ABSTRACT
Traditional screening using chemicals or flow cytometry (FCM) alone is not sufficient to isolate the high glutathione (GSH)-producing yeast strains used in food production. Therefore, to improve screening efficiency, we investigated a combination of both methods. A mutated Saccharomyces cerevisiae strain was labeled with 5-chloromethylfluorescein diacetate and sorted by FCM according to emitted fluorescence intensity. Moderate GSH (1%-2%)-producing mutants were isolated, whereas high GSH (>2%)-producing mutants were not. Traditional screening using cerulenin resulted in similar findings, but a combination of both methods resulted in a 40% increase in the screening yield of high GSH-producing mutants. An analysis of model strains indicated that the ratio of high GSH-producing cells in a sample affected the FCM results. By combining FCM with traditional screening using chemicals, we succeeded in isolating high GSH-producing mutants from several parental strains.
Subject(s)
Glutathione/biosynthesis , High-Throughput Screening Assays , Saccharomyces cerevisiae/isolation & purification , Cerulenin/pharmacology , Fatty Acid Synthesis Inhibitors/pharmacology , Flow Cytometry , Fluoresceins , Fluorescent Dyes , Food Technology , Glutathione/genetics , Mutation , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
In the present study, we clarified that transforming growth factor beta (TGF-beta) induces cellular senescence in human normal diploid cells, TIG-1, and identified protein kinase Cs (PKCs) as downstream mediators of TGF-beta-induced cellular senescence. Among PKCs, we showed that PKC-delta induced cellular senescence in TIG-1 cells and was activated in replicatively and prematurely senescent TIG-1 cells. The causative role of PKC-delta in cellular senescence programs was demonstrated using a kinase negative PKC-delta and small interfering RNA against PKC-delta. Furthermore, PKC-delta was shown to function in human telomerase reverse transcriptase (hTERT) gene repression. These results indicate that PKC-delta plays a key role in cellular senescence programs, and suggest that the induction of senescence and hTERT repression are coordinately regulated by PKC-delta.
Subject(s)
Cellular Senescence/physiology , Protein Kinase C-delta/physiology , Acetamides/pharmacology , Catalytic Domain/genetics , Cell Line , Cell Proliferation/drug effects , Diploidy , Enzyme Activators/pharmacology , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Expression Regulation, Enzymologic , Genes, ras , Humans , Kidney/cytology , Kidney/metabolism , Protein Kinase C-delta/antagonists & inhibitors , Protein Kinase C-delta/biosynthesis , Pyrans/pharmacology , RNA, Small Interfering , Recombinant Proteins/biosynthesis , Spiro Compounds/pharmacology , Telomerase/genetics , Transduction, Genetic , Transforming Growth Factor beta/pharmacology , beta-Galactosidase/biosynthesisABSTRACT
Escherichia coli has many periplasmic phosphatase activities. To test whether it can take up and excrete purine nucleotides, we attempted to completely disrupt periplasmic 5'-nucleotidase activity. A 5'-nucleotidase activity was induced in ushA knockout mutant cells, which lack major 5'-nucleotidase activity, when they were grown with purine nucleotides as the sole carbon source. Using DNA macroarrays to compare global gene expression in wild-type and ushA knockout mutant cells cultured with IMP or GMP as the sole carbon source, we identified two genes that were induced in the ushA knockout mutant cells and encoded signal sequence needed for secretion. One of the genes, aphA, encoded a 5'-nucleotidase activity and was induced by IMP or inosine. An ushA aphA double knockout mutant was shown to be unable to grow on purine nucleotides as the sole carbon source. To investigate the excretion of purine nucleotides, we constructed an ushAaphA double knockout mutant of an inosine-producing strain and found that it accumulated IMP in the medium. In addition, when the guaBA operon was introduced into the ushAaphA double knockout IMP producer, GMP was released into the medium. These observations imply the existence of efflux activity for purine nucleotides in E. coli.
Subject(s)
5'-Nucleotidase/metabolism , Escherichia coli/enzymology , Purine Nucleotides/metabolism , 5'-Nucleotidase/genetics , Acid Phosphatase/genetics , Acid Phosphatase/metabolism , Blotting, Northern , Cell Division/drug effects , Cell Division/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Deletion , Gene Expression Regulation, Bacterial/drug effects , Guanosine Monophosphate/metabolism , Guanosine Monophosphate/pharmacology , Hydrogen-Ion Concentration , Inosine Monophosphate/metabolism , Inosine Monophosphate/pharmacology , Models, Biological , Mutation , Oligonucleotide Array Sequence Analysis , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , Purine Nucleotides/pharmacologyABSTRACT
We have previously reported that transforming growth factor beta (TGF-beta) triggers two independent senescence programs, 1) replicative senescence dependent upon telomere shortening and 2) premature senescence independent of telomere shortening, in the cell line of A549 human lung adenocarcinoma. In this study, we examined the possibility that cancer cell tumor phenotypes could be suppressed by forced senescence. We used A549 cells treated with TGF-beta for a long time (over 50 days), where senescence was induced in a telomere-shortening-dependent or an independent way. Fully senescent A549 cells were elongated, acquired contact inhibition capabilities when reaching confluence, and secreted the senescence-associated cytokine IL-6. Furthermore, senescent A549 cells had no tumorigenicity in nude mice. These results indicate that the forced induction of senescence in cancer cells may be a novel and potentially powerful method for advancing anti-cancer therapy.
