Characterization of a novel manganese dependent endoglucanase belongs in GH family 5 from Phanerochaete chrysosporium.

The cDNA encoding a putative glycoside hydrolase family 5, which has been predicted to be an endoglucanase (PcEg5A), was cloned from Phanerochaete chrysosporium and expressed in Pichia pastoris. PcEg5A contains a carbohydrate-binding domain and two important amino acids, E209 and E319, playing as proton donor and nucleophile in substrate catalytic domain. SDS-PAGE analysis indicated that the recombinant endoglucanase 5A (rPcEg5A) has a molecular size of 43 kDa which corresponds with the theoretical calculation. Optimum pH and temperature were found to be 4.5-6.0, and 50°C-60°C, respectively. Moreover, rPcEg5A exhibited maximal activity in the pH range of 3.0-8.0, whereas over 50% of activity still remained at 20°C and 80°C. rPcEg5A was stable at 60°C for 12 h incubation, indicating that rPcEg5A is a thermostable enzyme. Manganese ion enhanced the enzyme activity by 77%, indicating that rPcEg5A is a metal dependent enzyme. The addition of rPcEg5A to cellobiase (cellobiohydrolase and ß-glucosidase) resulted in a 53% increasing saccharification of NaOH-pretreated barley straw, whereas the glucose release was 47% higher than that cellobiase treatment alone. Our study suggested that rPcEg5A is an enzyme with great potential for biomass saccharification.

Pyrosequencing reveals bacterial diversity in Korean traditional wheat-based nuruk.

The emerging global importance of Korea's alcoholic beverages emphasizes the need for quality enhancement of nuruk, a traditional Korean cereal starter that is used extensively in traditional brewing. Apart from fungi and yeasts, bacteria known to be ubiquitously present are also a part of the nuruk ecosystem and are known to influence fermentation activity by influencing fermentation favorable factors. In the current study, bacterial diversity and temporal variations in the traditional wheat-based nuruk, fermented at two representative temperature conditions for 30 days, along with two commercial wheat-based nuruk samples for comparison analysis were evaluated using libraries of PCR amplicons and 454 pyrosequencing targeting of the hypervariable regions V1 to V3 of the 16S rRNA gene. A total of 90,836 16S reads were analyzed and assigned to a total of 314, 321, and 141 Operational Taxonomic Units (OTUs) for nuruk A, B, and C, respectively. Diversity parameters clearly indicated nuruk B to be more diverse in terms of bacterial composition than nuruk A. Taxonomic assignments indicated that nuruk A was dominated by phylum Cyanobacteria, whereas nuruk B was dominated by phylum Actinobacteria. For both nuruk A and B, members of the phylum Firmicutes mostly converged into the family Bacillaceae; these microorganisms might be present in negligible numbers at the beginning but became significant as the fermentation progressed. The commercial samples were predominated by phylum Firmicutes, which is composed of Lactobacillaceae and Leoconostocaceae. The findings of this study provide new insights into understanding the changes in bacterial community structure during traditional nuruk starter production.

Bee venom ameliorates ovalbumin induced allergic asthma via modulating CD4+CD25+ regulatory T cells in mice.

Asthma is a potentially life-threatening inflammatory disease of the lung characterized by the presence of large numbers of CD4+ T cells. These cells produce the Th2 and Th17 cytokines that are thought to orchestrate the inflammation associated with asthma. Bee venom (BV) has traditionally been used to relieve pain and to treat chronic inflammatory diseases. Recent reports have suggested that BV might be an effective treatment for allergic diseases. However, there are still unanswered questions related to the efficacy of BV therapy in treating asthma and its therapeutic mechanism. In this study, we evaluated whether BV could inhibit asthma and whether BV inhibition of asthma could be correlated with regulatory T cells (Treg) activity. We found that BV treatment increased Treg populations and suppressed the production of Th1, Th2 and Th17-related cytokines in an in vitro culture system, including IL2, IL4, and IL17. Interestingly, production of IL10, an anti-inflammatory cytokine secreted by Tregs, was significantly augmented by BV treatment. We next evaluated the effects of BV treatment on allergic asthma in an ovalbumin (OVA)-induced mouse model of allergic asthma. Cellular profiling of the bronchoalveolar lavage (BAL) and histopathologic analysis demonstrated that peribronchial and perivascular inflammatory cell infiltrates were significantly lowered following BV treatment. BV also ameliorated airway hyperresponsiveness, a hallmark symptom of asthma. In addition, IL4 and IL13 levels in the BAL fluid were decreased in the BV treated group. Surprisingly, the beneficial effects of BV treatment on asthma were eradicated following Treg depletion by anti-CD25 antibody injection, suggesting that the major therapeutic targets of BV were Tregs. These results indicate that BV efficiently diminishes bronchial inflammation in an OVA-induced allergic asthma murine model, and that this effect might correlate with Tregs, which play an important role in maintaining immune homeostasis and suppressing the function of other T cells to limit the immune response. These results also suggest that BV has potential therapeutic value for controlling allergic asthma responses.

Anti-cancer effect of bee venom in prostate cancer cells through activation of caspase pathway via inactivation of NF-κB.

BACKGROUND: Bee venom has been used as a traditional medicine to treat arthritis, rheumatism, back pain, cancerous tumors, and skin diseases. However, the effects of bee venom on the prostate cancer and their action mechanisms have not been reported yet. METHODS: To determine the effect of bee venom and its major component, melittin on the prostate cancer cells, apoptosis is analyzed by tunnel assay and apoptotic gene expression. For xenograft studies, bee venom was administrated intraperitoneally twice per week for 4 weeks, and the tumor growth was measured and the tumor were analyzed by immunohistochemistry. To investigate whether bee venom and melittin can inactivate nuclear factor kappa B (NF-κB), we assessed NF-κB activity in vitro and in vivo. RESULTS AND CONCLUSIONS: Bee venom (1-10 µg/ml) and melittin (0.5-2.5 µg/ml) inhibited cancer cell growth through induction of apoptotic cell death in LNCaP, DU145, and PC-3 human prostate cancer cells. These effects were mediated by the suppression of constitutively activated NF-κB. Bee venom and melittin decreased anti-apoptotic proteins but induced pro-apoptotic proteins. However, pan caspase inhibitor abolished bee venom and melittin-induced apoptotic cell death and NF-κB inactivation. Bee venom (3-6 mg/kg) administration to nude mice implanted with PC-3 cells resulted in inhibition of tumor growth and activity of NF-κB accompanied with apoptotic cell death. Therefore, these results indicated that bee venom and melittin could inhibit prostate cancer in in vitro and in vivo, and these effects may be related to NF-κB/caspase signal mediated induction of apoptotic cell death.

Berberine inhibits p53-dependent cell growth through induction of apoptosis of prostate cancer cells.

Berberine has anti-tumor properties in some cancer cells including prostate cancer, but the exact mechanisms and in vivo effects are unclear. We investigated anti-cancer activity of berberine in vitro and in vivo, and possible mechanisms in prostate cancer cells. Berberine treatment inhibited cell cancer growth in a concentration (0-50 microM) and time- (0-48 h) dependent manner without any growth inhibition in normal human prostate epithelial PWR-1E cells. However, the p53 expressing LNCaP cells were more susceptible against berberine than the p53 lacking PC-3 cells. The cell arrest in G0/G1 phase, apoptotic cell death and the expression of apoptotic cell death proteins Bax and caspase-3 was much higher in berberine-treated LNCaP cells than those in PC-3 cells. Exploration of p53 siRNA or pifithrin-alpha, a p53 inhibitor to the LNCaP cells, suppressed berberine-induced cell death and expression of apoptosis-related proteins. In xenograft in vivo studies, berberine reduced tumor weights and volumes accompanied with apoptotic cell death and increased expression of apoptotic cell death proteins, however, the extent of inhibitory effect was more significant in LNCaP cell-bearing mice. Therefore, these results indicated that berberine inhibited p53-dependent prostate cancer cell death.

Inhibitory effects of Saururus chinensis (LOUR.) BAILL on the development of atopic dermatitis-like skin lesions in NC/Nga mice.

The present study was performed to examine whether the leaves of Saururus chinensis (LOUR.) BAILL (SC), an herb used for the management of various skin diseases including atopic dermatitis (AD) in Eastern countries, inhibited the development of AD-like skin lesions in NC/Nga mice which was induced by repeated application of picryl chloride (PiCl). The efficacy of SC was judged by measurement of skin severity, itching behavior, histological study, serum IgE levels, IL-4 and IFN-gamma in lymph nodes. Oral administration of SC extract to the PiCl-treated NC/Nga mice for 8 weeks (5 d per week) inhibited significantly the development of AD-like skin lesions macroscopically. Histologically, SC inhibited dermatitis changes like hypertrophy, hyperkeratosis, and infiltration of inflammatory cells into epidermis and dermis. The itching behavior and serum IgE level decreased significantly after SC administration. SC administration enhanced IFN-gamma mRNA expression but did not have an effect on IL-4 mRNA expression. These results suggest that SC could inhibit the development of AD-like skin lesions in NC/Nga mice possibly through modulating the Th1/Th2 imbalance by the promoting of Th1 cell response. Thus, SC may be an alternative substance for the management of AD patients.

The bark of Betula platyphylla var. japonica inhibits the development of atopic dermatitis-like skin lesions in NC/Nga mice.

The bark of Betula platyphylla Sukat. var. japonica Hara (Asian White Birch, AWB) is one of the herbs used in Eastern countries for the treatment of various inflammatory diseases including atopic dermatitis (AD). The present study was performed to examine if and how the bark of AWB inhibits the development of AD-like skin lesions in NC/Nga mice induced by repeated application of picryl chloride (PC). With this aim, we examined the skin symptom severity, itching behavior, serum immunoglobulin (Ig) E level and mRNA expression of cytokines at iliac and cervical lymph nodes in the mice. Oral administration of AWB extracts (25, 100 and 250 mg/kg) to the PC-treated mice inhibited the development of AD-like skin lesions as exemplified by a significant decrease in the total skin severity scores, itching behavior and a decrease in hypertrophy and infiltration of inflammatory cells into dermis. The serum IgE level was also significantly reduced by AWB extract. In the RT-PCR results, the expression of interleukin-4 mRNA was reduced by AWB extract, whereas the expression of interferon-gamma mRNA was not changed. These results suggest that AWB inhibits the development of AD-like skin lesions in NC/Nga mice through the suppression of the T-helper 2 cell response.

Berberine inhibits human neuroblastoma cell growth through induction of p53-dependent apoptosis.

Berberine, an alkaloid, has anti-tumor properties in some cancer cells, but action mechanisms are not clear yet. We here investigated the anticancer activity of berberine and possible mechanisms in human neuroblastoma SK-N-SH and SK-N-MC cells. The p53-expressing cells, SK-N-SH (IC50=37 microM) were more susceptible to berberine than the p53-deficient cells, SK-N-MC (IC50 > or =100 microM) without cytotoxic effect on the cortical neuronal cells. Berberine caused cell cycle arrest in G0/G1 phase and apoptotic cell death, and these effects were much greater in SK-N-SH cells than those in SK-N-MC cells. Berberine much greatly decreased G0/G1 phase-associated cyclin and cyclin-dependent kinase (cyclin D1, cyclin E, Cdk2, and Cdk4) expression, and increased apoptotic gene expression and activation of caspase-3 in SK-N-SH cells. Exploration of p53 siRNA or pifithrin-alpha (PFT-alpha), a p53 inhibitor, in the SK-N-SH cells resulted in increase of IC50 values for cell viability, and decreased apoptotic cell death, expression of p53 and activation of caspase-3. Therefore, these results showed that berberine causes p53-dependent apoptotic death of neuroblastoma cells, and suggested that berberine may be useful as an anticancer agent for neuroblastoma.

Increase of collagen synthesis by obovatol through stimulation of the TGF-beta signaling and inhibition of matrix metalloproteinase in UVB-irradiated human fibroblast.

BACKGROUND: Alterations of the extracellular matrix (ECM) is critical in the photo and age-damaged skin. Thus any compounds keep ECM can protected from photo and aged-damaged skin. ECM is predominantly composed of type I and type III collagens in the dermis. Transforming growth factor (TGF-beta)s play important roles in cellular biosynthesis of extracellular matrix. Activator protein 1 (AP-1) and Smad are significant factors that mediate TGF-beta. OBJECTIVE: We have investigated increasing effects of obovatol, a biphenolic compound isolated from leaves of Magnolia obovata on the collagen synthesis through stimulation of the TGF-beta signaling and inhibition of matrix metalloproteinase, thereby protect against from UV damages via maintain of collagen in the UVB irradiated human fibroblast cells. METHODS: The fibroblasts were pretreated with obovatol for 24h and then the cells were irradiated with UVB. UVB-exposed cells were further cultured for 24h. Type I procollagen, MMP-3, TGF-beta and Smad as well as phosphorylation of MAPK family expression were determined by Western blot. The activation of AP-1 was investigated using EMSA. The released type I procollagen and TGF-beta into cell culture medium were determined by Western blot after concentration of these proteins. RESULTS: The results showed that obovatol stimulated type I procollagen, TGF-beta, and Smad expression and inhibited matrix metalloproteinase-3 (MMP-3) in dose-dependent manner (1-5muM) in UVB-irradiated human fibroblast cells. Obovatol also inhibited UVB-induced activation of AP-1 and MAP kinases. CONCLUSION: These results suggest that obovatol increases collagen synthesis through stimulation of the TGF-beta signaling and inhibition of matrix metalloproteinase in UVB-irradiated human fibroblast, thus obovatol could be effective against photo-damaged skin.

Melittin inhibits inflammatory target gene expression and mediator generation via interaction with IkappaB kinase.

We previously found that bee venom (BV) and melittin (a major component of BV) has anti-inflammatory effect by reacting with the sulfhydryl group of p50 of NF-kappaB. Since the sulfhydryl group is present in IkappaB kinase (IKKalpha and IKKbeta), anti-inflammatory effect of melittin via interaction with IKKs was investigated. We first examined binding of melittin to IKKs using surface plasmon resonance analyzer. Melittin binds to IKKalpha (K(d) = 1.34 x 10(-9) M) and IKKbeta (K(d) = 1.01 x 10(-9) M). Consistent with the high binding affinity, melittin (5 and 10 microg/ml) and BV (0.5, 1 and 5 microg/ml) suppressed sodium nitroprusside, TNF-alpha and LPS induced-IKKbeta and IKKbeta activities, IkappaB release, and NF-kappaB activity as well as the expressions of iNOS and COX-2, and the generation of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in Raw 264.7 mouse macrophages and synoviocytes obtained from rheumatoid arthritis patients. The binding affinities of melittin to mutant IKKs, was reduced, and the inhibitory effect of melittin on IKK and NF-kappaB activities, and NO and PGE(2) generation were abrogated by the reducing agents or in Raw 264.7 transfected with mutant plasmid IKKalpha (C178A) or IKKbeta (C179A). These results suggest that melittin binding to the sulfhydryl group of IKKs resulted in reduced IKK activities, IkappaB release, NF-kappaB activity and generation of inflammatory mediators, indicating that IKKs may be also anti-inflammatory targets of BV.

Inhibitory effect of obovatol on nitric oxide production and activation of NF-kappaB/MAP kinases in lipopolysaccharide-treated RAW 264.7cells.

The components of Magnolia obovata are known to have many pharmacological activities. In this study, we investigated the effects of obovatol, a neolignan compound isolated from the leaves of M. obovata, on nitric oxide (NO) production and NF-kappaB activity in lipopolysaccharide (LPS)-activated RAW 264.7 cells. The results show that obovatol (1-5 microM) significantly inhibited LPS-induced NO production in a concentration-dependent manner (IC(50): 0.91 microM). Consistent with the inhibitory effect on NO production, obovatol inhibits the expression of inducible nitric oxide synthase and cyclooxygenase-2 expression. Furthermore, obovatol suppressed NF-kappaB (p50 and p65) translocation to the nucleus as well as IkappaB release resulting in the inhibition of the DNA binding activity of the NF-kappaB. Obovatol also inhibited c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signal, which are the most significantly involved signal in NO production and NF-kappaB activation. When the cells were treated with the combination of obovatol with U0126 (an ERK inhibitor) or SP600125 (a JNK inhibitor) as well as with SC-514 (an IKK2 inhibitor), much more inhibition of NO production was observed than that by obovatol alone. The present results suggest that obovatol has an inhibitory effect on NO production through the inhibition of NF-kappaB/MAPK activity, and thus can be used as an anti-inflammatory agent.

Induction of apoptotic cell death by 2'-hydroxycinnamaldehyde is involved with ERK-dependent inactivation of NF-kappaB in TNF-alpha-treated SW620 colon cancer cells.

2'-Hydroxycinnamaldehyde (HCA) inhibits cell growth of several human cancer cells with unknown mechanisms. We investigated the inhibitory effect of HCA on TNF-alpha-induced cell growth and possible signal pathway in SW620 colon cancer cells. HCA inhibited TNF-alpha-induced SW620 colon cell growth in time- and dose-dependent manner through induction of apoptotic cell death. Parallel with inhibitory effect on cell growth, HCA dose dependency inhibited TNF-alpha-induced activation of NF-kappaB accompanied with inhibition of the translocation of p50. HCA also induced expression of caspase-3 and Bax, but decreased Bcl-2. HCA furthermore activated ERK pathway, and ERK inhibitor reversed inhibitory effect of HCA on cell growth and transcriptional activation of NF-kappaB. These results demonstrate that HCA inhibits cell growth through induction of apoptotic cell death by ERK pathway-dependent NF-kappaB inactivation.