Mitochondrial DNA (mtDNA) mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia) due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA) also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J) nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ0) mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan.
We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene.
IL28B and ITPA genetic variants are associated with the outcome of pegylated-interferon and ribavirin (PEG-IFN/RBV) therapy. However, the significance of these genetic variants in cirrhotic patients following splenectomy has not been determined.
Thirty-seven patients with HCV-induced cirrhosis who underwent laparoscopic splenectomy (Spx group) and 90 who did not (non-Spx group) were genotyped for IL28B and ITPA. The outcome or adverse effects were compared in each group. Interferon-stimulated gene 15 (ISG15) and protein kinase R expression in the spleen was measured using total RNA extracted from exenterate spleen.
Sustained virological response (SVR) rate was higher in patients carrying IL28B major genotype following splenectomy (50% vs 27.3%) and in patients carrying minor genotype in the Spx group compared to non-Spx group (27.3% vs 3.6%, P < 0.05). Pretreatment splenic ISG expression was higher in patients carrying IL28B major. There was no difference in progression of anemia or thrombocytopenia between patients carrying each ITPA genotype in the Spx group. Although splenectomy did not increase hemoglobin (Hb) level, Hb decline tended to be greater in the non-Spx group. In contrast, splenectomy significantly increased platelet count (61.1 × 103/μl vs 168.7 × 103/μl, P < 0.01), which was maintained during the course of PEG-IFN/RBV therapy.
IL28B genetic variants correlated with response to PEG-IFN/RBV following splenectomy. Splenectomy improved SVR rate among patients carrying IL28B minor genotype and protected against anemia and thrombocytopenia during the course of PEG-IFN/RBV therapy regardless of ITPA genotype.
IL28B; ITPA; Splenectomy; Liver cirrhosis
Helicobacter pylori infection causes gastritis, peptic ulcers and gastric malignancies, and its eradication has been advocated by many groups. We determined the H. pylori carrier status and eradication rates of patients with chronic hepatitis C virus (HCV) infection.
In total, 76 chronically HCV-infected patients were enrolled for comparison with 228 HCV-noninfected, age- and sex-matched controls. H. pylori infection was confirmed by H. pylori antibody and urea breath testing.
The H. pylori infection rate was significantly higher for HCV-infected patients (67 of 76, 88.2%) than for HCV-noninfected controls (158 of 228, 69.3%). Endoscopic findings showed that the rates of gastric ulcers and gastritis were significantly higher for the 67 HCV-infected patients with H. pylori infection (34.3% and 77.6%) than for the 158 HCV-noninfected controls with H. pylori infection (15.2% and 57.6%). Treatment to eradicate H. pylori had a significantly higher success rate for HCV-infected patients (61 of 67, 91.0%) than for HCV-noninfected controls (115 of 158, 72.8%).
The markedly high H. pylori eradication rate observed in this study shows that eradication of H. pylori holds promise for the improvement of the long-term health condition of patients with chronic HCV infection.
Helicobacter pylori; Chronic hepatitis C
Mutations in mitochondrial DNA (mtDNA) might contribute to expression of the tumor phenotypes, such as metastatic potential, as well as to aging phenotypes and to clinical phenotypes of mitochondrial diseases by induction of mitochondrial respiration defects and the resultant overproduction of reactive oxygen species (ROS). To test whether mtDNA mutations mediate metastatic pathways in highly metastatic human tumor cells, we used human breast carcinoma MDA-MB-231 cells, which simultaneously expressed a highly metastatic potential, mitochondrial respiration defects, and ROS overproduction. Since mitochondrial respiratory function is controlled by both mtDNA and nuclear DNA, it is possible that nuclear DNA mutations contribute to the mitochondrial respiration defects and the highly metastatic potential found in MDA-MB-231 cells. To examine this possibility, we carried out mtDNA replacement of MDA-MB-231 cells by normal human mtDNA. For the complete mtDNA replacement, first we isolated mtDNA-less (ρ0) MDA-MB-231 cells, and then introduced normal human mtDNA into the ρ0 MDA-MB-231 cells, and isolated trans-mitochondrial cells (cybrids) carrying nuclear DNA from MDA-MB-231 cells and mtDNA from a normal subject. The normal mtDNA transfer simultaneously induced restoration of mitochondrial respiratory function and suppression of the highly metastatic potential expressed in MDA-MB-231 cells, but did not suppress ROS overproduction. These observations suggest that mitochondrial respiration defects observed in MDA-MB-231 cells are caused by mutations in mtDNA but not in nuclear DNA, and are responsible for expression of the high metastatic potential without using ROS-mediated pathways. Thus, human tumor cells possess an mtDNA-mediated metastatic pathway that is required for expression of the highly metastatic potential in the absence of ROS production.
AIM: To analyze the association between the emergence of tyrosine-methionine-asparatate-asparatate (YMDD) mutants (reverse transcription; rtM204I/V) and deterioration of liver function during long-term lamivudine treatment of Japanese patients with chronic hepatitis B virus (HBV) infection.
METHODS: The data of 61 consecutive Japanese patients with chronic hepatitis B who underwent continuous lamivudine treatment for more than 24 mo and had a virological response were analyzed. Analysis of YMDD mutants was done by real-time polymerase chain reaction with LightCycler probe hybridization assay for up to 90 mo (mean, 50.8 mo; range, 24-90 mo).
RESULTS: A mixed mutant-type (YMDD + tyrosine-isoleucine-asparatate-asparatate: YIDD or tyrosine-valine-asparatate-asparatate: YVDD) or a mutant-type (YIDD or YVDD) were found in 57.4% of 61 patients at 1 year, 78.7% of 61 patients at 2 years, 79.6% of 49 patients at 3 years, 70.5% of 34 patients at 4 years, 68.4% of 19 patients at 5 years, 57.1% of 14 patients at 6 years, and 33.3% of 6 patients at 7 years. Of the 61 patients, 56 (92%) had mixed mutant- or a mutant-type. Only 5 (8%) had no mutants at each observation point. Virological breakthrough was found in 26 (46.4%) of 56 patients with YMDD mutants, 20 of whom had a hepatitis flare-up: the remaining 30 (53.6%) had neither a virological breakthrough nor a flare-up. All 20 patients who developed a hepatitis flare-up had a biochemical and virological response after adefovir was added to the lamivudine treatment.
CONCLUSION: Our results suggest that it is possible to continue lamivudine treatment, even after the emergence of YMDD mutants, up to the time that the patients develop a hepatitis flare-up.
Tyrosine-methionine-asparatate-asparatate mutant; Hepatitis B virus; Lamivudine; Drug resistance
Tumors or embryonic stem cells bearing foreign mitochondrial DNA are rejected by the innate immune system via a mechanism that depends on MyD88.
Mitochondrial DNA (mtDNA) has been proposed to be involved in respiratory function, and mtDNA mutations have been associated with aging, tumors, and various disorders, but the effects of mtDNA imported into transplants from different individuals or aged subjects have been unclear. We examined this issue by generating trans-mitochondrial tumor cells and embryonic stem cells that shared the syngenic C57BL/6 (B6) strain–derived nuclear DNA background but possessed mtDNA derived from allogenic mouse strains. We demonstrate that transplants with mtDNA from the NZB/B1NJ strain were rejected from the host B6 mice, not by the acquired immune system but by the innate immune system. This rejection was caused partly by NK cells and involved a MyD88-dependent pathway. These results introduce novel roles of mtDNA and innate immunity in tumor immunology and transplantation medicine.
AIM: To analyze the efficacy and safety of a combination therapy of pegylated interferon (PEG-IFN) α-2b plus ribavirin (RBV) in older Japanese patients (65 years or older) infected with hepatitis C virus (HCV).
METHODS: This multicenter study included 938 patients with HCV genotype 1 who received 1.5 μg/kg per week PEG-IFN α-2b plus RBV 600-1000 mg/d for 48 wk and 313 HCV genotype 2 patients who received this treatment for 24 wk.
RESULTS: At 24 wk after the end of combination therapy, the overall sustained virological response (SVR) for genotypes 1 and 2 were 40.7% and 79.6%, respectively. The SVR rate decreased significantly with age in each genotype, and was markedly reduced in genotype 1 (P < 0.001). Moreover, the SVR was significantly higher in patients with genotype 1 who were less than 65 years (47.3% of 685) than in those 65 years or older (22.9% of 253) (P < 0.001) and was higher in patients with genotype 2 who were less than 65 years (82.9% of 252) than in those 65 years or older (65.6% of 61) (P = 0.004). When patients received a dosage at least 80% or more of the target dosage of PEG-IFN α-2b and 60% or more of the target dosage of RBV, the SVR rate significantly increased to 66.5% in patients less than 65 years and to 45.2% in those 65 years or older (P < 0.001). Adverse effects resulted in treatment discontinuation more often in patients with genotype 1 (14.4%) than in patients with genotype 2 (7.3%), especially by patients 65 years or older (24.1%).
CONCLUSION: PEG-IFN α-2b plus RBV treatment was effective in chronic hepatitis C patients 65 years or older who completed treatment with at least the minimum acceptable treatment dosage.
Hepatitis C virus; Gerontology; Pegylated interferon; Ribavirin
An early virological response (EVR) after the start of interferon (IFN) treatment for chronic hepatitis C leads to a successful virological outcome. To analyze an association between sustained virological response (SVR) and EVR by comparing TaqMan with Amplicor assays in HCV genotype 1-infected patients treated with pegylated (PEG)-IFN alpha-2b plus ribavirin (RBV).
We retrospectively analyzed a total of 80 HCV genotype 1 patients (39 SVR and 41 non-SVR patients), who received an enough dosage and a complete 48-week treatment of PEG-IFN alpha-2b plus RBV. Serum HCV RNA levels were measured by both TaqMan and Amplicor assays for each patients at Weeks 2, 4, 8 and 12 after the start of the antiviral treatment.
Of the 80 patients with undetectable HCV RNA by Amplicor, 17 (21.3%) patients were positive for HCV RNA by TaqMan at Weeks 12. The quantification results showed that no significant difference in the decline of HCV RNA level between TaqMan and Amplicor 10-fold method assays within the initial 12 weeks of the treatment was found. However, the qualitative analysis showed significant differences of the positive predictive rates for SVR were found between TaqMan (100% at weeks 4 and 100% at weeks 8) and Amplicor (80.0% and 69.6%, respectively).
The COBAS TaqMan HCV assay is very useful for monitoring HCV viremia during antiviral treatment to predict a SVR in HCV genotype 1 patients.
It has been controversial for many years of whether mtDNA mutations are involved in phenotypes related to cancer due to the difficulty in excluding possible involvement of nuclear DNA mutations in these phenotypes. We addressed this issue by complete trading of mtDNAs between tumor cells expressing different metastatic phenotypes. Resultant trans-mitochondrial cybrids share the same nuclear background, but possess mtDNA from tumor cells expressing different metastatic phenotypes, and thus can be used to uncover the role of mtDNA in these phenotypes. The results showed that mtDNA controls development of metastasis in tumor cells, while tumor development is controlled by nuclear genome.
pathogenic mtDNA mutations; respiration defects; enhanced glycolysis; ROS overproduction; rho-zero cells; mtDNA transfer technology; metastasis
In mammals, observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations have led to the creation of the bottleneck theory for the transmission of mtDNA. The bottleneck could be attributed to a marked decline of mtDNA content in germ cells giving rise to the next generation, to a small effective number of mtDNA segregation units resulting from homoplasmic nucleoids rather than the single mtDNA molecule serving as the units of segregation, or to the selective transmission of a subgroup of the mtDNA population to the progeny. We have previously determined mtDNA copy number in single germ cells and shown that the bottleneck occurs without the reduction in germline mtDNA content. Recently one study suggested that the bottleneck is driven by a remarkable decline of mtDNA copies in early primordial germ cells (PGCs), while another study reported that the mtDNA genetic bottleneck results from replication of a subpopulation of the mtDNA genome during postnatal oocyte maturation and not during embryonic oogenesis, despite a detected a reduction in mtDNA content in early PGCs. To clarify these contradictory results, we examined the mtDNA copy number in PGCs isolated from transgenic mice expressing fluorescent proteins specifically in PGCs as in the aforementioned two other studies. We provide clear evidence to confirm that no remarkable reduction in mtDNA content occurs in PGCs and reinforce that the bottleneck is generated without reduction of mtDNA content in germ cells.
Mutations of mtDNA are responsible for many types of mitochondrial diseases in humans, including myopathy and neurological disorders. Females carrying a mixture of mutant and wild-type mtDNA variants transmit a variable amount of mutant mtDNA to each offspring. The proportion of mutated mtDNA inherited from the mother determines the onset and severity of diseases. Studies have suggested that the mtDNA genome is transmitted through a bottleneck, but the underlying mechanism remains controversial. By detecting mtDNA copy number in single cells, we previously showed that the bottleneck occurs without reduction of mtDNA content in germline cells. However, recently a study reported a marked decline of mtDNA copies in embryonic germ cells and attributed this reduction to the creation of the bottleneck. Yet another study concluded that the bottleneck occurs during postnatal oocyte maturation and not during embryonic oogenesis. To resolve these controversies, we examined mtDNA copies in embryonic germ cells identified using the same methodology as in the other two studies. We show solid evidence to confirm our previous findings. This confirmation is important because the understanding of mtDNA content in female germ cells will facilitate the development of therapeutic strategies preventing the transmission of mitochondrial diseases from mother to offspring.
Mitochondrial DNA (mtDNA) with pathogenic mutations has been found in patients with cognitive disorders. However, little is known about whether pathogenic mtDNA mutations and the resultant mitochondrial respiration deficiencies contribute to the expression of cognitive alterations, such as impairments of learning and memory. To address this point, we used two groups of trans-mitochondrial mice (mito-mice) with heteroplasmy for wild-type and pathogenically deleted (Δ) mtDNA; the "low" group carried 50% or less ΔmtDNA, and the "high" group carried more than 50% ΔmtDNA.
Both groups had normal phenotypes for not only spatial learning, but also memory at short retention delays, indicating that ΔmtDNA load did not affect learning and temporal memory. The high group, however, showed severe impairment of memory at long retention delays. In the visual cortex and dentate gyrus of these mice, we observed mitochondrial respiration deficiencies, and reduced Ca2+/calmodulin-dependent kinase II-α (α-CaMKII), a protein important for the establishment of spatial remote memory.
Our results indicated that normal mitochondrial respiratory function is necessary for retention and consolidation of memory trace; deficiencies in this function due to high loads of pathogenically mutated mtDNA are responsible for the preferential impairment of spatial remote memory.
In the situation that it would not be able to produce model animals for mitochondrial diseases caused by mitochondrial DNA (mtDNA) with pathogenic mutations, we succeeded in generating mice with pathogenic deletion mutant mtDNA (ΔmtDNA), named “mito-mice”, by direct introduction of mitochondria with ΔmtDNA into mouse zygotes. In the mito-mice, accumulation of ΔmtDNA induced mitochondrial respiration defects in various tissues, resulting in mitochondrial disease phenotypes, such as low body weight, lactic acidosis, ischemia, myopathy, heart block, deafness, male infertility, and renal failure. Thus, mito-mice are the first model animal for mtDNA-based diseases, and the mice could be valuable for understanding precise pathogeneses and testing therapies of mitochondrial diseases. In the present review, we summarized reverse genetic studies using the mito-mice.
mouse model; mitochondrial DNA; large-scale deletion mutation; mitochondrial complementation; mitochondrial diseases
Lamivudine treatment has been recently demonstrated to increase the serum albumin levels in cirrhotic patients with hepatitis B virus (HBV) infection, but the precise mechanism remains unclear. We hypothesized that the improvement of hypoalbuminemia by lamivudine may be attributable to the reduction of HBV replication itself, rather than to cessation of hepatitis. In order to confirm this hypothesis, in this study we evaluated factors which correlated with the increase in serum albumin levels. Fifty-four patients (Child-Pugh A/B/C, 35/9/10) with HBV-related liver cirrhosis who had been treated with lamivudine for more than 12 months were evaluated. We analyzed the correlation between the increase in serum albumin levels at month 12 after starting treatment (Δ-albumin) and various pretreatment variables. We also analyzed the correlation between Δ-albumin and the reduction in serum levels of HBV-DNA (Δ-HBV-DNA) or alanine aminotransferase (Δ-ALT) at month 12.
The average Δ-albumin was 0.38 g/dL and only serum HBV-DNA levels before treatment correlated significantly with Δ-albumin. We also analyzed the correlation in patients whose alanine aminotransferase levels were normalized after 12 months so that the possible influence of breakthrough hepatitis could be excluded. Even among this subgroup of patients, there was no significant correlation between Δ-albumin and either pretreatment alanine aminotransferase levels or Δ-ALT. In contrast, in patients whose serum HBV-DNA was undetectable at month 12, we found a significant correlation between Δ-albumin and both pretreatment serum HBV-DNA levels and Δ-HBV-DNA.
Our results demonstrated that albumin levels are associated with pretreatment HBV-DNA but not with alanine aminotransferase levels.
Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) persistently maintains a plasmid containing the KSHV latent origin of replication (oriP) as a closed circular episome in dividing cells. In this study, we investigated the involvement of chromosome binding activity of LANA1 in persistent episome maintenance. Deletion of the N-terminal 22 amino acids of LANA1 (ΔN-LANA) inhibited the interaction with mitotic chromosomes in a human cell line, and the mutant concomitantly lost activity for the long-term episome maintenance of a plasmid containing viral oriP in a human B-cell line. However, a chimera of ΔN-LANA with histone H1, a cellular chromosome component protein, rescued the association with mitotic chromosomes as well as the long-term episome maintenance of the oriP-containing plasmid. Our results suggest that tethering of KSHV episomes to mitotic chromosomes by LANA1 is crucial in mediating the long-term maintenance of viral episomes in dividing cells.
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.
Based upon the nucleotide sequence of the relA gene from Escherichia coli, a gene fragment corresponding to the homologous gene from the pathogenic oral bacterium Porphyromonas gingivalis 381 was isolated by PCR and utilized to construct a relA mutant. The mutant, KS7, was defective in ribosome-mediated ppGpp formation and also in the stringent response.