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Horseradish Peroxidase (horseradish + peroxidase)
Selected AbstractsCyclodextrins in Polymer Synthesis: Enzymatic Polymerization of a 2,6-Dimethyl- , -Cyclodextrin/2,4-Dihydroxyphenyl-4,-Hydroxybenzylketone Host-Guest Complex Catalyzed by Horseradish Peroxidase (HRP)MACROMOLECULAR BIOSCIENCE, Issue 8 2003Lorenzo Mejias Abstract This paper reports the enzymatic polymerization of the inclusion complex 2,4-dihydroxyphenyl-4,-hydroxybenzylketone/2,6-dimethyl- , -cyclodextrin by horseradish peroxidase (HRP) in aqueous media. The structure of the complex was determined by means of NOESY-NMR and crystallographic analysis (indicating an orthorhombic structure). The enzymatic polymerization of the uncomplexed 2,4-dihydroxyphenyl-4,-hydroxybenzylketone yields oligomers with molecular weights up to in organic-aqueous media, but because of its poor solubility in aqueous systems, no polymerization is observed if water is used as solvent. An increase of the availability of the ketone in solution is achieved by complexing it with random-methylated , -cyclodextrin in water. We found that the use of methylated , -cyclodextrin in equimolar concentration to the monomer increases the polymerization yield and the average molecular weight. The polymers formed were analyzed by GPC and ATR-FTIR techniques. Representation from X-ray diffraction analysis of the 2,6-dimethyl- , -cyclodextrin/2,4-dihydroxyphenyl-4,-hydroxybenzylketone host-guest complex (3). [source] Flexibility in Proteins: Tuning the Sensitivity to O2 Diffusion by Varying the Lifetime of a Phosphorescent Sensor in Horseradish Peroxidase,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2004Janna Nibbs ABSTRACT The heme in horseradish peroxidase (HRP) was replaced by phosphorescent Pt-mesoporphyrin IX (PtMP), which acted as a phosphorescent marker of oxygen quenching and allowed comparison with another probe, Pd-mesoporphyrin IX (Khajehpour et al. (2003) Proteins 53, 656,666). Benzohydroxamic acid (BHA), a competitive inhibitor of the enzyme, was also used to monitor its effects on phosphorescence quenching. With the addition of BHA, in the presence of oxygen, the phosphorescence intensity of the protein increased. In contrast, the addition of BHA, in the absence of oxygen, reduced the phosphorescence intensity of the protein. Kd= 18 ,M when BHA binds to PtMP-HRP. The effect of BHA can be explained by two factors: (1) BHA reduces the accessibility of O2 to the protein interior and (2) BHA itself quenches the phosphorescence. Consistent with this, the oxygen quenching of the phosphorescence of PtMP-HRP gave a quenching constant of kq= 234 mm Hg,1 s,1 in the absence of BHA and kq= 28.7 mm Hg,1 s,1 in the presence of BHA. The quenching rate of BHA is 4000 s,1. The relative quantum yield of the phosphorescence of the Pt derivative is about six times that of the Pd derivative, whereas the phosphorescence lifetime is approximately eight times shorter. The high quantum yield and suitable lifetime make Pt-porphyrins appropriate as sensors of O2 diffusion and flexibility in heme proteins. [source] ICPLQuant , A software for non-isobaric isotopic labeling proteomicsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2010Achim Brunner Abstract The main goal of many proteomics experiments is an accurate and rapid quantification and identification of regulated proteins in complex biological samples. The bottleneck in quantitative proteomics remains the availability of efficient software to evaluate and quantify the tremendous amount of mass spectral data acquired during a proteomics project. A new software suite, ICPLQuant, has been developed to accurately quantify isotope-coded protein label (ICPL)-labeled peptides on the MS level during LC-MALDI and peptide mass fingerprint experiments. The tool is able to generate a list of differentially regulated peptide precursors for subsequent MS/MS experiments, minimizing time-consuming acquisition and interpretation of MS/MS data. ICPLQuant is based on two independent units. Unit 1 performs ICPL multiplex detection and quantification and proposes peptides to be identified by MS/MS. Unit 2 combines MASCOT MS/MS protein identification with the quantitative data and produces a protein/peptide list with all the relevant information accessible for further data mining. The accuracy of quantification, selection of peptides for MS/MS-identification and the automated output of a protein list of regulated proteins are demonstrated by the comparative analysis of four different mixtures of three proteins (Ovalbumin, Horseradish Peroxidase and Rabbit Albumin) spiked into the complex protein background of the DGPF Proteome Marker. [source] On-plate-selective enrichment of glycopeptides using boronic acid-modified gold nanoparticles for direct MALDI-QIT-TOF MS analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 22 2009Jia Tang Abstract In this study, an on-plate-selective enrichment method is developed for fast and efficient glycopeptide investigation. Gold nanoparticles were first spotted and sintered on a stainless-steel plate, then modified with 4-mercaptophenylboronic acid to provide porous substrate with large specific surface and dual functions. These spots were used to selectively capture glycopeptides from peptide mixtures and the captured target peptides could be analyzed by MALDI-MS simply by deposition of 2,5-dihydroxybenzoic acid matrix. Horseradish peroxidase was employed as a standard glycoprotein to investigate the enrichment efficiency. In this way, the enrichment, washing and detection steps can all be fulfilled on a single MALDI target plate. The relatively small sample amount needed, low detection limit and rapid selective enrichment have made this on-plate strategy promising for online enrichment of glycopeptides, which could be applied in high-throughput proteome research. [source] A New Amperometric Biosensor Based on HRP/Nano-Au/L -Cysteine/Poly(o -Aminobenzoic acid)-Membrane-Modified Platinum Electrode for the Determination of Hydrogen PeroxideCHINESE JOURNAL OF CHEMISTRY, Issue 11 2006Ming-Yu Tang Abstract The third generation amperometric biosensor for the determination of hydrogen peroxide (H2O2) has been described. For the fabrication of biosensor, o -aminobenzoic acid (oABA) was first electropolymerized on the surface of platinum (Pt) electrode as an electrostatic repulsion layer to reject interferences. Horseradish peroxidase (HRP) absorbed by nano-scaled particulate gold (nano-Au) was immobilized on the electrode modified with polymerized o -aminobenzoic acid (poABA) with L -cysteine as a linker to prepare a biosensor for the detection of H2O2. Amperometric detection of H2O2 was realized at a potential of +20 mV versus SCE. The resulting biosensor exhibited fast response, excellent reproducibility and sensibility, expanded linear range and low interferences. Temperature and pH dependence and stability of the sensor were investigated. The optimal sensor gave a linear response in the range of 2.99×10,6 to 3.55×10,3 mol·L,1 to H2O2 with a sensibility of 0.0177 A·L,1·mol,1 and a detection limit (S/N=3) of 4.3×10,7 mol·L,1. The biosensor demonstrated a 95% response within less than 10 s. [source] Bioelectrochemical Characterization of Horseradish and Soybean PeroxidasesELECTROANALYSIS, Issue 21 2009Marco Frasconi Abstract Heme peroxidase are ubiquitous enzymes catalyzing the oxidation of a broad range of substrates by hydrogen peroxide. In this paper the bioelectrochemical characterization of horseradish peroxidase (HRP) and soybean peroxidase (SBP), belonging to class III of the plant peroxidase superfamily, was studied. The homogeneous reactions between peroxidases and some common redox mediators in the presence of hydrogen peroxide have been carried out by cyclic voltammetry. The electrochemical characterization of the reactions involving enzyme, substrate and mediators concentrations allowed us to calculate the kinetic parameters for the substrate,enzyme reaction (KMS) and for the redox mediator,enzyme reaction (KMM). A full characterization of the direct electron transfer kinetic parameters between the electrode and enzyme active site was also performed by opportunely modeling data obtained from cyclic voltammetry and square wave voltammetry experiments. The experimental data obtained with immobilized peroxidases show enhanced direct electron transfer and excellent electrocatalytical performance for H2O2. Despite the structural similarities and common catalytic cycle, HRP and SBP exhibit differences in their substrate affinity and catalytic efficiency. Basing on our results, it can be concluded that peroxidase from soybean represents an interesting alternative to the classical and largely employed one obtained from horseradish as biorecognition element of electrochemical mediated biosensors. [source] Dendritic Silver/Silicon Dioxide Nanocomposite Modified Electrodes for Electrochemical Sensing of Hydrogen PeroxideELECTROANALYSIS, Issue 17 2008Peixi Yuan Abstract A novel biosensor for hydrogen peroxide was prepared by immobilizing horseradish peroxidase (HPR) on newly synthesized dendritic silver/silicon dioxide nanocomposites, which were coated on a glassy carbon electrode. The modified electrode was characterized with XPS, SEM, and electrochemical methods. This biosensor showed a very fast amperometric response to hydrogen peroxide with a linear range from 0.7 to 120,,M, a limit of detection of 0.05,,M and a sensitivity of 1.02,mA mM,1 cm,2. The Michaelis-Menten constant of the immobilized HRP was estimated to be 0.21,mM, indicating a high affinity of the HRP to H2O2 without loss of enzymatic activity. The preparation of the proposed biosensor was convenient, and it showed high sensitivity and good stability. [source] Improved Detection Limit and Stability of Amperometric Carbon Nanotube-Based Immunosensors by Crosslinking Antibodies with PolylysineELECTROANALYSIS, Issue 2 2008Vito Cataldo Abstract Amperometric immunosensor configurations featuring covalently bound anti-biotin antibodies (Ab) embedded into a polylysine (PLL)-single walled carbon nanotube (SWCNT) composite layer were evaluated. Assemblies were made by first oxidizing pyrolytic graphite (PG) electrodes to form surface carboxylic acid groups, to which PLL, SWCNTs and anti-biotin were covalently linked. Incorporating SWCNT into PLL-antibody assemblies improved the amperometric detection limit for biotin (Ag) labeled with horseradish peroxidase to 10,fmol mL,1. Anti-biotin embedded into the PLL matrix had improved thermal stability and retained its binding ability for biotin after exposure to temperatures of 42,°C for up to 3 hours, while the noncrosslinked antibody was inactivated at this temperature in several minutes. [source] Fabrication of Active Horseradish Peroxidase Micropatterns with a High Resolution by Scanning Electrochemical MicroscopyELECTROANALYSIS, Issue 16 2007Xuemei Li Abstract We used a new reactive species OH, to fabricate active horseradish peroxidase (HRP) micropatterns with a high resolution by scanning electrochemical microscopy (SECM) coupled with a carbon fiber disk electrode as the SECM tip. In this method, except for active HRP micropatterns predesigned other regions on a HRP-immobilized substrate were deactivated by OH, generated at the tip held at ,1.7,V in 1.0,mol/L KCl containing 2.0×10,3 mol/L benzoquinone (BQ) (pH,8.0). The feedback mode of SECM with a tip potential of ,0.2,V was used to characterize the active HRP micropatterns in 1.0,mol/L KCl containing 2.0×10,3 mol/L BQ and 2.0×10,3 mol/L H2O2. [source] A Peroxidase-Based Biosensor Supported by Nanoporous Magnetic Silica Microparticles for Acetaminophen Biotransformation and Inhibition StudiesELECTROANALYSIS, Issue 17 2006Donghui Yu Abstract Magnetized nanoporous silica based microparticles (MMPs) were used for horseradish peroxidase (HRP) immobilization and applied for amperometric peroxidase-based biosensor development. A magnetized carbon paste electrode permitted the MMPs attraction. The biosensor was applied to the investigation of the enzymatic oxidation of acetaminophen (paracetamol). The biosensor operated at low applied potential and the signal corresponded to the electroreduction of N -acetylbenzoquinoneimine (NAPQI) generated by the enzyme HRP in the presence of hydrogen peroxide. The biosensor allowed performing the quantitation of acetaminophen in the micromolar concentration range and the comparative study of thiols which inhibited the biosensor response. Distinct inhibition results were observed for HRP entrapped in the silica microparticles compared to the soluble HRP. [source] Electrochemical, Chemical and Enzymatic Oxidations of PhenothiazinesELECTROANALYSIS, Issue 17 2005B. Blankert Abstract The oxidation of several phenothiazine drugs (phenothiazine, promethazine hydrochloride, promazine hydrochloride, trimeprazine hydrochloride and ethopropazine hydrochloride) has been carried out in aqueous acidic media by electrochemical, chemical and enzymatic methods. The chemical oxidation was performed in acetic acid with hydrogen peroxide or in formate buffers using persulfate. The enzymatic oxidation was performed in acetate or ammonium formate buffer by the enzyme horseradish peroxidase in the presence of H2O2. Molecules with, in the lateral chain, two carbon atoms (2C) separating the ring nitrogen and the terminal nitrogen, showed two parallel oxidation pathways, that is (i) formation of the corresponding sulfoxide and (ii) cleavage of the lateral chain with liberation of phenothiazine (PHZ) oxidized products (PHZ sulfoxide and PHZ quinone imine). Molecules with three carbon atoms (3C) separating the two nitrogens were oxidized to the corresponding sulfoxide. The chemical oxidation of all the studied molecules by hydrogen peroxide resulted in the corresponding sulfoxide with no break of the lateral chain. Oxidation by persulfate yielded, for the 3C derivatives, only the corresponding sulfoxide, but it produced cleavage of the lateral chain for the 2C derivatives. The origin of the distinct oxidation pattern between 2C and 3C molecules might be related to steric effects due to the lateral chain. The data are of interest in drug metabolism studies, especially for the early search. In the case of 2C phenothiazines, the results predict the possibility of an in vivo cleavage of the lateral chain with liberation of phenothiazine oxidized products which are known to produce several adverse side effects. [source] A Tubulin-Based Quantitative Assay for Taxol (Paclitaxel) with Enzyme Channeling SensingELECTROANALYSIS, Issue 8 2004Shin-ichiro Suye Abstract We report the development and characterization of a biosensor for sensitive and rapid determination of the anticancer agent Taxol (paclitaxel). The sensor is based on the interaction of Taxol with its receptor protein tubulin in conjunction with the separation-free immunosensor technique of enzyme channeling. The sensor system consisted of a three electrode electrochemical cell containing a graphite carbon electrode modified with glucose oxidase and tubulin as working electrode poised at +40,mV (vs. Ag/AgCl). Addition of Taxol, horseradish peroxidase labeled Taxol, glucose and potassium iodide to the cell generated a cathodic current response that was proportional to the concentration of Taxol in the range of 10 to 1,000,pM. [source] Early neural activity and dendritic growth in turtle retinal ganglion cellsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2006Vandana Mehta Abstract Early neural activity, both prenatal spontaneous bursts and early visual experience, is believed to be important for dendritic proliferation and for the maturation of neural circuitry in the developing retina. In this study, we have investigated the possible role of early neural activity in shaping developing turtle retinal ganglion cell (RGC) dendritic arbors. RGCs were back-labelled from the optic nerve with horseradish peroxidase (HRP). Changes in dendritic growth patterns were examined across development and following chronic blockade or modification of spontaneous activity and/or visual experience. Dendrites reach peak proliferation at embryonic stage 25 (S25, one week before hatching), followed by pruning in large field RGCs around the time of hatching. When spontaneous activity is chronically blocked in vivo from early embryonic stages (S22) with curare, a cholinergic nicotinic antagonist, RGC dendritic growth is inhibited. On the other hand, enhancement of spontaneous activity by dark-rearing (Sernagor & Grzywacz (1996)Curr. Biol., 6, 1503,1508) promotes dendritic proliferation in large-field RGCs, an effect that is counteracted by exposure to curare from hatching. We also recorded spontaneous activity from individual RGCs labelled with lucifer yellow (LY). We found a tendency of RGCs with large dendritic fields to be spontaneously more active than small-field cells. From all these observations, we conclude that immature spontaneous activity promotes dendritic growth in developing RGCs. [source] Central sprouting of uninjured small fiber afferents in the adult rat spinal cord following spinal nerve ligationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2004Jian Hu Abstract Partial nerve injury results in chronic pain that is difficult to treat effectively. To investigate the anatomic basis of this phenomenon we used wheat germ agglutinin,horseradish peroxidase (WGA-HRP) to label the central projections of uninjured small fibers (A, and C) in a well-established model of neuropathic pain created by selective spinal nerve ligation in the adult. We found extensive sprouting of uninjured WGA-HRP-labeled afferents into the central termination field in lamina II of dorsal horn normally occupied by L5 afferents whose peripheral axons had been ligated distal to the dorsal root ganglion. The formation of new projections by uninjured fibers into a functionally but not anatomically deafferented field in the adult may play a role in the development of chronic pain. [source] N -methyl- d -aspartate-triggered neuronal death in organotypic hippocampal cultures is endocytic, autophagic and mediated by the c-Jun N-terminal kinase pathwayEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2003Tiziana Borsello Abstract Acute excitotoxic neuronal death was studied in rat organotypic hippocampal slices exposed to 100 µmN -methyl- d -aspartate. Fulgurant death of pyramidal neurons occurred in the CA1 and CA3 regions and was already detectable within 2 h of the N-methyl- d -aspartate administration. Morphologically, the neuronal death was neither apoptotic nor necrotic but had the hallmarks of autophagic neuronal death, as shown by acid phosphatase histochemistry in both CA1 and CA3 and by electron microscopy in CA1. The dying neurons also manifested strong endocytosis of horseradish peroxidase or microperoxidase, occurring probably by a fluid phase mechanism, and followed, surprisingly, by nuclear entry. In addition to these autophagic and endocytic characteristics, there were indications that the c-Jun N-terminal kinase pathway was activated. Its target c-Jun was selectively phosphorylated in CA1, CA3 and the dentate gyrus and c-Fos, the transcription of which is under the positive control of c-Jun N-terminal kinase target Elk1, was selectively up-regulated in CA1 and CA3. All these effects, the neuronal death itself and the associated autophagy and endocytosis, were totally prevented by a cell-permeable inhibitor of the interaction between c-Jun N-terminal kinase and certain of its targets. These results show that pyramidal neurons undergoing excitotoxic death in this situation are autophagic and endocytic and that both the cell death and the associated autophagy and endocytosis are under the control of the c-Jun N-terminal kinase pathway. [source] Novel Model Sulfur Compounds as Mechanistic Probes for Enzymatic and Biomimetic OxidationsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 1 2005Alicia B. Peñéñory Abstract To test for the intermediacy of sulfide radical cations in biomimetic and enzymatic oxidations, the sulfides PhSCH3 (1a), PhSCH2Ph (1b), PhSCHPh2 (1c), PhSCPh3 (1d), CH3SCHPh2 (2), PhSCH2CH=CH2 (3), PhSCH2CH=CHPh (4) and CH3SCH2CH=CHPh (5) were studied, and their results were compared to those obtained for the corresponding chemical electron transfer (CET) and photoinduced electron transfer (PET) oxidations. The radical cations generated from 3,5 by CET in the presence of cerium(IV) ammonium nitrate (CAN) yielded only fragmentation products from the alkyl cations and the thiyl radicals (RS·), whereas 2·+ afforded both fragmentation and mainly ,-deprotonation products. Photochemical treatment of the sulfides 1a and 1b with C(NO2)4 gave only the corresponding sulfoxides, while fragmentation was the main pathway for the photoreactions of 1c, 2 and 5, and for 1d only this latter process was observed. These results support our selection of the sulfides RSCHPh2, RSCH2CH=CHPh (R = Me, Ph) and PhSCPh3 as models for the biomimetic and enzymatic studies. As evidenced by the sulfoxides and sulfones detected as unique products both in protic and in aprotic solvents, it is proposed that the mechanism of the biomimetic sulfoxidations of sulfides 1c and 2,5 by TPPFeIIICl is direct oxygen transfer. Three enzymes , Coprinus cinereus peroxidase (CiP), horseradish peroxidase (HRP) and chloroperoxidase (CPO) , were studied in the oxidation of sulfides 1a, 2, 4 and 5. The use of a racemic alkyl hydroperoxide in the CiP enzymatic oxidation of sulfides 5 and 2 yielded the corresponding sulfoxides (23 and 29%) and the aldehyde or benzophenone (5%), respectively. These results suggest the involvement of an ET process for the CiP-catalysed oxidation. Fragmentation products were observed in the enzymatic oxidation of sulfide 4 with HRP, which confirms the previously proposed ET mechanism. On the other hand, the CPO-enzymatic oxidation of sulfide 5 yielded only the corresponding sulfoxide, as would be expected for a direct oxygen-transfer or oxene mechanism. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005) [source] Self-Assembled Graphene,Enzyme Hierarchical Nanostructures for Electrochemical BiosensingADVANCED FUNCTIONAL MATERIALS, Issue 19 2010Qiong Zeng Abstract The self-assembly of sodium dodecyl benzene sulphonate (SDBS) functionalized graphene sheets (GSs) and horseradish peroxidase (HRP) by electrostatic attraction into novel hierarchical nanostructures in aqueous solution is reported. Data from scanning electron microscopy, high-resolution transmission electron microscopy, and X-ray diffraction demonstrate that the HRP,GSs bionanocomposites feature ordered hierarchical nanostructures with well-dispersed HRP intercalated between the GSs. UV-vis and infrared spectra indicate the native structure of HRP is maintained after the assembly, implying good biocompatibility of SDBS-functionalized GSs. Furthermore, the HRP,GSs composites are utilized for the fabrication of enzyme electrodes (HRP,GSs electrodes). Electrochemical measurements reveal that the resulting HRP,GSs electrodes display high electrocatalytic activity to H2O2 with high sensitivity, wide linear range, low detection limit, and fast amperometric response. These desirable electrochemical performances are attributed to excellent biocompatibility and superb electron transport efficiency of GSs as well as high HRP loading and synergistic catalytic effect of the HRP,GSs bionanocomposites toward H2O2. As graphene can be readily non-covalently functionalized by "designer" aromatic molecules with different electrostatic properties, the proposed self-assembly strategy affords a facile and effective platform for the assembly of various biomolecules into hierarchically ordered bionanocomposites in biosensing and biocatalytic applications. [source] Thermally induced conformational changes in horseradish peroxidaseFEBS JOURNAL, Issue 1 2001David G. Pina Detailed differential scanning calorimetry (DSC), steady-state tryptophan fluorescence and far-UV and visible CD studies, together with enzymatic assays, were carried out to monitor the thermal denaturation of horseradish peroxidase isoenzyme c (HRPc) at pH 3.0. The spectral parameters were complementary to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while that in the Soret region, as well as changes in intrinsic tryptophan fluorescence emission, corresponded to changes in the tertiary structure of the enzyme. The results, supported by data about changes in enzymatic activity with temperature, show that thermally induced transitions for peroxidase are irreversible and strongly dependent upon the scan rate, suggesting that denaturation is under kinetic control. It is shown that the process of HRPc denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated. [source] Biotemplated Synthesis of Gold Nanoparticle,Bacteria Cellulose Nanofiber Nanocomposites and Their Application in BiosensingADVANCED FUNCTIONAL MATERIALS, Issue 7 2010Taiji Zhang Abstract Bacteria cellulose (BC) nanofibers are used as robust biotemplates for the facile fabrication of novel gold nanoparticle (NP),bacteria cellulose nanofiber (Au,BC) nanocomposites via a one-step method. The BC nanofibers are uniformly coated with Au NPs in aqueous suspension using poly(ethyleneimine) (PEI) as the reducing and linking agent. With the addition of different halides, Au,BC nanocomposites with different Au shell thicknesses are formed, and a possible formation mechanism is proposed by taking into account the special role played by PEI. A novel H2O2 biosensor is constructed using the obtained Au,BC nanocomposites as excellent support for horseradish peroxidase (HRP) immobilization, which allows the detection of H2O2 with a detection limit lower than 1,µM. The Au,BC nanocomposites could be further used for the immobilization of many other enzymes, and thus, may find potential applications in bioelectroanalysis and bioelectrocatalysis. [source] Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx miceGLIA, Issue 3 2003Beatrice Nico Abstract In this study, we investigated the involvement of the blood-brain barrier (BBB) in the brain of the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy (DMD). To this purpose, we used two tight junction markers, the Zonula occludens (ZO-1) and claudin-1 proteins, and a glial marker, the aquaporin-4 (AQP4) protein, whose expression is correlated with BBB differentiation and integrity. Results showed that most of the brain microvessels in mdx mice were lined by altered endothelial cells that showed open tight junctions and were surrounded by swollen glial processes. Moreover, 18% of the perivascular glial endfeet contained electron-dense cellular debris and were enveloped by degenerating microvessels. Western blot showed a 60% reduction in the ZO-1 protein content in mdx mice and a similar reduction in AQP4 content compared with the control brain. ZO-1 immunocytochemistry and claudin-1 immunofluorescence in mdx mice revealed a diffuse staining of microvessels as compared with the control ones, which displayed a banded staining pattern. ZO-1 immunogold electron microscopy showed unlabeled tight junctions and the presence of gold particles scattered in the endothelial cytoplasm in the mdx mice, whereas ZO-1 gold particles were exclusively located at the endothelial tight junctions in the controls. Dual immunofluorescence staining of ,-actin and ZO-1 revealed colocalization of these proteins. As in ZO-1 staining, the pattern of immunolabeling with anti,,-actin antibody was diffuse in the mdx vessels and pointed or banded in the controls. ,-actin immunogold electron microscopy showed gold particles in the cytoplasms of endothelial cells and pericytes in the mdx mice, whereas ,-actin gold particles were revealed on the endothelial tight junctions and the cytoskeletal microfilaments of pericytes in the controls. Perivascular glial processes of the mdx mice appeared faintly stained by anti-AQP4 antibody, while in the controls a strong AQP4 labeling of glial processes was detected at light and electron microscope level. The vascular permeability of the mdx brain microvessels was investigated by means of the horseradish peroxidase (HRP). After HRP injection, extensive perivascular areas of marker escape were observed in mdx mice, whereas HRP was exclusively intravascularly localized in the controls. Inflammatory cells, CD4-, CD8-, CD20-, and CD68-positive cells, were not revealed in the perivascular stroma of the mdx brain. These findings indicate that dystrophin deficiency in the mdx brain leads to severe injury of the endothelial and glial cells with disturbance in ,-actin cytoskeleton, ZO-1, claudin-1, and AQP4 assembly, as well as BBB breakdown. The BBB alterations suggest that changes in vascular permeability are involved in the pathogenesis of the neurological dysfunction associated with DMD. GLIA 42:235,251, 2003. © 2003 Wiley-Liss, Inc. [source] Effect of reactive oxygen intermediaries on the viability and infectivity of Mycobacterium lepraemuriumINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 3 2007Kendy Wek-Rodriguez Summary Murine leprosy is a natural disease of the mouse, the most popular model animal used in biomedical research; the disease is caused by Mycobacterium lepraemurium (MLM), a successful parasite of macrophages. The aim of the study was to test the hypothesis that MLM survives within macrophages because it highly resists the toxic effects of the reactive oxygen intermediaries produced by these cells in response to infection by the microorganism. MLM cells were incubated in the presence of horseradish peroxidase (HRPO),H2O2,halide for several periods of time. The peroxidative effect of this system was investigated by assessing the changes occurred in (a) lipid composition; (b) viability; and (c) infectivity of the microorganism. Changes in the lipid composition of peroxidated- vs. intact-MLM were analysed by thin layer chromatography. The effect of the peroxidative system on the viability and infectivity of MLM was measured by the alamar blue reduction assay and by its ability to produce an infection in the mouse, respectively. Peroxidation of MLM produced drastic changes in the lipid envelope of the microorganism, killed the bacteria and abolished their ability to produce an in vivo infection in the mouse. In vitro, MLM is highly susceptible to the noxious effects of the HRPO,H2O2,halide system. Although the lipid envelope of MLM might protect the microorganism from the peroxidative substances produced at ,physiological' concentrations in vivo, the success of MLM as a parasite of macrophages might rather obey for other reasons. The ability of MLM to enter macrophages without triggering these cells' oxidative response and the lack of granular MPO in mature macrophages might better explain its success as an intracellular parasite of these cells. [source] High-throughput enzyme kinetics using microarraysISRAEL JOURNAL OF CHEMISTRY, Issue 2 2007Guoxin Lu We report a microanalytical method to study enzyme kinetics. The technique involves immobilizing horseradish peroxidase on a poly-L-lysine (PLL)-coated glass slide in a microarray format, followed by applying substrate solution onto the enzyme microarray. Enzyme molecules are immobilized on the PLL-coated glass slide through electrostatic interactions, and no further modification of the enzyme or glass slide is needed. In situ detection of the products generated on the enzyme spots is made possible by monitoring the light intensity of each spot using a scientific-grade charged-coupled device (CCD). Reactions of substrate solutions of various types and concentrations can be carried out sequentially on one enzyme microarray. To account for the loss of enzyme from washing in between runs, a standard substrate solution is used for calibration. Substantially reduced amounts of substrate solution are consumed for each reaction on each enzyme spot. The Michaelis constant Km obtained by using this method is comparable to the result for homogeneous solutions. Absorbance detection allows universal monitoring, and no chemical modification of the substrate is needed. High-throughput studies of native enzyme kinetics for multiple enzymes are therefore possible in a simple, rapid, and low-cost manner. [source] Connections of the zona incerta to the reticular nucleus of the thalamus in the ratJOURNAL OF ANATOMY, Issue 2 2006Safiye Çavdar Abstract This study demonstrated that there is a pathway from the zona incerta to the thalamic reticular nucleus. Injections of horseradish peroxidase or Fluorogold were made, using stereotaxic coordinates, into the rostral, intermediate or caudal regions of the thalamic reticular nucleus of adult Sprague,Dawley rats. The results show that the different regions of the thalamic reticular nucleus have distinct patterns of connections with the sectors of the zona incerta. In terms of the relative strength of the connections, injections made into the rostral regions of the thalamic reticular nucleus showed the highest number of labelled cells within the rostral and ventral sectors of the zona incerta; injections made into the intermediate regions of the thalamic reticular nucleus showed labelled cells in the dorsal and ventral sectors; while injections to the caudal regions of the thalamic reticular nucleus showed only a few labelled cells in the caudal sector of the zona incerta. Previous studies have shown that the zona incerta projects to the higher order thalamic nuclei but not first order thalamic nuclei. The labelling observed in the present study may represent collaterals of zona incerta to higher order thalamic nuclei projections. [source] Impact of dissolved wastewater constituents on peroxidase-catalyzed treatment of phenolJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 4 2002Monika Wagner Abstract The impact of dissolved wastewater constituents on the treatment of synthetic phenol solutions using horseradish peroxidase (HRP) and hydrogen peroxide was investigated under a variety of reaction conditions. The constituents studied included various inorganic salts, organic compounds and heavy metals. Higher H2O2 doses were required to treat phenol in the presence of sodium sulfite, thiosulfate and sulfide; however, enhanced levels of phenol conversion were achieved once sufficient H2O2 was supplied. Sulfide and cyanide inhibited phenol transformation. The inhibition of sulfide was overcome by supplying sufficient H2O2 to oxidize the sulfide to sulfur. However, increasing the H2O2 dose was ineffective in attempting to overcome the strong inhibiting effect of cyanide. Among the heavy metal ions tested, only Mn(II) substantially inhibited phenol removal when it was present at a concentration of 1,mmol,dm,3. The presence of inorganic salts including NaCl, CaCl2, MgCl2, NH4Cl and (NH4)2SO4 reduced phenol conversion as compared with the treatment in distilled-deionized water. This can be attributed to the increased ionic strength of the solution. © 2002 Society of Chemical Industry [source] Measurement of gp210 autoantibodies in sera of patients with primary biliary cirrhosisJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 4 2007Alicja Bauer Abstract Primary biliary cirrhosis (PBC) is an autoimmune liver disease with unknown etiology. Patients with PBC have antimitochondrial autoantibodies (AMA) and additionally 50% of them have antinuclear antibodies (ANA). A 15,amino acid fragment (DRKASPPSGLWSPAY) from the C-terminal part of the nuclear envelope glycoprotein gp210 has been proposed as one of the antigenic targets for ANA. The aim of this work was to develop an immunoenzymatic assay for determination of gp210 autoantibodies using for its binding a synthetic pentadecapeptide derived from the gp210 amino acid sequence and to determine level of these autoantibodies in sera of patients with PBC and other autoimmune liver diseases from Poland. Polystyrene microtitration plates coated with the synthetic peptide were consecutively incubated with diluted sera, anti-human immunoglobulin G (IgG) antibodies conjugated with horseradish peroxidase, and with tetramethylobenzidine. Optical density (OD) was read at 450 nm. The mean values of the intra- and interassay of variation coefficients of the test were 4.1 and 10.2%, respectively. Anti-gp210 was detected in 44% of PBC patients and in 6% of patients with PSC. The results were negative for healthy blood donors and other controls. The specificity of the test was 99%, so the anti-gp-210 autoantibodies estimated on DRKASPPSGLWSPAY can be a reliable marker of PBC. J. Clin. Lab. Anal. 21:227,231, 2007. © 2007 Wiley-Liss, Inc. [source] The relationship of proliferating cell density at the invasive tumour front with prognostic and risk factors in human oral squamous cell carcinomaJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 4 2004Vijay Tumuluri BACKGROUND:, We hypothesise that the density of proliferating cells at the invasive tumour front (ITF) has a positive relationship with prognostic and risk factors in human oral squamous cell carcinoma (SCC). METHODS:, Tissues from 47 human oral SCC specimens were collected and stained with a monoclonal antibody directed against the Ki-67 antigen using a horseradish peroxidase based two-step immunostaining method. Counting was performed on two parallel sections at the ITF using an image analyser. The Ki-67 labelling index (LI) was determined by measuring the number of nuclei/mm2 of epithelium. RESULTS:, Our results show that the density of proliferating cells is related to clinical staging, with advanced stage of disease having a significantly higher Ki-67 LI compared with early stage of disease (2111 ± 905 vs. 1908 ± 913; P = 0.03). Importantly, this study shows that tumours that have metastasised have a significantly higher Ki-67 LI than tumours where distant metastasis was not detected (3257 ± 650 vs. 1966 ± 881; P < 0.0001). CONCLUSIONS:, Cell proliferation, as measured by the Ki-67 LI at the ITF, has a positive relationship with clinical staging, tumour thickness, smoking status of the patient and alcohol consumption. Further, we suggest that a multicenter study with a large cohort of patients is indicated to fully elucidate whether cell proliferation at the ITF is directly related to patient survival. [source] Asymmetric biomimetic oxidations of phenols using oxazolidines as chiral auxiliaries: the enantioselective synthesis of (+)- and (,)-dehydrodiconiferyl alcohol,JOURNAL OF PHYSICAL ORGANIC CHEMISTRY, Issue 8-9 2006Maurizio Bruschi Abstract Stereoselective bimolecular radical coupling reactions of phenylpropenoid phenols are described. Evans's 2-oxazolidinone 11a,d derivatives of ferulic acid were prepared and oxidized to give dimeric benzofuran neolignan structures 12,13a,d in 40,50% overall yields. The chiral phenols were dimerized either enzymatically with hydrogen peroxide and horseradish peroxidase (HRP) or with silver oxide. The enantioselectivity after reductive cleavage of the chiral auxiliaries to give dehydrodiconiferyl alcohol ranged from 18% to 62% enantiomeric excess. The conformational analysis and the activation energy using semiempirical PM3 calculations on the intermediate quinomethides is used to explain the observed stereoselectivity. Copyright © 2006 John Wiley & Sons, Ltd. [source] The effect of pH on horseradish peroxidase-catalyzed oxidation of melatonin: production of N1 -acetyl- N2 -formyl-5-methoxykynuramine versus radical-mediated degradationJOURNAL OF PINEAL RESEARCH, Issue 3 2007Valdecir F. Ximenes Abstract:, There is a growing body of evidence that melatonin and its oxidation product, N1 -acetyl- N2 -formyl-5-methoxykynuramine (AFMK), have anti-inflammatory properties. From a nutritional point of view, the discovery of melatonin in plant tissues emphasizes the importance of its relationship with plant peroxidases. Here we found that the pH of the reaction mixture has a profound influence in the reaction rate and products distribution when melatonin is oxidized by the plant enzyme horseradish peroxidase. At pH 5.5, 1 mm of melatonin was almost completely oxidized within 2 min, whereas only about 3% was consumed at pH 7.4. However, the relative yield of AFMK was higher in physiological pH. Radical-mediated oxidation products, including 2-hydroxymelatonin, a dimer of 2-hydroxymelatonin and O -demethylated dimer of melatonin account for the fast consumption of melatonin at pH 5.5. The higher production of AFMK at pH 7.4 was explained by the involvement of compound III of peroxidases as evidenced by spectral studies. On the other hand, the fast oxidative degradation at pH 5.5 was explained by the classic peroxidase cycle. [source] Development of a sensitivity-improved immunoassay for the determination of carbaryl in food samplesJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 7 2010Tingting Dong Abstract BACKGROUND: With the aim of developing a highly sensitive immunoassay for carbaryl, a hapten which had high similarity to carbaryl was synthesised using a safer and more practical approach. After it was conjugated to horseradish peroxidase, direct competitive heterologous enzyme-linked immunosorbent assay (CD-ELISA) was optimised and characterised. The assay performance conditions were investigated in details. Enhanced chemiluminescence ELISA (ECL-ELISA) was also used in preliminary studies. RESULTS: The assay obtained an IC50 value (the concentration causing 50% inhibition) of 2 µg kg,1, which was 12-fold more sensitive than previous results of homologous CD-ELISA. In ECL-ELISA, the IC50 was further decreased 10-fold to 0.2 µg kg,1. The CD-ELISA developed was applicable for broad conditions, and could be applied on various food samples with a more convenient pre-treatment. Average recoveries were in a range of 88.3,101.7%. The results correlated well with those obtained using high-performance liquid chromatography (HPLC) analysis (R2 = 0.989). CONCLUSION: The ELISA developed was a great improvement in the determination of carbaryl, showing that the immunoassay developed was a simple, rapid and efficient method that was reliable for the detection of carbaryl and suitable for rapid quantitative or qualitative determination in food samples. Copyright © 2010 Society of Chemical Industry [source] Sensitive enzyme-linked immunosorbent assay and rapid one-step immunochromatographic strip for fumonisin B1 in grain-based food and feed samplesJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 6 2010Chang-Min Shiu Abstract BACKGROUND: Maize contaminated with mycotoxin fumonisin B1 poses a global threat to agricultural production. In this study, polyclonal antibodies (pAb) specific to fumonisin B1 were generated from rabbits immunised with fumonisin B1,keyhole limpet haemocyanin (KLH). These antibodies were used to establish a sensitive competitive direct enzyme-linked immunosorbent assay (cdELISA) and gold nanoparticle immunochromatographic strip for detecting fumonisin B1 levels in maize-based foods and feeds. RESULTS: In cdELISA, fumonisins B1, B2 and B3 at concentrations of 0.42, 0.58 and 81.5 ng mL,1 respectively caused 50% inhibition (IC50) of binding of fumonisin B1,horseradish peroxidase (HRP) to the antibodies. Effective on-site detection of fumonisin B1 was achieved by developing a rapid and sensitive pAb-based gold nanoparticle immunochromatographic strip. This strip had a detection limit of 5 ng mL,1 for fumonisin B1 in maize-based samples. Additionally, the whole analytical process could be completed within 10 min. Close examination of 15 maize-based samples by cdELISA revealed that 11 were fumonisin-positive, with a mean concentration of 435 ± 20.1 ng g,1. These results correlated well with those obtained by immunochromatographic strip. CONCLUSION: Both cdELISA and immunochromatographic strip methods established in this study are sensitive for rapid detection of fumonisins in agricultural commodities. Copyright © 2010 Society of Chemical Industry [source] | |||||||||||||||||||||||||||||||||||||