Expansions of an intronic GAA repeat reduce the expression of frataxin and cause Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disease. Frataxin is a mitochondrial protein, and disruption of a frataxin homolog in yeast results in increased sensitivity to oxidant stress, increased mitochondrial iron and respiration deficiency. These previous data support the hypothesis that FRDA is a disease of mitochondrial oxidative stress, a hypothesis we have tested in cultured cells from FRDA patients. FRDA fibroblasts were hypersensitive to iron stress and significantly more sensitive to hydrogen peroxide than controls. The iron chelator deferoxamine rescued FRDA fibroblasts more than controls from oxidant-induced death, consistent with a role for iron in the differential kinetics of death; however, mean mitochondrial iron content in FRDA fibroblasts was increased by only 40%. Treatment of cells with the intracellular Ca2+chelator BAPTA-AM rescued both FRDA fibroblasts and controls from oxidant-induced death. Treatment with apoptosis inhibitors rescued FRDA but not control fibroblasts from oxidant stress, and staurosporine-induced caspase 3 activity was higher in FRDA fibroblasts, consistent with the possibility that an apoptotic step upstream of caspase 3 is activated in FRDA fibroblasts. These results demonstrate that FRDA fibroblasts are sensitive to oxidant stress, and may be a useful model in which to elucidate the FRDA mechanism and therapeutic strategies.

Bioactive compounds reported to stimulate mitochondrial biogenesis are linked to many health benefits such increased longevity, improved energy utilization, and protection from reactive oxygen species. Previously studies have shown that mice and rats fed diets lacking in pyrroloquinoline quinone (PQQ) have reduced mitochondrial content. Therefore, we hypothesized that PQQ can induce mitochondrial biogenesis in mouse hepatocytes. Exposure of mouse Hepa1-6 cells to 10 -30 M PQQ for 24 -48 h resulted in increased citrate synthase and cytochrome c oxidase activity, Mitotracker staining, mitochondrial DNA content, and cellular oxygen respiration. The induction of this process occurred through the activation of cAMP response element-binding protein (CREB) and peroxisome proliferator-activated receptor-␥ coactivator-1␣ (PGC-1␣), a pathway known to regulate mitochondrial biogenesis. PQQ exposure stimulated phosphorylation of CREB at serine 133, activated the promoter of PGC-1␣, and increased PGC-1␣ mRNA and protein expression. PQQ did not stimulate mitochondrial biogenesis after small interfering RNA-mediated reduction in either PGC-1␣ or CREB expression. Consistent with activation of the PGC-1␣ pathway, PQQ increased nuclear respiratory factor activation (NRF-1 and NRF-2) and Tfam, TFB1M, and TFB2M mRNA expression. Moreover, PQQ protected cells from mitochondrial inhibition by rotenone, 3-nitropropionic acid, antimycin A, and sodium azide. The ability of PQQ to stimulate mitochondrial biogenesis accounts in part for action of this compound and suggests that PQQ may be beneficial in diseases associated with mitochondrial dysfunction.Bioactive compounds, such as pyrroloquinoline quinone (PQQ), 2 resveratrol, genistein, hydroxy-tyrosol, and quercetin have been reported to improve mitochondrial respiratory control or stimulate mitochondrial biogenesis (1-5), which is potentially important to a number of health-related issues ranging from increased longevity, improved energy utilization, and protection from reactive oxygen species (6 -8). Furthermore, mitochondrial DNA depletion and mutations are associated with cardiomyopathy, lactic acidosis, developmental delay, failure to thrive, and impaired neurological function (9). The response to most biofactors is observed after pharmacological intervention or dietary supplementation, although often neargram amounts per kg of diet, or millimolar quantities, are needed for such responses in vivo. PQQ stimulates mitochondriogenesis with the addition of only milligram quantities of PQQ per kg of diet, or micromolar concentrations, in vivo. For example, PQQ deprivation depresses mitochondrial function, which is reversed when as little as 200 -300 g of PQQ/kg of diet are added (1, 10). PQQ also remains detectable in tissues when there is no or little dietary exposure (11), which has not been observed for other dietary polyphenolic compounds known to promote mitochondriogenesis.Recently, PQQ produced by rhizobacterium has been identified as an important plant growth factor (12) and is a...

Chronic myeloid leukemia (CML) starts with the acquisition of a BCR-ABL fusion gene in a single hematopoietic stem cell, but the time to progression is unpredictable. Although the tyrosine kinase inhibitor imatinib mesylate is highly effective in the treatment of CML, its continuous administration is associated with development of resistance, particularly in advanced phase or blast crisis. We investigate here whether a feature of disease progression (i.e., elevated expression of Bcr-Abl in CD34 + progenitor cells from CML patients in blast crisis) has any bearing on the kinetics of resistance to imatinib. By studying cell lines that exogenously express Bcr-Abl over the range found from chronic phase to blast crisis of CML, we show that cells expressing high amounts of Bcr-Abl, as in blast crisis, are much less sensitive to imatinib and, more significantly, take a substantially shorter time for yielding a mutant subclone resistant to the inhibitor than cells with low expression levels, as in chronic phase. Our data suggest that the differential levels of the Bcr-Abl oncoprotein expressed by CD34 + CML cells may reflect the extent and duration of their response to imatinib; the relatively high levels of oncoprotein in advanced-phase disease may underlie the observed rapid development of resistance.

eMSCs from BM possessed the highest in vitro osteogenic potential; eMSCs from adipose tissue also had robust osteogenic potential. The tuber coxae and the sternum were viable sources of BM-eMSCs in yearlings, and 60 mL of BM aspirate was sufficient for culture and expansion. Expanding BM-eMSCs in AS to avoid potential immunologic reactions decreased the total yield because BM-eMSCs grew significantly slower in AS than in fetal bovine serum. Additional studies are needed to determine optimal ex vivo eMSC culture and expansion conditions, including the timing and use of growth factor—supplemented AS.