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Biomedical subjects

Kazuto Nakada

Publications and source records attributed to Kazuto Nakada.

At least 19 recordsLinked to original sources
PubMed2006

Mitochondria-related male infertility.

Approximately 15% of human couples are affected by infertility, and about half of these cases of infertility can be attributed to men, through low sperm motility (asthenozoospermia) or/and numbers (oligospermia). Because mitochondrial genome (mtDNA) mutations are identified in patients with fertility problems, there is a possibility that mitochondrial respiration defects contribute to male infertility. To address this possibility, we used a transmitochondrial mouse model (mito-mice) carrying wild-type mtDNA and mutant mtDNA with a pathogenic 4,696-bp deletion (DeltamtDNA). Here we show that mitochondrial respiration defects caused by the accumulation of DeltamtDNA induced oligospermia and asthenozoospermia in the mito-mice. Most sperm from the infertile mito-mice had abnormalities in the middle piece and nucleus. Testes of the infertile mito-mice showed meiotic arrest at the zygotene stage as well as enhanced apoptosis. Thus, our in vivo study using mito-mice directly demonstrates that normal mitochondrial respiration is required for mammalian spermatogenesis, and its defects resulting from accumulated mutant mtDNAs cause male infertility.

Animals↗
PubMed2006

Suppression of disease phenotypes of adult mito-mice carrying pathogenic mtDNA by bone marrow transplantation.

For directly addressing the issue of gene therapy of adult patients with mitochondrial diseases, we carried out bone marrow transplantation to adult mito-mice with mutated mtDNA and expressing respiration defects for improvement of disease phenotypes. We supposed that bone marrow cells transdifferentiated into various tissues, so that their transplantation would suppress disease phenotypes. The results showed improvement of survival and delayed expression of renal failure. As most mito-mice without a transplant died due to renal failure, we examined whether transplanted bone marrow cells transdifferentiated into renal tissues carrying improved renal function. Histochemical analyses showed that the suppression of disease phenotypes was not due to transdifferentiation, but due to suppression of apoptosis of renal cells. Thus, bone marrow cells possess a novel function of supporting tissues by suppressing apoptosis induced by respiration defects.

Animals↗
PubMed2006

Mitochondrial dynamics and aging: Mitochondrial interaction preventing individuals from expression of respiratory deficiency caused by mutant mtDNA.

In mammalian cells, there is an extensive and continuous exchange of mitochondrial DNA (mtDNA) and its products between mitochondria. This mitochondrial complementation prevents individuals from expression of respiration deficiency caused by mutant mtDNAs. Thus, the presence of mitochondrial complementation does not support the generally accepted mitochondrial theory of aging, which proposes that accumulation of somatic mutations in mtDNA is responsible for age-associated mitochondrial dysfunction. Moreover, the presence of mitochondrial complementation enables gene therapy for mitochondrial diseases using nuclear transplantation of zygotes.

Aging↗
PubMed2006

Generation of trans-mitochondrial mice carrying homoplasmic mtDNAs with a missense mutation in a structural gene using ES cells.

Generation of various kinds of trans-mitochondrial mice, mito-mice, each carrying mtDNAs with a different pathogenic mutation, is required for precise investigation of the pathogenesis of mitochondrial diseases. This study used two respiration-deficient mouse cell lines as donors of mtDNAs with possible pathogenic mutations. One cell line expressed 45-50% respiratory activity due to mouse mtDNAs with a T6589C missense mutation in the COI gene (T6589C mtDNA) and the other expressed 40% respiratory activity due to rat (Rattus norvegicus) mtDNAs in mouse cells. By cytoplasmic transfer of these mtDNAs to mouse ES cells, we isolated respiration-deficient ES cells. We obtained chimeric mice and generated their F(6) progeny carrying mouse T6589C mtDNAs by its female germ line transmission. They were respiration-deficient and thus could be used as models of mitochondrial diseases caused by point mutations in mtDNA structural genes. However, chimeric mice and mito-mice carrying rat mtDNAs were not obtained, suggesting that significant respiration defects or some deficits induced by rat mtDNAs in mouse ES cells prevented their differentiation to generate mice carrying rat mtDNAs.

Animals↗
PubMed2005

Gene therapy for progeny of mito-mice carrying pathogenic mtDNA by nuclear transplantation.

Pathogenic mutations in mtDNAs have been shown to be responsible for expression of respiration defects and resultant expression of mitochondrial diseases. This study directly addressed the issue of gene therapy of mitochondrial diseases by using nuclear transplantation of zygotes of transmitochondria mice (mito-mice). Mito-mice expressed respiration defects and mitochondrial diseases due to accumulation of mtDNA carrying a large-scale deletion (DeltamtDNA). Second polar bodies were used as biopsy samples for diagnosis of mtDNA genotypes of mito-mouse zygotes. Nuclear transplantation was carried out from mito-mouse zygotes to enucleated normal zygotes and was shown to rescue all of the F(0) progeny from expression of respiration defects throughout their lives. This procedure should be applicable to patients with mitochondrial diseases for preventing their children from developing the diseases.

Aging↗
PubMed2005

Application of ES cells for generation of respiration-deficient mice carrying mtDNA with a large-scale deletion.

In a previous study, we used mouse zygotes as recipients of mtDNA with a large-scale deletion mutation (DeltamtDNA) and generated respiration-deficient mice (mito-mice) carrying DeltamtDNA. In this study, we used mouse ES cells as recipients of DeltamtDNA, and generated mito-mice with DeltamtDNA only when the ES cells carried 17% DeltamtDNA. No chimera mice or their F(1) progenies were obtained from ES cells carrying more than 61% DeltamtDNA. These observations suggest that respiratory defects of ES cells inhibit their normal differentiation into chimera mice and mito-mice, and that ES cells are more effective than zygotes for generation of mito-mice carrying mtDNAs without significant pathogenic mutations.

Animals↗
PubMed2005

Rare creation of recombinant mtDNA haplotypes in mammalian tissues.

The problem of whether recombinant mtDNAs are created in mammalian cells has been controversial for many years. We show convincing evidence for the very rare creation of recombinant mtDNA haplotypes by isolating human somatic hybrid cells and by generating mice carrying two different mtDNA haplotypes. To avoid misinterpretation of PCR-jumping products as recombinants, we used purified mtDNAs for cloning and sequencing. The results showed that only three of 318 clones of mtDNA purified from mouse tissues corresponded to recombinant mtDNA haplotypes, whereas no recombinants were found in human somatic hybrid cells. Such an extremely low frequency of mtDNA recombination does not require any revision of important concepts on human evolution that are based on its absence. Considering the high concentration of reactive oxygen species around the mtDNA and its frequent strand breakage, recombinant clones would correspond to gene conversion products created by repair of nucleotide mismatches.

Animals↗
PubMed2005

Nuclear DNA but not mtDNA controls tumor phenotypes in mouse cells.

Recent studies showed high frequencies of homoplasmic mtDNA mutations in various human tumor types, suggesting that the mutated mtDNA haplotypes somehow contribute to expression of tumor phenotypes. We directly addressed this issue by isolating mouse mtDNA-less (rho(0)) cells for complete mtDNA replacement between normal cells and their carcinogen-induced transformants, and examined the effect of the mtDNA replacement on expression of tumorigenicity, a phenotype forming tumors in nude mice. The results showed that genome chimera cells carrying nuclear DNA from tumor cells and mtDNA from normal cells expressed tumorigenicity, whereas those carrying nuclear DNA from normal cells and mtDNA from tumor cells did not. These observations provided direct evidence that nuclear DNA, but not mtDNA, is responsible for carcinogen-induced malignant transformation, although it remains possible that mtDNA mutations and resultant respiration defects may influence the degree of malignancy, such as invasive or metastatic properties.

Animals↗
PubMed2004

Accumulation of pathogenic DeltamtDNA induced deafness but not diabetic phenotypes in mito-mice.

Mito-mice carrying various proportions of deletion mutant mtDNA (DeltamtDNA) were generated by introduction of the DeltamtDNA from cultured cells into fertilized eggs of C57BL/6J (B6) strain mice. Great advantages of mito-mice are that they share exactly the same nuclear-genome background, and that their genetic variations are restricted to proportions of pathogenic DeltamtDNA. Since accumulation of DeltamtDNA to more than 75% induced respiration defects, the disease phenotypes observed exclusively in mito-mice carrying more than 75% DeltamtDNA should be due to accumulated DeltamtDNA. In this study, we focused on the expressions of hearing loss and diabetic phenotypes, since these common age-associated abnormalities have sometimes been reported to be inherited maternally and to be associated with pathogenic mutant mtDNAs. The results showed that accumulation of exogenously introduced DeltamtDNA was responsible for hearing loss, but not for expression of diabetic phenotypes in mito-mice.

Animals↗
PubMed2004

Presence of interaction but not complementation between human mtDNAs carrying different mutations within a tRNA(Leu(UUR)) gene.

This study provided answers to fundamental questions on mammalian mitochondrial genetics: Could respiratory function in mitochondria be maintained by their exchange of genetic contents even when mutations were created within the same genes in different mitochondrial DNA (mtDNA) molecules? Using cell fusion techniques, we created a chance to coexist two types of respiration-deficient syn(-) mitochondria carrying different mtDNA mutations within the same tRNA(Leu(UUR)) gene obtained from patients with mitochondrial diseases. The results showed that two syn(-) mitochondria exchanged their genetic contents, but did not restore respiration defects, suggesting that mitochondrial interaction could not complement the mutations created within the same gene in different mtDNA molecules.

DNA, Mitochondrial↗
PubMed2004

Determination of normal ranges of mitochondrial respiratory activities by mtDNA transfer from 54 Human subjects to mtDNA-less HeLa cells for identification of the pathogenicities of mutated mtDNAs.

To determine the pathogenicities of mutated mtDNAs in patients with respiration defects, the possible involvement of nuclear DNA mutations has to be excluded, since respiratory function is controlled by both nuclear DNA and mtDNA. This was achieved by showing that the mutated mtDNAs and respiration defects were co-transferred from patients to mtDNA-less human cells, and the resultant cybrid clones carrying mutated mtDNAs expressed respiration defects. To decide whether the cybrid clones expressed respiration defects, in this study the lowest limits of normal respiratory function were evaluated by transfer of mtDNAs from 54 normal subjects to mtDNA-less HeLa cells. The resultant cybrid clones showed that 71% respiratory function was the lowest limit of mtDNAs from normal subjects. On the other hand, cybrid clones carrying pathogenic mtDNAs from patients with mitochondrial diseases showed 0-64% respiratory function, suggesting that less than 71% respiratory function in cybrid clones should be a reliable indicator of whether the mutated mtDNAs of the patients were pathogenic.

Cell Respiration↗
PubMed2004

In vivo interaction between mitochondria carrying mtDNAs from different mouse species.

Mitochondrial disease model mice, mitomice, were created using zygotes of B6mtspr strain mice carrying mitochondrial DNA (mtDNA) from Mus spretus as recipients of exogenous mitochondria carrying wild-type and a deletion mutant mtDNA (DeltamtDNA) of M. musculus domesticus. In these experiments, mtDNAs from different mouse species were used for identification of exo- and endogenous wild-type mtDNAs in the mitomice. Results showed transmission of exogenous DeltamtDNA, but not exogenous wild-type mtDNA, of M. m. domesticus to following generations through the female germ line. Complete elimination of exogenous wild-type mtDNA would be due to stochastic segregation, whereas transmission of exogenous DeltamtDNA would be due to its smaller size leading to a propagational advantage. Tissues in mitomice of the F3 generation carrying exogenous DeltamtDNA showed protection from respiration defects until DeltamtDNA accumulated predominantly. This protection from expression of mitochondrial dysfunction was attained with the help of endogenous wild-type mtDNA of M. spretus, since mitomice did not possess exogenous wild-type mtDNA of M. m. domesticus. These observations provide unambiguous evidence for the presence of interaction between exogenous mitochondria carrying DeltamtDNA and endogenous mitochondria carrying M. spretus wild-type mtDNA.

Animals↗
PubMed2003

Ditercalinium chloride, a pro-anticancer drug, intimately associates with mammalian mitochondrial DNA and inhibits its replication.

Ditercalinium chloride was originally synthesized for use as an anticancer drug and was then found to deplete mitochondrial DNA. Ethidium bromide is widely used to deplete mitochondrial DNA and produce mitochondrial DNA-less cell lines. Although ethidium bromide is used in the case of human cell lines, it frequently fails to deplete mitochondrial DNA in mouse cells. In contrast, ditercalinium chloride can deplete mitochondrial DNA in both mouse and human cells. However, little is known of the mechanisms by which ditercalinium chloride depletes mitochondrial DNA. Here, we show that ditercalinium chloride inhibits human DNA polymerase gamma activity as efficiently as does ethidium bromide. Ethidium bromide accumulates much less in mouse B82 cells, as compared with findings in human HeLa cells, whereas ditercalinium chloride accumulates in both to a similar extent. This poor accumulation of ethidium bromide may, in part, account for the resistance. Ethidium bromide distributes diffusely in the mitochondria of HeLa cells, while ditercalinium chloride distributes granularly and hence may be strongly associated with mitochondrial DNA. Each granular spot presumably represents one mitochondrial DNA nucleoid. In support of this idea, ditercalinium chloride co-localizes with Twinkle, a mitochondrial helicase and is assumed to associate with mitochondrial DNA. This close association of ditercalinium chloride with mitochondrial DNA may contribute to the mitochondrial DNA-depleting activity.

Animals↗
PubMed2002

A novel defense system of mitochondria in mice and human subjects for preventing expression of mitochondrial dysfunction by pathogenic mutant mtDNAs.

Recently, we generated mtDNA-based disease mice (mito mice) by introduction of respiration-deficient mitochondria possessing pathogenic mutant mtDNA with a 4696 bp deletion (deltamtDNA4696) from somatic cells into mouse zygotes. Mito mice and cytochrome c oxidase (COX) electronmicrographs, that could identify the respiration enzyme activity at individual mitochondrial levels, enabled precise investigation of the pathogenesis of deltamtDNA4696. All the observations represented unambiguous evidence for the presence of extensive and continuous exchange of genetic contents between mitochondria. Thus, the inter-mitochondrial interaction could correspond to a very unique and effective defense system of the highly oxidative organelles for preventing mice and human subjects from expressing mitochondrial dysfunction caused by mtDNA lesions, which have been continuously created by oxidative stresses during aging. Here, we would like to propose a new hypothesis on mitochondrial biogenesis, 'the interaction theory of mammalian mitochondria': mitochondria exchange genetic contents, and thus lose individuality and function as a single dynamic cellular unit.

Journal Article↗
PubMed2002

Expression of multiple troponin T isoforms in chicken breast muscle regeneration induced by sub-serous implantation.

Chicken fast-muscle type (F-type) troponin T (TnT) isoforms are classified into two types, leg-muscle type (L-type) and breast-muscle type (B-type), which are generated by exclusion and inclusion of exon x series-derived sequences in mRNAs, respectively. The B-type isoforms are further classified into neonatal breast-muscle (BN), young chicken breast-muscle (BC), and adult chicken breast-muscle (BA) subtypes. It is known that the multiple F-type TnT isoforms are transiently expressed in the breast muscle tissue during normal development. To examine whether the transition of the isoforms was fixed in muscle cell lineage, breast muscle pieces (pectoralis major) of 1-day old chicks were cultured under gizzard serous membrane of the same chicks for 60 days at the longest. TnT isoform expression of the implants was monitored by immunoblotting and immunostaining using anti-F-type TnT against both L-type and B-type isoforms, anti-exon x3 against only B-type isoforms, and anti-S-type TnT against slow-muscle-type (S-type) isoforms. Muscle fibers in the implant degenerated first, and then new myotubes expressing L-type isoforms were formed by the fusion of myoblasts from surviving satellite cells. When the maturation of the myotubes into myofibers proceeded, BN-, BC-, and BA-subtype isoforms were expressed in the order of developmental stage specific-manner, indicating that the order of appearance of these isoforms was fixed in muscle cell lineage. In immunostaining of the implants recovered on the 60th day after implantation, at least three kinds of the regenerated myofibers were observed, expressing mainly B-type, both B-type and L-type, and only L-type isoforms. The immunohistochemical results suggested that the regulation of alternative splicing of F-type TnT pre-mRNAs was different among individual myofibers, and that the regulation was programmed in myogenic cells, probably satellite cells, which were the primary source of the fibers.

Animals↗
PubMed2002

Mammalian copper chaperone Cox17p has an essential role in activation of cytochrome C oxidase and embryonic development.

Cox17p is essential for the assembly of functional cytochrome c oxidase (CCO) and for delivery of copper ions to the mitochondrion for insertion into the enzyme in yeast. Although this small protein has already been cloned or purified from humans, mice, and pigs, the function of Cox17p in the mammalian system has not yet been elucidated. In vitro biochemical data for mammalian Cox17p indicate that the copper binds to the sequence -KPCCAC-. Although mouse embryos homozygous for COX17 disruption die between embryonic days E8.5 and E10, they develop normally until E6.5. This phenotype is strikingly similar to embryos of Ctr1(-/-), a cell surface copper transporter, in its lethality around the time of gastrulation. COX17-deficient embryos exhibit severe reductions in CCO activity at E6.5. Succinate dehydrogenase activity and immunoreactivities for anti-COX subunit antibodies were normal in the COX17(-/-) embryos, indicating that this defect was not caused by the deficiency of other complexes and/or subunits but was caused by impaired CCO activation by Cox17p. Since other copper chaperone (Atox1 and CCS)-deficient mice show a more moderate defect, the disruption of the COX17 locus causes the expression of only the phenotype of Ctr1(-/-). We found that the activity of lactate dehydrogenase was also normal in E6.5 embryos, implying that the activation of CCO by Cox17p may not be essential to the progress of embryogenesis before gastrulation.

Alleles↗