The human mitochondrial 12S ribosomal RNA (rRNA) A1555G mutation has been associated with aminoglycoside-induced and nonsyndromic deafness in many families worldwide. Our previous investigation revealed that the A1555G mutation is a primary factor underlying the development of deafness but is not sufficient to produce a deafness phenotype. However, it has been proposed that nuclear-modifier genes modulate the phenotypic manifestation of the A1555G mutation. Here, we identified the nuclear-modifier gene TRMU, which encodes a highly conserved mitochondrial protein related to transfer RNA (tRNA) modification. Genotyping analysis of TRMU in 613 subjects from 1 Arab-Israeli kindred, 210 European (Italian pedigrees and Spanish pedigrees) families, and 31 Chinese pedigrees carrying the A1555G or the C1494T mutation revealed a missense mutation (G28T) altering an invariant amino acid residue (A10S) in the evolutionarily conserved N-terminal region of the TRMU protein. Interestingly, all 18 Arab-Israeli/Italian-Spanish matrilineal relatives carrying both the TRMU A10S and 12S rRNA A1555G mutations exhibited prelingual profound deafness. Functional analysis showed that this mutation did not affect importation of TRMU precursors into mitochondria. However, the homozygous A10S mutation leads to a marked failure in mitochondrial tRNA metabolisms, specifically reducing the steady-state levels of mitochondrial tRNA. As a consequence, these defects contribute to the impairment of mitochondrial-protein synthesis. Resultant biochemical defects aggravate the mitochondrial dysfunction associated with the A1555G mutation, exceeding the threshold for expressing the deafness phenotype. These findings indicate that the mutated TRMU, acting as a modifier factor, modulates the phenotypic manifestation of the deafness-associated 12S rRNA mutations.

Oxidative stress is implicated as a major cause of aging and age-related diseases, such as Parkinson's and Alzheimer's, as well as ischemia-reperfusion injury in stroke. The mitochondrial electron transport chain is the principal source of reactive oxygen species within cells. Despite considerable medical interest, the molecular mechanisms that regulate reactive oxygen species formation within the mitochondrion remain poorly understood. Here, we report the isolation and characterization of a Drosophila mutant with a defect in subunit b of succinate dehydrogenase (SDH; mitochondrial complex II). The sdhB mutant is hypersensitive to oxygen and displays hallmarks of a progeroid syndrome, including early-onset mortality and age-related behavioral decay. Pathological analysis of the flight muscle, which is amongst the most highly energetic tissues in the animal kingdom, reveals structural abnormalities in the mitochondria. Biochemical analysis shows that, in the mutant, there is a complex II-specific respiratory defect and impaired complex II-mediated electron transport, although the other respiratory complexes remain functionally intact. The complex II defect is associated with an increased level of mitochondrial hydrogen peroxide production, suggesting a possible mechanism for the observed sensitivity to elevated oxygen concentration and the decreased lifespan of the mutant fly.aging ͉ hyperoxia ͉ reactive oxygen species ͉ sdhB ͉ succinate dehydrogenase

A reverse genetics approach was utilized to discover new proteins that interact with the mitochondrial fusion mediator mitofusin 2 (Mfn2) and that may participate in mitochondrial fusion. In particular, in vivo formaldehyde cross-linking of whole HeLa cells and immunoprecipitation with purified Mfn2 antibodies of SDS cell lysates were used to detect an ϳ42-kDa protein. Mammalian mitofusins Mfn1 and Mfn2 are large GTPases of the mitochondrial outer membrane that mediate mitochondrial fusion (1-3). They also contain two coiled-coil domains or heptad repeats (HR1 and HR2) 2 (3). The major portion of each of the proteins, including the N terminus-proximal GTPase domain and HR1 and the C terminus-proximal HR2, is exposed to the cytosol (4). The attachment to the outer membrane is mediated by two membrane-spanning segments that are separated by a small intermembrane space loop and that are located between the HR1 and HR2 repeats. Human Mfn1 and Mfn2 are highly homologous proteins with 62% identity at the amino acid level with both, however, being essential proteins. The homozygous Ϫ/Ϫ Mfn1-null and Ϫ/Ϫ Mfn2-null mice die during embryonic development (5). The mouse embryonic fibroblasts (MEF), derived from the Ϫ/Ϫ Mfn1-null or Ϫ/Ϫ Mfn2-null embryos, have distinct mitochondrial morphology defects, and exhibit severe reduction in mitochondrial fusion activity (5). Thus, Mfn1 and Mfn2 may have different functions in mitochondrial fusion. Unlike MEF lacking Mfn1 or Mfn2, MEF lacking both mitofusins completely lack mitochondrial fusion capacity and show severe cellular dysfunction (6). Functions of Mfn2 that are independent of its fusion activity, i.e. controlling mitochondrial metabolism and repressing vascular smooth muscle cell proliferation, have also been reported (7, 8).Mfn2 and Mfn1 have the capacity to form homo-and heterooligomers, as demonstrated by co-immunoprecipitation of tagged proteins (5, 9). At least one of the mitofusins is required on each of the adjacent mitochondria to promote mitochondrial fusion (10). The initial step of this process is characterized by tethering of two adjacent mitochondria via assembly of a mitofusin complex, which is mediated by HR2 forming a dimeric, antiparallel coiled-coil (10). The subsequent steps of mitochondrial fusion, including the exact function of the GTPase domains, are less well understood. In yeast, an interaction of the mitofusin homolog Fzo1, involving the GTPase domain and the HR domains, is essential for fusion (11). In the same organism, mitochondrial fusion has been reconstituted in vitro. Mitochondrial fusion proceeds through discreet and sequential mitochondrial outer and inner membrane fusion events, with both steps requiring GTP hydrolysis (12). A third GTPase, Opa1, appears also to be involved in the process of mitochondrial fusion (13). This GTPase is synthesized as a precursor, which is processed by a matrix-processing peptidase to a mature form, large Opa1 (l-Opa1). l-Opa1 is anchored to the inner mitochondrial membrane and can undergo another proteolyti...

The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK ؊ cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamatedependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK ؊ cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.

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We estimate and describe the incidence rates, mortality, and cost of CAP (community-acquired pneumonia), in both inpatient and outpatient settings, in the Czech Republic (CZ), Slovakia (SK), Poland (PL), and Hungary (HU). A retrospective analysis was conducted on administrative data from the health ministry and insurance reimbursement claims with a primary diagnosis of pneumonia in 2009 to determine hospitalization rates, costs, and mortality in adults ≥50 years of age. Patient chart reviews were conducted to estimate the number of outpatient cases. Among all adults ≥50 years, the incidence of hospitalized CAP per 100,000 person years was: 456.6 (CZ), 504.6 (SK), 363.9 (PL), and 845.3 (HU). The average fatality rate for all adults ≥50 is 19.1%, and for each country; 21.7% (CZ), 20.9% (SK), 18.6% (PL), 17.8% (HU). Incidence, fatality, and likelihood of hospitalization increased with advancing age. Total healthcare costs of CAP in EUR was 12,579,543 (CZ); 9,160,774 (SK); 22,409,085 (PL); and 18,298,449 (HU); with hospitalization representing over 90% of the direct costs of treatment. The burden of CAP increases with advancing age in four CEE countries, with hospitalizations driving the costs of CAP upwards in the elderly population. Mortality rates are generally higher than reported in Western EU countries.

No abstract

We have shown here that the apoptosis inducer staurosporine causes an early decrease in the endogenous respiration rate in intact 143B.TK ؊ cells. On the other hand, the activity of cytochrome c oxidase is unchanged for the first 8 h after staurosporine treatment, as determined by oxygen consumption measurements in intact cells. The decrease in the endogenous respiration rate precedes the release of cytochrome c from mitochondria. Moreover, we have ruled out caspases, permeability transition, and protein kinase C inhibition as being responsible for the decrease in respiration rate. Furthermore, overexpression of the gene for Bcl-2 does not prevent the decrease in respiration rate. The last finding suggests that Bcl-2 acts downstream of the perturbation in respiration. The evidence of normal enzymatic activities of complex I and complex III in staurosporinetreated 143B.TK ؊ osteosarcoma cells indicates that the cause of the respiration decrease is probably an alteration in the permeability of the outer mitochondrial membrane. Presumably, the voltage-dependent anion channel closes, thereby preventing ADP and oxidizable substrates from being taken up into mitochondria. This interpretation was confirmed by another surprising finding, namely that, in staurosporine-treated 143B.TK ؊ cells permeabilized with digitonin at a concentration not affecting the mitochondrial membranes in naive cells, the outer mitochondrial membrane loses its integrity; this leads to a reversal of its impermeability to exogenous substrates. The loss of outer membrane integrity leads also to a massive premature release of cytochrome c from mitochondria. Most significantly, Bcl-2 overexpression prevents the staurosporine-induced hypersensitivity of the outer membrane to digitonin. Our experiments have thus revealed early changes in the outer mitochondrial membrane, which take place long before cytochrome c is released from mitochondria in intact cells.