Human cells are protected from mitochondrial dysfunction by complementation of DNA products in fused mitochondria.

Extensive complementation between fused mitochondria is indicated by recombination of 'parental' mitochondrial (mt) DNA (ref. 1,2) of yeast and plant cells. It has been difficult, however, to demonstrate the occurrence of complementation between fused mitochondria in mammalian species through the presence of recombinant mtDNA molecules, because sequence of mtDNA throughout an individual tends to be uniform owing to its strictly maternal inheritance. We isolated two types of respiration-deficient cell lines, with pathogenic mutations in mitochondrial tRNAIle or tRNALeu(UUR) genes from patients with mitochondrial diseases. The coexistence of their mitochondria within hybrids restored their normal morphology and respiratory enzyme activity by 10-14 days after fusion, indicating the presence of an extensive and continuous exchange of genetic contents between the mitochondria. This complementation between fused mitochondria may represent a defence of highly oxidative organelles against mitochondrial dysfunction caused by the accumulation of mtDNA lesions with age.

Generation of mice with mitochondrial dysfunction by introducing mouse mtDNA carrying a deletion into zygotes.

Mice carrying mitochondrial DNA (mtDNA) with pathogenic mutations would provide a system in which to study how mutant mtDNAs are transmitted and distributed in tissues, resulting in expression of mitochondrial diseases. However, no effective procedures are available for the generation of these mice. Isolation of mouse cells without mtDNA (rho0) enabled us to trap mutant mtDNA that had accumulated in somatic tissues into rho0 cells repopulated with mtDNA (cybrids). We isolated respiration-deficient cybrids with mtDNA carrying a deletion and introduced this mtDNA into fertilized eggs. The mutant mtDNA was transmitted maternally, and its accumulation induced mitochondrial dysfunction in various tissues. Moreover, most of these mice died because of renal failure, suggesting the involvement of mtDNA mutations in the pathogeneses of new diseases.

Inter-mitochondrial complementation: Mitochondria-specific system preventing mice from expression of disease phenotypes by mutant mtDNA.

Here we investigated the pathogenesis of deletion mutant mitochondrial (mt)DNA by generating mice with mutant mtDNA carrying a 4696-basepair deletion (DeltamtDNA4696), and by using cytochrome c oxidase (COX) electron micrographs to identify COX activity at the individual mitochondrial level. All mitochondria in tissues with DeltamtDNA4696 showed normal COX activity until DeltamtDNA4696 accumulated predominantly; this prevented mice from expressing disease phenotypes. Moreover, we did not observe coexistence of COX-positive and -negative mitochondria within single cells. These results indicate the occurrence of inter-mitochondrial complementation through exchange of genetic contents between exogenously introduced mitochondria with DeltamtDNA4696 and host mitochondria with normal mtDNA. This complementation shows a mitochondria-specific mechanism for avoiding expression of deletion-mutant mtDNA, and opens the possibility of a gene therapy in which mitochondria possessing full-length DNA are introduced.

Nuclear but not mitochondrial genome involvement in 3-methylcholanthrene-induced expression of tumorigenicity in mouse somatic cells.

The involvement of heritable modifications of mitochondrial DNA (mtDNA) in chemical carcinogenesis was examined by studies on the effects on tumorigenicity of interchange of mtDNA between 3-methylcholanthrene (MCA)-induced mouse tumor cells and nontumorigenic mouse cells by the cytoplast-to-cell fusion technique. The difference in propagating abilities of two types of mouse mtDNA, type 1 mtDNA of B10mtJ strain and type 2 mtDNA of C57BL/10 strain, was applied successfully for complete replacement of the host cell mtDNA by cytoplasmically transmitted mtDNA. Tumorigenicity was assayed in nude mice by inoculating 5 x 10(6) cells s.c. into the backs of the mice. The results showed that tumorigenicity was not induced in nontumorigenic cells by replacement of their mtDNA by that from MCA-induced tumor cells. Moreover, the tumorigenicity of MCA-induced tumor cells was still expressed when their mtDNA was replaced by that from normal cells with a limited life span. These observations suggest that, even if MCA treatment causes heritable modifications of mtDNA, modified mtDNA cannot induce chemical carcinogenesis and that modifications of nuclear DNA alone are sufficient for the expression of tumorigenicity.

Interleukin-33 enhances programmed oncosis of ST2L-positive low-metastatic cells in the tumour microenvironment of lung cancer.

The proinflammatory interleukin-33 (IL-33) binds to its receptor ST2L on the surface of immune cells and stimulates the production of Th2 cytokines; however, the effects of IL-33 on tumour cells are poorly understood. Here we show that ST2 was significantly downregulated in human lung cancer tissues and cells compared with normal lung tissues and cells. IL-33 expression was also inversely correlated with the stages of human lung cancers. In accordance with this finding, low-metastatic cells but not high-metastatic cells derived from Lewis lung carcinoma expressed functional ST2L. IL-33 was abundantly present in the tumours established by the low-metastatic cells compared with those formed by the high-metastatic cells. Although the low-metastatic cells scarcely expressed IL-33 in vitro, these cells did expry 6ess this molecule in vivo, likely due to stimulation by intratumoural IL-1ß and IL-33. Importantly, IL-33 enhanced the cell death of ST2L-positive low-metastatic cells, but not of ST2L-negative high-metastatic cells, under glucose-depleted, glutamine-depleted and hypoxic conditions through p38 MAPK and mTOR activation, and in a mitochondria-dependent manner. The cell death was characterised by cytoplasmic blisters and karyolysis, which are unique morphological features of oncosis. Inevitably, the low-metastatic cells, but not of the high-metastatic cells, grew faster in IL-33(-/-) mice than in wild-type mice. Furthermore, IL-33 selected for the ST2L-positive, oncosis-resistant high-metastatic cells under conditions mimicking the tumour microenvironment. These data suggest that IL-33 enhances lung cancer progression by selecting for more malignant cells in the tumour microenvironment.

Existence of common antigenic sites in tropomyosins.

1. Tropomyosins were extracted from vertebrate and invertebrate muscles, and their immunolo;ical characteristics were compared using antisera against tropomyosins from chicken skeletal and cardiac muscles. 2. Antigenic sites common to those of chicken skeletal muscle tropomyosin were found in all the tropomyosins tested, although the reactions of these common antigenic sites in an immunodiffusion test were weak in tropomyosins from phylogenetically distant animals. 3. An immunological difference was found between alpha-tropomyosins from chicken cardiac muscle and rabbit cardiac muscle. Thus they had specific antigenic sites in addition to the common ones. 4. A component was found in a 1 M KCL extract of Tetrahymena pyriformis which reacted with antiserum against chicken skeletal muscle tropomyosin.

Positioning of the A . T-rich regions in rat mitochondrial DNA by electron microscopy and analysis of the hysteresis of denaturation.

Intramolecular heterogeneity in the base composition of rat mitochondrial DNA (mtDNA) was shown by a combination of an improved denaturation mapping technique using electron microscopy and analysis of high-resolution optical melting-renaturation profiles. Circular mtDNA starts to melt in one specific region and then forms loops in four other regions in random order. These five early melting regions are all located in one half of the molecule. The arrangement of the early melting regions in rat mtDNA bears a remarkable resemblance not only to those of mtDNAs from several species of Drosophila but also to those of several species of Drosophila but also to those of several plasmid DNAs and phage DNA.

Evolutionary aspects of variant types of rat mitochondrial DNA'S.

Mitochondrial DNA's (mtDNAs) were prepared from various kinds of individual Norway rats, Rattus norvegicus, and from three types of individual black rats, Rattus rattus, (Asian type, Ceylon type, and Oceanian type). Intra- and interspecies divergence of their mtDNA sequences were calculated based on changes in restriction endonuclease cleavage sites. The extent of intraspecies divergence of black rats (about 8%) is much larger than that of Norway rats (1%) and the mtDNA of Asian-type black rats resembles the mtDNA of Norway rats more closely than it resembles the mtDNA of other types of black rats. These results strongly suggest that during the course of intraspecies differentiation of black rats, probably long after the separation of the three types of black rats, some Asian-type black rats were isolated sexually and formed a new species, Norway rats. On the basis of our observations we propose a hypothetical process to explain the evolution of animal mtDNA.

A new mitochondrial DNA mutation at 14577 T/C is probably a major pathogenic mutation for maternally inherited type 2 diabetes.

From a family of 16 diabetic patients with typical maternal inheritance, we investigated a 69-year-old woman with type 2 diabetes. The proband showed no major deletions in the mitochondrial DNA (mtDNA). Direct sequencing revealed 7 missense and 5 ribosomal RNA homoplasmic nucleotide substitutions when compared with the Cambridge Sequence and its recent revision. When compared with the control cybrid cells, the proband cybrid cells showed 6 nucleotide substitutions. Among these, 14577 T/C, which turned out to be 98.9% heteroplasmic, is a new missense substitution in the NADH dehydrogenase 6 gene. We also observed 2 other patients with 14577 T/C substitution from another group of 252 unrelated diabetic patients, whereas no individual from a group of 529 control subjects had 14577 T/C substitution. Furthermore, these 6 substitutions were in linkage disequilibrium. Mitochondrial respiratory chain complex I activity and O2 consumption rates of the proband cybrid cells, which were obtained by the fusion of mtDNA-deleted (rho0) HeLa cells and mtDNA from the proband, showed 64.5 and 61.5% reductions, respectively, compared with control cybrid cells. The present study strongly indicates that the new mtDNA mutation at 14577 T/C is probably a major pathogenic mutation for type 2 diabetes in this family.

Maternal inheritance of mouse mtDNA in interspecific hybrids: segregation of the leaked paternal mtDNA followed by the prevention of subsequent paternal leakage.

The transmission profiles of sperm mtDNA introduced into fertilized eggs were examined in detail in F1 hybrids of mouse interspecific crosses by addressing three aspects. The first is whether the leaked paternal mtDNA in fertilized eggs produced by interspecific crosses was distributed stably to all tissues after the eggs' development to adults. The second is whether the leaked paternal mtDNA was transmitted to the subsequent generations. The third is whether paternal mtDNA continuously leaks in subsequent backcrosses. For identification of the leaked paternal mtDNA, we prepared total DNA samples directly from tissues or embryos and used PCR techniques that can detect a few molecules of paternal mtDNA even in the presence of 10(8)-fold excess of maternal mtDNA. The results showed that the leaked paternal mtDNA was not distributed to all tissues in the F1 hybrids or transmitted to the following generations through the female germ line. Moreover, the paternal mtDNA leakage was limited to the first generation of an interspecific cross and did not occur in progeny from subsequent backcrosses. These observations suggest that species-specific exclusion of sperm mtDNA in mammalian fertilized eggs is extremely stringent, ensuring strictly maternal inheritance of mtDNA.

Complete repopulation of mouse mitochondrial DNA-less cells with rat mitochondrial DNA restores mitochondrial translation but not mitochondrial respiratory function.

By the fusion of mtDNA-less (rho(0)) cells of Mus musculus domesticus with platelets from different species, mtDNA repopulated cybrids were obtained for finding the mtDNA species that could induce mitochondrial abnormalities. Expression of mitochondrial dysfunction might be expected in these cybrids due to incompatibility between nuclear and mitochondrial genomes from different species. The results showed that mouse rho(0) cells could receive mtDNA from a different mouse species, M. spretus, or even mtDNA from the rat, Rattus norvegicus, and that the introduced rat mtDNA, but not M. spretus mtDNA, caused mitochondrial dysfunction, even though rat mtDNA could restore normal mitochondrial translation in the cybrids. Considering that mitochondrial respiratory complexes consist of nuclear DNA- and mtDNA-coded polypeptides, these observations suggest that the nuclear and mitochondrial interactions required for replication, transcription, and translation of introduced rat mtDNA must be less stringently controlled than those required for formation of normal respiratory complexes. As no procedure for introduction of mutagenized mouse mtDNA into living cells has yet been established, these findings provide important insights into generating mtDNA-knockout mice.

Selective and continuous elimination of mitochondria microinjected into mouse eggs from spermatids, but not from liver cells, occurs throughout embryogenesis.

Exclusion of paternal mitochondria in fertilized mammalian eggs is very stringent and ensures strictly maternal mtDNA inheritance. In this study, to examine whether elimination was specific to sperm mitochondria, we microinjected spermatid or liver mitochondria into mouse embryos. Congenic B6-mt(spr) strain mice, which are different from C57BL/6J (B6) strain mice (Mus musculus domesticus) only in possessing M. spretus mtDNA, were used as mitochondrial donors. B6-mt(spr) mice and a quantitative PCR method enabled selective estimation of the amount of M. spretus mtDNA introduced even in the presence of host M. m. domesticus mtDNA and monitoring subsequent changes of its amount during embryogenesis. Results showed that M. spretus mtDNA in spermatid mitochondria was not eliminated by the blastocyst stage, probably due to the introduction of a larger amount of spermatid mtDNA than of sperm mtDNA into embryos on fertilization. However, spermatid-derived M. spretus mtDNA was eliminated by the time of birth, whereas liver-derived M. spretus mtDNA was still present in most newborn mice, even though its amount introduced was significantly less than that of spermatid mtDNA. These observations suggest that mitochondria from spermatids but not from liver have specific factors that ensure their selective elimination and resultant elimination of mtDNA in them, and that the occurrence of elimination is not limited to early stage embryos, but continues throughout embryogenesis.

Evolutionary relationships among five subspecies of Mus musculus based on restriction enzyme cleavage patterns of mitochondrial DNA.

The intra- and intersubspecific genetic distances between five subspecies of Mus musculus were estimated from restriction enzyme cleavage patterns of maps of mitochondrial DNA (mtDNA). The European subspecies, M. m. domesticus and Asian subspecies, M. m. bactrianus, M. m. castaneus, M. m. molossinus and M. m. urbanus were examined. For each subspecies, except M. m. urbanus, at least two local races from widely separated localities were examined. Intrasubspecific heterogeneity was found in the mtDNA cleavage patterns of M. m. bactrianus and M. m. castaneus. M. m. molossinus and M. m. domesticus, however, revealed no intrasubspecific heterogeneity. Four of the subspecies had distinct cleavage patterns. The fifth, M. m. urbanus, had cleavage patterns identical to those of M. m. castaneus with several enzymes. Estimates of genetic distances between the various races and subspecies were obtained by comparing cleavage maps of the mtDNAs with various restriction enzymes. Nucleotide sequence divergences of mtDNA between local races were estimated to be less than 0.4% in M. m. bactrianus and less than 0.3% in M. m. castaneus. The times of divergence of both subspecies were calculated to be 0.1--0.2 x 10(6) years. These values suggest that the intrasubspecific divergence began some 0.1--0.2 x 10(6) years ago. On the other hand, nucleotide sequence divergences between European subspecies M. m. domesticus and Asian subspecies M. m. bactrianus and M. m. castaneus were 7.1% ane 5.8%, respectively. The times of divergence were calculated to be 2.1--2.6 x 10(6) years. Further, the nucleotide sequence divergence and time of divergence between M. m. molossinus and the other two Asian subspecies were comparable to those between M. m. molossinus and M. m. domesticus (about 3% and 1 x 10(6) years, respectively). These results suggest that M. m. molossinus is situated in a unique evolutionary position among Asian subspecies.

Synergistic effect of anti-T cell receptor monoclonal antibody and 15-deoxyspergualin on cardiac xenograft survival in a mouse-to-rat model.

BACKGROUND: Successful xenograft transplantation faces several obstacles including the presence of xenoantibodies, natural killer cell- and macrophage-mediated rejection, and T lymphocyte activation. METHODS: A mouse-to-rat cardiac xenograft model was used to examine the synergistic effect of anti-T cell receptor (TCR) monoclonal antibodies (mAb) and 15-deoxyspergualin (DSG) on graft survival. RESULTS: Pretransplantation injections (days -5, -3, and -1) of anti-TCR mAb (500 microg/kg/day) combined with continuous i.p. infusion of DSG (5 mg/kg/day) from day -7 to 28 significantly prolonged graft survival compared to untreated controls (3.3+/-0.5 vs. 44.2+/-5.6 days, P<0.001). Postoperative splenectomy combined with discontinuation of all other treatment on day 28 enhanced graft survival in rats treated with anti-TCR mAb and DSG to 71.0+/-2.5 days. Histological examination of grafts showed characteristic signs of vascular rejection: interstitial edema and hemorrhage, and polymorphonuclear cell infiltration. Antimouse antibody titers in recipients were increased upon rejection in each group that received a xenograft. Flow cytometry analysis showed a markedly decreased T cell population and a relatively increased mature B cell population (IgM(bright)/IgD(dull)) in spleens of rats treated with anti-TCR mAb and DSG on day 28. CONCLUSIONS: The mechanism of prolonged xenograft survival in this model may include inhibition of antibody production by arrest of B-cell maturation during development from IgM(dull)/IgD(bright) mature B cells to antibody producing cells, and inhibition of T cell activation. The rejection seen in our model may be caused by xenoreactive antibodies and may be associated with T cells, natural killer cells, and macrophages.

The differences between the primary structures of mitochondrial DNAs from rat liver and ascites hepatoma (AH-130).

Rat mitochondrial DNAs (mtDNAs) of ascites hepatoma (AH-130) and normal liver cells (Donryu strain) were digested by various restriction endonucleases and the cleavage patterns compared by agarose gel electrophoresis. Different cleavage patterns were observed between AH-130 and liver mtDNAs when they were digested by HindII and EcoRI. The mtDNA of AH-130 lost one clevage site of HindII and one clevage site of EcoRI. The cleavage patterns of mtDNAs from other organs and strains tested were the same as that of liver mtDNA. From these observations we concluded that the molecular clone of AH-130 mtDNA was different from that of other mtDNAs.

Detection, localization, and sequence analyses of mitochondrial regulatory region RNAs in several mammalian species.

The mitochondrial regulatory region (mrr) located between the tRNAPhe and tRNAPro genes of mitochondrial DNA (mtDNA) is essential for regulation of replication and transcription of the mitochondrial genome. Polyadenylated short RNAs complementary to the L-strand of the mrr in human cells and similar RNAs (polyadenylation status unknown) in rat and mouse cells have been reported. We now report detection of ca. 0.2 kb polyadenylated mrrRNAs in cultured cells of Chinese hamster, African green monkey, mouse, rat, and human. We isolated a cDNA clone to a rat polyadenylated mrrRNA of 158 bp in length excluding the polyadenyl tail, which spans the region from the light strand promoter (LSP) to the origin of heavy strand replication (OriH). This cDNA contains both an open reading frame encoding a 26 amino acid polypeptide and a 12 nucleotide sequence complementary to the 3'-terminus of rat mitochondrial 12S rRNA. A cDNA clone to a human HeLa cell polyadenylated mrrRNA also contains a 12 nucleotide region complementary to the human mitochondrial 12S rRNA. We used a mitochondrial genome-deficient HeLa cell line, rho0 HeLa, and a derived cybrid cell line, HeEB, with a reconstituted mitochondrial genome, to demonstrate that the occurrence of the mrrRNA is dependent on the presence of a mitochondrial genome, and these polyadenylated mrrRNAs are transcribed from the mitochondrial genome. Our results further substantiate the common existence of polyadenylated mrrRNAs among mammals and support previously proposed hypotheses for the multi-functional nature of polyadenylated mrrRNA.

A rare case of lung cancer associated with renal cell cancer and benign histiocytoma of bone.

Lung cancer often metastasizes to organs outside the thorax, and consequently radiological evaluation of distant metastasis has become standard procedure prior to surgery. Although positive radiological findings generally suggest distant metastasis, the possibility of the co-existence of a benign tumor and primary malignancies must be considered. Herein we report a case of surgical resection of histologically confirmed lung cancer associated with renal cell cancer and benign histiocytoma of the humerus.

Protective mechanism of ultrafiltration against cardiopulmonary bypass-induced lung injury.

BACKGROUND: We previously demonstrated a negative effect of cardiopulmonary bypass (CPB) in a canine model of single-lung graft function and an improved effect with ultrafiltration during CPB. OBJECTIVE: To investigate the mechanism of these effects, focusing on cytokines and pulmonary surfactants using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). MATERIALS AND METHODS: Fifteen left-sided single-lung transplant procedures were performed in pairs of dogs. The animals were divided into 3 groups. In one group, transplantation was performed without CPB (non-CPB group); in a second group, transplantation was performed with CPB and CPB flow was decreased slowly and pulmonary artery pressure was controlled (CPB group; and in the third group, transplantation was performed with CPB and ultrafiltration (CPB+UF group). Grafted lung specimens were harvested for RT-PCR of cytokines (IL-6, IL-8, and IL-10) and surfactant proteins (SP-A, SP-B, and SP-C). RESULTS: Real-time quantitative RT-PCR demonstrated increased IL-6 expression in the CPB group compared with the non-CPB group. IL-6 gene expression was suppressed and pulmonary surfactant restored using ultrafiltration. Gene expression of surfactant protein (SP)-A, SP-B, and SP-C was decreased in the CPB group compared with normal lung and ultrafiltration groups, which demonstrated sustained gene expression of SP-A and SP-B. CONCLUSION: Cardiopulmonary bypass has negative effects on grafts; however, ultrafiltration attenuates acute lung dysfunction by decreasing the inflammatory response and increasing pulmonary surfactant.

Gene therapy of mitochondrial diseases using human cytoplasts.

Mitochondrial (mt) DNA is highly susceptible to mutation. A novel approach using cytoplasts is essential for gene therapy of the diseases caused by these mutations, because of their non-Mendelian genetics. In this method, mtDNA is transferred into the mutant cells by forming cybrids and can complement the defect correctly and safely.

An improved method for estimating sequence divergence between related DNAs from changes in restriction endonuclease cleavage sites.

We have developed a theory to estimate the degree of sequence divergence between related DNAs from the comparison of restriction endonuclease recognition sites. Two major improvements have been made upon a similar method reported by Upholt (1977). First, the most probable value is calculated by the collective use of all available data. This reduces intrinsic statistical error and extends the analyzable range of sequence divergence. Second, all variables are redefined so that they have strict mathematical implications. This corrects a serious error arising from the misinterpretation of the meaning of the fraction of conserved cleavage sites. With this refined method, sequence divergence between rat and mouse mitochondrial DNAs (mtDNAs) was calculated to be about 25% substitutions/nucleotide, which is in good agreement with the DNA-DNA hybridization data obtained by Jakovcic et al. (1975). It was also estimated that the three types of rat mtDNAs differ from one another by 0.3 approximately 1% of total base pairs. These values are 2 approximately 5 times smaller than those obtained with the conventional method.