Actin cytoskeletal architecture regulates nitric oxide-induced apoptosis,dedifferentiation, and cyclooxygenase-2 expression in articular chondrocytes via mitogen-activated protein kinase and protein kinase C pathways

Nitric oxide (NO) in articular chondrocytes regulates differentiation, survival, and inflammatory responses by modulating ERK-1 and -2, p38 kinase, and protein kinase C (PKC) alpha and zeta. In this study, we investigated the effects of the actin cytoskeletal architecture on NO-induced dedifferentiation, apoptosis, cyclooxygenase (COX)-2 expression, and prostaglandin E2 production in articular chondrocytes, with a focus on ERK-1/-2, p38 kinase, and PKC signaling. Disruption of the actin cytoskeleton by cytochalasin D (CD) inhibited NO-induced apoptosis, dedifferentiation, COX-2 expression, and prostaglandin E2 production in chondrocytes cultured on plastic or during cartilage explants culture. CD treatment did not affect ERK-1/-2 activation but blocked the signaling events necessary for NO-induced dedifferentiation, apoptosis, and COX-2 expression such as activation of p38 kinase and inhibition of PKCalpha and -zeta. CD also suppressed activation of downstream signaling of p38 kinase and PKC, such as NF-kappaB activation, p53 accumulation, and caspase-3 activation, which are necessary for NO-induced apoptosis. NO production in articular chondrocytes caused down-regulation of phosphatidylinositol (PI) 3-kinase and Akt activities. The down-regulation of PI 3-kinase and Akt was blocked by CD treatment, and the CD effects on apoptosis, p38 kinase, and PKCalpha and -zeta were abolished by the inhibition of PI 3-kinase with LY294002. Our results collectively indicate that the actin cytoskeleton mediates NO-induced regulatory effects in chondrocytes by modulating down-regulation of PI 3-kinase and Akt, activation of p38 kinase, and inhibition of PKCalpha and -zeta     

Retinoic acid inhibits chondrogenesis of mesenchymal cells by sustaining expression of N-cadherin and its associated proteins

Retinoic acid (RA) is a well-known regulator of chondrocyte phenotype. RA inhibits chondrogenic differentiation of mesenchymal cells and also causes loss of differentiated chondrocyte phenotype. The present study investigated the mechanisms underlying RA regulation of chondrogenesis. RA treatment in chondrifying mesenchymal cells did not affect precartilage condensation, but blocked progression from precartilage condensation to cartilage nodule formation. This inhibitory effect of RA was independent of protein kinase C and extracellular signal-regulated protein kinase, which are positive and negative regulators of cartilage nodule formation, respectively. The progression from precartilage condensation to cartilage nodule requires downregulation of N-cadherin expression. However, RA treatment caused sustained expression of N-cadherin and its associated proteins including alpha- and beta-catenin suggesting that modulation of expression of these molecules is associated with RA-induced inhibition of chondrogenesis. This hypothesis was supported by the observation that disruption of the actin cytoskeleton by cytochalasin D (CD) blocks RA-induced sustained expression of cell adhesion molecules and overcomes RA-induced inhibition of chondrogenesis. Taken together, our results suggest RA inhibits chondrogenesis by stabilizing cell-to-cell interactions at the post-precartilage condensation stage.     

Construction of dextrin and isomaltose-assimilating brewer’s yeasts for production of low-carbohydrate beer

Most Saccharomyces spp. cannot degrade or ferment dextrin, which is the second most abundant carbohydrate in wort for commercial beer production. Dextrin-degrading brewer’s bottom and top yeasts expressing the glucoamylase gene (GAM1) from Debaryomyces occidentalis were developed to produce low-carbohydrate (calorie) beers. GAM1 was constitutively expressed in brewer’s yeasts using a rDNA-integration system that contained yeast CUP1 gene coding for copper resistance as a selective marker. The recombinants secreted active glucoamylase, displaying both α-1,4- and α-1,6-debranching activities, that degraded dextrin and isomaltose and consequently grew using them as sole carbon source. One of the recombinant strains expressing GAM1 hydrolyzed 96 % of 2 % (w/v) dextrin and 98 % of 2 % (w/v) isomaltose within 5 days of growth. Growth, substrate assimilation, and enzyme activity of these strains were characterized.     

Bioethanol production from the hydrolysate of rape stem in a surface-aerated fermentor

In this study, we investigated the feasibility of producing bioethanol from the hydrolysate of rape stem. Specifically, the most ideal yeast strain was screened, and the microaeration was performed by surface aeration on a liquid medium surface. Among the yeast strains examined, Pichia stipitis CBS 7126 displayed the best performance in bioethanol production during the surface-aerated fermentor culture. Pichia stipitis CBS 7126 produced maximally 9.56 g/l of bioethanol from the initial total reducing sugars (about 28 g/l). The bioethanol yield was 0.397 (by the DNS method). Furthermore, this controlled surface aeration method holds promise for use in the bioethanol production from the xylose-containing lignocellulosic hydrolysate of biomass.     

Enhancement of xylitol production by attenuation of intracellular xylitol dehydrogenase activity in <Emphasis Type="Italic">Candida tropicalis</Emphasis>

To construct Candida tropicalis strains that produce a high yield of xylitol with no requirement for co-substrates, we engineered the yeast with an attenuated xylitol dehydrogenase (XDH) and then assessed the efficiency of xylitol production The mutants, strains XDH-5 (with only one copy of the XDH gene), and ARSdR-16 (with a mutated XDH gene) showed 70 and 40% of wild type (WT) XDH activity, respectively. Conversions of xylose to xylitol by WT, XDH-5, and ARSdR-16 were 62, 64, and 75%, respectively, with productivities of 0.52, 0.54, and 0.62 g l⁻¹ h⁻¹, respectively. The ARSdR-16 mutant strain produced xylitol with high yield and high productivity in a simple process that required no co-substrates, such as glycerol. This strain represents a promising alternative for efficient and cost-effective xylitol production.     

Reduction of PDC1 expression in S. cerevisiae with xylose isomerase on xylose medium

Ethanol production using hemicelluloses has recently become a focus of many researchers. In order to promote D: -xylose fermentation, we cloned the bacterial xylA gene encoding for xylose isomerase with 434 amino acid residues from Agrobacterium tumefaciens, and successfully expressed it in Saccharomyces cerevisiae, a non-xylose assimilating yeast. The recombinant strain S. cerevisiae W303-1A/pAGROXI successfully colonized a minimal medium containing D: -xylose as a sole carbon source and was capable of growth in minimal medium containing 2% xylose via aerobic shake cultivation. Although the recombinant strain assimilates D: -xylose, its ethanol productivity is quite low during fermentation with D: -xylose alone. In order to ascertain the key enzyme in ethanol production from D: -xylose, we checked the expression levels of the gene clusters involved in the xylose assimilating pathway. Among the genes classified into four groups by their expression patterns, the mRNA level of pyruvate decarboxylase (PDC1) was reduced dramatically in xylose media. This reduced expression of PDC1, an enzyme which converts pyruvate to acetaldehyde, may cause low ethanol productivity in xylose medium. Thus, the enhancement of PDC1 gene expression may provide us with a useful tool for the fermentation of ethanol from hemicellulose.     

Simultaneous degradation of phytic acid and starch by an industrial strain of Saccharomyces cerevisiae producing phytase and α-amylase

Phytase liberates inorganic phosphate from phytic acid (myo-inositol hexakisphosphate) which is the major phosphate reserve in plant-derived foods and feeds. An industrial strain of Saccharomyces cerevisiae expressing the Debaryomyces castellii phytase gene (phytDc) and D. occidentalis α-amylase gene (AMY) was developed. The phytDc and AMY genes were constitutively expressed under the ADC1 promoter in S. cerevisiae by using the δ-integration system, which contains DNA derived exclusively from yeast. The recombinant industrial strain secreted both phytase and α-amylase for the efficient degradation of phytic acid and starch as main components of plant seeds. This new strain hydrolyzed 90% of 0.5% (w/v) sodium phytate within 5 days of growth and utilized 100% of 2% (w/v) starch within 48 h simultaneously.