The Halophilic Bacterium Paracoccus haeundaensis for the Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) from Single Carbon Sources.

The study objective was to evaluate the potential production of polyhydroxyalkanoates (PHAs), a biodegradable plastic material, by Paracoccus haeundaensis for which PHA production has never been reported. To identify the most effective nitrogen-limited culture conditions for PHAs production from this bacterium, batch fermentation using glucose concentrations ranging from 4 g l-1 to 20 g l-1 with a fixed ammonium concentration of 0.5 g l-1 was carried out at 30°C and pH 8.0. A glucose supplement of 12 g l-1 produced the highest PHA concentration (1.6 g l-1) and PHA content (0.63 g g-1) thereby identifying the optimal condition for PHA production from this bacterium. Gas chromatography-mass spectrometry analysis suggests that P. haeundaensis mostly produced copolymer types of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from glucose concentrations at 12 g l-1 or higher under the nitrogen-limited conditions. When several other single carbon sources were evaluated for the most efficient PHA production, fructose provided the highest biomass (2.8 g l-1), and PHAs (1.29 g l-1) concentrations. Results indicated that this bacterium mostly produced the copolymers P(3HB-co-3HV) from single carbon sources composing a range of 93-98% of 3-hydroxybutyrate and 2-7% of 3-hydroxyvalerate, whereas mannose-supplemented conditions produced the only homopolymer type of P(3HB). However, when propionic acid as a secondary carbon source were supplemented into the media, P. haeundaensis produced the copolymer P(3HB-co-3HV), composed of a 50% maximum monomeric unit of 3-hydroxyvaleric acid (3HV). However, as the concentration of propionic acid increased, cell biomass and PHAs concentrations substantially decreased due to cell toxicity.

Particulate matter 2.5 promotes inflammation and cellular dysfunction via reactive oxygen species/p38 MAPK pathway in primary rat corneal epithelial cells.

PURPOSE: Numerous studies have linked particulate matter2.5 (PM2.5) to ocular surface diseases, but few studies have been conducted on the biological effect of PM2.5 on the cornea. The objective of this study was to evaluate the harmful effect of PM2.5 on primary rat corneal epithelial cells (RCECs) in vitro and identify the toxic mechanism involved. MATERIALS AND METHODS: Primary cultured RCECs were characterized by pan-cytokeratin (CK) staining. In PM2.5-exposed RCECs, cell viability, microarray gene expression, inflammatory cytokine levels, mitochondrial damage, DNA double-strand break, and signalling pathway were investigated. RESULTS: Exposure to PM2.5 induced cytotoxicity and morphological changes in RCECs. In addition, PM2.5 markedly up-regulated pro-inflammatory mediators but down-regulated the wound healing-related transforming growth factor-ß. Furthermore, PM2.5 promoted mitochondrial reactive oxygen species (ROS) production and mediated cellular damage to mitochondria and DNA, whereas these cellular alterations induced by PM2.5 were markedly suppressed by a potential ROS scavenger. Noteworthy, removal of ROS selectively down-regulated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and the activation of the nuclear factor-κB (NF-κB) p65 in PM2.5-stimulated cells. Additionally, SB203580, a p38 MAPK inhibitor, markedly suppressed these PM2.5-mediated cellular dysfunctions. CONCLUSIONS: Taken together, our findings show that PM2.5 can promote the ROS/p38 MAPK/NF-κB signalling pathway and lead to mitochondrial damage and DNA double-strand break, which is ultimately caused inflammation and cytotoxicity in RCECs. These findings indicate that the ROS/p38 MAPK/NF-κB signalling pathway is one mechanism involved in PM2.5-induced ocular surface disorders.

Modulation of Inflammatory Pathways and Adipogenesis by the Action of Gentisic Acid in RAW 264.7 and 3T3-L1 Cell Lines.

Gentisic acid (GA), a benzoic acid derivative present in various food ingredients, has been shown to have diverse pharmaceutical activities such as anti-carcinogenic, antioxidant, and hepatoprotective effects. In this study, we used a co-culture system to investigate the mechanisms of the anti-inflammatory and anti-adipogenic effects of GA on macrophages and adipocytes, respectively, as well as its effect on obesity-related chronic inflammation. We found that GA effectively suppressed lipopolysaccharide-stimulated inflammatory responses by controlling the production of nitric oxide and pro-inflammatory cytokines and modulating inflammation-related protein pathways. GA treatment also inhibited lipid accumulation in adipocytes by modulating the expression of major adipogenic transcription factors and their upstream protein pathways. Furthermore, in the macrophage-adipocyte co-culture system, GA decreased the production of obesity-related cytokines. These results indicate that GA possesses effective anti-inflammatory and anti-adipogenic activities and may be used in developing treatments for the management of obesity-related chronic inflammatory diseases.

Production of Ethanol from Agarose by Unified Enzymatic Saccharification and Fermentation in Recombinant Yeast.

The unified saccharification and fermentation (USF) system was developed for direct production of ethanol from agarose. This system contains an enzymatic saccharification process that uses three types of agarases and a fermentation process by recombinant yeast. The pGMFα-HGN plasmid harboring AGAH71 and AGAG1 genes encoding ß-agarase and the NABH558 gene encoding neoagarobiose hydrolase was constructed and transformed into the Saccharomyces cerevisiae 2805 strain. Three secretory agarases were produced by introducing an S. cerevisiae signal sequence, and they efficiently degraded agarose to galactose, 3,6-anhydro- L-galactose (AHG), neoagarobiose, and neoagarohexose. To directly produce ethanol from agarose, the S. cerevisiae 2805/pGMFα-HGN strain was cultivated into YP-containing agarose medium at 40°C for 48 h (for saccharification) and then 30°C for 72 h (for fermentation). During the united cultivation process for 120 h, a maximum of 1.97 g/l ethanol from 10 g/l agarose was produced. This is the first report on a single process containing enzymatic saccharification and fermentation for direct production of ethanol without chemical liquefaction (pretreatment) of agarose.

Acer okamotoanum Nakai Leaf Extract Inhibits Adipogenesis Via Suppressing Expression of PPAR γ and C/EBP α in 3T3-L1 Cells.

The genus Acer contains several species with various bioactivities including antioxidant, antitumor and anti-inflammatory properties. However, Acer okamotoanum Nakai, one species within this genus has not been fully studied yet. Therefore, in this study, we investigated the anti-adipogenic activities of leaf extract from A. okamotoanum Nakai (LEAO) on 3T3-L1 preadipocytes. Adipogenesis is one of the cell differentiation processes, which converts preadipocytes into mature adipocytes. Nowadays, inhibition of adipogenesis is considered as an effective strategy in the field of anti-obesity research. In this study, we observed that LEAO decreased the accumulation of lipid droplets during adipogenesis and down-regulated the expression of key adipogenic transcription factors such as peroxisome proliferator-activated receptor γ (PPAR γ) and CCAAT/enhancer binding protein α (C/EBP α). In addition, LEAO inactivated PI3K/Akt signaling and its downstream factors that promote adipogenesis by inducing the expression of PPAR γ. LEAO also activated ß-catenin signaling, which prevents the adipogenic program by suppressing the expression of PPAR γ. Therefore, we found that treatment with LEAO is effective for attenuating adipogenesis in 3T3-L1 cells. Consequently, these findings suggest that LEAO has the potential to be used as a therapeutic agent for preventing obesity.

Cloning, Expression, and Characterization of a Cold-Adapted Shikimate Kinase from the Psychrophilic Bacterium Colwellia psychrerythraea 34H.

Most cold-adapted enzymes possess higher Km and kcat values than those of their mesophilic counterparts to maximize the reaction rate. This characteristic is often ascribed to a high structural flexibility and improved dynamics in the active site. However, this may be less convincing to cold-adapted metabolic enzymes, which work at substrate concentrations near Km. In this respect, cold adaptation of a shikimate kinase (SK) in the shikimate pathway from psychrophilic Colwellia psychrerythraea (CpSK) was characterized by comparing it with a mesophilic Escherichia coli homolog (EcSK). The optimum temperatures for CpSK and EcSK activity were approximately 30°C and 40°C, respectively. The melting points were 33°C and 45°C for CpSK and EcSK, respectively. The ΔGH2O (denaturation in the absence of denaturing agent) values were 3.94 and 5.74 kcal/mol for CpSK and EcSK, respectively. These results indicated that CpSK was a cold-adapted enzyme. However, contrary to typical kinetic data, CpSK had a lower Km for its substrate shikimate than most mesophilic SKs, and the kcat was not increased. This observation suggested that CpSK may have evolved to exhibit increased substrate affinity at low intracellular concentrations of shikimate in the cold environment. Sequence analysis and homology modeling also showed that some important salt bridges were lost in CpSK, and higher Arg residues around critical Arg 140 seemed to increase flexibility for catalysis. Taken together, these data demonstrate that CpSK exhibits characteristics of cold adaptation with unusual kinetic parameters, which may provide important insights into the cold adaptation of metabolic enzymes.

The Anti-Adipogenic Activity of a New Cultivar, Pleurotus eryngii var. ferulae 'Beesan No. 2', through Down-Regulation of PPAR γ and C/EBP α in 3T3-L1 Cells.

Adipogenesis is one of the cellular processes and a highly controlled program. Nowadays, inhibition of adipogenesis has received attention as an effective way to regulate obesity. In the current study, we investigated the inhibition effect of a chloroform extract of Pleurotus eryngii var. ferulae 'Beesan No. 2' (CEBT) on adipogenesis in 3T3-L1 murine preadipocytes. Pleurotus eryngii var. ferulae is one of many varieties of King oyster mushroom and has been reported to have various biological activities, including antitumor and anti-inflammation effects. Biological activities of 'Beesan No. 2', a new cultivar of Pleurotus eryngii var. ferulae, have not yet been reported. In this study, we found that CEBT suppressed adipogenesis in 3T3-L1 cells through inhibition of key adipogenic transcription factors, such as peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α. Additionally, CEBT reduced the expression of the IRS/PI3K/Akt signaling pathway and its downstream factors, including mammalian target of rapamycin and p70S6 kinase, which stimulate adipogenesis. Furthermore, ß-catenin, a suppressor of adipogenesis, was increased in CEBT-treated cells. These results indicate that Pleurotus eryngii var. ferulae 'Beesan No. 2' effectively inhibited adipogenesis, so this mushroom has potential as an anti-obesity food and drug.

Molecular Cloning and Co-Expression of Phytoene Synthase Gene from Kocuria gwangalliensis in Escherichia coli.

A phytoene synthase gene, crtB, was isolated from Kocuria gwangalliensis. The crtB with 1,092 bp full-length has a coding sequence of 948 bp and encodes a 316-amino-acids protein. The deduced amino acid sequence showed a 70.9% identity with a putative phytoene synthase from K. rhizophila. An expression plasmid, pCcrtB, containing the crtB gene was constructed, and E. coli cells containing this plasmid produced the recombinant protein of approximately 34 kDa , corresponding to the molecular mass of phytoene synthase. Biosynthesis of lycopene was confirmed when the plasmid pCcrtB was co-transformed into E. coli containing pRScrtEI carrying the crtE and crtI genes encoding lycopene biosynthetic pathway enzymes. The results obtained from this study will provide a base of knowledge about the phytoene synthase of K. gwangalliensis and can be applied to the production of carotenoids in a non-carotenoidproducing host.

Selenium improves stem cell potency by stimulating the proliferation and active migration of 3T3-L1 preadipocytes.

Selenium is a trace nutrient element that protects cells against oxidative damage. In this study, the potential of selenium to improve stem cell potency through active proliferation and migration of 3T3-L1 preadipocytes was investigated, together with the underlying molecular mechanisms. The results indicated that selenium applied for 24 h stimulated cell proliferation up to 20% compared to untreated control cells. Selenium induced the expression of cyclin-dependent kinase (CDK) 1 and CDK2, which are known to regulate G2/M progression, and significantly downregulated the CDK inhibitors p21 and p27. Selenium also activated the expression of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, as well as extracellular signal-regulated kinase (ERK). Although LY294002, an inhibitor of PI3K, significantly inhibited the selenium-induced cell proliferation of the 3T3-L1 preadipocytes, PD98059, an inhibitor of ERK, did not affect selenium-induced active proliferation. These results clearly indicate that selenium stimulated cell proliferation through cell cycle progression and PI3K/Akt activation, but not through ERK activation. Furthermore, selenium increased 3T3-L1 cell migration, which was associated with the induction of matrix metalloproteinase (MMP)-2 and MMP-9. Taken together, the current findings suggest that selenium can stimulate stem cell potency by increasing the proliferation and active migration of 3T3-L1 cells.

Functional characterization of the α- and ß-subunits of a group II chaperonin from Aeropyrum pernix K1.

We isolated and functionally characterized the α- and ß- subunits (ApCpnA and ApCpnB) of a chaperonin from Aeropyrum pernix K1. The constructed vectors pET3d- ApCpnA and pET21a-ApCpnB were transformed into E. coli Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3) cells. The expression of ApCpnA (60.7 kDa) and ApCpnB (61.2 kDa) was confirmed by SDS-PAGE analysis. Recombinant ApCpnA and ApCpnB were purified by heat-shock treatment and anion-exchange chromatography. ApCpnA and ApCpnB were able to hydrolyze not only ATP, but also CTP, GTP, and UTP, albeit with different efficacies. Purified ApCpnA and ApCpnB showed the highest ATPase, CTPase, UTPase, and GTPase activities at 80°C. Furthermore, the addition of ApCpnA and ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at 43°C and 50°C, respectively. In particular, the addition of ATP or CTP to ApCpnA and ApCpnB resulted in the most effective prevention of thermal aggregation and inactivation of CS and ADH. The ATPase activity of the two chaperonin subunits was dependent on the salt concentration. Among the ions we examined, potassium ions were the most effective at enhancing the ATP hydrolysis activity of ApCpnA and ApCpnB.

Gastroprotective effect of selenium on ethanol-induced gastric damage in rats.

In the present study, we examined the gastroprotective effect of selenium against ethanol-induced gastric mucosal lesions in rats. The gastric mucosal lesions were produced by oral administration with various concentrations of ethanol for three days, and 80% ethanol treatment was determined to be the optimal condition for induction of gastric damage. To identify the protective effect of selenium on ethanol-induced gastric damage, various doses of selenium were given as pretreatment for three days, and then gastric damage was induced by 80% ethanol treatment. Selenium showed a protective effect against ethanol-induced gastric mucosal lesions in a dose dependent manner. Specifically, 100 µg/kg selenium showed the highest level of gastroprotection. In addition, selenium markedly attenuated ethanol-induced lipid peroxidation in gastric mucosa and increased activities of radical scavenging enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase in a dose-dependent manner. Histological data showed that 100 µg/kg selenium distinctly reduced the depth and severity of the ethanol induced gastric lesion. These results clearly demonstrate that selenium inhibits the formation of ethanol-induced gastric mucosal lesions through prevention of lipid peroxidation and activation of enzymatic radical scavenging.

Induction of apoptosis and autophagy by sodium selenite in A549 human lung carcinoma cells through generation of reactive oxygen species.

Selenium in the form of sodium selenite has been reported to exert anti-tumor effects in several cancer cell types by inducing autophagic cell death and apoptosis mediated by reactive oxygen species (ROS). However, the exact molecular pathways underlying these effects have not been fully established. The present study used A549 human lung carcinoma cells for further investigation of the anti-cancer mechanism of sodium selenite. We showed that sodium selenite modulated both the extrinsic and intrinsic apoptotic pathways, which were interconnected by Bid truncation. We used z-VAD-fmk, a pan-caspase inhibitor, to demonstrate that sodium selenite-induced apoptosis was dependent on the activation of caspases. Sodium selenite also increased autophagy, as indicated by an increase in microtubule-associated protein light chain-3 (LC3) puncta, accumulation of LC3II, and elevation of autophagic flux. Pretreatment with bafilomycin A1 enhanced sodium selenite-induced apoptosis, indicating that sodium selenite-induced autophagy functioned as a survival mechanism. Sodium selenite treatment also resulted in generation of ROS, which abrogated mitochondrial membrane potential (MMP) and regulated both apoptosis and autophagy. Phospho-nuclear factor erythroid 2-related factor 2 (p-Nrf2) showed a ROS-dependent translocation to the nucleus, which suggested that Nrf2 might increase cell survival by suppressing ROS accumulation and apoptosis mediated by oxidative stress. Sodium selenite treatment of A549 cells therefore appeared to trigger both apoptosis and cytoprotective autophagy, which were both mediated by ROS. The data suggest that regulation of ROS generation and autophagy can be a potential strategy for treating lung cancer that is resistant to pro-apoptotic therapeutics.

Coexpression of molecular chaperone enhances activity and export of organophosphorus hydrolase in Escherichia coli.

Periplasmic secretion has been used in attempts to construct an efficient whole-cell biocatalyst with greatly reduced diffusion limitations. Previously, we developed recombinant Escherichia coli that express organophosphorus hydrolase (OPH) in the periplasmic space using the twin-arginine translocation (Tat) pathway to degrade environmental toxic organophosphate compounds. This system has the advantage of secreting protein into the periplasm after folding in the cytoplasm. However, when OPH was expressed with a Tat signal sequence in E. coli, we found that the predominant OPH was an insoluble premature form in the cytoplasm, and thus, the whole-cell OPH activity was significantly lower than its cell lysate activity. In this work, we, for the first time, used a molecular chaperone coexpression strategy to enhance whole-cell OPH activity by improving the periplasmic translocation of soluble OPH. We found that the effect of GroEL-GroES (GroEL/ES) assistance on the periplasmic localization of OPH was secretory pathway dependent. We observed a significant increase in the amount of soluble mature OPH when cytoplasmic GroEL/ES was expressed; this increase in the amount of mature OPH might be due to enhanced OPH folding in the cytoplasm. Importantly, the whole-cell OPH activity of the chaperone-coexpressing cells was ∼5.5-fold greater at 12 h after induction than that of cells that did not express the chaperone as a result of significant Tat-based periplasmic translocation of OPH in the chaperone-coexpressing cells. Collectively, these data suggest that molecular chaperones significantly enhance the whole-cell activity of periplasmic OPH-secreting cells, yielding an effective whole-cell biocatalyst system with highly reduced diffusion limitations.

Widdrol blocks 3T3-L1 preadipocytes growth and differentiation due to inhibition of mitotic clonal expansion.

Adipocyte differentiation is strongly associated with obesity, which causes metabolic disorders. In this study, we investigated the inhibitory effects of widdrol on 3T3- L1 preadipocyte growth and differentiation. Widdrol decreased lipid droplet accumulation and down-regulated adipogenic transcription factors such as C/EBPalpha, C/EBPbeta, and PPARgamma. Widdrol blocked preadipocyte proliferation and differentiation through the inhibition of mitotic clonal expansion, which was accompanied by the failure of degradation of p21, a cyclin-dependent kinase inhibitor. Cell-cycle analysis clearly indicated that widdrol actively induces cell-cycle arrest at the G1-S phage transition, causing cells to remain in the preadipocyte state. Moreover, widdrol increased p21 expression and inhibited Rb phosphorylation in preadipocyte incubated in a hormone medium. Therefore, these findings clearly suggest that widdrol blocks preadipocyte growth and differentiation through the inhibition of mitotic clonal expansion by p21- and Rb-dependent G1 arrest and can be developed as a potent anti-adipogenic agent for reducing obesity.

Construction of an expression system for the secretory production of recombinant α-agarase in yeast.

α-Agarase hydrolyzes the α-1,3 linkage of agarose yielding agaro-oligosaccharides. It is less well characterized than ß-agarase. AgaA gene (2.3 kb ORF), encoding the α-agarase from Thalassomonas JAMB A33, was subcloned into both a constitutive and an inducible expression vector. Both the constructed plasmids, pVT-AgaA (ADH1 promoter) and pYInu-AgaA (GAL10 promoter), were transformed into Saccharomyces cerevisiae SEY2102 and FY833 and pPIC9-AgaA harboring the AOX1 promoter was transformed into Pichia pastoris GS115. The recombinant α-agarases were over-expressed with activities from 0.3 to 1.6 unit/ml, the highest being in the SEY2102/pYInu-AgaA transformant. Most of the recombinant α-agarase was extracellular because each plasmid possesses a signal sequence for the secretory production of α-agarase. In contrast, the Pichia host-vector expression system was unsuitable for the production of recombinant α-agarase. This is the first report of recombinant production of α-agarase in yeast for industrial use.

The effect of secretory factors of adipose-derived stem cells on human keratinocytes.

UNLABELLED: The beneficial effects of adipose-derived stem cell conditioned medium (ADSC-CM) on skin regeneration have been reported. Although the mechanism of how ADSC-CM promotes skin regeneration is unclear, ADSC-CM contained various growth factors and it is an excellent raw material for skin treatment. ADSC-CM produced in a hypoxia condition of ADSC-in other words, Advanced Adipose-Derived Stem cell Protein Extract (AAPE)-has great merits for skin regeneration. In this study, human primary keratinocytes (HKs), which play fundamental roles in skin tissue, was used to examine how AAPE affects HK. HK proliferation was significantly higher in the experimental group (1.22 µg/mL) than in the control group. DNA gene chip demonstrated that AAPE in keratinocytes (p < 0.05) notably affected expression of 290 identified transcripts, which were associated with cell proliferation, cycle and migration. More keratinocyte wound healing and migration was shown in the experimental group (1.22 µg/mL). AAPE treatment significantly stimulated stress fiber formation, which was linked to the RhoA-ROCK pathway. We identified 48 protein spots in 2-D gel analysis and selected proteins were divided into 64% collagen components and 30% non-collagen components as shown by the MALDI-TOF analysis. Antibody array results contained growth factor/cytokine such as HGF, FGF-1, G-CSF, GM-CSF, IL-6, VEGF, and TGF-ß3 differing from that shown by 2-D analysis. CONCLUSION: AAPE activates HK proliferation and migration. These results highlight the potential of the topical application of AAPE in the treatment of skin regeneration.

Curative effect of selenium against indomethacin-induced gastric ulcers in rats.

Indomethacin is a nonsteroid anti-inflammatory agent that is known to induce severe gastric mucosal lesions. In this study, we investigated the effect of selenium on gastric mucosal lesions in rats. To confirm the curative effect of selenium against indomethacin-induced gastric ulcers, gastric ulcers were induced by oral administration of 25 mg/kg indomethacin, and then different doses (10, 50, and 100 microgram/kg of body weight) of selenium or vehicle were treated by oral gavage for 3 days. Oral administration of indomethacin clearly increased the gastric ulcer area in the stomach, whereas selenium applied for 3 days significantly decreased the gastric ulcer area in a dose-dependent manner. In addition, selenium markedly reduced the increase of lipid peroxidation induced by indomethacin in the gastric mucosa and increased activities of radical scavenging enzymes such as superoxide dismutase, catalase, and glutathione peroxidase in a dose-dependent manner. These results reveal that selenium can heal indomethacininduced gastric ulcers through elimination of the lipid peroxides and activation of radical scavenging enzymes.

Curcumin stimulates proliferation, stemness acting signals and migration of 3T3-L1 preadipocytes.

In the present study, the potential of curcumin to stimulate proliferation, stemness acting signals and migration of 3T3-L1 preadipocytes and the associated molecular mechanisms were investigated. Low concentrations of curcumin stimulated cell proliferation, whereas high concentrations were cytotoxic to 3T3-L1 cells. In particular, application of 0.02 µM of curcumin for 24 h resulted in significantly increased cell proliferation and was determined to be the optimal treatment for this study. In a colony-forming cell assay, cells treated with 0.02 µM of curcumin showed an approximately 1.5-fold increase in colony formation. Curcumin treatment up-regulated the proliferation-related marker proteins coupled with increased cell growth, telomerase activity and overexpression of stemness acting signals, which was associated with activation of the phosphoinositide 3-kinase (PI3K) pathway. In addition, curcumin significantly inactivated p38 mitogen-activated protein kinases (MAPK) and stress-activated protein kinase/c-Jun N-terminal kinases (SARK/JNK), coupled with inhibition of p53 and p21 tumor suppressor gene products. In addition, curcumin significantly increased cell migration through activation of migration-associated transcription factors. Therefore, these results clearly show that activation of cell proliferation by curcumin is associated with improved stem cell potency in 3T3-L1 preadipocytes.

Cooperativity of alpha- and beta-subunits of group II chaperonin from the hyperthermophilic archaeum Aeropyrum pernix K1.

alpha- and beta-subunits (ApCpnA and ApCpnB) are group II chaperonins from the hyperthermophilic archaeum Aeropyrum pernix K1, specialized in preventing the aggregation and inactivation of substrate proteins under conditions of transient heat stress. In the present study, the cooperativity of alpha- and beta-subunits from the A. pernix K1 was investigated. The ApCpnA and ApCpnB chaperonin genes were overexpressed in E. coli Rosetta and Codonplus (DE3), respectively. Each of the recombinant alpha- and beta- subunits was purified to 92% and 94% by using anionexchange chromatography. The cooperative activity between purified alpha- and beta-subunits was examined using citrate synthase (CS), alcohol dehydrogenase (ADH), and malate dehydrogenase (MDH) as substrate proteins. The addition of both alpha- and beta-subunits could effectively protect CS and ADH from thermal aggregation and inactivation at 43 degreesC and 50 degreesC, respectively, and MDH from thermal inactivation at 80 degreesC and 85 degreesC. Moreover, in the presence of ATP, the protective effects of alpha- and beta-subunits on CS from thermal aggregation and inactivation, and ADH from thermal aggregation, were more enhanced, whereas cooperation between chaperonins and ATP in protection activity on ADH and MDH (at 85 degreesC) from thermal inactivation was not observed. Specifically, the presence of both alpha- and beta- subunits could effectively protect MDH from thermal inactivation at 80 degreesC in an ATP-dependent manner.

Astaxanthin improves the proliferative capacity as well as the osteogenic and adipogenic differentiation potential in neural stem cells.

In the present study, the effect of astaxanthin on improvement of the proliferative capacity as well as the osteogenic and adipogenic differentiation potential in neural stem cells (NSCs) was evaluated. Treatment of astaxanthin-induced actives cell growth in a dose-dependent and time-dependent manner. Results from a clonogenic assay clearly indicated that astaxanthin can actively stimulate proliferation of NSCs. Astaxanthin-induced improvement in the proliferative capacity of NSCs resulted in overexpression of several proliferation-related proteins. Astaxanthin-induced activation of PI3K and its downstream mediators, p-MEK, p-ERK, and p-Stat3 in NSCs resulted in subsequent induction of expression of proliferation-related transcription factors (Rex1, CDK1, and CDK2) and stemness genes (OCT4, SOX2, Nanog, and KLF4). Astaxanthin also improved the osteogenic and adipogenic differentiation potential of NSCs. Astaxanthin-treated NSCs showed prominent calcium deposits and fat formation. These results were consistent with overexpression of osteogenesis-related genes (osteonectin, RXR, and osteopontin) and adipogenesis-related genes (AP and PPAR-gamma) after astaxanthin treatment. These findings clearly demonstrated that astaxanthin acts synergistically on the regulatory circuitry that controls proliferation and differentiation of NSCs.