Acute oxidative stress induced by ischemia-reperfusion or inflammation causes serious damage to tissues, and persistent oxidative stress is accepted as one of the causes of many common diseases including cancer. We show here that hydrogen (H(2)) has potential as an antioxidant in preventive and therapeutic applications. We induced acute oxidative stress in cultured cells by three independent methods. H(2) selectively reduced the hydroxyl radical, the most cytotoxic of reactive oxygen species (ROS), and effectively protected cells; however, H(2) did not react with other ROS, which possess physiological roles. We used an acute rat model in which oxidative stress damage was induced in the brain by focal ischemia and reperfusion. The inhalation of H(2) gas markedly suppressed brain injury by buffering the effects of oxidative stress. Thus H(2) can be used as an effective antioxidant therapy; owing to its ability to rapidly diffuse across membranes, it can reach and react with cytotoxic ROS and thus protect against oxidative damage.

White adipocytes are unique in that they contain large unilocular lipid droplets that occupy most of the cytoplasm. To identify genes involved in the maintenance of mature adipocytes, we expressed dominant-negative PPARγ in 3T3-L1 cells and performed a microarray screen. The fat-specific protein of 27 kDa (FSP27) was strongly downregulated in this context. FSP27 expression correlated with induction of differentiation in cultured preadipocytes, and the protein localized to lipid droplets in murine white adipocytes in vivo. Ablation of FSP27 in mice resulted in the formation of multilocular lipid droplets in these cells. Furthermore, FSP27-deficient mice were protected from diet-induced obesity and insulin resistance and displayed an increased metabolic rate due to increased mitochondrial biogenesis in white adipose tissue (WAT). Depletion of FSP27 by siRNA in murine cultured white adipocytes resulted in the formation of numerous small lipid droplets, increased lipolysis, and decreased triacylglycerol storage, while expression of FSP27 in COS cells promoted the formation of large lipid droplets. Our results suggest that FSP27 contributes to efficient energy storage in WAT by promoting the formation of unilocular lipid droplets, thereby restricting lipolysis. In addition, we found that the nature of lipid accumulation in WAT appears to be associated with maintenance of energy balance and insulin sensitivity.

Mutant mitochondrial DNA with large-scale deletions (A-mtDNA) has been frequently observed in patients with chronic progressive external ophthalmoplegia (CPEO), a subgroup of the mitochondrial encephalomyopathies. To excdude involvement of the nuclear genome in expiression of the mitochondrial dysf~1nction characteristic of CPEO, we introduced the mtDNA of a CPEO patient into' clonal mtDNA-less HeLa cells and isolated cybrid clones. Quaqtitation' of A-mtDNA in the cybrids revealed that A-mtDNA was selectively propagated with higher levels of A-mtDNA correlating with slower cellular growth rate. In these cybrid clones, translational complementation of the missing tRNAs occurred only when A-mtDNA was <60% of the total mtDNA, whereas accumulation of A-mttDNA to >60% resulted in progressive inhibition of overall mitochondrial translation as well as reduction of cytochrome c oxidase a#ctivit'tthroughout the organelle population. Because these'cybrids shared the same nuclear background as HeLa cells, these results suggest that large-scale deletion mutations of mtDNA alone are sufficient for the mitochondrial dysfunction characteristic of CFEO. Recently, heteroplasmy of wt-mtDNA and mutant mtDNA with a point mutation in the mitochondrial tRNALYS and tRNAUbUR genes was shown to be closely associated with MERRF (9) and MELAS (10-12), respectively. However, there is as yet no convincing-evidence to explain how mutant mtDNA induces mitochondrial dysfunction, or whether the mutations of mtDNA alone are sufficient to cause mitochondrial dysfunction in these diseases.In the present study, by cytoplasmically transmitting CPEO-derived mtDNA to clonal mtDNA-less HeLa EB8 cells, we found that A-mtDNA had a clear propagational advantage over wt-mtDNA and that its consequent accumulation to over 60o of the'total mtDNA resulted in a progressive inhibition of overall mitochondrial translation as well as a reduction of COX activity. As-the mtDNA-recipient EB8 cells were clonal and thus shared the same nuclear background, we conclude that large-scale deletion mutations of mtDNA induced the mitochondrial dysfunction characteristic of the disease directly without the help of any defects in the nuclear genome. MATERIALS AND METHODSCell Culture. mtDNA-less HeLa EB8 cells were isolated by the procedure of King and Attardi (13) Proc. Natl. Acad. Sci. USA 88 (1991) 10615 nine. In this mtDNA transfer system, however, as no method for genetic selection to remove parental EB8 cells was available, the colonies that grew in the medium with 6-thioguanine were either cybrids or the parental EB8 cells. However, growth of cells with mtDNA (cybrids) is expected to be slightly better than that of mtDNA-less cells, so for preferential selection of cybrids we picked up 127 colonies growing relatively fast. Of 127 clones, 6 clones had mtDNA. mtDNA Analysis. The total DNA (2-5 jig) extracted from 2 x 105 cells was digested with the single-cut restriction enzyme Pvu II. The fragments separated by 0.8% agarose gel electrophoresis were then transferre...

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Molecular hydrogen (H2) has been accepted to be an inert and nonfunctional molecule in our body. We have turned this concept by demonstrating that H2 reacts with strong oxidants such as hydroxyl radical in cells, and proposed its potential for preventive and therapeutic applications. H2 has a number of advantages exhibiting extensive effects: H2 rapidly diffuses into tissues and cells, and it is mild enough neither to disturb metabolic redox reactions nor to affect signaling reactive oxygen species; therefore, there should be no or little adverse effects of H2. There are several methods to ingest or consume H2; inhaling H2 gas, drinking H2-dissolved water (H2-water), injecting H2-dissolved saline (H2-saline), taking an H2 bath, or dropping H2-saline into the eyes. The numerous publications on its biological and medical benefits revealed that H2 reduces oxidative stress not only by direct reactions with strong oxidants, but also indirectly by regulating various gene expressions. Moreover, by regulating the gene expressions, H2 functions as an anti-inflammatory and anti-apoptotic, and stimulates energy metabolism. In addition to growing evidence obtained by model animal experiments, extensive clinical examinations were performed or are under investigation. Since most drugs specifically act to their targets, H2 seems to differ from conventional pharmaceutical drugs. Owing to its great efficacy and lack of adverse effects, H2 has promising potential for clinical use against many diseases.

Point mutations in the mitochondrial (mt) tRNA Leu(UUR) gene are responsible for mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), a subgroup of mitochondrial encephalomyopathic diseases. We previously showed that mt tRNA Leu(UUR) with an A3243G or T3271C mutation derived from patients with MELAS are deficient in a normal taurinecontaining modification ( m 5 U; 5-taurinomethyluridine) at the anticodon wobble position. To examine decoding disorder of the mutant tRNA due to the wobble modification deficiency independent of the pathogenic point mutation itself, we used a molecular surgery technique to construct an mt tRNA Leu(UUR) molecule lacking the taurine modification but without the pathogenic mutation. This ''operated'' mt tRNA Leu(UUR) without the taurine modification showed severely reduced UUG translation but no decrease in UUA translation. We thus concluded that the UUG codon-specific translational defect of the mutant mt tRNAs Leu(UUR) is the primary cause of MELAS at the molecular level. This result could explain the complex I deficiency observed clinically in MELAS.