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Biomedical subjects

Jun-Ichi Hayashi

Publications and source records attributed to Jun-Ichi Hayashi.

At least 19 recordsLinked to original sources

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↗

Mitochondrial complex I activity is significantly decreased in a patient with maternally inherited type 2 diabetes mellitus and hypertrophic cardiomyopathy associated with mitochondrial DNA C3310T mutation: a cybrid study.

Mitochondrial respiratory function in a patient with maternally inherited type 2 diabetes mellitus and hypertrophic cardiomyopathy associated with heteroplasmic mitochondrial DNA (mtDNA) C3310T mutation, which replaces the second amino acid of NADH dehydrogenase 1 (ND1) from a hydrophobic Proline to a hydrophilic Serine, was investigated. Mitochondrial respiratory function solely due to mtDNA C3310T mutation was investigated in cybrid system by the fusion of mtDNA-deleted (rho(0)) HeLa cells and exogenous mtDNA either from the proband or from controls. Total oxygen consumption of the proband cybrid cells was significantly decreased compared with those of controls (2.468+/-0.475 versus 2.871+/-0.484 micromol/h/10(7) cells, p=0.0392). Mitochondrial respiratory chain complex I activity of the proband cybrid cells was also significantly decreased compared with those of controls (0.191+/-0.080 versus 0.288+/-0.113 micromol/h/mg protein, p=0.0223). Furthermore, ATP content in the proband cybrid cells was also significantly decreased compared with those in controls (1.119+/-0.344 versus 1.419+/-0.378 pmol/10(5) cells, p=0.044). The present study indicates that mtDNA C3310T mutation may be a pathogenic mutation of maternally inherited type 2 diabetes mellitus and hypertrophic cardiomyopathy in the proband and the family.

Blotting, Southern↗

Analysis of the effect of surgical lung biopsy on serum KL-6 Levels in patients with interstitial pneumonia: surgical lung biopsy does not elevate serum KL-6 levels.

OBJECTIVE: It is well known that the serum level of KL-6 can be an indicator of disease activity in patients with interstitial pneumonia (IP). However, surgical lung biopsy is often required for the diagnosis of IP, although this can result in IP exacerbation. METHODS: The effect of surgical lung biopsy on the serum level of KL-6 in patients with IP was analyzed. Thirty-two cases of IP were examined in this study. There were no cases showing exacerbation of IP. RESULTS: The serum level of KL-6 demonstrated 1067+/-550 U/ml (mean+/-SD) before lung biopsy, 991+/-471 U/ml a day, 824+/-377 U/ml 4 days and 826+/-384 U/ml 7 days after lung biopsy. The serum KL-6 levels on the 1st, 4th, 7th day after the lung biopsy were significantly lower than that before the lung biopsy (P<0.05, P<0.01 and P<0.01, respectively). The percent decrease of the serum KL-6 levels on the 4th day (the lowest level) was dependent on the urine volume, and the analysis of the urinary levels of KL-6 showed a transient increase in urinary KL-6 excretion, suggesting that the decrease in serum KL-6 levels associated with surgical lung biopsy may be caused by this increase in urinary KL-6 excretion. CONCLUSION: Surgical lung biopsy of patients with IP has little effect on the increase in serum KL-6 levels. An elevation of serum KL-6 after surgical lung biopsy may indicate exacerbation of IP.

Aged↗

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↗

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↗

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↗

A reduced priming system.

Cardiopulmonary bypass (CPB), an integral part of valve surgery, is known to cause hemodilution, which can lead to a need for blood transfusion as well as a systemic inflammatory reaction. Our aim was to evaluate a reduced priming (RP) system for CPB with regard to clinical outcomes in patients undergoing valve surgery. Eleven patients were assigned to the new system with an RP volume. The RP system allowed for reduced hemodilution and a possible reduction in the necessity for blood transfusions.

Adult↗

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↗

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↗

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↗

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↗