catalyzes the final step in mitochondrial electron transfer chain, and is regarded as one of the major regulation sites for oxidative phosphorylation. This enzyme is controlled by both nuclear and mitochondrial genomes. Among its 13 subunits, three are encoded by mitochondrial DNA and ten by nuclear DNA. In this work, an RNA interference approach was taken which led to the generation of mouse A9 cell derivatives with suppressed expression of nuclear-encoded subunit IV (COX IV) of this complex. The amounts of this subunit are decrease by 86% to 94% of normal level. A detail biosynthetic and functional analysis of several cell lines with suppressed COX IV expression revealed a loss of assembly of cytochrome c oxidase complex and, correspondingly, a reduction in cytochrome c oxidase-dependent respiration and total respiration. Furthermore, dysfunctional cytochrome c oxidase in the cells leads to a compromised mitochondrial membrane potential, a decreased ATP level, and failure to grow in galactose medium. Interestingly, suppression of COX IV expression also sensitizes the cells to apoptosis. These observations provide the evidence of the essential role of the COX IV subunit for a functional cytochrome c oxidase complex and also demonstrate a tight control of cytochrome c oxidase over oxidative phosphorylation. Finally, our results further shed some insights into the pathogenic mechanism of the diseases caused by dysfunctional cytochrome c oxidase complex.

[Therapeutic effect of dietary boron supplement on retinoic acid-induced osteoporosis in rats].

OBJECTIVE: To observe the therapeutic efficacy of dietary boron supplement on retinoic acid-induced osteoporosis in rats, so as to provide experimental evidence for clinical management of osteoporosis with boron. METHODS: Thirty-two SD rats were randomized into normal control group (8 rats) and osteoporotic group (24 rats), and osteoporosis was induced in rats of the latter group by intragastric retinoic acid administration at the daily dose of 80 mg/kg for 15 consecutive days. The osteoporotic rats were subdivided into control group (8 rats) without treatment, boron treatment group (8 rats) and estradiol treatment group (8 rats). After 30 days of treatment, the serum contents of Ca, P, boron and the activities of alkaline phosphatase (AKP) and tartrate-resistant acid phosphatase (TRAP) in the rats were assayed, the bone mineral density (BMD) of the whole body, lumbar vertebrae and tibia were determined, and the morphological changes of the femurs were observed. RESULTS: The serum contents of Ca and P in the rats of the 4 groups differed scarcely, but the content of boron in boron treatment group was markedly higher than that in the other three groups. In the osteoporotic control group, the activities of serum AKP and TRAP, the masses of spongy bone and cortical bone of the femurs, and the quantity of the osteoclasts were increased, with the BMD of the lumbar vertebrae and tibia decreased, suggesting osteoporotic conditions. The mean trabecular plate density and thickness, trabecular bone volume and cortical bone volume of the femurs in the osteoporotic rats treated with boron or estradiol were significantly increased, but the active osteoclast quantity in the spongy bone and serum TRAP activities were obviously decreased, and the bone quality was comparable with that of the normal group. In addition, the serum AKP activity and the active osteoblast quantity in the spongy bone were obviously increased in boron treatment group. CONCLUSION: The dietary boron supplement can increase the serum content of boron of osteoporotic rats to stimulate bone formation and inhibit bone resorption, producing therefore obvious therapeutical effect against osteoporosis.