The relationship between mitochondrial metabolism and cell viability and differentiation in stem cells (SCs) remains poorly understood. In the present study, we compared mitochondrial physiology and metabolism between P19SCs before/after differentiation and present a unique fingerprint of the association between mitochondrial activity, cell differentiation and stemness. In comparison with their differentiated counterparts, pluripotency of P19SCs was correlated with a strong glycolytic profile and decreased mitochondrial biogenesis and complexity: round, low-polarized and inactive mitochondria with a closed permeability transition pore. This decreased mitochondrial capacity increased their resistance against dichloroacetate. Thus, stimulation of mitochondrial function by growing P19SCs in glutamine/pyruvate-containing medium reduced their glycolytic phenotype, induced loss of pluripotent potential, compromised differentiation and became P19SCs sensitive to dichloroacetate. Because of the central role of this type of SCs in teratocarcinoma development, our findings highlight the importance of mitochondrial metabolism in stemness, proliferation, differentiation and chemoresistance. In addition, the present work suggests the regulation of mitochondrial metabolism as a tool for inducing cell differentiation in stem line therapies. Embryonal carcinoma cells, including the P19 cell line, are pluripotent cancer stem cells (CSCs) derived from pluripotent germ cell tumors called teratocarcinomas. These have been described as the malignant counterparts of embryonic stem cells (ESCs) and are considered a good model to study stem cell (SC) differentiation. The P19 cell line can be maintained as undifferentiated cells (P19SCs) or differentiated (P19dCs) to any cell type of the three germ layers. Similar to ESCs, P19 cells differentiate with retinoic acid (RA) in a dose-dependent manner and depending on growth conditions. 1 Although differentiation generally yields a mixed population of differentiated cells, P19 cells grown in monolayer and treated with 1 mM RA primarily differentiate in endoderm or mesoderm, while retaining their immortality. 2,3Although some therapeutic approaches for regenerative medicine and to targeting CSCs are based on differentiation 4 and mitochondrial-targeted therapies, 5,6 very little is known about the role of mitochondrial metabolism in SC maintenance and differentiation.7 Several mitochondrial characteristics that distinguish transformed cells from healthy cells have been described, 8 including increased mitochondrial transmembrane electric potential (Dcm), which may result from decreased mitochondrial ATP production under normoxia. Similarly, normal SCs primarily rely on glycolysis for energy supply, although the exact mechanism how this occurs in the presence of oxygen and the relationship between SC metabolism and cell fate control is not yet completely understood. 10Given the mitochondrial involvement in stemness and differentiation, 11 one can ask whether manipulation of mitochondrial physi...

Berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a)quinolizinium] is an alkaloid present in plant extracts and has a history of use in traditional Chinese and Native American medicine. Because of its ability to arrest the cell cycle and cause apoptosis of several malignant cell lines, it has received attention as a potential anticancer therapeutic agent. Previous studies suggest that mitochondria may be an important target of berberine, but relatively little is known about the extent or molecular mechanisms of berberine-mitochondrial interactions. The objective of the present work was to investigate the interaction of berberine with mitochondria, both in situ and in isolated mitochondrial fractions. The data show that berberine is selectively accumulated by mitochondria, which is accompanied by arrest of cell proliferation, mitochondrial fragmentation and depolarization, oxidative stress, and a decrease in ATP levels. Electron microscopy of berberine-treated cells shows a reduction in mitochondria-like structures, accompanied by a decrease in mitochondrial DNA copy number. Isolated mitochondrial fractions treated with berberine had slower mitochondrial respiration, especially when complex I substrates were used, and increased complex I-dependent oxidative stress. It is also demonstrated for the first time that berberine stimulates the mitochondrial permeability transition. Direct effects on ATPase activity were not detected. The present work demonstrates a number of previously unknown alterations of mitochondrial physiology induced by berberine, a potential chemotherapeutic agent, although it also suggests that high doses of berberine should not be used without a proper toxicology assessment.Berberine (Fig.

The MAL6 locus is one of five closely related unlinked loci, any one of which is sufficient for fermentation of maltose in Saccharomyces. Previous genetic analysis indicated that this locus is defined by two complementation groups, MALp and MALg. MALp reportedly is a regulatory gene required for inducible synthesis of the two enzymatic functions needed for fermentation: maltose permease and maltase. We have investigated the physical and genetic structure of the MAL6 locus, which has been isolated on a recombinant DNA plasmid. One subclone of the region, pDF-1, was found to encode a single transcribed region and to contain the MALp gene. A second subclone, pl, was shown to contain the MALg function but surprisingly had not one but two maltose-inducible transcripts. Subclones having only one of these transcribed regions lacked MALg activity. The three transcribed regions have been named MAL61 and MAL62, which correspond to MALg, and MAL63, which corresponds to MALp. This clustered arrangement of a regulatory gene adjacent to the sequences it controls has not previously been described in eukaryotes and is reminiscent of bacterial operons except that the messenger RNA molecules are not polycistronic.

Purpose Natural products represent a rich reservoir of potential small molecule inhibitors exhibiting antiproliferative and tumoricidal properties. An example is the isoquinoline alkaloid berberine, which is found in plants such as goldenseal (Hydrastis canadensis). Studies have shown that berberine is able to trigger apoptosis in diVerent malignant cell lines, and can also lead to cell cycle arrest at sub-apoptotic doses. A particularly interesting feature of berberine is the fact that it is a Xuorescent molecule, and its uptake and distribution in cells can be studied by Xow cytometry and epiXuorescence microscopy. To test the relationships between berberine uptake, distribution and cellular eVect in melanoma cells, K1735-M2 mouse and WM793 human melanoma cells were treated with diVerent concentrations of berberine, and alterations in cell cycle progression, DNA synthesis, cell proliferation, and cell death measured. Methods Cell proliferation was measured by sulforhodamine B assays, cell death by Xow cytometry, berberine uptake and distribution by laser scanning confocal microscopy and Xow cytometry, cell cycle progression by Xow cytometry, and DNA synthesis, M-phase, and mitochondrial eVects by immunolabeling and epiXuorescence microscopy methods. Results In these melanoma cell lines, berberine at low doses (12.5-50 M) is concentrated in mitochondria and promotes G1 arrest. In contrast, higher doses (over 50 M) result in cytoplasmic and nuclear berberine accumulation, and G2 arrest. DNA synthesis is not markedly aVected by low doses of berberine, but 100 M is strongly inhibitory. Even at 100 M, berberine inhibits cell growth with relatively little induction of apoptosis. Conclusion Berberine displays multiphasic eVects in these malignant cell lines, which are correlated with the concentration and intracellular distribution of this alkaloid. These results help explain some of the conXicting information in the literature regarding the eVects of berberine, and suggest that its use in clinical development may be more as a cytostatic agent than a cytotoxic compound.

BackgroundIn this commentary we present the findings from an international consortium on fish toxicogenomics sponsored by the U.K. Natural Environment Research Council (Fish Toxicogenomics—Moving into Regulation and Monitoring, held 21–23 April 2008 at the Pacific Environmental Science Centre, Vancouver, BC, Canada).ObjectivesThe consortium from government agencies, academia, and industry addressed three topics: progress in ecotoxicogenomics, regulatory perspectives on roadblocks for practical implementation of toxicogenomics into risk assessment, and dealing with variability in data sets.DiscussionParticipants noted that examples of successful application of omic technologies have been identified, but critical studies are needed to relate molecular changes to ecological adverse outcome. Participants made recommendations for the management of technical and biological variation. They also stressed the need for enhanced interdisciplinary training and communication as well as considerable investment into the generation and curation of appropriate reference omic data.ConclusionsThe participants concluded that, although there are hurdles to pass on the road to regulatory acceptance, omics technologies are already useful for elucidating modes of action of toxicants and can contribute to the risk assessment process as part of a weight-of-evidence approach.