Anti-inflammatory effects and mechanisms of Hizikia fusiformis via multicellular signaling pathways in lipopolysaccharide-induced RAW 264.7 cells.

In this study, we investigated the anti-inflammatory effects and mechanisms of Hizikia fusiformis (HF) extracts in lipopolysaccharide (LPS)-induced RAW 264.7 cells. We extracted HF using solvent and sub-critical water techniques. In results, HF extracts inhibited nitric oxide (NO) production in cell-free and LPS-stimulated RAW 264.7 cells. HF210 (extract prepared with sub critical water at 210oC) was most effective. The HF210 extract dose-dependently inhibited inducible nitric oxide synthase expression (iNOS) and nuclear factor kappa (NF-B) p65 translocation from cytosol to the nucleus. Furthermore, HF210 extract dose-dependently inhibited the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), Jun N-terminal kinase (JNK), and signal transducers and activators of transcription (STAT)-1in LPS-induced RAW 264.7 cells. Thus, our results suggest that anti-inflammatory effects of HF210 extract showed a noticeable distinction by regulation of multiple signaling pathways in LPS-induced RAW 264.7 cells.

Hyperglycemia induces apoptosis via CB1 activation through the decrease of FAAH 1 in retinal pigment epithelial cells.

Fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the main endocannabinoid, anandamide, and related fatty acid amides, has emerged as a regulator of endocannabinoid signaling. Retinal pigment epithelial (RPE) cells are believed to be important cells in the pathogenesis of diabetic retinopathy. However, the pathophysiology of FAAH in diabetic retinopathy has not been determined. Thus, we examined the effect of high glucose (HG) on the expression of FAAH and CB(1)R in the ARPE-19 human RPE cells. We found that HG downregulated the expression of FAAH 1 mRNA and protein in ARPE-19 cells. In contrast, it upregulated the expression of CB(1)R mRNA and protein. HG-induced internalization of CB(1)R in HEK 293 cells and ARPE-19 cells was blocked by overexpression of FAAH 1 and treatment with the CB(1)R blocker, AM 251. HG-induced generation of reactive oxygen species and lipid peroxide formation were blocked by the overexpression of FAAH 1. FAAH 1 overexpression also blocked HG-induced expression of CB(1)R in the cytosolic fraction. We also investigated whether the overexpression of FAAH 1 protected against HG-induced apoptosis. High glucose increased the Bax/Bcl-2 ratio and levels of cleaved PARP, cleaved caspase-9 and caspase-3, and reduced cell viability. HG-induced apoptotic effects were reduced by the overexpression of FAAH 1, treatment with the CB(1)R-specific antagonist AM 251 and CB(1)R siRNA transfection. In conclusion, HG-induced apoptosis in ARPE-19 cells by inducing CB(1)R expression through the downregulation of FAAH 1 expression. Our results provide evidence that CB(1)R blockade through the recovery of FAAH 1 expression may be a potential anti-diabetic therapy for the treatment of diabetic retinopathy.

Expression of the human soluble epoxide hydrolase in Escherichia coli by auto-induction for the study of high-throughput inhibition assays.

Soluble epoxide hydrolase (sEH) is a key enzyme involved in the metabolism of epoxy fatty acid mediators such as epoxyeicosatrienoic acids with emerging roles in the regulations of hypertension and inflammation. Inhibitors of human sEH (hsEH) are effective drug candidates for the treatment of cardiovascular diseases. Preparation of hsEH for enzyme inhibition studies has been carried out by using baculovirus expression system. We herein explored the feasibility of expression of hsEH in Escherichia coli cells for the study of high-throughput screening assays of enzyme inhibitors, because the bacterial expression system is easier to handle and more cost-effective than the baculovirus expression system. The functional target enzyme was successfully produced in prokaryotic expression system by an auto-induction method and exhibited comparable enzyme activity to that yielded in baculovirus expression system. The bacterial-hsEH showed similar sensitivity to the baculovirus-hsEH against six reported inhibitors. Overalls indicate that bacterial expression of hsEH employed in the present study is useful for preparing enzymatically active hsEH, leading to effective performance of high-throughput screening assay of hsEH inhibitors and to rapid identification of novel drug candidates for the treatment of cardiovascular diseases.

Development of sensitive immunoassays for the detection of the glucuronide conjugate of 3-phenoxybenzyl alcohol, a putative human urinary biomarker for pyrethroid exposure.

Pyrethroids are widely used in agriculture as insecticides. This study describes a sensitive enzyme-linked immunosorbent assay for the detection of the glucuronide conjugate of 3-phenoxybenzyl alcohol, a putative pyrethroid metabolite that may be used as a biomarker of exposure to pyrethroids. Four antisera were elicited against two different immunizing haptens. Antisera were characterized in combination with several coating haptens. The lowest IC50 value (0.5 ng/mL) was obtained with antiserum 1891 and 3-phenoxybenzoic acid-BSA conjugate as the coating antigen. Antiserum 1891 was highly selective for the target compound with an overall cross-reactivity of <0.3% to structurally related compounds. The assay sensitivity was negligibly affected by pH 4-9. A 5-fold improvement in IC50 was observed using a 10-fold concentrated phosphate-fuffered saline as the assay buffer. Compared to assays conducted in normal phosphate-fuffered saline, the maximal absorbance was almost identical. A good correlation (r 2 = 0.99 and 0.97 for urine samples A and B, respectively) was observed between spiked levels and the levels detected by the immunoassay.

An immunoassay for a urinary metabolite as a biomarker of human exposure to the pyrethroid insecticide permethrin.

Permethrin is the most popular synthetic pyrethroid insecticide used in agriculture and public health. For the assessment of human exposure to permethrin, a competitive indirect enzyme-linked immunosorbent assay (ELISA) for the detection of the glycine conjugate of a major metabolite, cis-/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (DCCA), of permethrin was developed based on a polyclonal antibody. An assay based on an antibody with a high sensitivity was optimized and characterized. The IC50 value and the detection range for trans-DCCA-glycine, in the assay buffer were 1.2 and 0.2-7.0 microg/L, respectively. The antibody recognized trans-DCCA-glycine and the mixture of cis-/trans-DCCA-glycine with an isomer range from 30:70 to 50:50 nearly equally. Little or no cross-reactivity to permethrin and its other free metabolites or glycine conjugates was measured. The integration of the ELISA and solid-phase extraction which was used to reduce the matrix effect from human urine samples provided for analysis of total cis-/trans-DCCA-glycine at low parts per billion levels in the samples. The limit of quantitation of the target analyte was 1.0 microg/L in urine with a limit of detection of 0.1 microg/L in buffer. This assay might be a useful tool for monitoring human exposure to permethrin.

Attenuation of tobacco smoke-induced lung inflammation by treatment with a soluble epoxide hydrolase inhibitor.

Changes in the lungs due to smoking include inflammation, epithelial damage, and remodeling of the airways. Airway inflammation is likely to play a critical role in the genesis and progression of tobacco smoke-induced airway disease. Soluble epoxide hydrolase (sEH) is involved in the metabolism of endogenous chemical mediators that play an important role in inflammation. Epoxyeicosatrienoic acids (EETs) have demonstrated antiinflammatory properties, and hydrolysis of these epoxides by sEH is known to diminish this activity. To examine whether acute tobacco smoke-induced inflammation could be reduced by a sEH inhibitor, 12-(3-adamantane-1-yl-ureido)-dodecanoic acid n-butyl ester was given by daily s.c. injection to spontaneously hypertensive rats exposed to filtered air or tobacco smoke for a period of 3 days (6 h/day). Acute exposure to tobacco smoke significantly increased by 3.2-fold (P <0.05) the number of cells recovered by bronchoalveolar lavage. The sEH inhibitor significantly decreased total bronchoalveolar lavage cell number by 37% in tobacco smoke-exposed rats with significant reductions noted in neutrophils, alveolar macrophages, and lymphocytes. A combination of sEH inhibitor and EETs was more significant in its ability to further reduce tobacco smoke-induced inflammation compared with the sEH inhibitor alone. The sEH inhibitor led to a shift in some plasma epoxides and diols that are consistent with the hypothetical action of these compounds. We conclude that an sEH inhibitor, in the presence or absence of EETs, can attenuate, in part, inflammation associated with acute exposure to tobacco smoke.

Cloning of beta-primeverosidase from tea leaves, a key enzyme in tea aroma formation.

A beta-primeverosidase from tea (Camellia sinensis) plants is a unique disaccharide-specific glycosidase, which hydrolyzes aroma precursors of beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides) to liberate various aroma compounds, and the enzyme is deeply concerned with the floral aroma formation in oolong tea and black tea during the manufacturing process. The beta-primeverosidase was purified from fresh leaves of a cultivar for green tea (C. sinensis var sinensis cv Yabukita), and its partial amino acid sequences were determined. The beta-primeverosidase cDNA has been isolated from a cDNA library of cv Yabukita using degenerate oligonucleotide primers. The cDNA insert encodes a polypeptide consisting of an N-terminal signal peptide of 28 amino acid residues and a 479-amino acid mature protein. The beta-primeverosidase protein sequence was 50% to 60% identical to beta-glucosidases from various plants and was classified in a family 1 glycosyl hydrolase. The mature form of the beta-primeverosidase expressed in Escherichia coli was able to hydrolyze beta-primeverosides to liberate a primeverose unit and aglycons, but did not act on 2-phenylethyl beta-D-glucopyranoside. These results indicate that the beta-primeverosidase selectively recognizes the beta-primeverosides as substrates and specifically hydrolyzes the beta-glycosidic bond between the disaccharide and the aglycons. The stereochemistry for enzymatic hydrolysis of 2-phenylethyl beta-primeveroside by the beta-primeverosidase was followed by (1)H-nuclear magnetic resonance spectroscopy, revealing that the enzyme hydrolyzes the beta-primeveroside by a retaining mechanism. The roles of the beta-primeverosidase in the defense mechanism in tea plants and the floral aroma formation during tea manufacturing process are also discussed.

A direct continuous spectrophotometric assay for glycosidases with 3-nitro-2-pyridyl glycosides by tautomerization of 2-hydroxy-3-nitropyridine.

Two kinds of 3-nitro-2-pyridyl glycosides were synthesized and evaluated as substrates for continuous spectrophotometric assay for glycosidases. The liberated aglycon, 2-hydroxy-3-nitropyridine, immediately tautomerized to 3-nitro-2(1H)-pyridone, causing an absorption shift of ca. 60 nm even under acidic conditions (pH 3-6). Consequently, the enzymatic hydrolysis of these glycosides was monitored continuously in the acidic to neutral pH range (pH 4-7), the optimum pH for most glycosidases. The absorbance of liberated aglycon increased linearly at 390 nm until 10% consumption of the substrate to enable the initial rate to be determined at once without terminating the reaction. The kinetic parameters for the hydrolysis of 3-nitro-2-pyridyl glycosides were obtained from the slopes of the progress curves and were compared with those obtained from the conventional discontinuous assay using p- and o-nitrophenyl glycosides as substrates. The kinetic parameters indicated that 3-nitro-2-pyridyl glycosides were more activated and specific substrates, but with less affinity to the enzymes than the corresponding nitrophenyl glycosides. Moreover, the absorbance shift by tautomerization should promise further applications to continuous spectrophotometric assays for other enzymes acting under acidic conditions, such as acid proteases and acid phosphatases.