DDX3 Upregulates Hydrogen Peroxide-Induced Melanogenesis in Sk-Mel-2 Human Melanoma Cells.

DDX3 is a DEAD-box RNA helicase with diverse biological functions through multicellular pathways. The objective of this study was to investigate the role of DDX3 in regulating melanogenesis by the exploring signaling pathways involved. Various concentrations of hydrogen peroxide were used to induce melanogenesis in SK-Mel-2 human melanoma cells. Melanin content assays, tyrosinase activity analysis, and Western blot analysis were performed to determine how DDX3 was involved in melanogenesis. Transient transfection was performed to overexpress or silence DDX3 genes. Immunoprecipitation was performed using an antityrosinase antibody. Based on the results of the cell viability test, melanin content, and activity of tyrosinase, a key melanogenesis enzyme, in SK-Mel-2 human melanoma cells, hydrogen peroxide at 0.1 mM was chosen to induce melanogenesis. Treatment with H2O2 notably increased the promoter activity of DDX3. After treatment with hydroperoxide for 4 h, melanin content and tyrosinase activity peaked in DDX3-transfected cells. Overexpression of DDX3 increased melanin content and tyrosinase expression under oxidative stress induced by H2O2. DDX3 co-immunoprecipitated with tyrosinase, a melanogenesis enzyme. The interaction between DDX3 and tyrosinase was strongly increased under oxidative stress. DDX3 could increase melanogenesis under the H2O2-treated condition. Thus, targeting DDX3 could be a novel strategy to develop molecular therapy for skin diseases.

Regulation of p21 expression for anti-apoptotic activity of DDX3 against sanguinarine-induced cell death on intrinsic pathway.

BACKGROUND: DDX3 plays a role in multicellular pathways, especially exerting an anti-apoptotic effect on extrinsic apoptosis. However, studies on the role of DDX3 in intrinsic apoptosis are lacking. PURPOSE: In this study, we aimed to study the bio-function of DDX3 anti-apoptotic activity in the intrinsic pathway using HeLa cells treated with sanguinarine. STUDY DESIGN: Screening of apoptosis-inducing agents found that sanguinarine was the most effective. After treatment with sanguinarine, cell viability, caspase-3 activity, and intrinsic gene expression were analyzed. FACS assays were used to analyze the effect of overexpression and knockdown of DDX3 to determine its role on intrinsic apoptosis. The relationship between DDX3 and the inhibition of p21 and apoptosis was investigated. RESULTS: Sanguinarine was determined to be the most effective intrinsic apoptosis-inducing agent in HeLa cervical cancer cells. DDX3 upregulated anti-apoptotic gene expression (Bcl-xL, cyclin D1, cyclin E, and cyclin B1) and downregulated pro-apoptotic gene expression (caspase-3, Bax) after sanguinarine treatment. The apoptotic cell death rate increased from 8.74% (sanguinarine-treated control) to 17.6% after the knockdown of DDX3 but decreased to 5.29% after DDX3 overexpression. The results implied that p21 might be involved in the toxicity of sanguinarine to HeLa cells. Overexpression and knockdown of DDX3 under sanguinarine-treated conditions showed that DDX3 inhibited p21 expression in sanguinarine-treated HeLa cells. Notably, when we tested p21 expression among eight mutants located in the functional residues of DDX3 (S90A, S90E, T204A, T204E, GET, NEAD, LAT, and HRISR) under sanguinarine-treated conditions, only the S90E mutation in DDX3 had an effect on the inhibition of p21 expression and levels of pro-apoptotic genes (Bax and caspase-3) and anti-apoptotic genes (Bcl-xL, cyclin D1, cyclin E, and cyclin B1), as well as DDX3. CONCLUSION: Taken together, the results suggest that the S90E residue is important for the regulation of p21 expression responsible for the anti-apoptotic activity of DDX3 in HeLa cells treated with sanguinarine. A model of the antiapoptotic function of DDX3 on sanguinarine-treated HeLa cells was proposed to understand the molecular mechanism of the intrinsic apoptosis inhibition in cervical cancer cells.

Stimulating DDX3 expression by serotonin 5-HT receptor 7 through phosphorylation of p53 via the AC-PKA-ERK signaling pathway.

DDX3 is a host viral factor that can inhibit the hepatitis B virus-induced innate immune responses. In this study, the 20 bioactive compounds have screened the effects on DDX3 and we found that 5-HT upregulated DDX3 promoter activity via the 5-HT7 receptor on liver hepatocellular cells (HepG2 cells) by using a luciferase assay, reverse transcription-polymerase chain reaction analysis, and Western blot analysis. Furthermore, we are trying to elucidate the pathways involved in the stimulating effect of 5-HT on DDX3 expression to induce innate immune responses against hepatitis B virus infection. A knockdown of the 5-HT7 receptor by transfection si-5-HT7 receptors or si-control into HepG2 cells treated by 5-HT (or 5-HT plus agonist) confirmed the role of the 5-HT7 receptor in DDX3 expression. The IFN-ß-Luc expression and level of hepatitis B virus surface Antigen (HBsAg) showed that DDX3 mediated by the 5-HT7 agonist (AS-19) increased IFN-ß expression and inhibited HBV replication. Luciferase assays showed the involvement of 5-HT7 receptors in DDX3 expression via cAMP/AC/PKA pathways by using protein kinase A (PKA) and adenylyl cyclase inhibitor (MDL 12330A). AS-19 mediated DDX3 promoter activated PKA extracellular signal-regulated kinase ERK signaling the p53 phosphorylation (-1080/-1070) resulted in upregulation of DDX3 promoter transactivation via the 5-HT7 receptors agonist. Overall, 5-HT7 was found to be a new potential target to inhibit hepatitis B infection by activating AC/PKA/ERK pathways by phosphorylating p53 via the 5-HT7 agonist response by mediating DDX3 expression.

Stimulation of DDX3 expression by ginsenoside Rg3 through the Akt/p53 pathway activates the innate immune response via TBK1/IKKε/IRF3 signalling.

DEAD-box RNA helicase DDX3 is a well-known host factor that inhibits hepatitis B viral proliferation and boosts innate immune responses via TANK-binding kinase 1 (TBK1)/IKKε-mediated and/or interferon (IFN)-ß promoter stimulator-1 (IPS-1)-mediated IFN-ß induction. Previously, we demonstrated the anti-hepatitis B activity of Rg3 via stimulation of TRAF6/TAK1 degradation and inhibition of JNK/AP-1 signaling. To determine the effects of Rg3 on innate immunity, an IFN-ß promoter assay was performed. Rg3 ameliorated IFN-ß expression via upregulation of both the TBK1/IKKε pathway and DDX3 expression. In addition, Rg3 induced the phosphorylation of IRF3 and its translocation into nucleus, which is a key molecule to induction of IFN-ß expression. To evaluate the molecular mechanism of Rg3 on DDX3 expression, the DDX3 promoter (-1406/+105) was subjected to luciferase assay and ChIP analysis. p53 phosphorylation resulted in upregulation of DDX3 expression, which enhanced DDX3 promoter transactivation activity. Transient transfection with wild-type p53 increased DDX3 promoter activity in Hep3B cells which have null mutant of p53, whereas knockdown p53 by si-p53 reduced DDX3 promoter activity in HepG2.2.15 and HepG2 cells, respectively. Rg3- mediated phosphorylation of p53 resulted in inhibition of Akt phosphorylation, which in turn reduced MDM2-mediated p53 degradation. An Akt inhibitor augmented DDX3 promoter activity and reduced the secretion of hepatitis B surface antigen. Our data indicate that Rg3 enhances innate immunity by inducing IFN-ß expression through upregulation of DDX3 promoter activity via p53-mediated transactivation and activation of the TBK1/IKKε/IRF3 pathway.

Stimulation of TRAF6/TAK1 degradation and inhibition of JNK/AP-1 signalling by ginsenoside Rg3 attenuates hepatitis B virus replication.

In present study, we investigated the effect of ginsenoside Rg3 on hepatitis B virus DNA replication and secretion of hepatitis B surface antigen and e antigen in HepG2.2.15 cells. Rg3 dose- and time-dependently inhibited hepatitis B surface antigen, e antigen, and hepatitis B viral particle secretion. To explore the effect of Rg3 on anti-hepatitis B activity, we analysed toll-like receptor-myeloid differentiation primary response gene 88 signalling. Rg3 did not affect the expression of toll-like receptors or myeloid differentiation primary response gene 88. However, it significantly inhibited the expression of TNF receptor-associated factor 6 and transforming growth factor ß activated kinase-1, which are adaptor molecules that signal through a toll-like receptor-myeloid differentiation primary response gene 88-dependent pathway. The inhibitory effect of Rg3 on TNF receptor-associated factor 6/transforming growth factor ß activated kinase-1 expression was caused by the downregulation of TNF receptor-associated factor 6 expression as well as the stimulation of ubiquitination and proteasomal degradation of TNF receptor-associated factor 6, followed by downregulation of transforming growth factor ß activated kinase-1. Furthermore, Rg3 inhibited mitogen-activated protein kinase signalling by inhibiting c-Jun N-terminal kinase phosphorylation, reduced the expression of AP-1 transcription factors (especially c-Jun and JunB), and inhibited AP-1 promoter activity. The inhibitory effect of Rg3 on c-Jun N-terminal kinase/AP-1 signalling showed anti-inflammatory activity based on the reduction in the expression of pro-inflammatory cytokines, IL-8 and TNF-α, at both the transcriptional and translational levels. Therefore, Rg3 showed anti-hepatitis B activity via the degradation of TNF receptor-associated factor 6/transforming growth factor ß activated kinase-1 and the inhibition of c-Jun N-terminal kinase/AP-1 signalling.

Induction of ATP synthase ß by H2O2 induces melanogenesis by activating PAH and cAMP/CREB/MITF signaling in melanoma cells.

Hydrogen peroxide (H2O2) production due to oxidative stress is associated with apoptosis and melanogenesis in melanocytes. Here, we analyzed the effects of H2O2 on melanogenesis by measuring the melanin content and analyzing the expression of melanogenesis-related proteins, including cAMP-responsive element binding protein (CREB), microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), and phenylalanine hydroxylase (PAH). Treatment with 1mM H2O2 increased the cellular melanin content; the expression of PAH, TYR, and MITF; and the phosphorylation of CREB in B16F10 and SK-Mel-2 cells. In addition, H2O2 increased the expression of ATP synthase ß (ATP5B), a mitochondrial F1 complex, and increased intracellular ATP levels. Studies using the ATP5B inhibitor oligomycin (OM) showed that the induction of cAMP resulted from an increase in ATP caused by the induction of ATP5B. OM treatment increased H2O2-mediated apoptosis via accelerated ATP depletion and apoptosis-related gene expressions. In summary, H2O2 may induce melanogenesis via the upregulation of PAH and activation of cAMP/p-CREB/MITF signaling by increasing intracellular cAMP levels through the induction of ATP5B.

Caffeoylserotonin protects human keratinocyte HaCaT cells against H2 O2 -induced oxidative stress and apoptosis through upregulation of HO-1 expression via activation of the PI3K/Akt/Nrf2 pathway.

Caffeoylserotonin (CaS) has strong radical scavenging activity as well as antioxidant activities, protecting cells from lipid peroxidation, intracellular reactive oxygen species generation, DNA damage, and cell death. The molecular mechanism by which CaS protects against oxidative stress is not well understood. Here, we analyzed the cytoprotective activity of CaS in hydrogen peroxide (H2 O2 )-treated keratinocyte HaCaT cells. H2 O2 induced apoptosis in the cells through activation of pro-apoptotic p21, Bax, and caspase-3. Pretreatment with CaS inhibited apoptotic gene expression and activated the anti-apoptotic gene, Bcl-xL. Although CaS did not directly affect heme oxygenase-1 (HO-1) expression, pretreatment with CaS augmented HO-1 expression through an increase in NF-E2-related factor (Nrf2) stability and stimulation of Nrf2 translocation to the nucleus upon H2 O2 exposure. H2 O2 also induced the phosphorylation and subsequent activation of ERK, p38 MAPK, and Akt. Analysis using specific inhibitors of p38 MAPK and Akt demonstrated that only Akt activation was involved in HO-1 and Nrf2 expressions. In addition, PI3K and PKC inhibitors suppressed HO-1/Nrf2 expression and Akt phosphorylation. These results demonstrate that CaS protects against oxidative stress-induced keratinocyte cell death in part through the activation of Nrf2-mediated HO-1 induction via the PI3K/Akt and/or PKC pathways, but not MAPK signaling.

The effects of Caffeoylserotonin on inhibition of melanogenesis through the downregulation of MITF via the reduction of intracellular cAMP and acceleration of ERK activation in B16 murine melanoma cells.

In this study, we evaluated the anti-melanogenesis effects of Caffeoylserotonin (CaS) in B16 melanoma cells. Treatment with CaS reduced the melanin content and tyrosinase (TYR) activity in B16 melanoma cells in a dose-dependent manner. CaS inhibited the expression of melanogenesis-related proteins, including microphthalmia- associated transcription factor (MITF), TYR, and tyrosinase-related protein-1 (TRP-1), but not TRP-2. α-MSH is known to interact with melanocortin 1 receptor (MC1R) thus activating adenylyl cyclase and increasing intracellular cyclic AMP (cAMP) levels. Furthermore, cAMP activates extracellular signal-regulated kinase 2 (ERK2) via phosphorylation, which phosphorylates MITF, thereby targeting the transcription factor to proteasomes for degradation. The CaS reduced intracellular cAMP levels to unstimulated levels and activated ERK phosphorylation within 30 min. The ERK inhibitor PD98059 abrogated the suppressive effect of CaS on α-MSH-induced melanogenesis. Based on this study, the inhibitory effects of CaS on melanogenesis are derived from the downregulation of MITF signaling via the inhibition of intracellular cAMP levels, as well as acceleration of ERK activation.

Caffeoylserotonin suppresses THP-1 monocyte adhesion and migration via inhibition of the integrin ß1/FAK/Akt signalling pathway.

We investigated the effect of caffeoylserotonin (CaS) on THP-1 monocyte migration and adhesion to fibronectin in response to MCP-1. CaS decreased monocyte adhesion and migration induced by MCP-1, together with CCR2 expression and α5ß1 integrin, and activated ß1 integrin expression on the cell surface. CaS also inhibited FAK and Akt phosphorylation. We found that CaS had anti-inflammatory activity based on inhibition of adhesion and migration via inhibition of the integrin ß1/FAK/Akt signalling pathway. Thus, the inhibitory effects of CaS on monocyte function may support the future development of this compound as a potential treatment for inflammation-dependent diseases.

Ginsenoside Rh1 suppresses matrix metalloproteinase-1 expression through inhibition of activator protein-1 and mitogen-activated protein kinase signaling pathway in human hepatocellular carcinoma cells.

Invasion and metastasis are the major causes of treatment failure in patients with cancer. Here, we investigated the effects of ginsenoside Rh1 on tumor invasion and metastasis in human hepatocellular carcinoma HepG2 cells and its possible mechanism of action. Rh1 showed concentration- and time-dependent inhibition of HepG2 cell migration and invasion. Matrix metalloproteinase-1 (MMP-1) gene expression and its promoter activity were also concentration-dependently inhibited by Rh1 treatment. The inhibitory effect of Rh1 on MMP-1 expression was due to inactivation of the mitogen-activated protein kinases (MAPKs) ERK, JNK, and p38 MAPK. By transient transfection analysis with the MMP-1 promoter (-2846 to -29 nt) and AP-1 promoter, MMP-1 and AP-1 promoter activities were induced by phorbol myristate acetate (PMA) but were significantly inhibited by PD98059 (ERK1/2 inhibitor) or SP600125 (JNK inhibitor). The induction of MMP-1 and AP-1 promoters by PMA was attenuated by Rh1, and both promoter activities were synergistically inhibited by co-treatment with PD98059. To evaluate the effects of Rh1 on AP-1 dimers, expression analysis and electrophoretic mobility shift (EMSA) assay using radiolabeled AP-1-specific oligomers at proximal site (-73 nt) and distal site (-1600 nt) of the MMP-1 promoter were performed. The results showed that Rh1 inhibited the expression of c-Jun and c-Fos but did not affect the DNA binding ability of AP-1-specific oligomers. However, Rh1 attenuated the stability of c-Jun. Therefore, Rh1 has potential for development of novel chemotherapeutic agents for treatment of malignant cancers, including early hepatocellular carcinoma related to MMP-1 expression.

Anti-metastatic effects of ginsenoside Rd via inactivation of MAPK signaling and induction of focal adhesion formation.

Ginsenoside Rd is a protopanaxadiol-type ginsenoside found in ginseng and is the active ingredient in several Oriental herbal medicines. We investigated the effects of ginsenoside Rd on tumor invasion and metastasis in the human hepatocellular carcinoma HepG2 and its possible mechanism of action. HepG2 cells were treated with ginsenoside Rd at different concentrations. Scratch wound and Boyden chamber assays were used to determine the effects of ginsenoside Rd on the migration and invasiveness of HepG2 cells, respectively. The molecular mechanisms by which ginsenoside Rd inhibited the invasion and migration of HepG2 cells were investigated by RT-PCR, Western blotting, gelatin zymography, promoter assay, and treatment with inhibitors of MAPK signaling. Immunofluorescence analysis was conducted to evaluate the effect of ginsenoside Rd on focal adhesion formation in HepG2 cells. Treatment with ginsenoside Rd dose- and time-dependently inhibited the migration and invasion of HepG2 cells. It achieved this by reducing the expression of MMP-1, MMP-2, and MMP-7, by blocking MAPK signaling by inhibiting the phosphorylation of ERK and p38 MAPK, by inhibition of AP-1 activation, and by inducing focal adhesion formation and modulating vinculin localization and expression. Treatment of HepG2 cells with ginsenoside Rd significantly inhibited metastasis, most likely by blocking MMP activation and MAPK signaling pathways involved in cancer cell migration. These findings may be useful for the development of novel chemotherapeutic agents for the treatment of malignant cancers.

The DEAD-box RNA helicase DDX3 interacts with DDX5, co-localizes with it in the cytoplasm during the G2/M phase of the cycle, and affects its shuttling during mRNP export.

DDX3 is involved in RNA transport, translational control, proliferation of RNA viruses, and cancer progression. From yeast two-hybrid screening using the C-terminal region of DDX3 as a bait, the DEAD-box RNA helicase DDX5 was cloned. In immunofluorescence analysis, DDX3 and DDX5 were mainly co-localized in the cytoplasm. Interestingly, cytoplasmic levels of DDX5 increased in the G(2) /M phase and consequently protein-protein interaction also increased in the cytoplasmic fraction. DDX3 was highly phosphorylated at its serine, threonine, and tyrosine residues in the steady state, but not phosphorylated at the serine residue(s) in the G(2) /M phase. DDX5 was less phosphorylated in the G(1) /S phase; however, it was highly phosphorylated at serine, threonine, and tyrosine residues in the G(2) /M phase. PP2A treatment of the cytoplasmic lysate from G(2) /M phase cells positively affected the interaction between DDX3 and DDX5, whereas, PTP1B treatment did not. In an analysis involving recombinant His-DDX3 and His-DDX5, PP2A pretreatment of His-DDX5 increased the interaction with endogenous DDX3, and vice versa. Furthermore, the results of GST pull-down experiments support the conclusion that dephosphorylation of serine and/or threonine residues in both proteins enhanced protein-protein interactions. UV cross-linking experiments showed that DDX3 and DDX5 are involved in mRNP export. Additionally, DDX3 knockdown blocked the shuttling of DDX5 to the nucleus. These data demonstrate a novel interaction between DDX3 and DDX5 through the phosphorylation of both proteins, especially in the G(2) /M phase, and suggest a novel combined mechanism of action, involving RNP remodeling and splicing, for DEAD-box RNA helicases involved in mRNP export.

Ginsenoside Rh1 inhibits the invasion and migration of THP-1 acute monocytic leukemia cells via inactivation of the MAPK signaling pathway.

Ginsenoside Rh1 has been reported to possess antiallergic and anti-inflammatory activities, but its effects on monocytes remain to be determined. Herein, we investigated the effects of Rh1 on the expression of MCP-1 and CCR2, activation of MAPK signaling, and chemotaxis of monocytes. Treatment of Rh1 decreased the levels of MCP-1 and CCR2 and the expression of VLA5 and activated ß1 integrin on the cell surface, and attenuated the phosphorylation of MAPKs. Based on these results, the inhibitory effects of Rh1 on monocyte function should be regarded as a promising new anti-inflammatory response with a potential therapeutic role against inflammation-dependent diseases.

Substrate specificity of the Bacillus licheniformis lyxose isomerase YdaE and its application in in vitro catalysis for bioproduction of lyxose and glucose by two-step isomerization.

Enzymatic processes are useful for industrially important sugar production, and in vitro two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded by ydaE of Bacillus licheniformis was found to have broad substrate specificities and has shown high catalytic efficiency on D-lyxose, suggesting that the enzyme is D-lyxose isomerase. Escherichia coli BL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn²+. The apparent K(m) values for D-lyxose and D-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (k(cat)/K(m)) for lyxose (3.2 ± 0.1 mM⁻¹ s⁻¹) was higher than that for D-mannose (1.6 mM⁻¹ s⁻¹). The purified protein was applied to the bioproduction of D-lyxose and D-glucose from d-xylose and D-mannose, respectively, along with the thermostable xylose isomerase of Thermus thermophilus HB08. From an initial concentration of 10 mM D-lyxose and D-mannose, 3.7 mM and 3.8 mM D-lyxose and D-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method for in vitro catalysis and can be applied to industrial production.

Antidiabetic effect of propolis: reduction of expression of glucose-6-phosphatase through inhibition of Y279 and Y216 autophosphorylation of GSK-3α/ß in HepG2 cells.

Propolis is a sticky, resinous material that honey bees collect from various plants, and mix with wax and other secretions. The aim of this study was to evaluate the antidiabetic effect of propolis through an analysis of the expression and enzyme activity of glucose-6-phosphatase (G6Pase) and to elucidate the mechanism by which propolis inhibits G6Pase gene expression. When HepG2 cells were incubated in high glucose media (25 mm), G6Pase expression was induced. Propolis significantly reduced the expression and enzyme activity of G6Pase; however, the hypoglycemic effect was not abolished by the phosphoinositide 3-kinase inhibitor, LY294002, and by the mitogen-activated protein kinase (MAPK) inhibitor, U0126. Propolis inhibited the activity of GSK3α and ß via the inhibition of serine and tyrosine phosphorylation, specifically, Y279 for GSK3α and Y216 for GSK3ß. The phosphorylations of Y279 and Y216 occur through autophosphorylation by GSK3α/ß and are involved in their own activity. Although propolis showed antioxidant activity, antidiabetic effect of propolis was not influenced by hydrogen peroxide and N-acetylcysteine. These results suggest that propolis inhibits the expression of G6Pase by inhibiting the autophosphorylation of Y279 and Y216 of GSK3α and ß, respectively, which are involved in the activation of GSK3. These findings suggest that propolis may be a potential antidiabetic agent for the treatment of insulin-insensitive diabetes.

Cytoprotective activities of hydroxycinnamic acid amides of serotonin against oxidative stress-induced damage in HepG2 and HaCaT cells.

Hydroxycinnamic acid amides of serotonin (HCAAS) [caffeoylserotonin (compound 1), p-coumaroylserotonin (compound 2), and feruloylserotonin (compound 3)] are secondary metabolites produced in plants. The aim of this study was to investigate the cytoprotective effects of HCAAS based on intracellular reactive oxygen radical (ROS) generation, lipid peroxidation, protein carbonylation, and phosphorylation of histone H2AX in H(2)O(2) treated-HepG2 and HaCaT cells. We have shown that HCAAS showed various strong antioxidant activities in hydrogen peroxide treated both cell lines, suggesting that these compounds may play as chemotherapeutic agents for preventing or reducing the oxidative stress-induced diseases.

Production of plant-specific tyramine derivatives by dual expression of tyramine N-hydroxycinnamoyltransferase and 4-coumarate:coenzyme A ligase in Escherichia coli.

Plant-specific bioactive compounds including feruloyltyramine (FT), 4-coumaroyltyramine (CT), and caffeoyltyramine (CaT) were simultaneously produced in Escherichia coli by heterologous expression of two biosynthetic genes encoding 4-coumarate:coenzyme A ligase and tyramine N-hydroxycinnamoyltransferase (THT) cloned from Arabidopsis thaliana and pepper, respectively. Simultaneous supplementation of substrates to the recombinant E. coli resulted in the secretion of multiple tyramine derivatives into the medium at high yield: CT (189 mg l(-1)), FT (135 mg l(-1)), CaT (40 mg l(-1)). In addition, the recombinant E. coli also produced, albeit at low concentration, a range of dopamine derivatives such as feruloyldopamine due to THT's ability to accept dopamine as a substrate.

Anti-inflammatory activity of a water extract of Acorus calamus L. leaves on keratinocyte HaCaT cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Acorus calamus L., sweet flag, is a well-known medicinal plant that grows worldwide wildly along swamps, rivers, and lakes. AIM OF THE STUDY: The aim of this study was to evaluate the anti-inflammatory activity of Acorus calamus leaf (ACL) extract and to explore its mechanism of action on human keratinocyte HaCaT cells. MATERIALS AND METHODS: HaCaT cells treated with polyinosinic:polycytidylic acid (polyI:C) and peptidoglycan (PGN) induced the inflammatory reactions. The anti-inflammatory activities of ACL were investigated using RT-PCR, ELISA assay, immunoblotting, and immunofluorescence staining. RESULTS: HaCaT cells induced the pro-inflammatory cytokines, interleukin-8 (IL-8) and/or interleukin-6 (IL-6) expressions after treatment with polyI:C or PGN. ACL inhibited the expression of IL-8 and IL-6 RNA and protein levels, and attenuated the activation of NF-kappaB and IRF3 after polyI:C treatment. ACL also inhibited expression of IL-8 and activation of NF-kappaB following PGN induction. CONCLUSIONS: These results suggest that ACL inhibits the production of pro-inflammatory cytokines through multiple mechanisms and may be a novel and effective anti-inflammatory agent for the treatment of skin diseases.

HPLC analysis of serotonin, tryptamine, tyramine, and the hydroxycinnamic acid amides of serotonin and tyramine in food vegetables.

Biogenic monoamines such as serotonin, tryptamine, and tyramine function as neurotransmitters and mitogenic factors in animals and are involved in flowering, morphogenesis, and protection from and adaptation to environmental changes in plants. In plants, serotonin and tyramine are conjugated to form phenolic compounds via thioester linkages during the synthesis of hydroxycinnamic acid amides, including p-coumaroylserotonin (CS), feruloylserotonin (FS), p-coumaroyltyramine (CT), and feruloyltyramine (FT). In this study, we determined the amounts of the biogenic monoamines CS, FS, CT, and FT in commonly consumed vegetables using high-performance liquid chromatography. Serotonin, tryptamine, and tyramine were detected in all vegetables tested. The serotonin levels ranged from 1.8 to 294 microg/g of dry weight, the tryptamine levels ranged from 0.8 to 372 microg/g of dry weight, and the tyramine levels ranged from 1.4 to 286 microg/g of dry weight. The highest serotonin and tryptamine contents were found in tomato and cherry tomato (140.3-222 microg/g of dry weight), while paprika and green pepper had higher tyramine contents than the other vegetables (286 and 141.5 microg/g of dry weight, respectively). Overall, the levels of CS, FS, CT, and FT ranged from 0.03 to 13.8 microg/g of dry weight, with green onion possessing the highest levels of CS (0.69 microg/g of dry weight), FT (1.99 microg/g of dry weight), and CT (13.85 microg/g of dry weight).

Gene-gene interaction between IL-13 and IL-13Ralpha1 is associated with total IgE in Korean children with atopic asthma.

Interleukin (IL)-13, which is essential for IgE synthesis, mediates its effects by binding with a receptor composed of IL-4Ralpha and IL-13Ralpha1. We investigated the effects of IL-13 and IL-13Ralpha1 polymorphisms in Korean children with asthma, and whether these have been associated with IgE production. We enrolled 358 atopic asthmatic, 111 non-atopic asthmatic, and 146 non-atopic healthy children. IL-13 and IL-13Ralpha1 genotypes were identified using the PCR-RFLP method. There was an association between the asthma susceptibility and homozygosity for risk allele of IL-13 G+2044A. In children with atopic asthma, risk alleles in IL-13 (A-1512C and C-1112T) and IL-13Ralpha1 (A+1398G) showed increased total IgE (P=0.012, 0.015 and 0.017, respectively). Three-loci haplotype analysis for IL-13 showed that the haplotype composed of -1512C, -1112T and +2044A was associated with higher total IgE than other tested haplotypes in children with atopic asthma (P=0.003). The gene-gene interaction between risk alleles of each IL-13 promoter polymorphism and IL-13Ralpha1 polymorphism was associated with higher total IgE in children with atopic asthma (P=0.002, 0.010). These findings indicate that the IL-13 G+2044A is associated with asthma development and the IL-13 and IL-13Ralpha1 polymorphisms may interact to enhance IgE production.