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Find video protocols related to scientific articles indexed in Pubmed.
[Cardiac protective effect of the autoantibody against ?3-adrenoceptor in rats with experimental heart failure].
Zhonghua Xin Xue Guan Bing Za Zhi
PUBLISHED: 07-22-2014
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To explore the effect of the autoantibody against the ?3-adrenoceptor on rats with experimental heart failure.
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High-yield phosphatidylserine production via yeast surface display of phospholipase D from Streptomyces chromofuscus on Pichia pastoris.
J. Agric. Food Chem.
PUBLISHED: 05-29-2014
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The gene encoding phospholipase D (PLD) from Streptomyces chromofuscus was displayed on the cell surface of Pichia pastoris GS115/pKFS-pldh using a Flo1p anchor attachment signal sequence (FS anchor). The displayed PLD (dPLD) showed maximum enzymatic activity at pH 6.0 and 55 °C and was stable within a broad range of temperatures (20-65 °C) and pHs (pH 4.0-11.0). In addition, the thermostability, acid stability and organic solvent tolerance of the dPLD were significantly enhanced compared with the secreted PLD (sPLD) from S. chromofuscus. Use of dPLD for conversion of phosphatidylcholine (PC) and l-serine to phosphatidylserine (PS) showed that 67.5% of PC was converted into PS at the optimum conditions. Moreover, the conversion rate of PS remained above 50% after 7 repeated batch cycles. Thus, P. pastoris GS115/pKFS-pldh shows the potential for viable industrial production of PS.
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Design and synthesis of lactam-thiophene carboxylic acids as potent hepatitis C virus polymerase inhibitors.
Bioorg. Med. Chem. Lett.
PUBLISHED: 04-23-2014
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Herein we report the successful incorporation of a lactam as an amide replacement in the design of hepatitis C virus NS5B Site II thiophene carboxylic acid inhibitors. Optimizing potency in a replicon assay and minimizing potential risk for CYP3A4 induction led to the discovery of inhibitor 22a. This lead compound has a favorable pharmacokinetic profile in rats and dogs.
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A novel approach for improving the yield of Bacillus subtilis transglutaminase in heterologous strains.
J. Ind. Microbiol. Biotechnol.
PUBLISHED: 04-21-2014
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The transglutaminase (BTG) from Bacillus subtilis is considered to be a new type of transglutaminase for the food industry. Given that the BTG gene only encodes a mature peptide, the expression of BTG in heterologous microbial hosts could affect their normal growth due to BTG's typical transglutaminase activity which can catalyze cross-linking of proteins in the cells. Therefore, we developed a novel approach to suppress BTG activity and reduce the toxicity on microbial hosts, thus improving BTG yield. Genes encoding the respective regions of transglutaminase propeptide from seven species of Streptomyces were fused to the N-terminal of the BTG gene to produce fusion proteins. We found that all the fused propeptides could suppress BTG activity. Importantly, BTG activity could be completely restored after the removal of the propeptides by proteolytic cleavage. Of the seven propeptides tested, the propeptide proD from Streptomyces caniferus had the strongest suppressive effect on BTG activity (70 % of the activity suppressed). Moreover, fusion protein proD-BTG (containing proD) also exhibited the highest yield which was more than twofold of the expression level of BTG in an active form in Escherichia coli. Secretion expression of BTG and proD-BTG in Corynebacterium glutamicum further showed that our novel approach was suitable for the efficient BTG expression, thus providing a valuable platform for further optimization of large-scale BTG production.
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A highly active alpha amylase from Bacillus licheniformis: directed evolution, enzyme characterization and structural analysis.
J. Microbiol. Biotechnol.
PUBLISHED: 04-12-2014
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The stability of Bacillus licheniformis alpha-amylase (BLA) under acid condition was enhanced through direct evolution using the error-prone polymerase chain reaction. One beneficial mutation site, H281I, was obtained in BLA. The specific activity of H281I was 161/352 U/mg, which was 62.6/27.5% higher than that of the wild-type (WT) (99/276 U/mg) at pH 4.5/6.5 and 95°C. The pH optimum for H281I was decreased about 1 unit, whereas no significant changes of optimum temperature and thermostability were observed compared with the wild type (WT). The kcat/Km value of H281I was 1.7-/1.4-fold higher at pH 4.5/6.5, respectively, than that of WT. The structure model analysis indicated that the H281I mutation altered the predicted interaction between the amino acid residues at 281 and 273, thus creating a conducive local environment for substrate binding, as reflected by its decreased Km, and consequently increased the specific activity.
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Nitrosative stress and nitrated proteins in trichloroethene-mediated autoimmunity.
PLoS ONE
PUBLISHED: 01-01-2014
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Exposure to trichloroethene (TCE), a ubiquitous environmental contaminant, has been linked to a variety of autoimmune diseases (ADs) including SLE, scleroderma and hepatitis. Mechanisms involved in the pathogenesis of ADs are largely unknown. Earlier studies from our laboratory in MRL+/+ mice suggested the contribution of oxidative/nitrosative stress in TCE-induced autoimmunity, and N-acetylcysteine (NAC) supplementation provided protection by attenuating oxidative stress. This study was undertaken to further evaluate the contribution of nitrosative stress in TCE-mediated autoimmunity and to identify proteins susceptible to nitrosative stress. Groups of female MRL +/+ mice were given TCE, NAC or TCE + NAC for 6 weeks (TCE, 10 mmol/kg, i.p., every 4th day; NAC, ? 250 mg/kg/day via drinking water). TCE exposure led to significant increases in serum anti-nuclear and anti-histone antibodies together with significant induction of iNOS and increased formation of nitrotyrosine (NT) in sera and livers. Proteomic analysis identified 14 additional nitrated proteins in the livers of TCE-treated mice. Furthermore, TCE exposure led to decreased GSH levels and increased activation of NF-?B. Remarkably, NAC supplementation not only ameliorated TCE-induced nitrosative stress as evident from decreased iNOS, NT, nitrated proteins, NF-?B p65 activation and increased GSH levels, but also the markers of autoimmunity, as evident from decreased levels of autoantibodies in the sera. These findings provide support to the role of nitrosative stress in TCE-mediated autoimmune response and identify specific nitrated proteins which could have autoimmune potential. Attenuation of TCE-induced autoimmunity in mice by NAC provides an approach for designing therapeutic strategies.
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N-Acetylcysteine protects against trichloroethene-mediated autoimmunity by attenuating oxidative stress.
Toxicol. Appl. Pharmacol.
PUBLISHED: 07-02-2013
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Exposure to trichloroethene (TCE), a ubiquitous environmental contaminant, is known to induce autoimmunity both in humans and animal models. However, mechanisms underlying TCE-mediated autoimmunity remain largely unknown. Previous studies from our laboratory in MRL+/+ mice suggest that oxidative stress may contribute to TCE-induced autoimmune response. The current study was undertaken to further assess the role of oxidative stress in TCE-induced autoimmunity by supplementing with an antioxidant N-acetylcysteine (NAC). Groups of female MRL+/+ mice were given TCE, NAC or TCE+NAC for 6 weeks (TCE, 10mmol/kg, i.p., every 4th day; NAC, 250mg/kg/day through drinking water). TCE exposure led to significant increases in serum levels of anti-nuclear, anti-dsDNA and anti-Sm antibodies. TCE exposure also led to significant induction of anti-malondiadelhyde (MDA)- and anti-hydroxynonenal (HNE)-protein adduct antibodies which were associated with increased ANA in the sera along with increased MDA-/HNE-protein adducts in the livers and kidneys, and increases in protein oxidation (carbonylation) in the sera, livers and kidneys, suggesting an overall increase in oxidative stress. Moreover, TCE exposure also resulted in increased release of IL-17 from splenocytes and increases in IL-17 mRNA expression. Remarkably, NAC supplementation attenuated not only the TCE-induced oxidative stress, IL-17 release and mRNA expression, but also the markers of autoimmunity, as evident from decreased levels of ANA, anti-dsDNA and anti-Sm antibodies in the sera. These results provide further support to a role of oxidative stress in TCE-induced autoimmune response. Attenuation of TCE-induced autoimmunity in mice by NAC provides an approach for preventive and/or therapeutic strategies.
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Solvothermal synthesis of antimony sulfide dendrites for electrochemical detection of dopamine.
Dalton Trans
PUBLISHED: 07-02-2013
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Sb2S3 dendrites composed of 1-dimensional rods were prepared by a facile solvothermal reaction. The dosage of the poly(acrylic acid) (PAA) morphology-controlling reagent and the reaction temperature are key factors determining the final morphology of the product. Temporal experiments revealed that the formation of Sb2S3 dendrites experienced successive stages including precipitate reaction, crystallization and tip splitting. The as-prepared Sb2S3 dendrites were further employed as sensing material for electrochemical detection of dopamine (DA). A cyclic voltammogram (CV) showed that an Sb2S3 dendrite modified electrode enables the selective electro-oxidation of DA in the presence of ascorbic acid (AA). The constructed biosensor demonstrated a linear response range of 0.125-160 ?M and a detection limit of 0.1 ?M, which suggests that the Sb2S3 dendrites are promising sensing materials in the electrochemical analysis of DA.
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Improvement of alkali stability and thermostability of Paenibacillus campinasensis Family-11 xylanase by directed evolution and site-directed mutagenesis.
J. Ind. Microbiol. Biotechnol.
PUBLISHED: 06-22-2013
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The extreme process condition of high temperature and high alkali limits the applications of most of natural xylanases in pulp and paper industry. Recently, various methods of protein engineering have been used to improve the thermal and alkalic tolerance of xylanases. In this work, directed evolution and site-directed mutagenesis were performed to obtain a mutant xylanase improved both on alkali stability and thermostability from the native Paenibacillus campinasensis Family-11 xylanase (XynG1-1). Mutant XynG1-1B43 (V90R/P172H) with two units increased in the optimum pH (pH 7.0-pH 9.0) and significant improvement on alkali stability was selected from the second round of epPCR library. And the further thermoduric mutant XynG1-1B43cc16 (V90R/P172H/T84C-T182C/D16Y) with 10 °C increased in the optimum temperature (60-70 °C) was then obtained by introducing a disulfide bridge (T84C-T182C) and a single amino acid substitution (D16Y) to XynG1-1B43 using site-directed mutagenesis. XynG1-1B43cc16 also showed higher thermostability and catalytic efficiency (k cat /K m ) than that of wild-type (XynG1-1) and XynG1-1B43. The attractive improved properties make XynG1-1B43cc16 more suitable for bioleaching of cotton stalk pulp under the extreme process condition of high temperature (70 °C) and high alkali (pH 9.0).
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Advances in nano-scaled biosensors for biomedical applications.
Analyst
PUBLISHED: 06-10-2013
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Recently, a growing amount of attention has been focused on the utility of biosensors for biomedical applications. Combined with nanomaterials and nanostructures, nano-scaled biosensors are installed for biomedical applications, such as pathogenic bacteria monitoring, virus recognition, disease biomarker detection, among others. These nano-biosensors offer a number of advantages and in many respects are ideally suited to biomedical applications, which could be made as extremely flexible devices, allowing biomedical analysis with speediness, excellent selectivity and high sensitivity. This minireview discusses the literature published in the latest years on the advances in biomedical applications of nano-scaled biosensors for disease bio-marking and detection, especially in bio-imaging and the diagnosis of pathological cells and viruses, monitoring pathogenic bacteria, thus providing insight into the future prospects of biosensors in relevant clinical applications.
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[Rapid analysis of 28 primary aromatic amines in aqueous food simulants by high performance liquid chromatography-tandem mass spectrometry].
Se Pu
PUBLISHED: 05-15-2013
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A novel method for rapid analysis of the migration amounts of 28 primary aromatic amines (PAAs) in aqueous food simulants (10% ethanol, 30 g/L acetic acid and 20% ethanol aqueous solution) was developed using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). After the migration test, the soaking solution was cooled down from 100 degrees C, vortexed, filtered through a hydrophilic polytetrafluoroethylene filter with a disposable syringe, and then the filtrate was analyzed by HPLC-MS/MS. A Zorbax SB-Phenyl column (250 mm x 4.6 mm, 5 microm) was selected for chromatography. The mobile phase consisted of water and 0.1% formic acid-25% acetonitrile-methanol solution with gradient elution. The 28 PAAs in aqueous food simulants were detected by tandem mass spectrometer operated in positive electrospray ionization (ESI+) and multiple reaction monitoring (MRM) mode. The limits of quantification for the 28 PAAs were between 0.002 microg/L and 10 microg/L. The linearity of the method was good with correlation coefficients (r2) greater than 0.995 over the concentration range from 5 microg/L or 10 microg/L to 100 microg/L. The average recoveries of the 28 PAAs were between 76.6% and 114% with the relative standard deviations between 1.53% and 8.97% at the levels of 10, 20, and 40 microg/L. The method shows rapid pretreatment, the lower limits of quantification, good recoveries and accuracies, and meets the requirement of European Union (EU) No 10/2011 regulation for the specific migration of PAAs. The method has been applied to analyze the 28 PAAs in different aqueous food simulants from the migration test of 30 batches of food contact material samples exported to EU.
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Construction of engineered Arthrobacter simplex with improved performance for cortisone acetate biotransformation.
Appl. Microbiol. Biotechnol.
PUBLISHED: 05-09-2013
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Arthrobacter simplex 156 is a microorganism that is used for steroid drug biotransformation of cortisone acetate (CA) to prednisone acetate (PA). The enzyme 3-ketosteroid-?(1)-dehydrogenase encoded by the ksdD gene plays an important role in the bioconversion process. To further improve the biotransformation efficiencies of the industrial strain, a genetic manipulation system for A. simplex 156 was developed. Additional copies of the ksdD gene under the control of the cat promoter (from pXMJ19) were transferred into the strain A. simplex 156 and integrated into the 16S rDNA sites, yielding a series of recombinant strains. One of these recombinant strains, designated A. simplex M158, exhibited superior properties for CA biotransformation. At the substrate concentration of 83.6 g/l, the highest PA production of the recombinant strain reached 66.7 g/l, which is approximately 32.9 % higher than that of wild-type strains, and the incubation time for CA to PA bioconversion was reduced by 20 h. Southern blotting analysis of the recombinant strain indicated two copies of deregulated ksdD genes were integrated into the 16S rDNA sites, which means two of five 16S rRNA operons were insertionally disrupted in the recombinant strain. However, the disruption of the two 16S rRNA operons did not affect the growth rate of the recombinant strain, which survived and thrived under desired conditions. In addition, the new strain was genetically stable for more than 100 generations without the use of antibiotics for selection. These superior characteristics make the new strain more suitable than the wild-type strain for PA production.
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Permeability diagnosis model in drug discovery: a diagnostic tool to identify the most influencing properties for gastrointestinal permeability.
Curr Top Med Chem
PUBLISHED: 04-22-2013
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Permeability is important in governing the ability of drug substances to transport across gastrointestinal membrane and also crucial for proper drug distribution to pharmacological target organs and cells, and is therefore frequently utilized in drug discovery and development. In this report, we have performed a systematic analysis, using principal component analysis on the historically measured permeability data from in-house Caco-2 and parallel artificial membrane permeability assays on discovery new chemical entities from multiple projects. This work allows for establishment of a permeability diagnosis model by purposefully identifying most influencing physicochemical properties of the permeability issues, including polarity-lipophilicity line contributed primarily by polar surface area and LogP, number of rotation bond, fractional ionization at neutral pH and efflux ratio. A number of cases were also shown to demonstrate the applicability of the current model. The analysis of the model over internal drug discovery compounds exhibited promising diagnostic and predictive power of the model. The advantages and limitation of the model as well as the integral strategy to apply it in drug discovery to guide projects for permeability-related optimization were also presented.
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Mononuclear leukocyte infiltrate in extraplacental membranes and preterm delivery.
Am. J. Epidemiol.
PUBLISHED: 02-20-2013
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Large numbers of polymorphonuclear leukocytes in the amnion and chorion define histological chorioamnionitis (HCA), a condition linked to spontaneous preterm delivery (PTD). Less is known about placental patterns of mononuclear leukocyte (MNL) density and PTD. In this prospective study (1998-2004), women were sampled from 52 clinics in 5 Michigan communities and enrolled at 16-27 weeks gestation. HCA and MNL distributions in delivered placentas were evaluated microscopically in a subcohort (290 preterm, 823 term). Midpregnancy biomarkers from maternal blood (i.e., C-reactive protein (CRP), corticotropin-releasing hormone, and cytokines) were compared among term and PTD subjects grouped by presence/absence of HCA and high MNL density. A density of more than 10 MNLs per high-power field in the chorion of the membrane roll, referred to as MNL-CMR, was associated with medically indicated PTD (odds ratio = 2.2, 95% confidence interval: 1.3, 3.6) and spontaneous PTD (odds ratio = 2.5, 95% confidence interval: 1.7, 3.7). Associations persisted after removal of women with HCA-positive placentas, abruption, hypertensive disorders, or obesity. HCA-associated PTD showed higher CRP and cytokine levels. MNL-CMR-associated PTD showed higher CRP and corticotropin-releasing hormone levels. These data suggest that an MNL infiltrate in the chorion of the membrane roll marks PTD pathways that are distinct from HCA and not entirely explained by pregnancy complications.
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In vivo self-bio-imaging of tumors through in situ biosynthesized fluorescent gold nanoclusters.
Sci Rep
PUBLISHED: 01-29-2013
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Fluorescence imaging in vivo allows non-invasive tumor diagnostic thus permitting a direct monitoring of cancer therapies progresses. It is established herein that fluorescent gold nanoclusters are spontaneously biosynthesized by cancerous cell (i.e., HepG2, human hepatocarcinoma cell line; K562, leukemia cell line) incubated with micromolar chloroauric acid solutions, a biocompatible molecular Au(III) species. Gold nanoparticles form by Au(III) reduction inside cells cytoplasms and ultimately concentrate around their nucleoli, thus affording precise cell imaging. Importantly, this does not occur in non-cancerous cells, as evidenced with human embryo liver cells (L02) used as controls. This dichotomy is exploited for a new strategy for in vivo self-bio-imaging of tumors. Subcutaneous injections of millimolar chloroauric acid solution near xenograft tumors of the nude mouse model of hepatocellular carcinoma or chronic myeloid leukemia led to efficient biosynthesis of fluorescent gold nanoclusters without significant dissemination to the surrounding normal tissues, hence allowing specific fluorescent self-bio-marking of the tumors.
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Discovery and mechanism study of SIRT1 activators that promote the deacetylation of fluorophore-labeled substrate.
J. Med. Chem.
PUBLISHED: 01-28-2013
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SIRT1 is an NAD(+)-dependent deacetylase, whose activators have potential therapeutic applications in age-related diseases. Here we report a new class of SIRT1 activators. The activation is dependent on the fluorophore labeled to the substrate. To elucidate the activation mechanism, we solved the crystal structure of SIRT3/ac-RHKK(ac)-AMC complex. The structure revealed that the fluorophore blocked the H-bond formation and created a cavity between the substrate and the Rossmann fold. We built the SIRT1/ac-RHKK(ac)-AMC complex model based on the crystal structure. K(m) and K(d) determinations demonstrated that the fluorophore decreased the peptide binding affinity. The binding modes of SIRT1 activators indicated that a portion of the activators interacts with the fluorophore through ?-stacking, while the other portion inserts into the cavity or interacts with the Rossmann fold, thus increasing the substrate affinity. Our study provides new insights into the mechanism of SIRT1 activation and may aid the design of novel SIRT1 activators.
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Induction of base excision repair enzymes NTH1 and APE1 in rat spleen following aniline exposure.
Toxicol. Appl. Pharmacol.
PUBLISHED: 01-09-2013
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Mechanisms by which aniline exposure elicits splenotoxicity, especially a tumorigenic response, are not well-understood. Earlier, we have shown that aniline exposure leads to oxidative DNA damage and up-regulation of OGG1 and NEIL1/2 DNA glycosylases in rat spleen. However, the contribution of endonuclease III homolog 1 (NTH1) and apurinic/apyrimidinic endonuclease 1 (APE1) in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on examining whether NTH1 and APE1 contribute to the repair of oxidative DNA lesions in the spleen, in an experimental condition preceding tumorigenesis. To achieve this, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. By quantitating the cleavage products, the activities of NTH1 and APE1 were assayed using substrates containing thymine glycol (Tg) and tetrahydrofuran, respectively. Aniline treatment led to significant increases in NTH1- and APE1-mediated BER activity in the nuclear extracts of spleen of aniline-treated rats compared to the controls. NTH1 and APE1 mRNA expression in the spleen showed 2.9- and 3.2-fold increases, respectively, in aniline-treated rats compared to the controls. Likewise, Western blot analysis showed that protein expression of NTH1 and APE1 in the nuclear extracts of spleen from aniline-treated rats was 1.9- and 2.7-fold higher than the controls, respectively. Immunohistochemistry indicated that aniline treatment also led to stronger immunoreactivity for both NTH1 and APE1 in the spleens, confined to the red pulp areas. These results, thus, show that aniline exposure is associated with induction of NTH1 and APE1 in the spleen. The increased repair activity of NTH1 and APE1 could be an important mechanism for the removal of oxidative DNA lesions. These findings thus identify a novel mechanism through which NTH1 and APE1 may regulate the repair of oxidative DNA damage in aniline-induced splenic toxicity.
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Mid-pregnancy levels of angiogenic markers as indicators of pathways to preterm delivery.
J. Matern. Fetal. Neonatal. Med.
PUBLISHED: 10-24-2011
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To determine whether mid-pregnancy levels of angiogenic markers were associated with increased risk of preterm delivery (PTD).
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Protein adducts of malondialdehyde and 4-hydroxynonenal contribute to trichloroethene-mediated autoimmunity via activating Th17 cells: dose- and time-response studies in female MRL+/+ mice.
Toxicology
PUBLISHED: 09-22-2011
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Trichloroethene (TCE), a common occupational and environmental toxicant, is known to induce autoimmunity. Previous studies in our laboratory showed increased oxidative stress in TCE-mediated autoimmunity. To further establish the role of oxidative stress and to investigate the mechanisms of TCE-mediated autoimmunity, dose- and time-response studies were conducted in MRL+/+ mice by treating them with TCE via drinking water at doses of 0.5, 1.0 or 2.0mg/ml for 12, 24 or 36 weeks. TCE exposure led to dose-related increases in malondialdehyde (MDA)-/hydroxynonenal (HNE)-protein adducts and their corresponding antibodies in the sera and decreases in GSH and GSH/GSSG ratio in the kidneys at 24 and 36 weeks, with greater changes at 36 weeks. The increases in these protein adducts and decreases in GSH/GSSG ratio were associated with significant elevation in serum anti-nuclear- and anti-ssDNA-antibodies, suggesting an association between TCE-induced oxidative stress and autoimmune response. Interestingly, splenocytes from mice treated with TCE for 24 weeks secreted significantly higher levels of IL-17 and IL-21 than did splenocytes from controls after stimulation with MDA-mouse serum albumin (MSA) or HNE-MSA adducts. The increased release of these cytokines showed a dose-related response and was more pronounced in mice treated with TCE for 36 weeks. These studies provide evidence that MDA- and or HNE-protein adducts contribute to TCE-mediated autoimmunity, which may be via activation of Th17 cells.
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Predicting phospholipidosis: a fluorescence noncell based in vitro assay for the determination of drug-phospholipid complex formation in early drug discovery.
Anal. Chem.
PUBLISHED: 08-19-2011
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This paper describes for the first time, a high-throughput fluorescence noncell based assay to screen for the drug-phospholipid interaction, which correlates to phospholipidosis. Anionic amphiphilic phospholipids can form complexes in aqueous solution, and its critical micelle concentration (CMC) can be determined using the fluorescence probe N,N-dimethyl-6-propionyl-2-naphthylamine (Prodan). Upon interaction with drug candidates, this CMC may shift to a lower value due to the association between lipids and drug candidates, the stronger the interaction, the greater the shift. Metabolism of a drug can change the degree of phospholipidosis depending on the rate of metabolism and the nature of the metabolite(s). Our data from 45 drugs and metabolites of 10 drugs using this fluorescence approach demonstrate a good correlation with phospholipidosis as reported with human studies, in vivo testing, and cellular assays. This assay therefore offers a fast, reliable, and cost-effective screening tool for early prediction of the phospholipidosis-inducing potential of drug candidates.
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Imidazolopiperazines: hit to lead optimization of new antimalarial agents.
J. Med. Chem.
PUBLISHED: 06-23-2011
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Starting from a hit series from a GNF compound library collection and based on a cell-based proliferation assay of Plasmodium falciparum, a novel imidazolopiperazine scaffold was optimized. SAR for this series of compounds is discussed, focusing on optimization of cellular potency against wild-type and drug resistant parasites and improvement of physiochemical and pharmacokinetic properties. The lead compounds in this series showed good potencies in vitro and decent oral exposure levels in vivo. In a Plasmodium berghei mouse infection model, one lead compound lowered the parasitemia level by 99.4% after administration of 100 mg/kg single oral dose and prolonged mice survival by an average of 17.0 days. The lead compounds were also well-tolerated in the preliminary in vitro toxicity studies and represents an interesting lead for drug development.
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Efficient expression of an alkaline pectate lyase gene from Bacillus subtilis and the characterization of the recombinant protein.
Biotechnol. Lett.
PUBLISHED: 06-06-2011
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The gene encoding a novel alkaline pectate lyase (Apel) from Bacillus subtilis was cloned and expressed in B. subtilis WB600. Apel contained an ORF of 1,260 bp, encoding a signal peptide of 21 amino acids and a mature protein of 399 amino acids with a calculated molecular mass of 45497.9 Da. The mature Apel was structurally related to the enzymes in the polysaccharide lyase family 1. After purification, the recombinant Apel had a specific activity of 445 U mg(-1). The enzyme was optimally active at 50°C and pH 9.
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Aniline-induced nitrosative stress in rat spleen: proteomic identification of nitrated proteins.
Toxicol. Appl. Pharmacol.
PUBLISHED: 05-03-2011
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Aniline exposure is associated with toxicity to the spleen which is characterized by splenomegaly, hyperplasia, fibrosis, and a variety of sarcomas on chronic exposure in rats. However, mechanisms by which aniline elicits splenotoxic responses are not well understood. Earlier we have shown that aniline exposure leads to increased nitration of proteins in the spleen. However, nitrated proteins remain to be characterized. Therefore, in the current study using proteomic approaches, we focused on characterizing the nitrated proteins in the spleen of aniline-exposed rats. Aniline exposure led to increased tyrosine nitration of proteins, as determined by 2D Western blotting with anti-3-nitrotyrosine specific antibody, compared to the controls. The analyzed nitrated proteins were found in the molecular weight range of 27.7 to 123.6kDa. A total of 37 nitrated proteins were identified in aniline-treated and control spleens. Among them, 25 were found only in aniline-treated rats, 11 were present in both aniline-treated and control rats, while one was found in controls only. The nitrated proteins identified mainly represent skeletal proteins, chaperones, ferric iron transporter, enzymes, nucleic acids binding protein, and signaling and protein synthesis pathways. Furthermore, aniline exposure led to significantly increased iNOS mRNA and protein expression in the spleen, suggesting its role in increased reactive nitrogen species formation and contribution to increased nitrated proteins. The identified nitrated proteins provide a global map to further investigate alterations in their structural and functional properties, which will lead to a better understanding of the role of protein nitration in aniline-mediated splenic toxicity.
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Maximizing the outcome of early ADMET models: strategies to win the drug-hunting battles?
Expert Opin Drug Metab Toxicol
PUBLISHED: 03-19-2011
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Despite substantial changes in the drug discovery paradigm leveraged from the advancement of early ADMET technologies, an open debate remains on how full the return on investment is, along with where to balance risks to costs of lost opportunities in the clinic. Here, the recent advancement of ADMET tools, the areas where they seem to work and where their application and connection with physiology in man remain challenging are briefly reviewed. While the more is better type of box-checking profiling strategy is no longer viable, the key to success lies in an intelligent integration of existing in silico, in vitro and in vivo ADMET data to help generate and test hypotheses that are critical for projecting the benefits and risks of a drug candidate in the clinic. The improvement of in silico, in vitro and in vivo correlations (ISIVIVC) and best utilization of early ADMET data are far more critical and urgent than expanding capacity and portfolio in leveraging ADMET to win the drug-hunting battles in the post-genome era.
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Late-preterm birth and its association with cognitive and socioemotional outcomes at 6 years of age.
Pediatrics
PUBLISHED: 11-22-2010
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Late-preterm birth (34-36 weeks gestation) has been associated with a risk for long-term cognitive and socioemotional problems. However, many studies have not incorporated measures of important contributors to these outcomes, and it is unclear whether effects attributed to gestational age are separate from fetal growth or its proxy, birth weight for gestational age.
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Attenuation of intestinal absorption by major efflux transporters: quantitative tools and strategies using a Caco-2 model.
Drug Metab. Dispos.
PUBLISHED: 11-04-2010
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Efflux transporters expressed in the apical membrane of intestinal enterocytes have been implicated in drug oral absorption. The current study presents a strategy and tools to quantitatively predict the impact of efflux on oral absorption for new chemical entities (NCEs) in early drug discovery. Sixty-three marketed drugs with human absorption data were evaluated in the Caco-2 bidirectional permeability assay and subjected to specific transporter inhibition. A four-zone graphical model was developed from apparent permeability and efflux ratios to quickly identify compounds whose efflux activity may distinctly influence human absorption. NCEs in "zone 4" will probably have efflux as a barrier for oral absorption and further mechanistic studies are required. To interpret mechanistic results, we introduced a new quantitative substrate classification parameter, transporter substrate index (TSI). TSI allowed more flexibility and considered both in vitro and in vivo outcomes. Its application ranged from addressing the challenge of overlapping substrate specificity to projecting the role of transporter(s) on exposure or potential drug-drug interaction risk. The potential impact of efflux transporters associated with physicochemical properties on drug absorption is discussed in the context of TSI and also the previously reported absorption quotient. In this way, the chemistry strategy may be differentially focused on passive permeability or efflux activity or both.
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Induction of NEIL1 and NEIL2 DNA glycosylases in aniline-induced splenic toxicity.
Toxicol. Appl. Pharmacol.
PUBLISHED: 09-22-2010
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The mechanisms by which aniline exposure elicits splenotoxic response, especially the tumorigenic response, are not well-understood. Earlier, we have shown that aniline-induced oxidative stress is associated with increased oxidative DNA damage in rat spleen. The base excision repair (BER) pathway is the major mechanism for the repair of oxidative DNA base lesions, and we have shown an up-regulation of 8-oxoguanine glycosylase 1 (OGG1), a specific DNA glycosylase involved in the removal of 8-hydroxy-2-deoxyguanosine (8-OHdG) adducts, following aniline exposure. Nei-like DNA glycosylases (NEIL1/2) belong to a family of BER proteins that are distinct from other DNA glycosylases, including OGG1. However, contribution of NEIL1/2 in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on evaluating if NEILs also contribute to the repair of oxidative DNA lesions in the spleen following aniline exposure. To achieve that, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. The BER activity of NEIL1/2 was assayed using a bubble structure substrate containing 5-OHU (preferred substrates for NEIL1 and NEIL2) and by quantitating the cleavage products. Aniline treatment led to a 1.25-fold increase in the NEIL1/2-associated BER activity in the nuclear extracts of spleen compared to the controls. Real-time PCR analysis for NEIL1 and NEIL2 mRNA expression in the spleen revealed 2.7- and 3.9-fold increases, respectively, in aniline-treated rats compared to controls. Likewise, Western blot analysis showed that protein expression of NEIL1 and NEIL2 in the nuclear extract of spleens from aniline-treated rats was 2.0- and 3.8-fold higher than controls, respectively. Aniline treatment also led to stronger immunoreactivity for NEIL1 and NEIL2 in the spleens, confined to the red pulp areas. These studies, thus, show that aniline-induced oxidative stress is associated with an induction of NEIL1/2. The increased NIEL-mediated BER activity is another indication of aniline-induced oxidative damage in the spleen and could constitute another important mechanism of removal of oxidative DNA lesions, especially in transcribed DNA following aniline insult.
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Enhanced expression of cyclins and cyclin-dependent kinases in aniline-induced cell proliferation in rat spleen.
Toxicol. Appl. Pharmacol.
PUBLISHED: 08-27-2010
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Aniline exposure is associated with toxicity to the spleen leading to splenomegaly, hyperplasia, fibrosis and a variety of sarcomas of the spleen on chronic exposure. In earlier studies, we have shown that aniline exposure leads to iron overload, oxidative stress and activation of redox-sensitive transcription factors, which could regulate various genes leading to a tumorigenic response in the spleen. However, molecular mechanisms leading to aniline-induced cellular proliferation in the spleen remain largely unknown. This study was, therefore, undertaken on the regulation of G1 phase cell cycle proteins (cyclins), expression of cyclin-dependent kinases (CDKs), phosphorylation of retinoblastoma protein (pRB) and cell proliferation in the spleen, in an experimental condition preceding a tumorigenic response. Male SD rats were treated with aniline (0.5 mmol/kg/day via drinking water) for 30 days (controls received drinking water only), and splenocyte proliferation, protein expression of G1 phase cyclins, CDKs and pRB were measured. Aniline treatment resulted in significant increases in splenocyte proliferation, based on cell counts, cell proliferation markers including proliferating cell nuclear antigen (PCNA), nuclear Ki67 protein (Ki67) and minichromosome maintenance (MCM), MTT assay and flow cytometric analysis. Western blot analysis of splenocyte proteins from aniline-treated rats showed significantly increased expression of cyclins D1, D2, D3 and E, as compared to the controls. Similarly, real-time PCR analysis showed significantly increased mRNA expression for cyclins D1, D2, D3 and E in the spleens of aniline-treated rats. The overexpression of these cyclins was associated with increases in the expression of CDK4, CDK6, CDK2 as well as phosphorylation of pRB protein. Our data suggest that increased expression of cyclins, CDKs and phosphorylation of pRB protein could be critical in cell proliferation, and may contribute to aniline-induced tumorigenic response in the spleen.
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Midpregnancy levels of angiogenic markers in relation to maternal characteristics.
Am. J. Obstet. Gynecol.
PUBLISHED: 06-14-2010
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The purpose of this study was to describe relations among maternal demographic and lifestyle characteristics and midpregnancy levels of angiogenic markers (soluble Fms-like tyrosine kinase-1, placental growth factor, soluble endoglin).
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[Construction of eukaryotic expression vector of EGFRi-IL-24 recombinant gene].
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi
PUBLISHED: 05-21-2010
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The epithelial growth factor receptor interference (EGFRi) was obtained by synthetic primers. Overlapping PCR was used to produce EGFRi-IL-24 fusion gene, which is linked by Gly4Ser3. After sequence analysis, EGFRi-IL-24 was cloned into expression vector pPIC9k; EGFRi-IL-24/pPIC9k was linearized with SacI,and then transformed to electroporated pastoris GS115. Subsequently, positive clone was selected by G418 and PCR, and its phenotype was determined by SDS-PAGE and MTT assay. The results demonstrated that EGFRi-IL-24 protein was expressed and shown to have the potential for use in researches of its biological function and in clinical application.
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Towards prediction of in vivo intestinal absorption using a 96-well Caco-2 assay.
J Pharm Sci
PUBLISHED: 02-19-2010
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We systematically validated a robust 96-well Caco-2 assay via an extended set of 93 marketed drugs with diverse transport mechanisms and quantified by LC/MS/MS, to investigate its predictive utility while dealing with challenging discovery compounds. Utilizing nonlinear fit, the validation led to a good correlation (R(2) = 0.76) between absorptive permeability, log P(app)(A-B), from in vitro Caco-2 assay and reported human fraction of dose absorbed. We observed that paracellular compounds could be flagged by log P(app)(A-B) (<-5.5 cm/s) and physicochemical property space (c log P < 1). Of 8000 Novartis discovery compounds examined 13% were subject to low recovery (<30%). Compound loss was investigated by comparing cell monolayer and artificial membrane, while 0.5% bovine serum albumin (in both donor and acceptor compartments) was utilized to improve recovery. The second focus of this study was to investigate the advantages and limitations of the current Caco-2 assay for predicting in vivo intestinal absorption. Caco-2 measurements for compounds with high aqueous solubility and low in vitro metabolic clearance were compared to 88 in vivo rat bioavailability studies. Despite the challenges posed by discovery compounds with suboptimal physicochemical properties, Caco-2 data successfully projected low intestinal absorption. This platform sets the stage for mechanistically evaluating compounds towards improving in vitro-in vivo correlations.
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Mitigating permeability-mediated risks in drug discovery.
Expert Opin Drug Metab Toxicol
PUBLISHED: 01-13-2010
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Adequate permeability is essential for good oral absorption and proper interpretation of pharmacokinetic and pharmacological data. Permeability-limiting absorption and distribution, however, are complex phenomena involving multiple mechanisms and organs ranging from gastro-intestine, liver, kidney to BBB, while the options for pharmaceutical improvement of permeability are quite limited.
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Recent advances in physicochemical and ADMET profiling in drug discovery.
Chem. Biodivers.
PUBLISHED: 11-26-2009
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The drastic increase in the cost for discovering and developing a new drug along with the high attrition rate of development candidates led to shifting drug-discovery strategy to parallel assessment of comprehensive drug physicochemical, and absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties alongside efficacy. With the proposal of a profiling paradigm and utilization of integrated risk assessment, one can exponentially enhance the predictive power of in vitro tools by taking into consideration the interplay among profiling parameters. In particular, this article will review recent advances in accurate assessment of solubility and other physicochemical parameters. The proper interpretation of these experimental data is crucial for rapid and meaningful risk assessment and rational optimization of drug candidates in drug discovery. The impact of these tools on assisting drug-discovery teams in establishing in vitro-in vivo correlation (IVIVC) as well as structure-property relationship (SPR) will be presented.
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Up-regulation of heme oxygenase-1 in rat spleen after aniline exposure.
Free Radic. Biol. Med.
PUBLISHED: 09-21-2009
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The splenic toxicity of aniline is characterized by vascular congestion, hyperplasia, fibrosis, and the development of a variety of sarcomas in rats. However, the underlying mechanisms by which aniline elicits splenotoxic response are not well understood. Previously we have shown that aniline exposure causes oxidative damage to the spleen. To further explore the oxidative mechanism of aniline toxicity, we evaluated the potential contribution of heme oxygenase-1 (HO-1), which catalyzes heme degradation and releases free iron. Male SD rats were given 1 mmol/kg/day aniline in water by gavage for 1, 4, or 7 days, and respective controls received water only. Aniline exposure led to significant increases in HO-1 mRNA expression in the spleen (2-and 2.4-fold at days 4 and 7, respectively) with corresponding increases in protein expression, as confirmed by ELISA and Western blot analysis. Furthermore, immunohistochemical assessment of spleen showed stronger immunostaining for HO-1 in the spleens of rats treated for 7 days, confined mainly to the red pulp areas. No changes were observed in mRNA and protein levels of HO-1 after 1 day exposure. The increase in HO-1 expression was associated with increases in total iron (2.4-and 2.7-fold), free iron (1.9-and 3.5-fold), and ferritin levels (1.9-and 2.1-fold) at 4 and 7 days of aniline exposure. Our data suggest that HO-1 up-regulation in aniline-induced splenic toxicity could be a contributing pro-oxidant mechanism, mediated through iron release, and leading to oxidative damage.
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[Effect of Panax notoginseng saponins on vascular intima hyperplasia and PCNA expression in rat aorta after balloon angioplasty].
Zhongguo Zhong Yao Za Zhi
PUBLISHED: 07-24-2009
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To explore the effect and the mechanism of panax notoginseng saponins (PNS) on the vascular intima hyperplasia and expression of proliferating cell nuclear antigen (PCNA) after aortic intima injury induced ballcon in rats.
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Comprehensive assessment of ADMET risks in drug discovery.
Curr. Pharm. Des.
PUBLISHED: 07-16-2009
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The high attrition rate in drug development and the deteriorated drug ability as a result of the shifted chemical space of new therapeutic target for unmet medical needs have posed drastic challenges in current drug discovery. It has triggered the strategic transition in the past decade into parallel assessment of efficacy and comprehensive ADMET (absorption, distribution, metabolism, elimination and toxicity) properties of new chemical entities (NCEs) in the lead selection and optimization stages, to convert chemically a problematic NCE to an "all-around" candidate. This review summarizes multiple in silico, in vitro and in vivo ADMET filters developed and implemented in various stages of drug discovery to flag potential ADMET issues in the clinic. The full awareness of the benefits and limitations of each tool assures right questions to be answered using right tools at right time. The integrated ADMET risk assessment will allow project teams to have a clear vision in terms of the competitive position of own NCEs against comparable marketed drugs.
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High-level expression, purification and characterization of a recombinant medium-temperature alpha-amylase from Bacillus subtilis.
Biotechnol. Lett.
PUBLISHED: 05-08-2009
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Alpha-amylases are important industrial enzymes with a wide range of applications. Although medium-temperature alpha amylase (AmyE) has some practical advantages, its low yield has limited its applications. When an amyE gene from Bacillus subtilis BF768 was cloned into vector pWB980 and over-expressed in B. subtilis WB600, high activities (723 U ml(-1)) of secreted AmyE were produced. Recombinant AmyE was purified to a specific activity of 36 U mg(-1) having optimal activity at pH 6.0 and 60 degrees C.
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Increased nitration and carbonylation of proteins in MRL+/+ mice exposed to trichloroethene: potential role of protein oxidation in autoimmunity.
Toxicol. Appl. Pharmacol.
PUBLISHED: 01-16-2009
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Even though reactive oxygen and nitrogen species (RONS) are implicated as mediators of autoimmune diseases (ADs), little is known about contribution of protein oxidation (carbonylation and nitration) in the pathogenesis of such diseases. The focus of this study was, therefore, to establish a link between protein oxidation and induction and/or exacerbation of autoimmunity. To achieve this, female MRL +/+ mice were treated with trichloroethene (TCE), an environmental contaminant known to induce autoimmune response, for 6 or 12 weeks (10 mmol/kg, i.p., every 4(th) day). TCE treatment resulted in significantly increased formation of nitrotyrosine (NT) and induction of iNOS in the serum at both 6 and 12 weeks of treatment, but the response was greater at 12 weeks. Likewise, TCE treatment led to greater NT formation, and iNOS protein and mRNA expression in the livers and kidneys. Moreover, TCE treatment also caused significant increases ( approximately 3 fold) in serum protein carbonyls (a marker of protein oxidation) at both 6 and 12 weeks. Significantly increased protein carbonyls were also observed in the livers and kidneys (2.1 and 1.3 fold, respectively) at 6 weeks, and to a greater extent at 12 weeks (3.5 and 2.1 fold, respectively) following TCE treatment. The increases in TCE-induced protein oxidation (carbonylation and nitration) were associated with significant increases in Th1 specific cytokine (IL-2, IFN-gamma) release into splenocyte cultures. These results suggest an association between protein oxidation and induction/exacerbation of autoimmune response. The results present a potential mechanism by which oxidatively modified proteins could contribute to TCE-induced autoimmune response and necessitates further investigations for clearly establishing the role of protein oxidation in the pathogenesis of ADs.
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Layer-by-layer assembly of graphene, Au and poly(toluidine blue O) films sensor for evaluation of oxidative stress of tumor cells elicited by hydrogen peroxide.
Biosens Bioelectron
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High level of oxidative stress is involved in formation of incipient tumor and carcinomatous cells. Here in this contribution we have explored a facile strategy to assess the oxidative stress elicited by hydrogen peroxide (H(2)O(2)) in cells with amperometric current-time technique in vitro. An electrochemical biosensor exhibiting high sensitivity and selectivity to H(2)O(2) is fabricated by integration of graphene with gold nanoparticles and poly(toluidine blue O) films. The efflux of H(2)O(2) from several representative tumor cells and normal cells on exposure to ascorbic acid could be detected by using the graphene-based nanocomposite films. The results indicate that tumor cells release much more H(2)O(2) than do the normal cells. The novel sensor raises the possibility for clinical diagnostic application to evaluate the higher level of intracellular oxidative stress of tumor cells in comparison with normal cells.
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Overexpression of a Paenibacillus campinasensis xylanase in Bacillus megaterium and its applications to biobleaching of cotton stalk pulp and saccharification of recycled paper sludge.
Bioresour. Technol.
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A xylanase gene (xynG1-1) from Paenibacillus campinasensis G1-1 was expressed in Bacillus megaterium MS941 and a high level of extracellular xylansae activity (304.26 IU/mL) was achieved after induction with 0.5% xylose. The purified recombinant xylanase (XynG1-1R) revealed optimal activity at 60°C and pH 7.0 and retained 79% and 81% activity after incubation without substrate at 60°C, pH 5.0 and pH 8.0 for 3h, respectively. Application of XynG1-1R (15 IU/g pulp) to cotton stalk pulp bleaching increased brightness by 3.65% over that of the control without the xylanase and reduced the need for chlorine compounds by 50% without loss of brightness and pulp fiber qualities. When XynG1-1R (80 IU/g paper sludge) was used in combination with mixed cellulolytic enzymes, the saccharification efficiency of recycled paper sludge was increased by 10%. These results indicated that XynG1-1R is a promising candidate for various industrial applications such as biobleaching and bioenergy conversion.
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Photoelectrocatalytic oxidation of glutathione based on porous TiO2-Pt nanowhiskers.
Langmuir
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The performance of TiO(2) nanoparticles is extremely attractive in various areas of chemical and biochemical engineering as they can effectively work by combining the photocatalytic property with various superior properties of the related nanostructure. The relevant photoelectrochemical detection has attracted considerable interest and shown potential applications in a wide range of areas. In this study, we have prepared new nanowhiskers of platinum-doped titanium dioxide (TiO(2)-Pt), which could be further used to fabricate a novel nanointerface for the sensitive detection of biomolecules including glutathione (GSH). Our observations demonstrate that the sensitive TiO(2)-Pt nanowhiskers biointerface could be readily fabricated by casting the TiO(2)-Pt nanowhiskers suspension on a glassy carbon electrode (GCE), which could readily combine the photocatalytic and eletrocatalytic properties of TiO(2) nanocomposites to introduce a novel photoelectrocatalytic biosensor for GSH detection in real samples. Compared to other analysis strategies, the TiO(2)-Pt nanowhiskers-modified GCE showed a considerably high sensitivity for the detection of GSH due to the excellent photoelectrocatalytic ability of the porous TiO(2)-Pt nanowhiskers. Scanning electron microscopy (SEM), Raman spectroscopy, and electrochemical impedance spectroscopy have shown that Pt can readily blend with porous TiO(2) nanowhiskers and facilitate the relevant catalysis property of TiO(2), resulting in the enhanced photoelectrocatalytic effect. Thus, through the new strategy of the utilization of the excellent photoelectrocatalytic property of TiO(2)-Pt nanocomposites, it is possible to realize the rapid electrochemical detection of glutathione with high sensitivity, low cost, and good reproducibility.
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Probe ADME and test hypotheses: a PATH beyond clearance in vitro-in vivo correlations in early drug discovery.
Expert Opin Drug Metab Toxicol
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In vitro cytochrome P450 (CYP450) metabolic profiling is pursued extensively to optimize drug properties. Still, the in vivo clearance of half of all new chemical entities (NCEs) remains poorly predicted by CYP450 metabolism, based on Novartis rat pharmacokinetic data. The conventional route to illuminating key drivers of in vivo clearance beyond hepatic metabolism is, frequently, the process of elimination, a time-consuming and sometimes resource-intensive practice. A more nimble and efficient diagnosis of drug clearance is imperative to support todays chemistry optimization.
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Isolation, purification, and characterization of a thermostable xylanase from a novel strain, Paenibacillus campinasensis G1-1.
J. Microbiol. Biotechnol.
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High levels of xylanase activity (143.98 IU/ml) produced by the newly isolated Paenibacillus campinasensis G1-1 were detected when it was cultivated in a synthetic medium. A thermostable xylanase, designated XynG1-1, from P. campinasensis G1-1 was purified to homogeneity by Octyl-Sepharose hydrophobic-interaction chromatography, Sephadex G75 gel-filter chromatography, and Q-Sepharose ion-exchange chromatography, consecutively. By multistep purification, the specific activity of XynG1-1 was up to 1,865.5 IU/mg with a 9.1-fold purification. The molecular mass of purified XynG1-1 was about 41.3 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sequence analysis revealed that XynG1-1 containing 377 amino acids encoded by 1,134 bp genomic sequences of P. campinasensis G1-1 shared 96% homology with XylX from Paenibacillus campinasensis BL11 and 77%~78% homology with xylanases from Bacillus sp. YA- 335 and Bacillus sp. 41M-1, respectively. The activity of XynG1-1 was stimulated by Ca2+, Ba2+, DTT, and beta- mercaptoethanol, but was inhibited by Ni2+, Fe2+, Fe3+, Zn2+, SDS, and EDTA. The purified XynG1-1 displayed a greater affinity for birchwood xylan, with an optimal temperature of 60 degrees C and an optimal pH of 7.5. The fact that XynG1-1 is cellulose-free, thermostable (stability at high temperature of 70 degrees C~80 degrees C), and active over a wide pH range (pH 5.0~9.0) suggests that the enzyme is potentially valuable for various industrial applications, especially for pulp bleaching pretreatment.
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Imidazolopiperazines: lead optimization of the second-generation antimalarial agents.
J. Med. Chem.
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On the basis of the initial success of optimization of a novel series of imidazolopiperazines, a second generation of compounds involving changes in the core piperazine ring was synthesized to improve antimalarial properties. These changes were carried out to further improve the potency and metabolic stability of the compounds by leveraging the outcome of a set of in vitro metabolic identification studies. The optimized 8,8-dimethyl imidazolopiperazine analogues exhibited improved potency, in vitro metabolic stability profile and, as a result, enhanced oral exposure in vivo in mice. The optimized compounds were found to be more efficacious than the current antimalarials in a malaria mouse model. They exhibit moderate oral exposure in rat pharmacokinetic studies to achieve sufficient multiples of the oral exposure at the efficacious dose in toxicology studies.
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Assessment of biliary clearance in early drug discovery using sandwich-cultured hepatocyte model.
J Pharm Sci
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It is challenging to predict biliary clearance (CL(b) ) for new chemical entities (NCEs) in early drug discovery. Although sandwich-cultured hepatocyte (SCH) model has offered a valuable tool for characterizing hepatobiliary disposition and drug-drug interaction potential of NCEs, no comprehensive study was reported to project in vivo biliary clearance (in vivo CL(b,observed) ) potential using in vitro SCH model during the drug discovery stage. In this study, the CL(b) of 110 discovery compounds was evaluated using rat SCH model. Parallel artificial membrane permeability assay, Caco-2, and rat liver microsomes were employed in parallel to explore the interplay of biliary excretion with cellular permeability and liver metabolism. Selected compounds were further tested in bile-duct-cannulated rats, confirming the value of the SCH model for ranking and predicting in vivo CL(b,observed) during drug discovery. For compounds with extremely low passive permeability and metabolism, rat SCH may underestimate in vivo CL(b,observed) . The combination of passive permeability, metabolic intrinsic clearance, and the SCH model could serve as an initial screening platform for biliary excretion potential as well as a means for improving compound liabilities and properties. A preliminary evaluation strategy was proposed to highlight biliary excretion risk evaluation during the drug discovery process.
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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.