Search

The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene promoter was used to drive the homologous expression of the lignin peroxidase (LiP) isozyme H2 gene in primary metabolic cultures of Phanerochaete chrysosporium. The molecular mass, pI, and optical absorption spectra of purified recombinant LiPH2 (rLiPH2) were essentially identical to those of wild-type LiPH2 (wtLiPH2). wtLiPH2 was prepared by growing cells in the absence of MnII, conditions under which P. chrysosporium manganese peroxidase (MnP) is not expressed, ensuring that wtLiPH2 was not contaminated with MnP. The kinetics of veratryl alcohol (VA) oxidation were essentially identical for rLiPH2 and wtLiPH2. The rLiPH2, wtLiPH2, and wild-type LiP isozyme H8 (wtLiPH8) enzymes were used to reexamine previous claims that LiPH2 can oxidize MnII at a rate sufficient to promote catalytic turnover of the enzyme. Our results demonstrate that rLiPH2, wtLiPH2, and LiPH8 do not turn over under steady-state conditions, when MnII is the sole reducing substrate. Furthermore, transient-state kinetic analyses show that the reduction rate of the catalytic intermediate, LiP compound I, by VA was at least 2 × 103-fold higher than the rate of reduction in the presence of MnII. No reduction of LiP compound II was observed in the presence of MnII. In contrast to previous claims, these data strongly suggest that MnII is not a productive substrate for LiPH2 or LiPH8.

The imperfect ascomycetous yeastsCandida parapsilosis andArxula adeninivorans degraded 3-hydroxybenzoic acid via gentisate which was the cleavage substrate. 4-Hydroxybenzoic acid was metabolized via protocatechuate. No cleavage enzyme for the latter was detected. In stead of this NADH- and NADPH-dependent monooxygenases were present. In cells grown at the expense of hydroquinone and 4-hydroxygenzoic acid, enzymes of the hydroxyhydroquinone variant of the 3-oxoadipate pathway were demonstrated, which also took part in the degradation of 2,4-dihydroxybenzoic acid byC. parapsilosis.

Cultures ofCryptococcus elinovii CBS 7051 grown at the expense of cinnamic acid as the sole source of carbon and energy partially converted this substrate into styrene. The latter is toxic and eventually kills the culture. Cell-free extracts of cultures grown on cinnamic acid produced styrene from cinnamate. Other basidiomycetous yeasts tested did not produce styrene from cinnamic acid.

The present study addresses, by transcriptomics and quantitative stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, the estrogen receptor α (ERα) and β (ERβ)-mediated effects on gene and protein expression in T47D breast cancer cells exposed to the phytoestrogen genistein. Using the T47D human breast cancer cell line with tetracycline-dependent ERβ expression (T47D-ERβ), the effect of a varying intracellular ERα/ERβ ratio on genistein-induced gene and protein expression was characterized. Results obtained reveal that in ERα-expressing T47D-ERβ cells with inhibited ERβ expression genistein induces transcriptomics and proteomics signatures pointing at rapid cell growth and migration by dynamic activation of cytoskeleton remodeling. The data reveal an interplay between integrins, focal adhesion kinase, CDC42, and actin cytoskeleton signaling cascades, occurring upon genistein treatment, in the T47D-ERβ breast cancer cells with low levels of ERα and no expression of ERβ. In addition, data from our study indicate that ERβ-mediated gene and protein expression counteracts ERα-mediated effects because in T47D-ERβ cells expressing ERβ and exposed to genistein transcriptomics and proteomics signatures pointing at a clear down-regulation of cell growth and induction of cell cycle arrest and apoptosis were demonstrated. These results suggest that ERβ decreases cell motility and metastatic potential as well as cell survival of the breast cancer cell line. It is concluded that the effects of genistein on proteomics and transcriptomics end points in the T47D-ERβ cell model are comparable with those reported previously for estradiol with the ultimate estrogenic effect being dependent on the relative affinity for both receptors and on the receptor phenotype (ERα/ERβ ratio) in the cells or tissue of interest.

The present study addresses, by transcriptomics and quantitative stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, the estrogen receptor α (ERα) and β (ERβ)-mediated effects on gene and protein expression in T47D breast cancer cells exposed to the phytoestrogen genistein. Using the T47D human breast cancer cell line with tetracycline-dependent ERβ expression (T47D-ERβ), the effect of a varying intracellular ERα/ERβ ratio on genistein-induced gene and protein expression was characterized. Results obtained reveal that in ERα-expressing T47D-ERβ cells with inhibited ERβ expression genistein induces transcriptomics and proteomics signatures pointing at rapid cell growth and migration by dynamic activation of cytoskeleton remodeling. The data reveal an interplay between integrins, focal adhesion kinase, CDC42, and actin cytoskeleton signaling cascades, occurring upon genistein treatment, in the T47D-ERβ breast cancer cells with low levels of ERα and no expression of ERβ. In addition, data from our study indicate that ERβ-mediated gene and protein expression counteracts ERα-mediated effects because in T47D-ERβ cells expressing ERβ and exposed to genistein transcriptomics and proteomics signatures pointing at a clear down-regulation of cell growth and induction of cell cycle arrest and apoptosis were demonstrated. These results suggest that ERβ decreases cell motility and metastatic potential as well as cell survival of the breast cancer cell line. It is concluded that the effects of genistein on proteomics and transcriptomics end points in the T47D-ERβ cell model are comparable with those reported previously for estradiol with the ultimate estrogenic effect being dependent on the relative affinity for both receptors and on the receptor phenotype (ERα/ERβ ratio) in the cells or tissue of interest.