Molecular cloning and expression of a laccase from Ganoderma lucidum, and its antioxidative properties

Laccases are multicopper-containing oxidases that catalyze the oxidation of many aromatic compounds with concomitant reduction of oxygen to water. Interest in this enzyme has arisen in many fields of industry, including detoxification, wine stabilization, paper processing, and enzymatic conversion of chemical intermediates. In this study, we cloned a laccase gene (GLlac1) from the white-rot fungus Ganoderma lucidum. The cloned gene consists of 4,357 bp, with its coding region interrupted by nine introns, and the upstream region has putative CAAT and TATA boxes as well as several metal responsive elements (MREs). We also cloned a full-length cDNA of GLlac1, which contains an uninterrupted open reading frame (ORF) of 1,560 bp coding for 520 amino acids with a putative 21-residue signal sequence. The DNA and deduced amino acid sequences of GLlac1 were similar but not identical to those of other fungal laccases. GLlac1 was released from the cells when expressed in P. pastoris, and had high laccase activity. In addition, GLlac1 conferred antioxidative protection from protein degradation, and thus may be useful in bio-medical applications.     

House dust mite, Dermatophagoides pteronissinus increases expression of MCP-1, IL-6, and IL-8 in human monocytic THP-1 cells

The house dust mite (Dermatophagoides pteronissinus) plays an important role in the pathogenesis of allergic diseases, including atopic dermatitis, and asthma. Monocyte chemotactic protein 1 (MCP-1/CCL2)/IL-6/IL-8 (CXCL8) plays a pivotal role in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis. The aim of this study was to investigate the effect of D. pteronissinus extract (DpE) on expression of MCP-1/IL-6/IL-8 mRNA and protein and the signal transduction in the human monocytic cell line, THP-1. The mRNA and protein expression of MCP-1/CCL2, IL-6, and IL-8 were elevated by DpE in a time and dose-dependent manner in THP-1 cells. The increased expression of MCP-1, IL-6, and IL-8 was not affected by aprotinin (serine protease inhibitor) or E64 (cysteine protease inhibitor). We found that MCP-1 and IL-6 expression due to DpE was related to Src, protein kinase C δ (PKC δ), extracellular-signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and IL-8 expression was involved in Src family tyrosine kinase, PKC δ, ERK. DpE increased the phosphorylation of ERK and p38 MAPK after 5 min and peaked at 30 min. The activation was significantly blocked by PP2, an inhibitor of Src family tyrosine kinase and rottlerin, an inhibitor of PKC δ (p < 0.01). DpE increases MCP-1, IL-6, and IL-8 expression and transduces its signal via Src family tyrosine kinase, PKC, and ERK in a protease-independent manner. This finding may contribute to the elucidation of the pathogenic mechanism triggered by DpE .     

Phospholipase D activity regulates integrin-mediated cell spreading and migration by inducing GTP-Rac translocation to the plasma membrane

Small GTPase Rac is a crucial regulator of actin cytoskeletal rearrangement, and it plays an important role in cell spreading, migration, mitogenesis, phagocytosis, superoxide generation, and axonal growth. It is generally accepted that Rac activity is regulated by the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle. But, it is suggested that in addition to Rac-GTP loading, membrane localization is required for the initiation of downstream effector signaling. However, the molecular mechanisms that control the targeting of GTP-Rac to the plasma membrane remain largely unknown. Here, we have uncovered a signaling pathway linking phospholipase D (PLD) to the localized functions of Rac1. We show that PLD product phosphatidic acid (PA) acts as a membrane anchor of Rac1. The C-terminal polybasic motif of Rac1 is responsible for direct interaction with PA, and Rac1 mutated in this region is incapable of translocating to the plasma membrane and of activating downstream target p21-activated kinase upon integrin activation. Finally, we show that PA induces dissociation of Rho-guanine nucleotide dissociation inhibitor from Rac1 and that PA-mediated Rac1 localization is important for integrin-mediated lamellipodia formation, cell spreading, and migration. These results provide a novel molecular mechanism for the GTP-Rac1 localization through the elevating PLD activity, and they suggest a general mechanism for diverse cellular functions that is required localized Rac activation.     

Activation of NF-κB by alloferon through down-regulation of antioxidant proteins and IκBα

Alloferon is a 13-amino acid peptide isolated from the bacteria-challenged larvae of the blow fly Calliphora vicina. The pharmaceutical value of the peptide has been well demonstrated by its capacity to stimulate NK cytotoxic activity and interferon (IFN) synthesis in animal and human models, as well as to enhance antiviral and antitumor activities in mice. Antiviral and the immunomodulatory effectiveness of alloferon have also been supported clinically proved in patients suffering with herpes simplex virus (HSV) and human papilloma virus (HPV) infections. To elucidate molecular response to alloferon treatment, we initially screened a model cell line in which alloferon enhanced IFN synthesis upon viral infection. Among the cell lines tested, Namalva was chosen for further proteomic analysis. Fluorescence difference gel electrophoresis (DIGE) revealed that the levels of a series of antioxidant proteins decreased after alloferon treatment, while at least three glycolytic enzymes and four heat-shock proteins were increased in their expression levels. Based on the result of our proteomic analysis, we speculated that alloferon may activate the NF-kappaB signaling pathway. IkappaB kinase (IKK) assay, Western blot analysis on IkappaBalpha and its phosphorylated form at Ser 32, and an NF-kappaB reporter assay verified our proteomics-driven hypothesis. Thus, our results suggest that alloferon potentiates immune cells by activating the NF-kappaB signaling pathway through regulation of redox potential. Since NF-kappaB activation is involved in IFN synthesis, our results provide further clues as to how the alloferon peptide may stimulate IFN synthesis.     

Inhibitory effects on mushroom tyrosinase by flavones from the stem barks of Morus lhou (S.) Koidz

Five flavones displaying tyrosinase inhibitory activity were isolated from the stem barks of Morus lhou (S.) Koidz., a cultivated edible plant. The isolated compounds were identified as mormin (1), cyclomorusin (2), morusin (3), kuwanon C (4), and norartocarpetin (5). Mormin (1) was characterized as a new flavone possesing a 3-hydroxymethyl-2-butenyl at C-3. The inhibitory potencies of these flavonoids toward monophenolase activity of mushroom tyrosinase were investigated. The IC50 values of compounds 1-5 for monophenolase activity were determined to be 0.088, 0.092, 0.250, 0.135 mM, and 1.2 microM, respectively. Mormin (1), cyclomorusin (2), kuwanon C (4) and norartocarpetin (5) exhibited competitive inhibition characteristics. Interestingly norartocarpetin (5) showed a time-dependent inhibition against oxidation of L-tyrosine: it also operated under the enzyme isomerization model (k5 = 0.8424 min(-1), k6 = 0.0576 min(-1), K(app)(i) = 1.354 microM).     

Control of the harmful alga Cochlodinium polykrikoides by the naked ciliate Strombidinopsis jeokjo in mesocosm enclosures

Red tides dominated by the harmful dinoflagellate Cochlodinium polykrikoides have caused annual losses of USD $5–60 million to the Korean aquaculture industry annually since 1995 and a loss of USD $3 million during a 1999 net-pen fish mortality event in Canada. In order to evaluate the potential to control C. polykrikoides red tides dominated by using mass-cultured heterotrophic protistan grazers, we monitored the abundance of Strombidinopsis jeokjo (a naked ciliate) and C. polykrikoides after mass-cultured S. jeokjo was introduced into mesocosms (ca. 60 l) deployed in situ and containing natural red tide waters dominated by C. polykrikoides. Water temperature, salinity, and pH, as well as the abundance of co-occurring other protists and metazooplankton were measured concurrently. To compare the growth and ingestion rates of S. jeokjo feeding on cultured versus natural populations of C. polykrikoides, we also monitored the abundance of cultured C. polykrikoides and S. jeokjo in bottles during laboratory grazing experiments. S. jeokjo introduced into the mesocosms grew well, effectively reducing natural populations of C. polykrikoides from approximately 1000 cells ml⁻¹ to below 10 cells ml⁻¹ within 2 days. The growth and ingestion rates of cultured S. jeokjo on natural populations of C. polykrikoides in the mesocosms for the first 30 h (0.72 day⁻¹ and 51 ng C grazer⁻¹ day⁻¹) were 84% and 44%, respectively, of those measured in the laboratory during bottle incubations with similar initial prey concentrations. The calculated grazing impact of S. jeokjo on natural populations of C. polykrikoides suggests that large-scale cultures of this ciliate could be used for controlling red tides by C. polykrikoides in small areas.     

Quantification of roxatidine in human plasma by liquid chromatography electrospray ionization tandem mass spectrometry: application to a bioequivalence study

A sensitive and specific method using a one-step liquid-liquid extraction (LLE) with ethyl acetate followed by high-performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed and validated for the determination of roxatidine in human plasma using famotidine as an internal standard (IS). Data acquisition was carried out in multiple reaction monitoring (MRM) mode, by monitoring the transitions m/z 307.3-->107.1 for roxatidine and m/z 338.4-->189.1 for famotidine. Chromatographic separation was performed on a reverse phase Hydrosphere C(18) column at 0.2 mL min(-1) using a mixture of methanol-ammonium formate buffer as mobile phase (20:80, v/v; adjusted to pH 3.9 with formic acid). The achieved lower limit of quantification (LLOQ) was 1.0 ng mL(-1) and the standard calibration curve for roxatidine was linear (r(2)=0.998) over the studied range (1-1000 ng mL(-1)) with acceptable accuracy and precision. Roxatidine was found to be stable in human plasma samples under short-, long-term storage and processing conditions. The developed method was validated and successfully applied to the bioequivalence study of roxatidine administrated as a single oral dose (75 mg as roxatidine acetate hydrochloride) to healthy female Korean volunteers.     

Comparative study of Helicobacter pylori eradication rates of twice-versus four-times-daily amoxicillin administered with proton pump inhibitor and clarithromycin: a randomized study

Background: Proton pump inhibitor (PPI)-containing triple therapy with clarithromycin and amoxicillin is now a standard regimen for Helicobacter pylori eradication in Korea. Amoxicillin has time-dependent bactericidal activity against H. pylori ; we therefore assumed a dosing schedule of amoxicillin would affect the eradication rate of H. pylori . The purpose of this study was to evaluate and compare the efficacy of different amoxicillin dosing schedules for the eradication of H. pylori .
Materials and Methods: One hundred and eighty-six patients with H. pylori infection were eligible for this study. Patients were randomly assigned to one of two regimens: amoxicillin 1000 mg with clarithromycin 500 mg and omeprazole 20 mg twice daily for 2 weeks (BID group, n = 93), or amoxicillin 500 mg four times daily with clarithromycin 500 mg and omeprazole 20 mg twice daily for 2 weeks (QID group, n = 93). The success of H. pylori eradication was evaluated 4–5 weeks after completing treatment.
Results: Overall eradication rate was 90.3%, and eradication rates were 91.4% in the BID group and 89.2% in the QID group ( p  = 0.62). Compliances was 95.7% in the BID group and 93.5% in the QID group ( p  = 0.516); this was the only factor that significantly affected H. pylori eradication in this study. Side effects in both groups were generally mild.
Conclusions: Amoxicillin regimens with PPI and clarithromycin are found to be equally effective and safe in both the BID and QID groups for H. pylori eradication. Therefore, considering patient's comfort, we recommend a twice daily amoxicillin regimen.     

Imagine Jane and identify John: face identity aftereffects induced by imagined faces

It is not known whether prolonged exposure to perceived and imagined complex visual images produces similar shifts in subsequent perception through selective adaptation. This question is important because a positive finding would suggest that perception and imagery of visual stimuli are mediated by shared neural networks. In this study, we used a selective adaptation procedure designed to induce high-level face-identity aftereffects--a phenomenon in which extended exposure to a particular face facilitates recognition of subsequent faces with opposite features while impairing recognition of all other faces. We report here that adaptation to either real or imagined faces produces a similar shift in perception and that identity boundaries represented in real and imagined faces are equivalent. Together, our results show that identity information contained in imagined and real faces produce similar behavioral outcomes. Our findings of high-level visual aftereffects induced by imagined stimuli can be taken as evidence for the involvement of shared neural networks that mediate perception and imagery of complex visual stimuli.