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.

Spontaneous glomerular and tubulointerstitial lesions in common marmosets (Callithrix jacchus).

Spontaneous progressive nephropathy dominated by glomerular lesions in common marmosets has been reported. However, the histopathologic characteristics, including the relationship between glomerular and tubulointerstitial lesions, have not been described in detail. In the present study, the authors examined the histopathologic characteristics of the background renal lesions in common marmosets (3 males and 9 females, 3 to 8 years old). The severity of glomerular lesions was graded into 3 classes: grade I, no alteration; grade II, hilar/focal increase of mesangial matrix; grade III, global/diffuse increase of mesangial matrix. Tubulointerstitial lesions (tubular regeneration and hyperplasia and interstitial inflammation and fibrosis) were scored according to the area of each lesion. The renal lesions were characterized by enlargement of glomeruli, expanded mesangial area with increase of periodic acid-Schiff reaction-positive matrix, tubular regeneration and hyperplasia, and interstitial inflammation and fibrosis. Glomerular lesions progressed with increasing mesangial matrix and aging. Additionally, the tubulointerstitial lesions became exacerbated with progressing glomerular lesions. Tubular hyperplasia was divided into 4 types according to the structure of the cell layer (simple or stratified-like), the area of increased lining cells (partial or entire), cytoplasmic staining (eosinophilic or basophilic), brush border and thickness of basement membrane, and the activity of cell proliferation. In conclusion, the background renal lesions in common marmosets were characterized by glomerular lesions with increase of mesangial matrix, which progressed with aging, and secondary tubulointerstitial lesions, including tubular hyperplasia. Those lesions were thus diagnosed as progressive glomerulonephropathy in common marmosets.

Dextran sulphate sodium increases splenic Gr1(+)CD11b(+) cells which accelerate recovery from colitis following intravenous transplantation.

While Gr1(+)CD11b(+) cells are known to regulate immune responses and accumulate in most cancer tissues, the function of Gr1(+)CD11b(+) cells in inflammation is poorly understood. We investigated the role of Gr1(+)CD11b(+) cells in a dextran sulphate sodium (DSS)-treated mouse model of ulcerative colitis (UC). C57BL/6 mice were treated with 2% DSS in drinking water for 5 days. Disease progression and recovery were assessed by body weight, disease activity index score (DAI) score and colon length. Splenic Gr1(+)CD11b(+) cell number was greatly increased during the recovery phase of DSS-induced colitis. DSS-derived splenic Gr1(+)CD11b(+) cells were administered intravenously to recipient (C57BL/6) mice during the early phase of DSS treatment. The transplanted splenic DSS-induced Gr1(+)CD11b(+) cells improved DSS-induced colitis and promoted efficient colonic mucosal healing. We found that the CD11b(+) single positive cells increased in the course of DSS-induced colitis in lamina propria. The transplantation of splenic Gr1(+)CD11b(+) cells induced feedback suppression of myeloid-lineage cell development. Namely, the transplantation of splenic Gr1(+)CD11b(+) cells greatly suppressed the migration of CD11b(+) single positive cells to the lamina propria. Further, transplantation of Gr-1(+)CD11b(+) cells greatly suppressed the increase of the same population, especially during the late phase of DSS colitis both in spleen and bone marrow.

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.

Delta15N of soil N and plants in a N-saturated, subtropical forest of southern China.

We investigated the delta(15)N profile of N (extractable NH(4)(+), NO(3)(-), and organic N (EON)) in the soil of a N-saturated subtropical forest. The order of delta(15)N in the soil was EON > NH(4)(+) > NO(3)(-). Although the delta(15)N of EON had been expected to be similar to that of bulk soil N, it was higher than that of bulk soil N by 5 per thousand. The difference in delta(15)N between bulk soil N and EON (Delta(15)N(bulk-EON)) was correlated significantly with the soil C/N ratio. This correlation implies that carbon availability, which determines the balance between N assimilation and dissimilation of soil microbes, is responsible for the high delta(15)N of EON, as in the case of soil microbial biomass delta(15)N. A thorough delta(15)N survey of available N (NH(4)(+), NO(3)(-), and EON) in the soil profiles from the organic layer to 100 cm depth revealed that the delta(15)N of the available N forms did not fully overlap with the delta(15)N of plants. This mismatch in delta(15)N between that of available N and that of plants reflects apparent isotopic fractionation during N uptake by plants, emphasizing the high N availability in this N-saturated forest.

[Six cases of spontaneous pneumomediastinum].

We report 6 cases of spontaneous pneumomediastinum. It is defined not to have clear etiology such as trauma, and is comparatively rare disease developing suddenly. Our patients complained of chest or neck pain, dyspnea and pain when swallowing. They were 3 men and 3 women, who were young and did not have causal disease. Chest X-ray films and computed tomography (CT) scans revealed pneumomediastinum. All of them were treated conservatively and recovered completely within 10 days hospitalization. Spontaneous pneumomediastinum is uncommon and usually benign. Most patients require only conservative treatment. However, since it possibly develops tension pneumothorax, pneumothorax, or mediastinitis, careful observation is recommended never to overlook life-threatening condition. In addition, it is important to distinguish from Boerhaave syndrome, tracheal trauma and so on.

IL23 differentially regulates the Th1/Th17 balance in ulcerative colitis and Crohn's disease.

BACKGROUND: A novel T helper (Th) cell lineage, Th17, that exclusively produces the proinflammatory cytokine interleukin 17 (IL17) has been reported to play important roles in various inflammatory diseases. IL23 is also focused upon for its potential to promote Th17. Here, the roles of the IL23/IL17 axis in inflammatory bowel diseases such as ulcerative colitis (UC) and Crohn's disease (CD) were investigated. METHODS: Mucosal samples were obtained from surgically resected specimens (controls, n = 12; UC, n = 17; CD, n = 22). IL17 production by isolated peripheral blood (PB) and lamina propria (LP) CD4(+) cells was examined. Quantitative PCR amplification was performed to determine the mRNA expression levels of IL17, interferon gamma (IFNgamma), IL23 receptor (IL23R) and retinoic acid-related orphan receptor gamma (RORC) in LP CD4(+) cells, and IL12 family members, such as IL12p40, IL12p35 and IL23p19, in whole mucosal specimens. The effects of exogenous IL23 on IL17 production by LP CD4(+) cells were also examined. RESULTS: IL17 production was higher in LP CD4(+) cells than in PB. Significant IL17 mRNA upregulation in LP CD4(+) cells was found in UC, while IFNgamma was increased in CD. IL23R and RORC were upregulated in LP CD4(+) cells isolated from both UC and CD. IL17 production was significantly increased by IL23 in LP CD4(+) cells from UC but not CD. Upregulated IL23p19 mRNA expression was correlated with IL17 in UC and IFNgamma in CD. CONCLUSIONS: IL23 may play important roles in controlling the differential Th1/Th17 balance in both UC and CD, although Th17 cells may exist in both diseases.

Concurrent daily cisplatin and extended-field radiation therapy for carcinoma of the cervix.

The aim of this study was to assess acute toxicities of concurrent low-dose daily cisplatin and extended-field radiation therapy (EFRT) for carcinoma of the uterine cervix. Fifteen women with cervical cancer who were treated with concurrent daily low-dose cisplatin and EFRT were analyzed. Daily cisplatin dose was fixed to 8 mg/m(2), which was determined in the preceding phase I study using pelvic radiotherapy. Twelve patients underwent either combined external beam radiation therapy and intracavitary brachytherapy or external beam radiation therapy alone. Three other patients were treated with adjuvant chemoradiotherapy after surgery. A total dose of EFRT ranged from 40 to 45 Gy, with an additional boost to the gross tumor volume up to 50.4-55 Gy. A median total dose of cisplatin during entire radiation therapy course was 224 mg/m(2) (range, 200-240 mg/m(2)). In 14 of 15 patients (93%), daily cisplatin could be delivered continuously as planned without any modification. Administration of cisplatin had to be interrupted in only one patient for only 3 days. Fourteen patients developed grade 2 or worse leukopenia including five after treatment, grade 2 in four, grade 3 in eight, and grade 4 in two. Grade 3 thrombocytopenia was observed in three patients. Grade 2 or worse anemia was observed in 12. Three patients had grade 3 nonhematologic toxicities, diarrhea in two, and nausea/vomiting in one. Although moderate to severe hematologic toxicities are common, this study suggests that concurrent low-dose daily cisplatin and EFRT are feasible. A cumulative cisplatin dose of greater than 200 mg/m(2) during radiation therapy could be achieved by using daily cisplatin dose of 8 mg/m(2).

5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside stimulates tyrosine hydroxylase activity and catecholamine secretion by activation of AMP-activated protein kinase in PC12 cells.

The activity of AMP-activated protein kinase (AMPK) is regulated by the metabolic and nutritional state of the cell. 5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) is transformed into riboside monophosphate (ZMP) via phosphorylation by adenosine kinase inside the cell and exerts it effect by stimulating AMPK. AICAR significantly induces an increase in AMPK activity in a dose- and time-dependent manner in the rat pheochromocytoma cell line PC12. In addition, compound C, an AMPK inhibitor, as well as 5'-amino-5'-dAdo, an adenosine kinase inhibitor, inhibits the AICAR-induced AMPK activity. AICAR significantly stimulates tyrosine hydroxylase (TH) (the rate-limiting enzyme in the biosynthesis of catecholamine) activity and the corresponding mRNA level, which closely matches with the TH protein level. In addition, AICAR provokes a rapid and long-lasting increase in the phosphorylation of TH at Ser19, Ser31 and Ser40. AICAR also markedly activates ERKs, JNK and p38. The MEK-1-inhibitor (PD-098059) causes a partial, but significant, inhibition of AICAR-induced TH enzyme activity by phosphorylation of Ser31 without affecting phosphorylation at the two other sites. By contrast, neither the JNK-inhibitor nor the p38-inhibitor affects TH enzyme activity and phosphorylation. Similarly, PD-098059 partially, but significantly, inhibits the AICAR-induced increase in the TH mRNA level. Furthermore, AICAR increases the level of cAMP in PC12 cells. The present study also shows that H89, a protein kinase A inhibitor, abolishes the AICAR-induced increase in the level of TH mRNA, as well as the corresponding enzyme activity and Ser40 phosphorylation. Finally, AICAR significantly increases dopamine secretion from PC12 cells. These findings indicate that AICAR activates catecholamine synthesis and secretion through AMPK activation in chromaffin cells.

Transcriptional repression by Drosophila methyl-CpG-binding proteins.

C methylation at genomic CpG dinucleotides has been implicated in the regulation of a number of genetic activities during vertebrate cell differentiation and embryo development. The methylated CpG could induce chromatin condensation through the recruitment of histone deacetylase (HDAC)-containing complexes by methyl-CpG-binding proteins. These proteins consist of the methylated-DNA binding domain (MBD). Unexpectedly, however, several studies have identified MBD-containing proteins encoded by genes of Drosophila melanogaster, an invertebrate species supposed to be void of detectable m(5)CpG. We now report the genomic structure of a Drosophila gene, dMBD2/3, that codes for two MBD-containing, alternatively spliced, and developmentally regulated isoforms of proteins, dMBD2/3 and dMBD2/3Delta. Interestingly, in vitro binding experiments showed that as was the case for vertebrate MBD proteins, dMBD2/3Delta could preferentially recognize m(5)CpG-containing DNA through its MBD. Furthermore, dMBD2/3Delta as well as one of its orthologs in mouse, MBD2b, could function in human cells as a transcriptional corepressor or repressor. The activities of HDACs appeared to be dispensable for transcriptional repression by dMBD2/3Delta. Finally, dMBD2/3Delta also could repress transcription effectively in transfected Drosophila cells. The surprisingly similar structures and characteristics of the MBD proteins as well as DNA cytosine (C-5) methyltransferase-related proteins in Drosophila and vertebrates suggest interesting scenarios for their roles in eukaryotic cellular functions.

Effects of exercise on adiponectin and adiponectin receptor levels in rats.

Adiponectin reportedly reduces insulin-resistance. Exercise has also been shown to lessen insulin-resistance, though it is not known whether exercise increases levels of adiponectin and/or its receptors or whether its effects are dependent on exercise intensity and/or frequency. Catecholamine levels have been shown to increase during exercise and to fluctuate based on exercise intensity and duration. In light of this information, we examined the effects of exercise on catecholamine, adiponectin, and adiponectin receptor levels in rats. Our data showed that blood adiponectin levels increased by 150% in animals that exercised at a rate of 30 m/min for 60 min 2 days per week, but not 5 days, per week; no such increase was observed in rats that exercised at a rate of 25 m/min for 30 min. The effects of exercise on adiponectin receptor mRNA were variable, with adiponectin receptor 1 (AdipoR1) levels in muscle increasing up to 4 times while adiponectin receptor 2 (AdipoR2) levels in liver fell to below half in response to exercise at a rate of 25 m/min for 30 min 5 days per week. We also observed that urinary epinephrine levels and plasma lipids were elevated by exercise at a rate of 25 m/min for 30 min 2 days per week. Exercise frequency at a rate of 25 m/min for 30 min correlated with AdipoR1 and AdipoR2 mRNA expression in the muscle and liver, respectively (r=0.640, p<0.05 and r=-0.808, p<0.0005, respectively). Urinary epinephrine levels correlated with AdipoR2 mRNA expression in liver tissues (r=-0.664, p<0.05) in rats that exercised at a rate of 25 m/min for 30 min. Thus, exercise may regulate adiponectin receptor mRNA expression in tissues, which might cause increases in glucose uptake and fatty acid oxidation in the muscle. The effect of exercise on adiponectin levels depends on the specific conditions of the exercise.

Induction of antitumor immunity in mice by allo-major histocompatibility complex class I gene transfectant with strong antigen expression.

An allo-major histocompatibility complex class I gene (H-2Kb) was transfected to murine mastocytoma P1.HTR (P815 subline) cells, after which several transfectant clones were obtained. Two clones, which expressed a low level of H-2Kb antigen, grew well and killed the syngeneic DBA/2 mice when they were inoculated ip. These mice lived longer than the mice given injections of the parental P1.HTR tumor. However, one clone, which expressed a high level of H-2Kb antigen, was rejected completely by the syngeneic DBA/2 mice and induced a generation of H-2Kb-specific cytotoxic T cells. Interestingly, the mice that had rejected the clone with high H-2Kb expression received strong anti-tumor immunity for rejection of the parental P1.HTR tumor challenged at the high dose.

Dynamics of generation of antigen loss variants from L1210 murine leukemia clones detected by a tumor-specific T-cell clone.

Originally T-cell clone K7L-sensitive L1210 murine leukemia clones were tested for their capacity to generate K7L-insensitive variants at various times after cloning. All of the L1210 clones (L1210/1, -2, -4, and -7) maintained in vitro for 1 month were severely inhibited in their growth in the culture in which K7L was added and in mice given injections of K7L at the initial stage. This indicated that any L1210 clone tested was not a mixture of K7L-sensitive and K7L-insensitive clones at the time of cloning. By both in vivo and in vitro K7L-mediated tumor suppression assays, K7L-insensitive antigen loss variants were then found to be generated from some (L1210/4, L1210/7) but not other (L1210/1, L1210/2) originally K7L-sensitive L1210 clones during 1 month of maintenance. Ratios of variant cells to total clone cells 1 month after cloning were estimated around 0.1% for L1210/7, 0.01% for L1210/4, and less than 0.001% (undetectable) for L1210/1 and L1210/2. Neither L1210/1 nor L1210/2 generated detectable K7L-insensitive variant cells during long-term (14-month) maintenance. All of the ten subclones of L1210/7 which were obtained 7 or 11 months after the initial cloning of L1210/7 were K7L sensitive, and not all the subclones generated K7L-insensitive variants in 1-2 months of maintenance after recloning. However, all of the subclones of L1210/7 which were maintained for 7 months generated antigen loss variants. All eight clone cells obtained from original L1210 and K7L-insensitive L1210 expressed H-2Kd and H-2Dd antigens detected by H-2Kd or Dd-specific cytotoxic T-lymphocyte clones or monoclonal antibodies. These results suggest that the antigen loss variants arise in originally K7L-sensitive L1210 clones at different times after cloning, and the probability of generation of the variants is clonally determined. The antigen loss variants seem to be generated by rare (once per 1 to 2 months or less frequent) chance with unproportionally rapid growth rather than by more frequent development for simple accumulation. The ratio of K7L-insensitive variant cells to total L1210/7 cells did not increase progressively during long-term (13 months or more) maintenance in vivo or in vitro and was always below 0.1%. It was suggested that the population size of antigen loss variants was controlled biphasically.

Identification and characterization of a unique tumor-associated surface antigen on L1210 leukemia cells recognized by semisyngeneic antisera.

The tumor-associated surface antigen on L1210 leukemia cells was studied by immunofluorescence staining and immunoprecipitation. Anti-L1210 serum was prepared in BALB/c X DBA/2 F1 mice by priming with a hybrid of L1210 and human Lesch-Nyhan fibroblast cells and hyperimmunizing with L1210 leukemia cells. This hyperimmune serum was able to demonstrate specific surface fluorescence on L1210 cells, while the antiserum did not react with various mouse tumor cell lines, normal lymphoid tissues, or mitogen-activated lymphoid cells. The anti-L1210 serum immunoprecipitated a single polypeptide with a molecular weight of 90,000 from 125I-labeled L1210 cells. The expression of this antigen was enhanced by tumor-promoting agent and heat shock treatment. The biological significance of the L1210-specific cell surface antigen is discussed.

GADD34 suppresses lipopolysaccharide-induced sepsis and tissue injury through the regulation of macrophage activation.

Growth arrest and DNA damage inducible protein 34 (GADD34) is induced by various cellular stresses, such as DNA damage, endoplasmic reticulum stress, and amino-acid deprivation. Although the major roles of GADD34 are regulating ER stress responses and apoptosis, a recent study suggested that GADD34 is linked to innate immune responses. In this report, we investigated the roles of GADD34 in inflammatory responses against bacterial infection. To explore the effects of GADD34 on systemic inflammation in vivo, we employed a lipopolysaccharide (LPS)-induced murine sepsis model and assessed the lethality, serum cytokine levels, and tissue injury in the presence or absence of GADD34. We found that GADD34 deficiency increased the lethality and serum cytokine levels in LPS-induced sepsis. Moreover, GADD34 deficiency enhanced tissue destruction, cell death, and pro-inflammatory cytokine expression in LPS-induced acute liver injury. Pro-inflammatory cytokine production after LPS stimulation is regulated by the Toll-like receptor 4 (TLR4)-mediated NF-κB signaling pathway. In vitro experiments revealed that GADD34 suppressed pro-inflammatory cytokine production by macrophages through dephosphorylation of IKKß. In conclusion, GADD34 attenuates LPS-induced sepsis and acute tissue injury through suppressing macrophage activation. Targeting this anti-inflammatory role of GADD34 may be a promising area for the development of therapeutic agents to regulate inflammatory disorders.

Linkage between melanocytic tumor development and early burst of Ret protein expression for tolerance induction in metallothionein-I/ret transgenic mouse lines.

We examined the basis of the all or none difference in inducing melanocytic tumor development among three transgenic mouse lines (304, 192 and 242) to which the same promoter-enhancer (metallothionein-I) and oncogene (ret) were introduced. We initially demonstrated that both skin melanosis and Ret protein expression in skin, thymus and brain first became detectable before or immediately after birth in the mice of the tumor developing lines (304 and 192), whereas they became detectable a few days after birth in the mice of the non-tumor developing line (242) by Western blotting and immunohistochemical analysis. Interestingly, the Ret protein expression in skin developed rapidly after birth as a burst with peak levels on 0.5-1.5 day newborns of lines 304 and 192 and on 7.0-7.5 day-old mice of line 242. The levels of autophosphorylation of Ret kinase in skin were, however, invariable among these three transgenic mouse lines. The mice of line 242, but not those of lines 192 and 304, responded to Ret protein immunization by increased antigen-dependent lymphocyte proliferation and T-cell-mediated tumor growth suppression in vitro. Furthermore, ret-transgenic mice of line 242, but not line 304, rejected the subcutaneously transplanted tumors that had originally developed in a mouse of line 304. These results suggest that whether oncogene product-specific-tolerance is established or not to antitumor immunity may be decided by the dynamics of ret oncogene expression before and after delivery and this is the primary factor determining development or non-development of melanoma.

Oncogenicity of the ret transforming gene in MMTV/ret transgenic mice.

We have successfully produced transgenic mice that carry the ret oncogene driven by a mouse mammary tumor virus promoter/enhancer. Mammary and salivary gland adenocarcinomas were developed in a stochastic fashion in these mice. Moreover, premalignant tumors with hyperplastic and dysplastic lesions of Harderian glands and male reproductive tracts frequently occurred at young ages. High expression of the transgene was closely associated with the development of these tumors although the levels of the transgene expression were variable among individuals. In addition, large amounts of phosphotyrosine-containing proteins were detected in cell lysates from mammary and salivary adenocarcinomas by immunoblotting with the anti-phosphotyrosine antibody.

Evidence for the interaction between Translin and GADD34 in mammalian cells.

To examine the function of GADD34, we used the yeast two-hybrid system to clone the protein that interacts with the murine GADD34 gene product. We utilized, as bait, the product of the GADD34 cDNA deletions including the PEST region and the gamma(1)34.5 domain. One of the cDNAs cloned encoded murine Translin which is known to bind to the DNA sequence detected in the DNA translocation. The interaction between GADD34 and Translin was also confirmed by an in vitro binding assay and in vivo two-hybrid analysis in NIH 3T3 cells. Although GADD34 expression was significantly elevated with methyl methanesulfonate treatment, we could not detect the induction of Translin mRNA.