Derivatization of Small Biomolecules for Optimized Matrix-assisted Laser Desorption/ionization Mass Spectrometry

Journal of Mass Spectrometry : JMS. Sep, 2002  |  Pubmed ID: 12271439

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is a powerful tool for the measurement of low molecular mass compounds of biological interest. The limitations for this method are the volatility of many analytes, possible interference with matrix signals or bad ionization or desorption behavior of the compounds. We investigated the application of well-known and straightforward one-pot derivatization procedures to circumvent these problems. The derivatizations tested allow the measurement and the labeling of alcohols, aldehydes and ketones, carboxylic acids, alpha-ketocarboxylic acids and amines.

Activating Signal Cointegrator 2 Belongs to a Novel Steady-state Complex That Contains a Subset of Trithorax Group Proteins

Molecular and Cellular Biology. Jan, 2003  |  Pubmed ID: 12482968

Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. These include activating signal cointegrator 2 (ASC-2), a recently isolated transcriptional coactivator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors and numerous other transcription factors. In this report, we show that ASC-2 belongs to a steady-state complex of approximately 2 MDa (ASC-2 complex [ASCOM]) in HeLa nuclei. ASCOM contains retinoblastoma-binding protein RBQ-3, alpha/beta-tubulins, and trithorax group proteins ALR-1, ALR-2, HALR, and ASH2. In particular, ALR-1/2 and HALR contain a highly conserved 130- to 140-amino-acid motif termed the SET domain, which was recently implicated in histone H3 lysine-specific methylation activities. Indeed, recombinant ALR-1, HALR, and immunopurified ASCOM exhibit very weak but specific H3-lysine 4 methylation activities in vitro, and transactivation by retinoic acid receptor appears to involve ligand-dependent recruitment of ASCOM and subsequent transient H3-lysine 4 methylation of the promoter region in vivo. Thus, ASCOM may represent a distinct coactivator complex of nuclear receptors. Further characterization of ASCOM will lead to a better understanding of how nuclear receptors and other transcription factors mediate transcriptional activation.

Identification of Monohydroxy Progesterones Produced by CYP106A2 Using Comparative HPLC and Electrospray Ionisation Collision-induced Dissociation Mass Spectrometry

Biochemical and Biophysical Research Communications. Jun, 2004  |  Pubmed ID: 15178459

Two previously uncharacterised products, produced by recombinant CYP106A2 of Bacillus megaterium ATCC 13368 using progesterone as substrate, were identified. For this purpose a combination of comparative HPLC and electrospray ionisation collision induced dissociation mass spectrometry (ESI CID MS) was established and applied for rapid identification of the steroids, which were identified as 11alpha-hydroxyprogesterone and 9alpha-hydroxyprogesterone. The pharmaceutical relevance of these steroids is discussed. Furthermore, the hydroxylation activity was quantified for all monohydroxylation products (15beta-hydroxyprogesterone, 6beta-hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone). The V(max) values for 15beta-hydroxyprogesterone, 6beta-hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone were determined as 337.3+/-43.7, 22.3+/-0.9, 17.5+/-0.9, and 6.5+/-0.3nmol product/min/nmol CYP106A2, respectively.

Quantitative Analysis of 5 Alpha-reductase Inhibitors in DU145 Cells Using Matrix-assisted Laser Desorption/ Ionization Time-of-flight Mass Spectrometry and High-performance Liquid Chromatography/tandem Mass Spectrometry

Journal of Mass Spectrometry : JMS. Jul, 2004  |  Pubmed ID: 15282755

N-(Dicyclohexyl)acetylpiperidine-4-benzylidene-4-carboxylic acid (1) is an excellent in vitro inhibitor of 5 alpha-reductase (5 alpha R). Compound 1 showed, however, much lower inhibition activity of 5 alpha R in vivo than in vitro, which might be caused by poor membrane permeability. The methyl ester of 1 (1a) was therefore tested as a model prodrug to see if it has better permeability properties than the corresponding acid 1. It was also monitored that this methyl ester was cleaved into the active compound 1 within the DU145 cells. Quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) methods were established with reliable linearity factors (0.996 for MALDI-TOFMS and 0.998 for HPLC/MS/MS) and reproducibility (relative standard deviation = 6.5% for MALDI-TOFMS and 2.8% for HPLC/MS/MS). The samples for MS analysis were effectively prepared from the cell homogenates using solid-phase extraction, with a high recovery of 90% on average. The intracellular amount of 1a (1.7 nmol) was much higher than that of 1 (0.032 nmol) in DU145 cells after 6 h of incubation. After incubation with the ester (1a), the cleaved acid (1) was detected within the cells. The concentration of acid 1 (0.045 nmol) in this experiment was higher than the acid content (0.032 nmol) after direct incubation with 1. Surprisingly, high amounts of the cleaved compound 1 were found outside the cells after 6 h of incubation with 1a.

Influence of the Physical Form of Processed Rice Products on the Enzymatic Hydrolysis of Rice Starch in Vitro and on the Postprandial Glucose and Insulin Responses in Patients with Type 2 Diabetes Mellitus

Bioscience, Biotechnology, and Biochemistry. Sep, 2004  |  Pubmed ID: 15388956

The manufacturing processes used determined the physicochemical properties of the three kinds of rice food, garaeduk, bagsulgi, and cooked rice. The initial rate of hydrolysis by porcine pancreatic alpha-amylase (PPA) was affected by the food form. The firmer structure of garaeduk was apparently responsible for the difficulty in maceration, resulting in less digestion than with easily digestible food for the same maceration time. The initial rate of hydrolysis of each rice product by PPA increased with increasing maceration time in a Waring Blender for all of the processed rice products. The postprandial glucose and insulin responses to the three processed rice products were also studied in ten patients with type 2 diabetes mellitus (4 men and 6 women aged 56.8 +/- 2.3 yr; duration of diabetes, 3.6 +/- 1.2 yr; body mass index (BMI), 23.7 +/- 2.6 kg/m2; fasting serum glucose, 143.9 +/- 5.1 mg/dl; serum insulin, 20.8 +/- 2.2 microU/ml). Each subject ingested of the three rice foods after a 12-h overnight fast, and the serum glucose and insulin levels were measured over a 0-240 min period. The postprandial serum glucose and insulin levels at 90 min after ingesting bagsulgi and cooked rice were less than those at 60 min, while the levels at 90 min after ingesting garaeduk were higher than those at 60 min. Garaeduk also significantly decreased the incremental responses of glucose and insulin when compared with bagsulgi and cooked rice. The results suggest that garaeduk would be the most unlikely to increase the postprandial serum glucose and insulin levels among the three rice foods. The food form, which eventually differentiated each food by its specific surface area with the same degree of maceration because of the characteristic physical strength, therefore affected the rate of rice starch hydrolysis both in vitro and in vivo.

Use of High-performance Liquid Chromatography/electrospray Ionization Collision-induced Dissociation Mass Spectrometry for Structural Identification of Monohydroxylated Progesterones

Rapid Communications in Mass Spectrometry : RCM. 2004  |  Pubmed ID: 15508138

For the structural identification of monohydroxylated progesterones synthesized by microorganisms, a method was developed using a combination of high-performance liquid chromatography and electrospray ionization collision-induced dissociation mass spectrometry (HPLC/ESI-CIDMS). The retention times and MS/MS spectra of 11 different standards at 30 eV were collected and compared. The identification of D-ring-hydroxylated progesterones (15beta-, 16alpha-, 17alpha- and 21-OH-P) using ESI-CIDMS was not possible. However, they were separated chromatographically using a 65:35 mixture of water and acetonitrile containing 0.5% acetic acid. The other hydroxylated progesterones (2alpha-, 6beta-, 7beta-, 9alpha-, 11alpha-, 11beta-, and 19-OH-P) could be identified by comparison of eight fragments. The complete separation of 11 standards was achieved chromatographically. The developed assay was evaluated by the identification of monohydroxylated progesterones produced by CYP106A2 from Bacillus megaterium ATCC 13368.

Evaluation of Cell Permeation of a Potent 5alpha-reductase Inhibitor Using MALDI-TOF MS

Journal of Enzyme Inhibition and Medicinal Chemistry. Oct, 2004  |  Pubmed ID: 15648657

N-(Dicyclohexyl)acetyl-piperidine-4-benzylidene-4-carboxylic acid (1), although a very potent in vitro 5alpha-steroid reductase (5alphaR) type 2 inhibitor, showed only marginal in vivo activity in rats. Since this could be due to hindered cellular uptake of the carboxylic acid, acid (1) and its corresponding methyl ester (1a) were compared with respect to their permeation properties. In the parallel artificial membrane permeation assay (PAMPA), 1a showed a higher %flux of 55 versus 6 for 1. Considering the high potency of 1 and better permeation of 1a, the use of 1a as a prodrug for 1 was explored using the human prostate carcinoma cell line DU145. Esterase activity, a prerequisite for this prodrug concept was detected employing 4-nitrophenyl acetate (4-NPA) as a substrate. After incubation of DU145 cells with 1 and 1a, respectively, permeated 1a and its hydrolysis to 1 were unequivocally observed by MALDI-TOF MS analyses, whereas 1 could not be detected inside the cells above the detection limit. Regarding biological activity, 1a showed a stronger inhibition of 5alphaR in intact DU145 cells than 1 (IC50 values, 4 microM and > 10 microM for 1a and 1, respectively). These results suggest that the in vivo activity of 1 might be increased by the use of its methyl ester prodrug 1a.

Identification of Genes Affecting Lycopene Accumulation in Escherichia Coli Using a Shot-gun Method

Biotechnology and Bioengineering. Sep, 2005  |  Pubmed ID: 15898075

Genes enhancing lycopene production in Escherichia coli were identified through colorimetric screening of shot-gun library clones constructed with E. coli chromosomal DNA. These E. coli cells had been engineered to produce lycopene, a red-colored carotenoid, which enabled screening for genes that enhance lycopene production. Six clones with enhanced lycopene production were isolated. Among 13 genes in these clones, dxs, appY, crl, and rpoS were found to be involved in enhanced lycopene production. While dxs and rpoS have been already reported to enhance lycopene production, appY and crl have not. DXP (1-deoxy-D-xylulose-5-phosphate) synthase is encoded by dxs and participates in the rate-limiting step in the synthesis of isopentenyl pyrophosphate (IPP), a building block of lycopene. Sigma S factor, encoded by rpoS, regulates transcription of genes induced at the stationary phase. The appY and crl genes encode transcriptional regulators related to anaerobic energy metabolism and the formation of curli surface fibers, respectively. E. coli harboring appY plasmids produced 2.8 mg lycopene/g dry cell weight (DCW), the same amount obtained with dxs despite the fact that appY is not directly involved in the lycopene synthesis pathway. The co-expression of appY, crl, and rpoS with dxs synergistically enhanced lycopene production. The co-expression of appY with dxs produced eight times the amount of lycopene (4.7 mg/g DCW) that was produced without expression of both genes (0.6 mg/g DCW).

Efficient Prefractionation of Low-abundance Proteins in Human Plasma and Construction of a Two-dimensional Map

Proteomics. Aug, 2005  |  Pubmed ID: 16047310

Human plasma is the most clinically valuable specimen, containing not only a dynamic concentration range of protein components, but also several groups of high-abundance proteins that seriously interfere with the detection of low-abundance potential biomarker proteins. To establish a high-throughput method for efficient depletion of high-abundance proteins and subsequent fractionation, prior to molecular analysis of proteins, we explored how coupled immunoaffinity columns, commercially available as multiple affinity removal columns (MARC) and free flow electrophoresis (FFE), could apply to the HUPO plasma proteome project. Here we report identification of proteins and construction of a human plasma 2-DE map devoid of six major abundance proteins (albumin, transferrin, IgG, IgA, haptoglobin, and antitrypsin) using MARC. The proteins were identified by PMF, matching with various internal 2-DE maps, resulting in a total of 144 nonredundant proteins that were identified from 398 spots. Tissue plasminogen activator, usually present at 10-60 ng/mL plasma, was also identified, indicative of a potentially low-abundance biomarker. Comparison of representative 2-D gel images of three ethnic groups (Caucasian, Asian-American, African-American) plasma exhibited minor differences in certain proteins between races and sample pretreatment. To establish a throughput fractionation of plasma samples by FFE, either MARC flow-through fractions or untreated samples of Korean serum were subjected to FFE. After separation of samples on FFE, an aliquot of each fraction was analyzed by 1-D gel, in which MARC separation was a prerequisite for FFE work. Thus, a working scheme of MARC --> FFE --> 1-D PAGE --> 2-D-nanoLC-MS/MS may be considered as a widely applicable standard platform technology for fractionation of complex samples like plasma.

In Vivo and in Vitro Studies of Mgs1 Suggest a Link Between Genome Instability and Okazaki Fragment Processing

Nucleic Acids Research. 2005  |  Pubmed ID: 16251400

The non-essential MGS1 gene of Saccharomyces cerevisiae is highly conserved in eukaryotes and encodes an enzyme containing both DNA-dependent ATPase and DNA annealing activities. MGS1 appears to function in post-replicational repair processes that contribute to genome stability. In this study, we identified MGS1 as a multicopy suppressor of the temperature-sensitive dna2Delta405N mutation, a DNA2 allele lacking the N-terminal 405 amino acid residues. Mgs1 stimulates the structure-specific nuclease activity of Rad27 (yeast Fen1 or yFen1) in an ATP-dependent manner. ATP binding but not hydrolysis was sufficient for the stimulatory effect of Mgs1, since non-hydrolyzable ATP analogs are as effective as ATP. Suppression of the temperature-sensitive growth defect of dna2Delta405N required the presence of a functional copy of RAD27, indicating that Mgs1 suppressed the dna2Delta405N mutation by increasing the activity of yFen1 (Rad27) in vivo. Our results provide in vivo and in vitro evidence that Mgs1 is involved in Okazaki fragment processing by modulating Fen1 activity. The data presented raise the possibility that the absence of MGS1 may impair the processing of Okazaki fragments, leading to genomic instability.

Rapid Quantitative Determination of L-FMAU-TP from Human Peripheral-blood Mononuclear Cells of Hepatitis B Virus-infected Patients Treated with L-FMAU by Ion-pairing, Reverse-phase, Liquid Chromatography/electrospray Tandem Mass Spectrometry

Therapeutic Drug Monitoring. Feb, 2006  |  Pubmed ID: 16418707

The purpose of this study was to develop an analytical method for the determination of 2'-fluoro-5-methyl-beta-l-arabinofuranosyl uracil triphosphate (L-FMAU-TP) in human peripheral blood mononuclear cells (PBMCs), and its application in the determination of cellular levels of L-FMAU-TP in PBMCs isolated from patients treated with 2'-fluoro-5-methyl-beta-l-arabinofuranosyl uracil (L-FMAU). An ion-pairing liquid chromatography (IPC) method, coupled with negative ion electrospray ionization tandem mass spectrometry (ESI-MS/MS), was developed for the accurate and repeatable detection of L-FMAU-TP, with a limit of detection of 1.6 pmol/10 cells. The calibration curve for L-FMAU-TP was linear over the concentration range 1.6 to 80 pmol/10(6) cells. The intra- and inter-day precision was lower than 11.2%, and the accuracy was between 97.1 and 106.9%. When applied to the determination of L-FMAU-TP in PBMCs isolated from HBV-infected patients undergoing L-FMAU treatment, the levels reached a steady state concentration 4 weeks after daily single oral administration of 20 mg L-FMAU, and these levels were maintained for up to 12 weeks, but then decreased 12 weeks after drug cessation. The terminal half-life of L-FMAU-TP in PBMCs after drug cessation was estimated to be 15.6 days.

The Metabolism and Excretion of 2-methylaminoethoxycarbonyl-4,4'-dimethoxy-5,6,5',6'-dimethylenedioxybiphenyl-2'-carboxylic Acid (DDB-S) in Rats and Human

Rapid Communications in Mass Spectrometry : RCM. 2006  |  Pubmed ID: 16755608

The metabolism and excretion of 2-methylaminoethoxycarbonyl-4,4'-dimethoxy-5,6,5',6'-dimethylenedioxybiphenyl-2'-carboxylic acid (DDB-S) were investigated in both rats and humans using liquid chromatography/electrospray ion trap mass spectrometry (LC/ESI-MS/MS). In rats, DDB-S was rapidly eliminated from the body after a single 50 mg/kg intravenous injection, with urine being a major excretion route. DDB-S was metabolically stable; approximately 96% of the administered dose was recovered in the form of the parent compound. Nevertheless, 12 metabolites were detected in the urine and feces collected from DDB-S-treated rats. The structural characterizations of the metabolites were elucidated from the MS(n) spectral analysis. Because DDB-S has a pseudo-symmetrical methylenedioxy biphenyl structure, regioselective deuterium-substituted DDB-S (d(5)-DDB-S) was used to assign the metabolic modification. The major metabolic pathways of DDB-S were identified as demethylenation of the methylenedioxy moiety, O-demethylation of the methoxy moiety and glucuronidation. In addition, N-demethylation of the methylaminoethyl group was also detected as a minor reaction.

Pinitol from Soybeans Reduces Postprandial Blood Glucose in Patients with Type 2 Diabetes Mellitus

Journal of Medicinal Food. 2006  |  Pubmed ID: 16822203

The effect of 3-O-methyl-D-chiro-inositol (D-pinitol), purified from soybean, on the postprandial blood glucose response in patients with type 2 diabetes mellitus was examined. Fifteen Korean subjects with type 2 diabetes mellitus (seven men, eight women; 60.3 +/- 3.1 years old) ingested cooked white rice containing 50 g of available carbohydrate with or without prior ingestion of soy pinitol. Pinitol was given either as a 1.2 g dose at 0, 60, 120, or 180 minutes prior to rice ingestion, or as a 0.6 g dose at 60 minutes prior to rice ingestion. Capillary blood glucose levels were monitored for 4 hours after rice consumption. The ingestion of 1.2 g of pinitol 60 minutes prior to rice consumption controlled postprandial capillary blood glucose most effectively, significantly diminishing the postprandial increase in plasma glucose levels measured at 90 and 120 minutes after rice consumption (P < .05). The incremental area under the plasma glucose response curve for subjects who consumed both pinitol and rice was significantly lower than that for subjects who consumed only rice (P < .05), but pinitol had no apparent effect on postprandial insulin levels. Therefore, soybean-derived pinitol may be useful in controlling postprandial increases in blood glucose in patients with type 2 diabetes.

Etanercept-induced Systemic Lupus Erythematosus in a Patient with Rheumatoid Arthritis

Journal of Korean Medical Science. Oct, 2006  |  Pubmed ID: 17043436

Tumor necrosis factor (TNF) is known to play a critical role in the pathogenesis of rheumatoid arthritis (RA). Etanercept is a recombinant soluble fusion protein of TNF alpha type II receptor and IgG, which acts as a specific TNF-alpha antagonist. Anti-TNF-alpha therapy has been an important advance in the treatment of RA. However, induction of autoantibodies in some proportion of patients treated with TNF alpha inhibitors raised concerns for development of systemic autoimmune diseases such as systemic lupus erythematosus (SLE). Although new autoantibody formation is common with anti-TNF alpha therapy, there are only rare reports of overt SLE, most of which manifested without major organ involvement and resolved shortly after discontinuation of the therapy. We describe a 55-yr-old Korean woman who developed overt life threatening SLE complicated by pneumonia and tuberculosis following etanercept treatment for RA. This case is to our knowledge, the first report of etanercept-induced SLE in Korea.

Validation and Application of a Screening Method for Beta2-agonists, Anti-estrogenic Substances and Mesocarb in Human Urine Using Liquid Chromatography/tandem Mass Spectrometry

Rapid Communications in Mass Spectrometry : RCM. 2007  |  Pubmed ID: 17171780

Electrospray ionization (ESI) mass spectra of 15 anti-estrogenic substances, beta2-agonists and mesocarb were investigated in terms of fragmentation patterns. On the basis of this product ion information, a simultaneous screening method for anti-estrogenic substances, beta2-agonists and mesocarb was developed for doping control purposes. After hydrolysis, liquid-liquid extraction was adopted for the sample preparation. The recoveries for all compounds were 30 and 96%. A single liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis could be performed in 13 min for the analysis of 15 anti-estrogenic substances, beta2-agonists and mesocarb. A quantitative analysis was also validated. Inaccuracies were below +/-12% and precisions varied from 0 to 15.8%. The limit of detection was below 10 ng/mL except formestane (300 ng/mL) and aminoglutethimide (100 ng/mL). The validated method was applied for the analysis of excretion samples.

Synthesis, Characterization, and Reactivities of Manganese(V)-oxo Porphyrin Complexes

Journal of the American Chemical Society. Feb, 2007  |  Pubmed ID: 17263410

The reactions of manganese(III) porphyrin complexes with terminal oxidants, such as m-chloroperbenzoic acid, iodosylarenes, and H(2)O(2), produced high-valent manganese(V)-oxo porphyrins in the presence of base in organic solvents at room temperature. The manganese(V)-oxo porphyrins have been characterized with various spectroscopic techniques, including UV-vis, EPR, 1H and 19F NMR, resonance Raman, and X-ray absorption spectroscopy. The combined spectroscopic results indicate that the manganese(V)-oxo porphyrins are diamagnetic low-spin (S = 0) species with a longer, weaker Mn-O bond than in previously reported Mn(V)-oxo complexes of non-porphyrin ligands. This is indicative of double-bond character between the manganese(V) ion and the oxygen atom and may be attributed to the presence of a trans axial ligand. The [(Porp)Mn(V)=O](+) species are stable in the presence of base at room temperature. The stability of the intermediates is dependent on base concentration. In the absence of base, (Porp)Mn(IV)=O is generated instead of the [(Porp)Mn(V)=O](+) species. The stability of the [(Porp)Mn(V)=O](+) species also depends on the electronic nature of the porphyrin ligands: [(Porp)Mn(V)=O](+) complexes bearing electron-deficient porphyrin ligands are more stable than those bearing electron-rich porphyrins. Reactivity studies of manganese(V)-oxo porphyrins revealed that the intermediates are capable of oxygenating PPh(3) and thioanisoles, but not olefins and alkanes at room temperature. These results indicate that the oxidizing power of [(Porp)Mn(V)=O](+) is low in the presence of base. However, when the [(Porp)Mn(V)=O](+) complexes were associated with iodosylbenzene in the presence of olefins and alkanes, high yields of oxygenated products were obtained in the catalytic olefin epoxidation and alkane hydroxylation reactions. Mechanistic aspects, such as oxygen exchange between [(Porp)Mn(V)=16O](+) and H(2)(18)O, are also discussed.

Adenoviral Pneumonia During Etanercept Treatment in a Patient with Rheumatoid Arthritis

The Korean Journal of Internal Medicine. Mar, 2007  |  Pubmed ID: 17427651

Inhibitors of tumor necrosis factor-alpha (TNF-alpha) have been approved for treating rheumatoid arthritis. As one of the biological response modifiers, etanercept has also been used in the treatment of psoriatic arthritis and inflammatory bowel disease. While etanercept is effective, certain infectious complications, such as tuberculosis, fungus, and cytomegalovirus, have been reported. We report the first Korean case of adenoviral pneumonia in a 55-year-old female who developed disseminated adenoviral infection following etanercept treatment, which resolved after anti-TNF-alpha discontinuation.

Mechanistic Insight into the Aromatic Hydroxylation by High-valent Iron(IV)-oxo Porphyrin Pi-cation Radical Complexes

The Journal of Organic Chemistry. Aug, 2007  |  Pubmed ID: 17622172

Mechanistic studies of the aromatic hydroxylation by high-valent iron(IV)-oxo porphyrin pi-cation radicals revealed that the aromatic oxidation involves an initial electrophilic attack on the pi-system of the aromatic ring to produce a tetrahedral radical or cationic sigma-complex. The mechanism was proposed on the basis of experimental results such as a large negative Hammett rho value and an inverse kinetic isotope effect. By carrying out isotope labeling studies, the oxygen in oxygenated products was found to derive from the iron-oxo porphyrin intermediates.

Analytic Evaluation of the Beta-human Chorionic Gonadotropin Assay on the Abbott IMx and Elecsys2010 for Its Use in Doping Control

Clinical Biochemistry. Nov, 2007  |  Pubmed ID: 17884034

The principal objective of this study was to compare the analytical performance of the Elecsys2010 (Roche Diagnostics) system with the IMx (Abbott laboratories) system for beta-hCG assay in order to assess its possible utility as a confirmation test for the quantitative measurement of beta-hCG in urine for doping control purposes.

Primary Esophageal Adenoid Cystic Carcinoma

Gut and Liver. Dec, 2007  |  Pubmed ID: 20485637

Adenoid cystic carcinoma (ACC) is common in the salivary glands but rare in the esophagus. Routine esophagogastroscopy performed in a 54-year-old woman as part of a medical check-up revealed a submucosal tumor (1.5x1.0 cm) at the mid-esophagus. Endoscopic ultrasonography revealed a lesion with mixed echogenicity in the submucosal layer. The submucosal mass was removed by incisional endoscopic enucleation, and pathological analysis revealed epithelial cells with small hyperchromatic angular nuclei in tubular and cribriform patterns. The lesion was pathologically confirmed as an ACC of the esophagus.

Hypoglycemic and Hypolipidemic Effects of Saururus Chinensis Baill in Streptozotocin-induced Diabetic Rats

Nutrition Research and Practice. 2007  |  Pubmed ID: 20535394

Saururus chinensis Baill was reported to inhibit alpha-glucosidase in vitro and flatten postprandial increase in blood glucose in streptozotocin (STZ)-induced diabetic rats. We studied the effect of chronic consumption of S. chinensis Baill on blood glucose and lipid profile in STZ-induced diabetic male rats fed high fat diet. Male rats weighing 100-120 g were fed 30% fat diet with and without 10% freeze-dried leaves of S. chinensis Baill for 7 weeks after 1 week of adaptation. The rats were rendered diabetic by intravenous injection of STZ (60 mg/kg) after 6-week feeding of the assigned diets. At 1 week after the injection, the rats were sacrificed after an overnight fast. Plasma glucose (380.2 +/- 14.4 mg/dL), total cholesterol (93.9 +/- 7.9 mg/dL) and triglyceride levels (123.6 +/- 7.5 mg/dL) of the S. chinensis Baill group were significantly lower than those of the control group (418.1 +/- 12.0 mg/dL, 119.9 +/- 9.4 mg/dL, 152.0 +/- 10.3 mg/dL, respectively, p<0.05). Chronic consumption of S. chinesis Baill significantly decreased maltase activity of the small intestinal mucosa (120.1 +/- 8.7 U/g protein) compared with the control group (96.8 +/- 7.0 U/g protein, p<0.05). These results suggest that S. chinensis Baill have hypoglycemic and hypolipidemic effects by inhibiting alpha-glucosidase activity in the animal model of diabetes mellitus.

Pancreatic Lipase Inhibitory Activity of Taraxacum Officinale in Vitro and in Vivo

Nutrition Research and Practice. 2008  |  Pubmed ID: 20016719

Obesity has become a worldwide health problem. Orlistat, an inhibitor of pancreatic lipase, is currently approved as an anti-obesity drug. However, gastrointestinal side effects caused by Orlistat may limit its use. In this study the inhibitory activities of dandelion (Taraxacum officinale) against pancreatic lipase in vitro and in vivo were measured to determine its possible use as a natural anti-obesity agent. The inhibitory activities of the 95% ethanol extract of T. officinale and Orlistat were measured using 4-methylumbelliferyl oleate (4-MU oleate) as a substrate at concentrations of 250, 125, 100, 25, 12.5 and 4 microg/ml. To determine pancreatic lipase inhibitory activity in vivo, mice (n=16) were orally administered with corn oil emulsion (5 ml/kg) alone or with the 95% ethanol extract of T. officinale (400 mg/kg) following an overnight fast. Plasma triglyceride levels were measured at 0, 90, 180, and 240 min after treatment and incremental areas under the response curves (AUC) were calculated. The 95% ethanol extract of T. officinale and Orlistat, inhibited, porcine pancreatic lipase activity by 86.3% and 95.7% at a concentration of 250 microg/ml, respectively. T. officinale extract showed dose-dependent inhibition with the IC(50) of 78.2 microg/ml. A single oral dose of the extract significantly inhibited increases in plasma triglyceride levels at 90 and 180 min and reduced AUC of plasma triglyceride response curve (p<0.05). The results indicate that T. officinale exhibits inhibitory activities against pancreatic lipase in vitro and in vivo. Further studies to elucidate anti-obesity effects of chronic consumption of T. officinale and to identify the active components responsible for inhibitory activity against pancreatic lipase are necessary.

Enzymatic C-demethylation of 1-[2-(5-tert-butyl-[1,3,4] Oxadiazole-2-carbonyl)-4-fluoro-pyrrolidin-1-yl]-2-(2-hydroxy-1,1-dimethyl-ethylamino)-ethanone (LC15-0133) in Rat Liver Microsomes

Drug Metabolism and Disposition: the Biological Fate of Chemicals. Mar, 2008  |  Pubmed ID: 18057116

The in vitro metabolism of 1-[2-(5-tert-butyl-[1,3,4] oxadiazole-2-carbonyl)-4-fluoro-pyrrolidin-1-yl]-2-(2-hydroxy-1,1-dimethyl-ethylamino)-ethanone (LC15-0133), a novel dipeptidyl peptidase-4 inhibitor, was investigated using a hepatic microsomal system. The structures of the metabolites were characterized using mass spectral analysis and by comparison with synthetic references. The in vitro incubation of LC15-0133 with rat liver microsomes resulted in the formation of six metabolites, with the major metabolic reactions being hydroxylation and carbonyl reduction. Of the metabolites, a C-demethylated metabolite (M4) was identified, but was only detected in rat liver microsomes; experimental evidence revealed that the C-demethylated metabolite was generated by nonenzymatic decarboxylation of the carboxyl metabolite (M1). Nonenzymatic decarboxylation is postulated to occur due to the resonance stabilization by the oxadiazole ring attached to the tert-butyl moiety.

Esophageal Tuberculosis Presenting As a Submucosal Tumor

Clinical Gastroenterology and Hepatology : the Official Clinical Practice Journal of the American Gastroenterological Association. Feb, 2008  |  Pubmed ID: 18237860

Protein Profiling of Human Plasma Samples by Two-dimensional Electrophoresis

Methods in Molecular Biology (Clifton, N.J.). 2008  |  Pubmed ID: 18287768

Human plasma is regarded the most complex and well-known clinical specimen that can be easily obtained; alterations in the levels of plasma proteins or their corresponding enzyme activities may reflect either a healthy or a diseased state. Given that there is no defined genomic information as to the intact protein components in plasma, protein profiling could be the first step toward its molecular characterization. Several problems exist in the analysis of plasma proteins, however. For example, the widest dynamic range of protein concentrations, the presence of high-abundance proteins, and post-translational modifications need to be considered before proteomic studies are undertaken. In particular, efficient depletion or pre-fractionation of high-abundance proteins is crucial for the identification of low-abundance proteins that may contain potential biomarkers. After the removal of high-abundance proteins, protein profiling can be initiated using two-dimensional electrophoresis (2DE), which has been widely used for displaying the differential proteome under specific physiological conditions. Here, we describe a typical 2DE procedure for plasma proteome under either a healthy or a diseased state (e.g., liver cancer) in which pre-fractionation and depletion are integral steps in the search for disease biomarkers.

[Efficacy of Levofloxacin-based Triple Therapy As Second-line Helicobacter Pylori Eradication]

The Korean Journal of Gastroenterology = Taehan Sohwagi Hakhoe Chi. May, 2008  |  Pubmed ID: 18516012

Bismuth-based quadruple therapy for second-line eradication treatment achieves the eradication rate ranging from 70% to 81% due to antimicrobial resistance and poor compliance. The aim of this study was to compare the eradication rate of levofloxacin-based triple therapy with that of bismuth-based quadruple therapy in second-line Helicobacter pylori (H. pylori) eradication therapy.

LC-MS/MS Method for Determination of Hederacolchiside E, a Neuroactive Saponin from Pulsatilla Koreana Extract in Rat Plasma for Pharmacokinetic Study

Journal of Pharmaceutical and Biomedical Analysis. Dec, 2008  |  Pubmed ID: 18947958

A simple, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was applied to pharmacokinetic study of a neuroactive oleanolic-glycoside saponin, hederacolchiside E from SK-PC-B70M, a standardized extract of Pulsatilla koreana in rat. Rat plasma samples were pretreated by protein precipitation with acetonitrile, eluted from C(18) column, and analyzed using electrospray ionization (ESI)-MS/MS in negative ion mode. Digoxin was used as an internal standard. The standard curves were linear (r>0.997) over the concentration ranges of 2-500 ng/mL. The intra- and inter-day precisions were measured to be below 9% and accuracy between 90 and 111% for all quality control samples at 2, 20, 100, and 500 ng/mL (n=5). The lower limits of quantification (LLOQ) for hederacolchiside E was 2 ng/mL and the limit of detection (LOD) 0.5 ng/mL using 20 microL of plasma sample. Subsequently, hederacolchiside E was determined in rat plasma samples after oral administration of SK-PC-B70M. The mean maximum plasma concentrations of hederacolchiside E were 0.07, 0.13, and 0.36 microg/mL and the mean areas under the plasma concentration versus time curve 0.56, 1.27, and 6.46 microg h/mL at doses of 100, 200, and 400 mg/kg, respectively, which indicated non-linear pharmacokinetic pattern. In conclusion, this method was successfully applied to the pharmacokinetic study of hederacolchiside E after an oral administration of SK-PC-B70M to rats.

The MPH1 Gene of Saccharomyces Cerevisiae Functions in Okazaki Fragment Processing

The Journal of Biological Chemistry. Apr, 2009  |  Pubmed ID: 19181670

Saccharomyces cerevisiae MPH1 was first identified as a gene encoding a 3' to 5' DNA helicase, which when deleted leads to a mutator phenotype. In this study, we isolated MPH1 as a multicopy suppressor of the dna2K1080E helicase-negative lethal mutant. Purified Mph1 stimulated the endonuclease activities of both Fen1 and Dna2, which act faithfully in the processing of Okazaki fragments. This stimulation required neither ATP hydrolysis nor the helicase activity of Mph1. Multicopy expression of MPH1 also suppressed the temperature-sensitive growth defects in cells expressing dna2Delta405N, which lacks the N-terminal 405 amino acids of Dna2. However, Mph1 did not stimulate the endonuclease activity of the Dna2Delta405N mutant protein. The stimulation of Fen1 by Mph1 was limited to flap-structured substrates; Mph1 hardly stimulated the 5' to 3' exonuclease activity of Fen1. Mph1 binds to flap-structured substrate more efficiently than to nicked duplex structures, suggesting that the stimulatory effect of Mph1 is exerted through its binding to DNA substrates. In addition, we found that Mph1 reversed the inhibitory effects of replication protein A on Fen1 activity. Our biochemical and genetic data indicate that the in vivo suppression of Dna2 defects observed with both dna2K1080E and dna2Delta405N mutants occur via stimulation of Fen1 activity. These findings suggest that Mph1 plays an important, although not essential, role in processing of Okazaki fragments by facilitating the formation of ligatable nicks.

[Comparison of Clinical Characteristics and Outcomes Between Geriatric and Non-geriatric Patients in Peptic Ulcer Bleeding]

The Korean Journal of Gastroenterology = Taehan Sohwagi Hakhoe Chi. May, 2009  |  Pubmed ID: 19458466

In geriatric patients with peptic ulcer, the use of NSAID and prevalence of chronic illness have been increased, but the Helicobacter pylori (H. pylori) infected portion decreased. The aim of this study was to evaluate the clinical characteristics and outcomes of geriatric patients (aged 65 or older) with peptic ulcer bleeding and compare with non-geriatric patients (less than 65 years old).

Nevus-like Appearance of Primary Malignant Melanoma of the Esophagus

Gastroenterology Research and Practice. 2009  |  Pubmed ID: 19644559

The primary malignant melanoma of the esophagus (PMME) is a rare malignant disease, accounting for only 0.1-0.2% of all esophageal neoplasms, and the majority of the patients are diagnosed at advanced stages with poor prognosis. We present here a case of 56-year-old woman with epigastric pain and her endoscopic finding revealed several flat and black pigmented mucosal lesions within the distal portion of the esophagus which looked like flat nevus. The histopathology and immunohistochemical profile of the tissue specimens were diagnostic of malignant melanoma.

Effects of Dimyristoylphosphatidylethanol and Ethanol on Thickness of Neuronal Membrane Lipid Bilayers

Archives of Pharmacal Research. Oct, 2009  |  Pubmed ID: 19898812

The aim of this study was to provide a basis for examining the molecular mechanism for the pharmacological action of ethanol. Energy transfer between the surface fluorescent probe 1-anilinonaphthalene-8-sulfonic acid and hydrophobic fluorescent probe 1,3-di(1-pyrenyl)propane was used to examine the effect of both dimyristoylphosphatidylethanol (DMPEt) and ethanol on the thickness (D) of the synaptosomal plasma membrane vesicles (SPMV) isolated from the bovine cerebral cortex. The thickness (D) of the intact SPMV was 1.044 +/- 0.008 (arbitrary units, n=5) at 37 degrees C (pH 7.4). Both DMPEt and ethanol decreased the thickness of the SPMV lipid bilayer in a dose-dependent manner with a significant decrease in thickness observed at 5 microM and 25 mM, respectively. It was assumed that both ethanol and DMPEt cause interdigitation in the SPMV lipid bilayers. The effects of ethanol on the neuronal membranes were attributed to its direct and indirect actions. The indirect action of ethanol refers to the action of phosphatidylethanol, which is an ethanol abnormal metabolite, on the neuronal membranes. The decrease in membrane thickness by both DMPEt and ethanol might be responsible for some, but not all of its anesthetic actions.

Infectious Mononucleosis Hepatitis in Young Adults: Two Case Reports

The Korean Journal of Internal Medicine. Dec, 2009  |  Pubmed ID: 19949739

Infectious mononucleosis due to Epstein-Barr virus (EBV) infection sometimes causes acute hepatitis, which is usually self-limiting with mildly elevated transaminases, but rarely with jaundice. Primary EBV infection in children is usually asymptomatic, but in a small number of healthy individuals, typically young adults, EBV infection results in a clinical syndrome of infectious mononucleosis with hepatitis, with typical symptoms of fever, pharyngitis, lymphadenopathy, and hepatosplenomegaly. EBV is rather uncommonly confirmed as an etiologic agent of acute hepatitis in adults. Here, we report two cases: the first case with acute hepatitis secondary to infectious mononucleosis and a second case, with acute hepatitis secondary to infectious mononucleosis concomitantly infected with hepatitis A. Both cases involved young adults presenting with fever, pharyngitis, lymphadenopathy, hepatosplenomegaly, and atypical lymphocytosis confirmed by serologic tests, liver biopsy and electron microscopic study.

Antioxidant Effect of Garlic and Aged Black Garlic in Animal Model of Type 2 Diabetes Mellitus

Nutrition Research and Practice. 2009  |  Pubmed ID: 20016716

Hyperglycemia in the diabetic state increases oxidative stress and antioxidant therapy can be strongly correlated with decreased risks for diabetic complications. The purpose of this study is to determine antioxidant effect of garlic and aged black garlic in animal model of type 2 diabetes. The antioxidant activity of garlic and aged black garlic was measured as the activity in scavenging free radicals by the trolox equivalent antioxidant capacity (TEAC) assay. Three week-old db/db mice were fed AIN-93G diet or diet containing 5% freeze-dried garlic or aged black garlic for 7 weeks after 1 week of adaptation. Hepatic levels of lipid peroxides and activities of antioxidant enzymes were measured. TEAC values of garlic and aged black garlic were 13.3 +/- 0.5 and 59.2 +/- 0.8 micromol/g wet weight, respectively. Consumption of aged black garlic significantly decreased hepatic thiobarbituric acid reactive substances (TBARS) level compared with the garlic group which showed lower TBARS level than control group (p<0.05). Activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) of garlic and aged black garlic group were significantly elevated compared to the control group. Catalase (CAT) activity of aged black garlic group was increased compared with the control group. These results show that aged black garlic exerts stronger antioxidant activity than garlic in vitro and in vivo, suggesting garlic and aged black garlic, to a greater extent, could be useful in preventing diabetic complications.

[Analysis of Risk Factors for Low Bone Mineral Density in Patients with Inflammatory Bowel Disease]

The Korean Journal of Gastroenterology = Taehan Sohwagi Hakhoe Chi. Apr, 2010  |  Pubmed ID: 20389177

Several clinical risk factors for low bone mineral density (BMD) in the patients with inflammatory bowel disease (IBD) have been suggested. However, its prevalence and pathophysiology in Korean population have not been fully studied. The aim of this study was to investigate the prevalence and risk factors for low BMD in Korean IBD patient.

Genetic and Functional Interactions Between Mus81-Mms4 and Rad27

Nucleic Acids Research. Nov, 2010  |  Pubmed ID: 20660481

The two endonucleases, Rad27 (yeast Fen1) and Dna2, jointly participate in the processing of Okazaki fragments in yeasts. Mus81-Mms4 is a structure-specific endonuclease that can resolve stalled replication forks as well as toxic recombination intermediates. In this study, we show that Mus81-Mms4 can suppress dna2 mutational defects by virtue of its functional and physical interaction with Rad27. Mus81-Mms4 stimulated Rad27 activity significantly, accounting for its ability to restore the growth defects caused by the dna2 mutation. Interestingly, Rad27 stimulated the rate of Mus81-Mms4 catalyzed cleavage of various substrates, including regressed replication fork substrates. The ability of Rad27 to stimulate Mus81-Mms4 did not depend on the catalytic activity of Rad27, but required the C-terminal 64 amino acid fragment of Rad27. This indicates that the stimulation was mediated by a specific protein-protein interaction between the two proteins. Our in vitro data indicate that Mus81-Mms4 and Rad27 act together during DNA replication and resolve various structures that can impede normal DNA replication. This conclusion was further strengthened by the fact that rad27 mus81 or rad27 mms4 double mutants were synergistically lethal. We discuss the significance of the interactions between Rad27, Dna2 and Mus81-Mms4 in context of DNA replication.

Hypoglycemic Effects of Welsh Onion in an Animal Model of Diabetes Mellitus

Nutrition Research and Practice. Dec, 2010  |  Pubmed ID: 21286406

Tight control of blood glucose is the most important strategy for the treatment of diabetes mellitus. Here, we investigated the beneficial effects of Welsh onion on fasting and postprandial hyperglycemia. Inhibitory activities of hot water extracts from the green stalk and white bulb, which are the edible portions of the Welsh onion, and the fibrous root extract against yeast α-glucosidase were measured in vitro. To study the effects of Welsh onion on postprandial hyperglycemia, a starch solution (1 g/kg) with and without Welsh onion fibrous root extract (500 mg/kg) or acarbose (50 mg/kg) was administered to streptozotocin-induced diabetic rats after an overnight fast. Postprandial plasma glucose levels were measured and incremental areas under the response curve were calculated. To study the hypoglycemic effects of chronic feeding of Welsh onion, five-week-old db/db mice were fed an AIN-93G diet or a diet containing either Welsh onion fibrous root extract at 0.5% or acarbose at 0.05% for 7 weeks after 1 week of adaptation. Fasting plasma glucose and blood glycated hemoglobin were measured. Compared to the extract from the edible portions of Welsh onion, the fibrous root extract showed stronger inhibition against yeast α-glucosidase, with an IC(50) of 239 µg/mL. Oral administration of Welsh onion fibrous root extract (500 mg/kg) and acarbose (50 mg/kg) significantly decreased incremental plasma glucose levels 30-120 min after oral ingestion of starch as well as the area under the postprandial glucose response curve, compared to the control group (P < 0.01). The plasma glucose and blood glycated hemoglobin levels of the Welsh onion group were significantly lower than those of the control group (P < 0.01), and were not significantly different from those fed acarbose. Thus, we conclude that the fibrous root of Welsh onion is effective in controlling hyperglycemia in animal models of diabetes mellitus.

Involvement of Vts1, a Structure-specific RNA-binding Protein, in Okazaki Fragment Processing in Yeast

Nucleic Acids Research. Mar, 2010  |  Pubmed ID: 20007605

The non-essential VTS1 gene of Saccharomyces cerevisiae is highly conserved in eukaryotes and encodes a sequence- and structure-specific RNA-binding protein. The Vts1 protein has been implicated in post-transcriptional regulation of a specific set of mRNAs that contains its-binding site at their 3'-untranslated region. In this study, we identified VTS1 as a multi-copy suppressor of dna2-K1080E, a lethal mutant allele of DNA2 that lacks DNA helicase activity. The suppression was allele-specific, since overexpression of Vts1 did not suppress the temperature-dependent growth defects of dna2Delta405N devoid of the N-terminal 405-amino-acid residues. Purified recombinant Vts1 stimulated the endonuclease activity of wild-type Dna2, but not the endonuclease activity of Dna2Delta405N, indicating that the activation requires the N-terminal domain of Dna2. Stimulation of Dna2 endonuclease activity by Vts1 appeared to be the direct cause of suppression, since the multi-copy expression of Dna2-K1080E suppressed the lethality observed with its single-copy expression. We found that vts1Delta dna2Delta405N and vts1Deltadna2-7 double mutant cells displayed synergistic growth defects, in support of a functional interaction between two genes. Our results provide both in vivo and in vitro evidence that Vts1 is involved in lagging strand synthesis by modulating the Dna2 endonuclease activity that plays an essential role in Okazaki fragment processing.

Associations Between Single Nucleotide Polymorphisms of MMP2, VEGF, and HIF1A Genes and the Risk of Developing Colorectal Cancer

Anticancer Research. Feb, 2011  |  Pubmed ID: 21378341

The aim of this study was to investigate the association between the risk for colorectal cancer and single nucleotide polymorphisms (SNP) of matrix metalloproteinase-2 (MMP2) -1306C/T, vascular endothelial growth factor (VEGF) 936C/T and hypoxia inducible factor-1α (HIF1A) 1772C/T.

Flagellin Promotes the Proliferation of Gastric Cancer Cells Via the Toll-like Receptor 5

International Journal of Molecular Medicine. Jul, 2011  |  Pubmed ID: 21455558

Signaling of the Toll-like receptor (TLR) is closely associated with tumor development and progression processes including cell proliferation, angiogenesis, metastasis, and immunosuppression. In this study, we examined the expression of TLR5 in gastric cancer cells and its function in cell proliferation. RT-PCR revealed that the TLR5 gene was expressed in all gastric cancer cell lines examined, SNU638, SNU601, SNU216, and AGS. The TLR5 agonist, flagellin, induced IL-8 production and NF-κB activation in the gastric cancer cell lines. In addition, flagellin enhanced the proliferation of all gastric cancer cells examined, whereas LPS did not affect that of SNU638 cells. Blockade of TLR5 using an antibody, restored the proliferation of SNU638 cells enhanced by flagellin, indicating that TLR5 is essential for cell proliferation by flagellin. Flagellin also led to phosphorylation of ERK in SNU638 cells. The ERK inhibitor, PD98059, restored the proliferation ability of SNU638 cells enhanced by flagellin, suggesting that ERK may play an important role in the proliferation of gastric cancer cells. These findings suggest that TLR5 may play an important role in tumor progression of gastric cancer via the regulation of cell proliferation.

Quercetin Attenuates Fasting and Postprandial Hyperglycemia in Animal Models of Diabetes Mellitus

Nutrition Research and Practice. Apr, 2011  |  Pubmed ID: 21556223

The objective of this study was to investigate the hypoglycemic effects of quercetin (QE) in animal models of diabetes mellitus (DM). A starch solution (1 g/kg) with and without QE (100 mg/kg) or acarbose (40 mg/kg) was orally administered to streptozotocin (STZ)-induced diabetic rats after an overnight fast. Postprandial plasma glucose levels were measured and incremental areas under the response curve were calculated. To study the effects of chronic feeding of QE, five-week-old db/db mice were fed an AIN-93G diet, a diet containing QE at 0.08%, or a diet containing acarbose at 0.03% for 7 weeks after 1 week of adaptation. Plasma glucose and insulin, blood glycated hemoglobin, and maltase activity of the small intestine were measured. Oral administration of QE (100 mg/kg) or acarbose (40 mg/kg) to STZ-treated rats significantly decreased incremental plasma glucose levels 30-180 min after a single oral dose of starch and the area under the postprandial glucose response, compared with the control group. QE (0.08% of diet) or acarbose (0.03% of diet) offered to db/db mice significantly reduced both plasma glucose and blood glycated hemoglobin compared to controls without significant influence on plasma insulin. Small intestine maltase activities were significantly reduced by consumption of QE or acarbose. Thus, QE could be effective in controlling fasting and postprandial blood glucose levels in animal models of DM.

Renoprotective and Antioxidant Effects of Saururus Chinensis Baill in Rats Fed a High-fructose Diet

Nutrition Research and Practice. Aug, 2011  |  Pubmed ID: 21994532

This study investigated the preventive effect of Saururus chinensis Baill against renal damage induced by a high-fructose diet in rats. The rats (n = 30) were fed either a cornstarch-based (65%), high-fructose (65%), or high-fructose (64.5%) diet with 0.5% S. chinensis Baill extract for 10 weeks. Twenty-four hour urine collections were obtained and the animals were sacrificed after an overnight fast. Serum urea and creatinine and urine albumin were measured using colorimetric methods, and creatinine clearance was determined. In addition, thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), and the activity of superoxide dismutase (SOD) in the kidney were determined. Kidney samples were also examined histologically. The fructose-fed rats showed renal dysfunction, indicated by decreased creatinine clearance, increased albumin in the urine, and increased urea and creatinine in the serum. These renal function parameters were comparable to control levels in rats that consumed S. chinensis Baill. Fructose consumption increased renal TBARS and reduced GSH and SOD activity, whereas these levels were near-normal in the rats consuming S. chinensis Baill. The kidneys of fructose-fed rats showed glomerular basement membrane thickening, mesangial matrix expansion, and tubule dilation. These pathological changes were not seen in the rats that consumed S. chinensis Baill. Therefore, S. chinensis Baill effectively alleviated fructose-induced renal damage in these rats, at least partially due to antioxidant activity.

Autodisplay of Streptavidin

Enzyme and Microbial Technology. Apr, 2011  |  Pubmed ID: 22112942

Streptavidin was expressed on the outer membrane of E. coli as a recombinant fusion protein with an autotransporter domain called AIDA-I (adhesin involved in diffuse adherence) using autodisplay technology. The autodisplay of streptavidin was confirmed by SDS-PAGE of the outer membrane proteins, and the number of autodisplayed streptavidin molecules on a single E. coli cell was evaluated with densitometric analysis. The biotin-binding activity of the autodisplayed streptavidin was estimated after treatment with fluorescently labeled biotin by fluorescence microscopy and flow cytometry. The biotin-binding activity of the E. coli with autodisplayed streptavidin was compared with the activity of streptavidin immobilized on magnetic beads. Finally, the outer membrane presenting autodisplayed streptavidin was isolated and layered on a 96-well microplate for an immunoassay.

Quantification of Trace-level DNA by Real-time Whole Genome Amplification

PloS One. 2011  |  Pubmed ID: 22174862

Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, -2.1%, and -13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.

Activation of Nod1 and Nod2 Induces Innate Immune Responses of Prostate Epithelial Cells

The Prostate. Jan, 2012  |  Pubmed ID: 22228081

BACKGROUND: Nod1 and Nod2 are cytosolic receptors which are responsible for sensing bacterial peptidoglycan derivatives. In this study, we determined whether Nod1 and Nod2 are involved in the innate immune responses of prostate epithelial cells. METHODS: The expression of Nod1 and Nod2 was examined by RT-PCR and immunohistochemistry. ELISA was performed to determine the production of cytokines/chemokines. Activation of NF-κB and MAPK was examined using western blot analysis. RESULTS: The Nod1 gene was distinctly expressed in all tested cells including DU145, PC3, and TRAMP-C2 cells, whereas Nod2 expression was weak. Both Nod1 and Nod2 proteins were expressed in normal mouse prostate epithelia with difference of expression levels. Tri-DAP (Nod1 agonist), but not MDP (Nod2), increased the production of IL-8 (or KC) and IL-6 in prostate epithelial cells. Tri-DAP and MDP could upregulate the gene expression of COX-2 and activate NF-κB and MAPK. In addition, Tri-DAP and MDP synergized with TLR agonists to induce the production of IL-8/KC or IL-6 in PC3 and TRAMP-C2 cells. We finally showed that Nod1 and Nod2 were also expressed in a wide range of prostate lesions including prostate intraepithelial neoplasm (PIN), phyllodes-like tumor, and adenocarcinoma in TRAMP (transgenic adenocarcinoma of the mouse prostate) mice, even though the expression level of Nod1 and Nod2 was different. CONCLUSION: These results indicate that Nod1 and Nod2 may play important roles in the innate immune response of prostate epithelial cells and the development and progression of prostate cancer. Prostate © 2012 Wiley Periodicals, Inc.

Gustatory Receptors Required for Avoiding the Insecticide L-canavanine

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jan, 2012  |  Pubmed ID: 22279227

Insect survival depends on contact chemosensation to sense and avoid consuming plant-derived insecticides, such as l-canavanine. Members of a family of ∼60 gustatory receptors (GRs) comprise the main peripheral receptors responsible for taste sensation in Drosophila. However, the roles of most Drosophila GRs are unknown. In addition to GRs, a G protein-coupled receptor, DmXR, has been reported to be required for detecting l-canavanine. Here, we showed that GRs are essential for responding to l-canavanine and that flies missing DmXR displayed normal l-canavanine avoidance and l-canavanine-evoked action potentials. Mutations disrupting either Gr8a or Gr66a resulted in an inability to detect l-canavanine. We found that l-canavanine stimulated action potentials in S-type sensilla, which were where Gr8a and Gr66a were both expressed, but not in Gr66a-expressing sensilla that did not express Gr8a. l-canavanine-induced action potentials were also abolished in the Gr8a and Gr66a mutant animals. Gr8a was narrowly required for responding to l-canavanine, in contrast to Gr66a, which was broadly required for responding to other noxious tastants. Our data suggest that GR8a and GR66a are subunits of an l-canavanine receptor and that GR8a contributes to the specificity for l-canavanine.

Growing Trend of CE at the Omics Level: the Frontier of Systems Biology--an Update

Electrophoresis. Jan, 2012  |  Pubmed ID: 22139583

Omics is the study of proteins, peptides, genes, and metabolites in living organisms. Systems biology aims to understand the system through the study of the relationship between elements such as genes and proteins in biological system. Recently, systems biology emerged as the result of the advanced development of high-throughput analysis technologies such as DNA sequencers, DNA arrays, and mass spectrometry for omics studies. Among a number of analytical tools and technologies, CE and CE coupled to MS are promising and relatively rapidly developing tools with the potential to provide qualitative and quantitative analyses of biological molecules. With an emphasis on CE for systems biology, this review summarizes the method developments and applications of CE for the genomic, transcriptomic, proteomic, and metabolomic studies focusing on the drug discovery and disease diagnosis and therapies since 2009.

Expression and Activation of Matrix Metalloproteinase-9 and NADPH Oxidase in Tissues and Plasma of Experimental Autoimmune Encephalomyelitis in Mice

Experimental and Toxicologic Pathology : Official Journal of the Gesellschaft Für Toxikologische Pathologie. Jan, 2012  |  Pubmed ID: 20810258

Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for multiple sclerosis (MS) that can be induced by immunization with myelin antigens such as myelin oligodendrocyte glycoprotein (MOG). The objective of this study was (i) to investigate how matrix metalloproteinase-9 (MMP-9) and NADPH oxidase enzymes are affected in the EAE mouse model and (ii) to know whether peripheral organs also express these enzymes in the EAE model. MOG(33-55) was administered subcutaneously on two sites over the back. Pertussis toxin was administered intraperitoneally immediately after MOG and again two days later. A significant difference was observed in body weights and clinical signs of EAE-induced mice. MMP-9 and NADPH oxidase enzymes were measured in central nervous system (CNS) tissues, peripheral tissues and plasma of EAE-induced mice. The primary findings include the distribution pattern of MMP-9 in CNS and peripheral tissues, and alterations in the enzymatic expression of MMP-9 and NADPH oxidase in the CNS tissues, spleen and plasma of EAE-induced mice. From these results, it can be considered that the spleen as well as the CNS can act as target organs in EAE disease, and plasma MMP-9 and NADPH oxidase may contribute to the pathogenesis of the disease.