Pap1p-dependent upregulation of thioredoxin 3 and thioredoxin reductase genes from the fission yeast under nitrosative stress.

The thioredoxin system, consisting of thioredoxin, thioredoxin reductase, and NADPH, is involved in the response against a variety of stresses. The TRX3(+) and TrxR(+) genes encode thioredoxin 3 and thioredoxin reductase, respectively, in the fission yeast Schizosaccharomyces pombe . Their transcriptional regulations were studied using the lacZ fusion genes. Synthesis of ß-galactosidase from the TRX3(+)-lacZ fusion gene was markedly enhanced by nitric-oxide-generating sodium nitroprusside in the Pap1p-positive cells but not in the Pap1p-negative cells. Similarly, synthesis of ß-galactosidase from the TrxR(+)-lacZ fusion gene was upregulated by sodium nitroprusside in a Pap1p-dependent manner. Synthesis of ß-galactosidase from the TRX3(+)-lacZ and TrxR(+)-lacZ fusion genes was also enhanced by S-nitrosoglutathione in the Pap1p-positive cells but not in the Pap1p-negative cells. In brief, the S. pombe genes encoding thioredoxin 3 and thioredoxin reductase are upregulated under nitrosative stress in a Pap1p-dependent manner.

Anti-inflammatory activity of n-propyl gallate through down-regulation of NF-κB and JNK pathways.

The present study aimed to assess anti-inflammatory activity and underlying mechanism of n-propyl gallate, the n-propyl ester of gallic acid. n-Propyl gallate was shown to contain anti-inflammatory activity using two experimental animal models, acetic acid-induced permeability model in mice, and air pouch model in rats. It suppressed production of nitric oxide and induction of inducible nitric oxide synthase and cyclooxygenase-2 in the lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. It was able to diminish reactive oxygen species level elevated in the LPS-stimulated RAW264.7 macrophage cells. It also suppressed gelatinolytic activity of matrix metalloproteinase-9 enhanced in the LPS-stimulated RAW264.7 macrophage cells. It inhibited inhibitory κB-α degradation and enhanced NF-κB promoter activity in the stimulated macrophage cells. It was able to suppress phosphorylation of c-Jun NH(2)-terminal kinase 1/2 (JNK1/2) and activation of c-Jun promoter activity in the stimulated macrophage cells. In brief, n-propyl gallate possesses anti-inflammatory activity via down-regulation of NF-κB and JNK pathways.

Zinc metal solubilization by Gluconacetobacter diazotrophicus and induction of pleomorphic cells.

Gluconacetobacter diazotrophicus strain PAl5 exhibited a minimum inhibitory concentration value of 11 mM in an LGI medium amended with ZnCl2. When an LGI medium was amended with Zn metal, solubilization halos were observed in a plate assay, and further solubilization was confirmed in a broth assay. The maximum solubilization was recorded after 120 h with a 0.1% Zn metal amendment. During solubilization, the culture growth and pH of the broth were indirectly correlated. Using a Fourier Transform Infrared Spectroscopy analysis, one of the agents solubilizing the Zn metal was identified as gluconic acid. When the Zn-amended broth was observed under a bright field microscope, long involution cells were observed, and further analysis with Atomic Force Microscopy revealed highly deformed, pleomorphic, aggregate-like cells.

Expression, characterization and regulation of a Saccharomyces cerevisiae monothiol glutaredoxin (Grx6) gene in Schizosaccharomyces pombe.

Glutaredoxins (Grxs), also known as thioltransferases (TTases), are thiol oxidoreductases that regulate cellular redox state in a variety of organisms. In the budding yeast Saccharomyces cerevisiae, Grx1 and 2 are cytosolic dithiol Grxs, while Grx3, 4 and 5 are monothiol Grxs. A gene encoding a new monothiol Grx, Grx6, was cloned from the genomic DNA of S. cerevisiae by PCR. Its DNA sequence contains 1,080 bp, and encodes a putative protein of 203 amino acid residues containing Cys-Phe-Tyr-Ser at the active site. Grx6 is similar to other monothiol Grxs in the same organism and to Grx3 in the fission yeast Schizosaccharomyces pombe. and its predicted three-dimensional structure resembles that of S. pombe Grx3. S. pombe cells harboring plasmid pFGRX6 containing the Grx6 gene had about 1.3-fold elevated Grx activity in the exponential phase, and grew better than the control cells under some stressful conditions. Synthesis of beta-galactosidase from a Grx6-lacZ fusion gene in S. pombe was enhanced by potassium chloride, aluminum chloride and heat (37 degrees C) treatment. S. pombe cells harboring plasmid pFGRX6 had elevated ROS levels whereas S. pombe cells harboring extra copies of Grx3 had reduced ROS levels.

Effects of heterologous expression of thioredoxin reductase on the level of reactive oxygen species in COS-7 cells.

Thioredoxin reductase (TrxR), a component of the redox control system involving thioredoxin (Trx), is implicated in defense against oxidative stress, control of cell growth and proliferation, and regulation of apoptosis. In the present study a stable transfectant was made by introducing the vector pcDNA3.0 harboring the fission yeast TrxR gene into COS-7 African green monkey kidney fibroblast cells. The exogenous TrxR gene led to an increase in TrxR activity of up to 3.2-fold but did not affect glutathione (GSH) content, or glutaredoxin and caspase-3 activities. Levels of reactive oxygen species (ROS), but not those of nitric oxide (NO), were reduced. Conversely, 1-chloro-2,4-dinitrobezene (CDNB), an irreversible inhibitor of mammalian TrxR, enhanced ROS levels in the COS-7 cells. After treatment with hydrogen peroxide, the level of intracellular ROS was lower in the transfectants than in the vector control cells. These results confirm that TrxR is a crucial determinant of the level of cellular ROS during oxidative stress as well as in the normal state.

Up-regulation of defense enzymes is responsible for low reactive oxygen species in malignant prostate cancer cells.

Reactive oxygen species (ROS) are involved in a diversity of important phenomena in the process of tumor development. To investigate the alterations of oxidative stress and their related systems in tumor progression, a variety of components in the antioxidative stress defense system were examined in prostate cancer cell lines, PC3 and LNCaP. Cell surface molecules involved in metastasis were expressed highly in PC3 cells compared with LNCaP cells, and strong invasion ability was shown in PC3 cells only. ROS level in LNCaP cells was twice higher than that in PC3 cells, although nitric oxide (NO) level was similar between the two cell lines. The content of GSH increased up to about 2-fold in PC3 compared with LNCaP. Activities of glutathione reductase, thioredoxin reductase, and glutathione S-transferase except catalase are significantly higher in PC3 cells than in LNCaP cells. Furthermore, oxidative stress-inducing agents caused down-regulation of GSH and glutathione S-transferase much more significantly in LNCaP cells than in PC3 cells. These results imply that malignant tumor cells may maintain low ROS content by preserving relatively high anti-oxidative capacity, even in the presence of stressful agents.

The gene encoding gamma-glutamyl transpeptidase II in the fission yeast is regulated by oxidative and metabolic stress.

gamma-Glutamyl transpeptidase (GGT, EC 2.3.2.2.) catalyzes the transfer of the gamma-glutamyl moiety from gamma-glutamylcontaining compounds, notably glutathione (GSH), to acceptor amino acids and peptides. A second gene (GGTII) encoding GGT was previously isolated and characterized from the fission yeast Schizosaccharomyces pombe. In the present work, the GGTII-lacZ fusion gene was constructed and used to study the transcriptional regulation of the S. pombe GGTII gene. The synthesis of beta-galactosidase from the GGTII-lacZ fusion gene was significantly enhanced by NO-generating SNP and hydrogen peroxide in the wildtype yeast cells. The GGTII mRNA level was increased in the wild-type S. pombe cells treated with SNP. However, the induction by SNP was abolished in the Pap1-negative S. pombe cells, implying that the induction by SNP of GGTII is mediated by Pap1. Fermentable carbon sources, such as glucose (at low concentrations), lactose and sucrose, as a sole carbon source, enhanced the synthesis of beta-galactosidase from the GGTII-lacZ fusion gene in wildtype KP1 cells but not in Pap1-negative cells. Glycerol, a non-fermentable carbon source, was also able to induce the synthesis of beta-galactosidase from the fusion gene, but other non-fermentable carbon sources such as acetate and ethanol were not. Transcriptional induction of the GGTII gene by fermentable carbon sources was also confirmed by increased GGTII mRNA levels in the yeast cells grown with them. Nitrogen starvation was also able to induce the synthesis of beta-galactosidase from the GGTII-lacZ fusiongene in a Pap1-dependent manner. On the basis of the results, it is concluded that the S. pombe GGTII gene is regulated by oxidative and metabolic stress.

The Schizosaccharomyces pombe gene encoding gamma-glutamyl transpeptidase I is regulated by non-fermentable carbon sources and nitrogen starvation.

In our previous study, the first structural gene (GGTI) encoding g-glutamyl transpeptidase was cloned and characterized from the fission yeast Schizosaccharomyces pombe, and its transcription, using the GGTI-lacZ fusion gene, containing the 1,085 bp upstream region from the translational initiation point, was found to be enhanced by sodium nitroprusside and L-buthionine-(S,R)-sulfoximine (BSO). In the present work, regulation of the GGTI gene was further elucidated. Non-fermentable carbon sources, such as acetate and ethanol, markedly enhanced the synthesis of beta-galactosidase from the GGTI-lacZ fusion gene. However, its induction by non-fermentable carbon sources appeared to be independent of the presence of the Pap1 protein. Nitrogen starvation also gave rise to induction of GGTI gene expression in a Pap1-independent manner. The three additional fusion plasmids, carrying 754, 421 and 156 bp regions, were constructed. The sequence responsible for the induction by non-fermentable carbon sources and nitrogen starvation was identified to exist within a -421 bp region of the GGTI gene. Taken together, the S. pombe GGTI gene is regulated by non-fermentable carbon sources and nitrogen starvation.

Differential regulation of three genes encoding glutathione S-transferases in Schizosaccharomyces pombe.

Glutathione S-transferases (GSTs) are detoxifying enzymes that catalyze the conjugation of glutathione with a variety of reactive electrophilic compounds. Three GST genes were previously characterized in the fission yeast Schizosaccharomyces pombe. In this work, we examined the transcriptional regulation of these genes using individual GST-lacZ fusions and RT-PCR. Basal synthesis of beta-galactosidase from the GSTII-lacZ fusion was higher than from the GSTI-lacZ and GSTIII-lacZ fusion. Diethylmaleate (0.2 mM) greatly enhanced the synthesis of beta-galactosidase from the GSTII-lacZ fusion, but did not affect synthesis from the other two fusion genes. A switch to 0.3% glucose or 0.3% sucrose as sole carbon source enhanced expression from the GSTIII-lacZ fusion gene, while sodium nitroprusside (1.5 mM), tert-butylhydroquinone (0.2 mM), and L-buthionine-[S,R]-sulfoximine (0.01 mM) increased expression of the GSTII gene. The effects of these agents on GST mRNA levels were confirmed by measurements employing RT-PCR. Our results suggest that transcription of the three S. pombe GST genes is subjected to differential regulation under various stress conditions, and may be linked to their different physiological functions.

Regulation of the manganese-containing superoxide dismutase gene from fission yeast.

The manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that dismutates a potentially toxic superoxide radical into hydrogen peroxide and dioxygen. To study the regulation of the Schizosaccharomyces pombe MnSOD gene, the 943 bp upstream region was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357, which resulted in the fusion plasmid pMS14. Restriction mapping and nucleotide sequencing confirmed its construction. The synthesis of beta-galactosidase from the fusion plasmid was induced by aluminum chloride, menadione, cadmium chloride, manganese chloride, and hydrogen peroxide. It was also induced by NO-generating S-nitroso-N-acetylpenicillamine (SNAP). However, cupric chloride and zinc chloride did not affect the synthesis of beta-galactosidase from the fusion plasmid. The beta-galactosidase synthesis appeared to be independent of the Pap1 protein. These results suggest that some metals, oxidative stress, and nitric oxide regulate the S. pombe MnSOD gene.