Chemical and physiological functions of molecular oxygen and reactive oxygen species (ROS) and existing equilibrium between pools of pro-oxidants and anti-oxidants providing steady state ROS level vital for normal mitochondrial and cell functioning are reviewed. The presence of intracellular oxygen and ROS sensors is postulated and few candidates for this role are suggested. Possible involvement of ROS in the process of fragmentation of mitochondrial reticulum made of long mitochondrial filaments serving in the cell as "electric cables", as well as the role of ROS in apoptosis and programmed mitochondrial destruction (mitoptosis) are reviewed. The critical role of ROS in destructive processes under ischemia/reoxygenation and ischemic preconditioning is discussed. Mitochondrial permeability transition gets special consideration as a possible component of the apoptotic cascade, resulting in excessive "ROS-induced ROS release".
To extend previous observations demonstrating differences in number, morphology, and activity of mitochondria in spontaneously immortalized vim(+) and vim(-) fibroblasts derived from wild-type and vimentin knockout mice, some structural and functional aspects of mitochondrial genome performance and integrity in both types of cells were investigated. Primary Vim(+/+) and Vim(-/-) fibroblasts, which escaped terminal differentiation by immortalization were characterized by an almost twofold lower mtDNA content in comparison to that of their primary precursor cells, whereby the average mtDNA copy number in two clones of vim(+) cells was lower by a factor of 0.6 than that in four clones of vim(-) cells. However, during serial subcultivation up to high passage numbers, the vim(+) and vim() fibroblasts increased their mtDNA copy number 1.5- and 2.5-fold, respectively. While early-passage cells of the vim(+) and vim(-) fibroblast clones differed only slightly in the ratio between mtDNA content and mitochondrial mass represented by mtHSP70 protein, after ca. 300 population doublings the average mtDNA/mtmass ratio in the vim(+) and vim() cells was increased by a factor of 2 and 4.5, respectively. During subcultivation, both types of cells acquired the fully transformed phenotype. These findings suggest that cytoskeletal vimentin filaments exert a strong influence on the mechanisms controlling mtDNA copy number during serial subcultivation of immortalized mouse embryo fibroblasts, and that vimentin deficiency causes a disproportionately enhanced mtDNA content in high-passage vim(-) fibroblasts. Such a role of vimentin filaments was supported by the stronger retention potential for mtDNA and mtDNA polymerase (gamma) detected in vim(+) fibroblasts by Triton X-100 extraction of mitochondria and agaroseembedded cells. Moreover, although the vim(+) and vim(-) fibroblasts were equally active in generating free radicals, the vim(-) cells exhibited higher levels of immunologically detectable 8-oxoG and mismatch repair proteins MSH2 and MLH1 in their mitochondria. Because in vim(-) fibroblasts only one point mutation was detected in the mtDNA D-loop control region, these cells are apparently able to efficiently remove oxidatively damaged nucleobases. On the other hand, a number of large-scale mtDNA deletions were found in high-passage vim(-) fibroblasts, but not in low-passage vim(-) cells and vim(+) cells of both low and high passage. Large mtDNA deletions were also induced in young vim(-) fibroblasts by treatment with the DNA intercalator ethidium bromide, whereas no such deletions were found after treatment of vim(+) cells. These results indicate that in immortalized vim(-) fibroblasts the mitochondrial genome is prone to large-scale rearrangements, probably due to insufficient control of mtDNA repair and recombination processes in the absence of vimentin.
Results and discussion Fig. 1. Genetic map of the pPICZαA:IFNα2blinker-ApoA-I expression vector. IFN-a2b-ApoAI 432 aa IFN-a2b ApoA-I Linker (Ser-Gly)10 Fig. 2. Scheme of the chimeric fusion protein IFN-ApoA-I Fig. 3. Representation of the tertiary structure of the chimeric protein IFN-ApoA-I. Clinical use of recombinant human interferon alpha-2b (rIFN) is limited by its short halflife 1 and toxicity. Currently, there are two main modifications of long-acting rIFN-pegylated rIFN 2,3 and rIFN genetically fused with albumin 4. The use of human apolipoprotein A-I (apoA-I) as a protector protein seems also promising, since apoA-I has a long half-life in the body, is not immunogenic, is capable of natural formation of lipoprotein complexes, and allows improve the pharmacokinetics of therapeutic proteins fused with it. In this work, a recombinant strain of Pichia pastoris X33, producing a chimeric protein consist of rIFN, fused with human apoA-I through a flexible linker, was obtained. The IFN and apoA-I genes were optimized for expression in P. pastoris. The cultivation of a yeast strain producing a chimera was carried out in an orbital shaker. The protein yield was 30 mg/L. The chimera was purified by reverse phase chromatography and its purity was about 90%. The primary structure of the chimera was confirmed by MALDI-TOF. IFN-apoA-I exhibited high specific antiviral activity comparable to that of rIFN and equal to 1.6 × 10 8 IU/mg. The chimera showed a 1.8-fold longer half-life in comparison with rIFN аfter a single subcutaneous injection in mice.
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