Modulation of oxidative phosphorylation of human kidney 293 cells by transfection with the internal rotenone-insensitive NADH–quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae

AbstractIn contrast to the mitochondrial proton-translocating NADH–quinone oxidoreductase (complex I), which consists of at least 43 different subunits, the internal rotenone-insensitive NADH–quinone oxidoreductase (Ndi1) of Saccharomyces cerevisiae is a single polypeptide enzyme. The NDI1 gene was stably transfected into the human embryonal kidney 293 (HEK 293) cells. The transfected NDI1 gene was then transcribed and translated in the HEK 293 cells to produce the functional enzyme. The immunochemical and immunofluorescence analyses indicated that the expressed Ndi1 polypeptide was located to the inner mitochondrial membranes. The expression of Ndi1 did not alter the content of existing complex I in the HEK 293 mitochondria, suggesting that the expressed Ndi1 enzyme does not displace the endogenous complex I. The NADH oxidase activity of the NDI1-transfected HEK 293 cells was not affected by rotenone but was inhibited by flavone. The ADP/O ratios coupled to NADH oxidation were lowered from 2.4 to 1.8 by NDI1-transfection while the ADP/O ratios coupled to succinate oxidation (1.6) were not changed. The NDI1-transfected HEK 293 cells were able to grow in media containing a complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion. The potential usefulness of incorporating the Ndi1 protein into mitochondria of human cells is discussed

Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene

The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency

No Immune Responses by the Expression of the Yeast Ndi1 Protein in Rats

Background: The rotenone-insensitive internal NADH-quinone oxidoreductase from yeast, Ndi1, has been shown to work as a replacement molecule for complex I in the respiratory chain of mammalian mitochondria. In the so-called transkingdom gene therapy, one major concern is the fact that the yeast protein is foreign in mammals. Long term expression of Ndi1 observed in rodents with no apparent damage to the target tissue was indicative of no action by the host’s immune system. Methodology/Principal Findings: In the present study, we examined rat skeletal muscles expressing Ndi1 for possible signs of inflammatory or immune response. In parallel, we carried out delivery of the GFP gene using the same viral vector that was used for the NDI1 gene. The tissues were subjected to H&E staining and immunohistochemical analyses using antibodies specific for markers, CD11b, CD3, CD4, and CD8. The data showed no detectable signs of an immune response with the tissues expressing Ndi1. In contrast, mild but distinctive positive reactions were observed in the tissues expressing GFP. This clear difference most likely comes from the difference in the location of the expressed protein. Ndi1 was localized to the mitochondria whereas GFP was in the cytosol. Conclusions/Significance: We demonstrated that Ndi1 expression did not trigger any inflammatory or immune response in rats. These results push forward the Ndi1-based molecular therapy and also expand the possibility of using foreign protein

Protection by the NDI1 Gene against Neurodegeneration in a Rotenone Rat Model of Parkinson's Disease

It is widely recognized that mitochondrial dysfunction, most notably defects in the NADH-quinone oxidoreductase (complex I), is closely related to the etiology of sporadic Parkinson's disease (PD). In fact, rotenone, a complex I inhibitor, has been used for establishing PD models both in vitro and in vivo. A rat model with chronic rotenone exposure seems to reproduce pathophysiological conditions of PD more closely than acute mouse models as manifested by neuronal cell death in the substantia nigra and Lewy body-like cytosolic aggregations. Using the rotenone rat model, we investigated the protective effects of alternative NADH dehydrogenase (Ndi1) which we previously demonstrated to act as a replacement for complex I both in vitro and in vivo. A single, unilateral injection of recombinant adeno-associated virus carrying the NDI1 gene into the vicinity of the substantia nigra resulted in expression of the Ndi1 protein in the entire substantia nigra of that side. It was clear that the introduction of the Ndi1 protein in the substantia nigra rendered resistance to the deleterious effects caused by rotenone exposure as assessed by the levels of tyrosine hydroxylase and dopamine. The presence of the Ndi1 protein also prevented cell death and oxidative damage to DNA in dopaminergic neurons observed in rotenone-treated rats. Unilateral protection also led to uni-directional rotation of the rotenone-exposed rats in the behavioral test. The present study shows, for the first time, the powerful neuroprotective effect offered by the Ndi1 enzyme in a rotenone rat model of PD

Successful Amelioration of Mitochondrial Optic Neuropathy Using the Yeast NDI1 Gene in a Rat Animal Model

Background: Leber’s hereditary optic neuropathy (LHON) is a maternally inherited disorder with point mutations in mitochondrial DNA which result in loss of vision in young adults. The majority of mutations reported to date are within the genes encoding the subunits of the mitochondrial NADH-quinone oxidoreductase, complex I. Establishment of animal models of LHON should help elucidate mechanism of the disease and could be utilized for possible development of therapeutic strategies. Methodology/Principal Findings: We established a rat model which involves injection of rotenone-loaded microspheres into the optic layer of the rat superior colliculus. The animals exhibited the most common features of LHON. Visual loss was observed within 2 weeks of rotenone administration with no apparent effect on retinal ganglion cells. Death of retinal ganglion cells occurred at a later stage. Using our rat model, we investigated the effect of the yeast alternative NADH dehydrogenase, Ndi1. We were able to achieve efficient expression of the Ndi1 protein in the mitochondria of all regions of retinal ganglion cells and axons by delivering the NDI1 gene into the optical layer of the superior colliculus. Remarkably, even after the vision of the rats was severely impaired, treatment of the animals with the NDI1 gene led to a complete restoration of the vision to the normal level. Control groups that received either empty vector or the GFP gene had no effects

Gel-type autologous chondrocyte (Chondron™) implantation for treatment of articular cartilage defects of the knee

<p>Abstract</p> <p>Background</p> <p>Gel-type autologous chondrocyte (Chondron™) implantations have been used for several years without using periosteum or membrane. This study involves evaluations of the clinical results of Chondron™ at many clinical centers at various time points during the postoperative patient follow-up.</p> <p>Methods</p> <p>Data from 98 patients with articular cartilage injury of the knee joint and who underwent Chondron™ implantation at ten Korean hospitals between January 2005 and November 2008, were included and were divided into two groups based on the patient follow-up period, i.e. 13~24-month follow-up and greater than 25-month follow-up. The telephone Knee Society Score obtained during telephone interviews with patients, was used as the evaluation tool.</p> <p>Results</p> <p>On the tKSS-A (telephone Knee Society Score-A), the score improved from 43.52 ± 20.20 to 89.71 ± 13.69 (P < 0.05), and on the tKSS-B (telephone Knee Society Score-B), the score improved from 50.66 ± 20.05 to 89.38 ± 15.76 (P < 0.05). The total improvement was from 94.18 ± 31.43 to 179.10 ± 24.69 (P < 0.05).</p> <p>Conclusion</p> <p>Gel-type autologous chondrocyte implantation for chondral knee defects appears to be a safe and effective method for both decreasing pain and improving knee function.</p

Sperm motility and viability of experimental animals using different cryopreservatives

In this study, an experiment was conducted in order to determine what cryopreservatives (CPVs) were more effective in supporting the motility and viability of sperm from experimental animals. The sperm of mice, rats, beagle dogs, and rabbits were frozen using different CPVs, including DMSO, TYB, and Sperm CryoProtec. The results from freezing the sperm of each laboratory animal in Sperm CryoProtec showed a high level of sperm motility and viability in sperm samples from mice, rats, and beagle dogs melted at the end of the first week. For rabbits, a high level of motility was observed in sperm thawed during the first week, whereas a high level of viability was observed in sperm thawed during the second week. The results of analysis of sperm motility and viability using different CPVs according to laboratory animals showed a significantly higher level of sperm motility (26.28%) and viability (36.20%) for mice in Sperm CryoProtec and the lowest levels of motility and viability were observed in DMSO (p < 0.05). Significantly higher levels of motility (27.94%) and viability (37.94%) were observed for rats in Sperm CryoProtec compared with TYB, which showed the lowest levels of motility and viability (p < 0.05). The study findings described above suggest that the selection of appropriate cryopreservatives is required for each experimental animal. This is because there are differences in the levels of sperm motility and viability of experimental animals depending on the CPVs that are typically used for freezing human sperm, including Sperm CryoProtec and TYB

Effects of various freezing containers for vitrification freezing on mouse oogenesis

Abstract Background In the present study, various freezing containers were tested for mouse embryos of respective developmental stages; embryos were vitrified and then their survival rate and developmental rate were monitored. Mouse two cell, 8 cell, and blastula stage embryos underwent vitrification freezing-thawing and then their recovery rate, survival rate, development rate, and hatching rate were investigated. Methods EM-grid, OPS, and cryo-loop were utilized for vitrification freezing-thawing of mouse embryos. Results It was found that recovery rate and survival rate were higher in the group of cryo-loop compared to those of EM-grid (p < 0.05). Embryonic development rate, two cell embryos to blastocyst, as well as hatching rate were higher in the control group compared to the EM-grid group and OPS group (p < 0.05), yet no difference was noted between the control group and cryo-loop group. Development rate and hatching rate of eight cell morulae and blastocysts were all lower in the treatment groups than the control group whilst hatching rate of blastocysts was higher in the control group compared to the groups of EM-grid and OPS (p < 0.05); although the cryo-loop group was shown to be slightly higher than other groups, it was not statistically significant. Conclusions In the study, we investigate effects of freezing containers on vitrified embryos of respective developmental stages; it was demonstrated that higher developmental rate was shown in more progressed (or developed) embryos with more blastomeres. There was however, no difference in embryonic development rate was shown amongst containers. Taken together, further additional studies are warranted with regards to 1) manipulation techniques of embryos for various vitrification freezing containers and 2) preventive measures against contamination via liquid nitrogen

Effects of culture media conditions on production of eggs fertilized in vitro of embryos derived from ovary of high grade Hanwoo

Abstract Background This study was investigated the effects of culture media conditions on production of eggs fertilized in vitro of embryos from ovaries of high grade Korean native cow, Hanwoo. Methods The IVMD 101 and IVF 100 were used for in vitro maturation of selected Hanwoo oocytes and In vitro embryo culture after in vitro fertilization, respectively. The IVMD 101 and IVD 101 were used for in vitro culture and completely free of serum. Results The cleavage rates of 2-cell embryos in reference to Hanwoo oocytes were 86.7, 92.9 , and 90.1 % in the control group, IVDM101 medium and IVD101 medium, respectively which indicates that the IVDM101 medium and IVD101 medium may result favorable outcomes. The in vitro development rates of blastocysts were 12.4, 38.4 and 32.4 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. For hatched blastocysts, it was 5.3, 33.9, and 28.6 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. Hence, more favorable results were expected for the hatched blastocysts in which the IVMD101 medium and IVD101 medium were used than the control group. Average cell numbers of blastocysts were 128.3, 165.7, and 163.6 in the groups of TCM-199 + 10 % FBS medium, IVMD 101 medium, and IVD 101 medium, respectively which clearly show that the IVMD 101 and IVD 101 medium consequence significantly higher cell numbers compared to the control group (i.e., TCM-199 + 10 % FBS medium). Pregnancy rate after embryo transfer was 39.6 % when the serum free medium was used which is higher than that of the medium supplemented with serum (32.8 %). In addition, stillbirth rates were 4.9 % in the group of serum free medium whereas it was 13.6 % in the serum supplemented medium (13.6 %). Conclusions Taken altogether, serum free media, the IVMD 101 and IVD 101 represented more favorable results in the embryo development rate of embryos, cell numbers of blastocyst, and pregnancy rate. Of note, the IVMD 101 medium showed better outcomes hence, it might be a better option for future applications for in vitro culture of bovine embryos

Protective Role of rAAV-NDI1, Serotype 5, in an Acute MPTP Mouse Parkinson's Model

Defects in mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) have been implicated in a number of acquired and hereditary diseases including Leigh's syndrome and more recently Parkinson's disease. A limited number of strategies have been attempted to repair the damaged complex I with little or no success. We have recently shown that the non-proton-pumping, internal NADH-ubiquinone oxidoreductase (Ndi1) from Saccharomyces cerevisiae (baker's yeast) can be successfully inserted into the mitochondria of mice and rats, and the enzyme was found to be fully active. Using recombinant adenoassociated virus vectors (serotype 5) carrying our NDI1 gene, we were able to express the Ndi1 protein in the substantia nigra (SN) of C57BL/6 mice with an expression period of two months. The results show that the AAV serotype 5 was highly efficient in expressing Ndi1 in the SN, when compared to a previous model using serotype 2, which led to nearly 100% protection when using an acute MPTP model. It is conceivable that the AAV-serotype5 carrying the NDI1 gene is a powerful tool for proof-of-concept study to demonstrate complex I defects as the causable factor in diseases of the brain