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Abstracts / Mitochondrion 7 (2007) 404–433

7 Translocator protein 18 kDa (TSPO) endogenous ligand affect metabolic activity and cell cycle of human osteoblast-like cell Rosenberg Nahum *, Rosenberg Orit, Leschiner Svetlana, Soudry Michael, Weizman Abraham, Veenman Leo, Gavish Moshe Bruce and Ruth Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel Introduction: The mitochondrial Translocator Protein 18 kDa (TSPO, previously named as the peripheral benzodiazepine receptor – PBR) is involved in cellular respiration, steroidogenesis and apoptosis. In our recent study we reported on the role of the synthetic pharmacological ligands to the TSPO in enhancing human osteoblast catabolism. There is also a previous evidence of the existence of an endogenous ligands to the TSPO, but their role in the human osteoblast physiology has not been verified yet. porphyrine IX has been found having affinity to the TSPO. Therefore we hypothesize that human osteoblast metabolism might be mediated by the porphyrine IX and the mode of its action is similar the synthetic ligand to the TSPO. Methods: Cell cycle of the cultured human derived osteoblast-like cells, following exposure to porphyrine IX, endogenous ligand to TSPO, and N,N-di-n-hexyl 2-(4-fluorophenyl)indole-3-acetamide (FGIN-1-27), synthetic ligand to the TSPO, was determined by flow cytometry (FACS). These ligands’ affect on cell number, metabolic activity, i.e., cellular fluorodeoxyglucose ([18F]-FDG) incorporation and alkaline phosphatase activity, and cell death rate, i.e., LDH activity in the culture media, were assayed. The semi-quantitative response of TSPO to exposure to these ligands was estimated by Western blotting. Six samples of cultured cells for each condition were used. The t test was implemented for the statistical analyses. P values below .05 considered as statistically significant. Results: Cell count significantly decreased following exposure to FGIN-1-27 or porphyrine IX. Cellular [18F]-FDG incorporation and alkaline phosphatase activity were suppressed by both ligands. Cell cycle analysis showed a significant decrease in the fraction of cells in the G1 and G2/M phases when exposed to each ligand with a higher proportion of necrotic and apoptotic cells. Western blotting showed a decrease in TSPO abundance following treatment by both ligands. LDH activity in culture media significantly increased following exposure to FGIN-1-27 or porphyrine IX. Discussion: We show that FGIN-1-27 and porphyrine IX have a similar cell death inducing affect on human osteoblast-like cell in vitro. This affect is parallel to the inhibition of the cellular metabolism. Since both ligands similarly reduce the availability of TSPO we postulate that their mode of action is similar by affecting this mitochondrial structure with sub sequential induction of cell death. Therefore we suggest that human osteoblast metabolism and cell cycle are mediated through TSPO and that porphyrine IX might be an active endogenous ligand to the TSPO having a regulatory affect on the cell cycle. doi:10.1016/j.mito.2007.08.011

8 Internalization of isolated mitochondria by mammalian cells Volkmar Weissig a,*, Eyad Katrangi a, Gerard D’Souza a, Sarathi V. Boddapati a, Mariola Kulawiec b, Keshav Singh b, Brian Bigger c a Northeastern University, Bouve College of Health Sciences, School of Pharmacy, Department of Pharmaceutics, Boston, MA, USA; b Roswell Park Cancer Institute, Department of Cancer Genetics, Buffalo, NY, USA; c University of Manchester, Royal Manchester Children’s Hospital, Manchester, UK The co-culturing of mesenchymal stem cells with rho(0) cells was recently reported to produce clones of rescued cells with functional mitochondria (Spees, 2006). The authors’ suggestion that mitochondria can move between cells has triggered discussions about a putative physiological significance of such process and about its mechanisms (Csordas, 2006).

Co-incubation of rho(0) cells with isolated mitochondria, however, did not result in rescue of cells lacking mtDNA. This demonstrated lack of passive cellular uptake of exogenous isolated mitochondria appears to be in disparity with data published in a Nature paper a quarter of a century ago (Clark and Shay, 1982), in which the authors demonstrated the transfer of chloramphenicol resistance to cells sensitive to this antibiotic by incubating the chloramphenicol sensitive cells with isolated mitochondria carrying the chloramphenicol resistant gene. In view of the contradicting reports with respect to the ability of mammalian cells for internalizing exogenous isolated mitochondria, we resumed earlier, unpublished work from our own laboratory (Katrangi, 2005), during which we found supporting evidence for the ability of mammalian cells to internalize isolated mitochondria. Here, we demonstrate first the physical presence of internalized mitochondria within recipient cells using confocal fluorescence microscopy. Second we verify functionality of internalized mitochondria by measuring their contribution to respiration in cells lacking mtDNA. Third, utilizing the recently demonstrated natural competence of isolated mitochondria to take up linear DNA molecules (Koulintchenko, 2006) we demonstrate the feasibility of using cellular internalization of isolated exogenous mitochondria as a potential tool for studying mitochondrial genetics in living mammalian cells. doi:10.1016/j.mito.2007.08.012

References Spees, J.L. et al., 2006]. Proc. Natl. Acad. Sci. USA 103, 1283–1288. Csordas, A., 2006]. Rejuvenation Res. 9, 450–454. Clark, M.A., Shay, J.W., 1982]. Nature 295, 605–607. Katrangi, E. et al., 2005]. Intl. Symp. Control. Rel. Bioact. Mater. 32. Koulintchenko, M. et al., 2006]. Hum. Mol. Genet. 15, 143–154.

9 Single fibers from myopathic Tfam/ mice display alterations in Ca2+ handling Jan Aydin *, Daniel Andersson, Anna Wredenberg, Joseph Bruton, NilsGo¨ran Larsson, Ha˚kan Westerblad Institution of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden Mitochondrial transcription factor A (Tfam) is essential for maintenance of mitochondrial DNA (mtDNA) by playing critical roles in transcription, replication, damage sensing and DNA repair. Skeletal muscle tissue specific knockout of Tfam leads to development of a myopathy characterized by ragged-red fibers, accumulation of abnormally functioning mitochondria, together with a progressively deteriorating respiratory chain function. It has been proposed that the increase in mitochondrial mass may be a compensatory effect to counteract the respiratory deficiency, and a previous study has showed that the muscles from mice devoid of Tfam (Tfam/) do not fatigue more rapidly than their wildtype counterparts. The absolute forces in Tfam/ were however lower, and more so at lower frequencies. This reduction was believed to most likely be caused by a deficient in the force generating cross bridges and/ or a changed Ca2+ homeostasis. We have studied the mechanisms underlying the reduction in force using single, intact muscle fibers. As in whole muscles, Tfam/ single fibers did not fatigue more rapidly but they displayed lower absolute forces when compared to controls. (Tfam/ 288 ± 13 kPa, WT: 355 ± 13 kPa, p < 0.05 at 70 Hz). We also found that tetanic Ca2+ was lower in Tfam/ compared to WT (0.9 ± 0.05 lM vs. 1.6 ± 0.2 lM, p < 0.05 at 70 Hz). Tetanic Ca2+ in the presence of 5 mM caffeine, which reflects the SR Ca2+ load, was also lower in Tfam/ (3 ± 0.5 lM, vs. 7 ± 1.5 lM, p < 0.05). Furthermore, quantitative PCR showed a reduced mRNA-expression of the SR-calcium buffering protein calsequestrin-1 in Tfam/ muscle. Western blots confirmed this result by

Abstracts / Mitochondrion 7 (2007) 404–433 showing that Tfam/ have a decreased expression of calsequestrin-1 protein. In conclusion, the force decrease in mouse mitochondria myopathy muscle is due to decreased SR-Ca2+. This finding indicates an intricate SR-mitochondria interplay in the control genes encoding for Ca2+ handling proteins. doi:10.1016/j.mito.2007.08.013

10 A novel quantitative denaturing high performance-liquid chromatography assay based on heteroduplex analysis Robert K. Naviaux, Richard H. Haas, Kok Seong Lim * University of California, San Diego Objective: Denaturing high performance liquid chromatography (DHPLC) is a highly sensitive tool for detecting mutation based on heteroduplex analysis. Heteroduplex that results from heteroplasmic mutation, a common characteristic of mitochondrial DNA mutation, is therefore easily detected using DHPLC. As DHPLC is increasingly utilized in the studies involving screening the whole mitochondrial genome or specific regions of the genome for mutation, it would be valuable to devise a quantitative DHPLC method to determine the heteroplasmy level of mutation. Methodology: We generated site-directed mutants containing a common mutation in the mtDNA genome – A3243G. We used PCR to amplify the gene segment of interest in these mutants and performed restriction digestion. The mutants were mixed with wild-type sample to produce different mutation loads and the final samples were subjected to slow reannealing to induce heteroduplex formation before DHPLC analysis. Results: We observed a quadratic relationship between peak areas of heteroduplex and mutant loads and then developed a quantitative method based on this relationship to determine the heteroplasmy levels in plasmid DNA samples of known mutant loads. We found that this method was able to detect a mutation at a level as low as 1% and gave reproducible measurements of the mutations in the range of 2.5–97.5% with a R2 of approximately 0.99. Conclusion: The quantitative DHPLC method offers high accuracy and sensitivity and would thus be a suitable routine assay for the analysis of mitochondrial mutation in a clinical laboratory setting. doi:10.1016/j.mito.2007.08.014

11 Nuclear mitochondrial pseudogenes are amplified by tRNA Leu (UUR) primers but do not interfere with PCR-DHPLC analysis of mitochondrial DNA mutation Kok Seong Lim *, Robert K. Naviaux, Richard H. Haas University of California, San Diego School of Medicine, Department of Medicine, San Diego, CA, USA Objective: To determine if nuclear mitochondrial DNA pseudogenes (NUMTs) amplification by tRNA Leu (UUR)-specific PCR primers interferes with DHPLC analysis of the mitochondrial mutations. Methodology: We amplified the DNA region containing tRNA Leu (UUR) region by PCR using primers 5 0 -CTCACTGTCAACCCAACAC AGG-3 0 (2415–2436 bp) and 5 0 -TGTGTTGTGATAAGGGTGGAG AG-3 0 (3790–3812 bp) and performed a restriction digest to cut the amplicons into fragments of appropriate sizes (<600 bp) suitable for DHPLC analysis. These samples were all subjected to denaturation and slow reannealing before DHPLC analysis at 59 °C. This DNA region contains several mitochondrial mutation hotspots including A3243G, which causes mitochondrial encephalomyopathy, lactic acidemia, and stroke-like episodes (MELAS). Plasmid DNA containing homoplasmic A3243G

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mutation was generated through cloning and site-directed mutagenesis, and pure nuclear DNA (nDNA) was isolated from 143B osteosarcoma rho-0 cells. Total cellular DNA was obtained from whole blood of a healthy volunteer. Results: Using rho-0 cell DNA, we observed that the PCR generated several amplicons from NUMTs with sizes ranging from 200 to 400 bp, which might interfere with the desired digested amplicon of interest, which has a size of 342 bp. To investigate if these NUMT amplicons interfered with the DHPLC analysis of 342 bp fragment, we studied the formation of heteroduplexes in a DNA mixture (mtDNA and nDNA) containing 0 or 25% A3243G mutation, both with increasing amount of nuclear DNA. We observed no increase in the heteroduplex formation in these DNA samples. We also generated these fragments from whole blood total cellular DNA of a healthy normal subject, and mixed it with those generated from the wild-type plasmid DNA. If pseudogenes were amplified from the blood DNA heteroduplex species should be seen. We found no formation of heteroduplex in this mixture. This result taken with the finding from the above experiments with pure rho-0 cell nuclear DNA demonstrate that pseudogene amplification did not pose a problem in the DHPLC analysis of the mtDNA fragment (3193–3534 bp) which contains the A3243G and 8 other confirmed pathogenic mtDNA mutations. Conclusion: Amplification of pseudogenes occurred in the PCR but did not interfere with DHPLC analysis of A3243G mutation in our experiments. doi:10.1016/j.mito.2007.08.015

12 Tissue-distribution and possible functional roles of AK4 Takafumi Noma *, Keiko Miyoshi, Yuki Akazawa, Taigo Horiguchi Department of Molecular Biology, Institute of Health Biosciences Adenylate kinase (AK) is a key enzyme of the high-energy phosphoryl transfer reaction in living cells. AK4, the fourth isoform, has a similar sequence and subcellular localization to that of AK3 in the mitochondrial matrix. While AK3 is expressed ubiquitously with GTP:AMP phosphotransferase activity, AK4 is expressed in a tissue-specific manner without any enzymatic activity. To elucidate the physiological role of AK4, we determined the cellular localization of AK4 protein in mouse tissues by immunohistochemical analysis, comparing it with that of Tom20, a mitochondrial marker. AK4 expression was detected in kidney, liver, brain, heart, gastrointestinal tract, testis, ovary, and oviduct, but not in lung and spleen. The staining patterns of AK4 and Tom20 were classified into three groups: completely the same pattern, different patterns despite the same staining area, and reciprocal expression patterns. The expression pattern of AK4 in oocytes indicated that the regulation of AK4 expression is independent of mitochondriogenesis. Furthermore, the expression pattern of AK4 in combination with its tissue-specific physiological role suggests that AK4 may be a nucleotide reservoir involved in energy metabolism, and also a structural component protecting the mitochondrial architecture from environmental stress. doi:10.1016/j.mito.2007.08.016

13 Analysis of naturally occurring mutations in pyruvate dehydrogenase complex J.Gordon Lindsay, Alison Prior, Geetanjali Singh * University of Glasgow, Scotland, UK The pyruvate dehydrogenase complex (PDC) is a nuclear-encoded mitochondrial multi-enzyme assembly that provides the primary link between glycolysis and the TCA cycle by catalyzing the irreversible