Analysis of adaptation to high ethanol concentration in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using DNA microarray

In industrial process, yeast cells are exposed to ethanol stress that affects the cell growth and the productivity. Thus, investigating the intracellular state of yeast cells under high ethanol concentration is important. In this study, using DNA microarray analysis, we performed comprehensive expression profiling of two strains of Saccharomyces cerevisiae, i.e., the ethanol-adapted strain that shows active growth under the ethanol stress condition and its parental strain used as the control. By comparing the expression profiles of these two strains under the ethanol stress condition, we found that the genes related to ribosomal proteins were highly up-regulated in the ethanol-adapted strain. Further, genes related to ATP synthesis in mitochondria were suggested to be important for growth under ethanol stress. We expect that the results will provide a better understanding of ethanol tolerance of yeast. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tectoridin,a poor ligand of estrogen receptor α, exerts its estrogenic effects via an ERK-dependent pathway

Phytoestrogens are the natural compounds isolated from plants, which are structurally similar to animal estrogen, 17β-estradiol. Tectoridin, a major isoflavone isolated from the rhizome of Belamcanda chinensis. Tectoridin is known as a phytoestrogen, however, the molecular mechanisms underlying its estrogenic effect are remained unclear. In this study we investigated the estrogenic signaling triggered by tectoridin as compared to a famous phytoestrogen, genistein in MCF-7 human breast cancer cells. Tectoridin scarcely binds to ER α as compared to 17β-estradiol and genistein. Despite poor binding to ER α, tectoridin induced potent estrogenic effects, namely recovery of the population of cells in the S-phase after serum starvation, transactivation of the estrogen response element, and induction of MCF-7 cell proliferation. The tectoridin-induced estrogenic effect was severely abrogated by treatment with U0126, a specific MEK1/2 inhibitor. Tectoridin promoted phosphorylation of ERK1/2, but did not affect phosphorylation of ER α at Ser¹¹⁸. It also increased cellular accumulation of cAMP, a hallmark of GPR30-mediated estrogen signaling. These data imply that tectoridin exerts its estrogenic effect mainly via the GPR30 and ERK-mediated rapid nongenomic estrogen signaling pathway. This property of tectoridin sets it aside from genistein where it exerts the estrogenic effects via both an ER-dependent genomic pathway and a GPR30-dependent nongenomic pathway.     

Isolation and characterization of alpha-glucosidase inhibitor from the fungus Ganoderma lucidum

An alpha-glucosidase inhibitor, SKG-3, was isolated from the fruiting bodies of Ganoderma lucidum and its physico-chemical properties were characterized. It was a highly specific and effective reversible inhibitor of alpha-glucosidase. It showed very potent inhibitory activity against alpha-glucosidase with an IC50 value of 4.6 micro g/ml, but no activity for any other glycosidases tested. Enzyme activity could be recovered upon dialysis, thus providing evidence for the reversibility of the inhibition. A Lineweaver-Burk plot indicated that the SKG-3 inhibition of alpha-glucosidase was competitive.     

Adaptation of Saccharomyces cerevisiae cells to high ethanol concentration and changes in fatty acid composition of membrane and cell size

Background

Microorganisms can adapt to perturbations of the surrounding environment to grow. To analyze the adaptation process of the yeast Saccharomyces cerevisiae to a high ethanol concentration, repetitive cultivation was performed with a stepwise increase in the ethanol concentration in the culture medium.

Methodology/Principal Findings

First, a laboratory strain of S. cerevisiae was cultivated in medium containing a low ethanol concentration, followed by repetitive cultivations. Then, the strain repeatedly cultivated in the low ethanol concentration was transferred to medium containing a high ethanol concentration and cultivated repeatedly in the same high-ethanol-concentration medium. When subjected to a stepwise increase in ethanol concentration with the repetitive cultivations, the yeast cells adapted to the high ethanol concentration; the specific growth rate of the adapted yeast strain did not decrease during repetitive cultivation in the medium containing the same ethanol concentration, while that of the non-adapted strain decreased during repetitive cultivation. A comparison of the fatty acid composition of the cell membrane showed that the contents in oleic acid (C_18:1) in ethanol-adapted and non-adapted strains were similar, but the content of palmitic acid (C_16:0) in the ethanol-adapted strains was lower than that in the non-adapted strain in media containing ethanol. Moreover, microscopic observation showed that the mother cells of the adapted yeast were significantly larger than those of the non-adapted strain.

Conclusions

Our results suggest that activity of cell growth defined by specific growth rate of the yeast cells adapted to stepwise increase in ethanol concentration did not decrease during repetitive cultivation in high-ethanol-concentration medium. Moreover, fatty acid content of cell membrane and the size of ethanol-adapted yeast cells were changed during adaptation process. Those might be the typical phenotypes of yeast cells adapted to high ethanol concentration. In addition, the difference in sizes of the mother cell between the non-adapted and ethanol strains suggests that the cell size, cell cycle and adaptation to ethanol are thought to be closely correlated.     

PTEN regulates fibroblast elimination during collagen matrix contraction

During tissue repair, excess fibroblasts are eliminated by apoptosis. This physiologic process limits fibrosis and restores normal anatomic patterns. Replicating physiologic apoptosis associated with tissue repair, fibroblasts incorporated into type I collagen matrices undergo apoptosis in response to collagen matrix contraction. In this in vitro model of wound repair, fibroblasts first attach to collagen via alpha2beta1 integrin. This provides a survival signal via activation of the phosphatidylinositol 3-kinase/Akt signal pathway. However, during subsequent collagen matrix contraction, the level of phosphorylated Akt progressively declines, triggering apoptosis. The mechanism underlying the fall in phosphorylated Akt is incompletely understood. Here we show that PTEN phosphatase becomes activated during collagen matrix contraction and is responsible for antagonizing phosphatidylinositol 3-kinase activity and promoting a decline in phosphorylated Akt and fibroblast apoptosis in response to collagen contraction. PTEN null fibroblasts displayed enhanced levels of phosphorylated Akt and were resistant to collagen matrix contraction-induced apoptosis. Reconstitution of PTEN in PTEN null cells conferred susceptibility to apoptosis in response to contraction of collagen matrices. Consistent with this, knockdown of PTEN in PTEN(+/+) embryonic fibroblasts by small interfering RNA augmented Akt activity and suppressed apoptosis in contractile collagen matrices. Furthermore, inhibition of Akt activity restored the sensitivity of PTEN null cells to collagen contraction-induced apoptosis, indicating that the mechanism by which PTEN alters fibroblast viability is through modulation of phosphorylated Akt levels. Our work suggests that collagen matrix contraction activates PTEN by a mechanism involving cytoskeletal disassembly. Our studies indicate a key role for PTEN in regulating fibroblast viability during tissue repair.     

Complete genome sequence and immunoproteomic analyses of the bacterial fish pathogen Streptococcus parauberis

Although Streptococcus parauberis is known as a bacterial pathogen associated with bovine udder mastitis, it has recently become one of the major causative agents of olive flounder (Paralichthys olivaceus) streptococcosis in northeast Asia, causing massive mortality resulting in severe economic losses. S. parauberis contains two serotypes, and it is likely that capsular polysaccharide antigens serve to differentiate the serotypes. In the present study, the complete genome sequence of S. parauberis (serotype I) was determined using the GS-FLX system to investigate its phylogeny, virulence factors, and antigenic proteins. S. parauberis possesses a single chromosome of 2,143,887 bp containing 1,868 predicted coding sequences (CDSs), with an average GC content of 35.6%. Whole-genome dot plot analysis and phylogenetic analysis of a 60-kDa chaperonin-encoding gene and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-encoding gene showed that the strain was evolutionarily closely related to Streptococcus uberis. S. parauberis antigenic proteins were analyzed using an immunoproteomic technique. Twenty-one antigenic protein spots were identified in S. parauberis, by reaction with an antiserum obtained from S. parauberis-challenged olive flounder. This work provides the foundation needed to understand more clearly the relationship between pathogen and host and develops new approaches toward prophylactic and therapeutic strategies to deal with streptococcosis in fish. The work also provides a better understanding of the physiology and evolution of a significant representative of the Streptococcaceae.     

Gymnaster koraiensis and its major components, 3,5-di-O-caffeoylquinic acid and gymnasterkoreayne B,reduce oxidative damage induced by tert-butyl hydroperoxide or acetaminophen in HepG2 cells

We investigated the protective effects of Gymnaster koraiensis against oxidative stress-induced hepatic cell damage. We used two different cytotoxicity models, i.e., the administration of tert-butyl hydroperoxide (t-BHP) and acetaminophen, in HepG2 cells to evaluate the protective effects of G. koraiensis. The ethyl acetate (EA) fraction of G. koraiensis and its major compound, 3,5-di-O-caffeoylquinic acid (DCQA), exerted protective effects in the t-BHP-induced liver cytotoxicity model. The EA fraction and DCQA ameliorated t-BHP-induced reductions in GSH levels and exhibited free radical scavenging activity. The EA fraction and DCQA also significantly reduced t-BHP-induced DNA damage in HepG2 cells. Furthermore, the hexane fraction of G. koraiensis and its major compound, gymnasterkoreayne B (GKB), exerted strong hepatoprotection in the acetaminopheninduced cytotoxicity model. CYP 3A4 enzyme activity was strongly inhibited by the extract, hexane fraction, and GKB. The hexane fraction and GKB ameliorated acetaminophen-induced reductions in GSH levels and protected against cell death. [BMB Reports 2013; 46(10): 513-518]