Mitochondrial metabolism directs stemness and differentiation in P19 embryonal carcinoma stem cells.

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.

Water interaction in paper cellulose fibres as investigated by NMR pulsed field gradient.

In this paper, water diffusion coefficients were measured using NMR pulsed field gradient, on a variety of paper materials made from predominantly cellulose fibre and nanofibres, derived from wood, with different dimensions, internal porosity, and chemical composition. The moisture content ranged from 0.2 to 1.2g of water/g of dry fibre. Diffusion measurements were made both in the plane and through the thickness of the sheet. All data was generally well fitted by a simple two component diffusion model. For moisture contents less than 0.55 and 0.85g/g for measurements in the plane and through the thickness, respectively, it was found that both diffusion components increased approximately linearly with moisture content, with the faster diffusion coefficient being approximately five times larger than the smaller. The water appeared, within errors, to be evenly split between two components. The measured diffusion coefficients were not affected by fibre dimensions, internal structure or chemical composition, but were consistently higher when measured in the plane.

Yeast and human genes that affect the Escherichia coli SOS response.

The sequencing of the human genome has led to the identification of many genes whose functions remain to be determined. Because of conservation of genetic function, microbial systems have often been used for identification and characterization of human genes. We have investigated the use of the Escherichia coli SOS induction assay as a screen for yeast and human genes that might play a role in DNA metabolism and/or in genome stability. The SOS system has previously been used to analyze bacterial and viral genes that directly modify DNA. An initial screen of meiotically expressed yeast genes revealed several genes associated with chromosome metabolism (e.g., RAD51 and HHT1 as well as others). The SOS induction assay was then extended to the isolation of human genes. Several known human genes involved in DNA metabolism, such as the Ku70 end-binding protein and DNA ligase IV, were identified, as well as a large number of previously unknown genes. Thus, the SOS assay can be used to identify and characterize human genes, many of which may participate in chromosome metabolism.

Yeast RNC1 encodes a chimeric protein, RhoNUC, with a human rho motif and deoxyribonuclease activity.

The yeast Saccharomyces cerevisiae contains an endoexonuclease yNucR that has been implicated in both recombination and repair. We describe the isolation and characterization of the corresponding gene. Within the predicted N-terminal half of the protein there is extensive homology (approximately 50%) with human rho genes, which are related to the ras oncogene, particularly in the proposed GTP-binding region. The C-terminal region, which is related to the Escherichia coli recC protein, presumably encodes the endoexonuclease activity. The yNucR may thus represent a new class of GTP-binding proteins. Because of the chimeric nature of the polypeptide, this protein is renamed RhoNUC (rather than the original yNucR) and the gene is RNC1 for Rho-associated-NuClease. Over expression of the gene leads to altered cell growth and nuclear morphology. We propose that the gene plays an important role in cell development as well as DNA repair/recombination.

MAL64c is a global regulator of alpha-glucoside fermentation: identification of a new gene involved in melezitose fermentation.

Maltase constitutive mutants at the MAL6 locus have been mapped to the newly identified regulatory gene MAL64c. We show here that MAL64c has in addition pleiotropic effects on sugar fermentation: MAL64c strains constitutively synthesize an alpha-methylglucosidase and can complement a new gene, MTP1, for the fermentation of melezitose and alpha-methylglucoside. MTP1, maps near MAL1, and either encodes a permease which transports melezitose, alpha-methylglucoside, and maltose or regulates the activity of such a permease. This work shows that MAL64c, a trans-acting regulatory gene, is a global regulatory gene affecting several different pathways of alpha-glucoside metabolism.

Identification of a second trans-acting gene controlling maltose fermentation in Saccharomyces carlsbergensis.

Maltose fermentation in Saccharomyces spp. requires the presence of a dominant MAL locus. The MAL6 locus has been cloned and shown to encode the structural genes for maltose permease (MAL61), maltase (MAL62), and a positively acting regulatory gene (MAL63). Induction of the MAL61 and MAL62 gene products requires the presence of maltose and the MAL63 gene. Mutations within the MAL63 gene produce nonfermenting strains unable to induce the two structural gene products. Reversion of these mal63 nonfermenters to maltose fermenters nearly always leads to the constitutive expression of maltase and maltose permease, and constitutivity is always linked to MAL6. We demonstrated that for one such revertant, strain C2, constitutivity did not require the MAL63 gene, since deletion disruption of this gene did not affect the constitutive expression of the structural genes. In addition, constitutivity was trans acting. Deletion disruption of the MAL6-linked structural genes for maltase and maltose permease in this strain did not affect the constitutive expression of a second, unlinked maltase structural gene. We isolated new maltose-fermenting revertants of a nonfermenting strain which carried a deletion disruption of the MAL63 gene. All 16 revertants isolated expressed maltase constitutively. In one revertant studied in detail, strain R10, constitutive expression was demonstrated to be linked to MAL6, semidominant, trans acting, and residing outside the MAL63-MAL61-MAL62 genes. From these studies we propose the existence of a second trans-acting regulatory gene at the MAL6 locus. We call this new gene MAL64. We mapped the MAL64 gene 2.3 centimorgans to the left of MAL63. The role of the MAL64 gene product in maltose fermentation is discussed.

MAL6 of Saccharomyces: a complex genetic locus containing three genes required for maltose fermentation.

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, p1, 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.