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Identification of hormone-producing cells of the endocrine pancreas of the sea bass, Dicentrarchus labrax, by ultrastructural immunocytochemistry.

The hormone-producing cells of the endocrine pancreas of the sea bass Dicentrarchus labrax have been identified by ultrastructural immunocytochemistry. The glucagon cells have "clear" cytoplasm and contain characteristic electron-dense polygonal granules surrounded by a "halo" of electron-lucent material. The insulin cells have numerous, tightly packed, electron-dense granules that are almost twice as large as the peripherally located granules of the somatostatin cells. The pancreatic polypeptide cells have granules with variable electron density. When specific antisera are applied in the peroxidase-anti-per-oxidase immunocytochemical method at the electron microscope level, each of the four types of granule is identified by the resultant overlying immunoreaction deposit. Especially in older fish, a fifth, nonclassified type of cell has been identified within the endocrine pancreatic tissue. These cells have many ramifying processes and contain a mixture of the granules of the four endocrine cell types as well as granules from the exocrine tissue. It is suggested that these cells may be undertaking macrophage activities. A distinct patterned arrangement of the endocrine cell types in both small pancreatic islets and Brockmann bodies is observed. There is a central core of insulin and somatostatin cells surrounded by an outer peripheral layer of glucagon and pancreatic polypeptide cells. A definite functional interrelationship is suggested by this arrangement.

Animals↗

Enzyme-based biosensor as a selective detection unit in column liquid chromatography.

A reagentless enzyme electrode based on co-immobilized alcohol oxidase and horseradish peroxidase was used as the working electrode in an amperometric flow-through cell connected to a column liquid chromatographic (CLC) system for the selective detection of methanol and ethanol. The enzymes were covalently immobilized in carbon paste (graphite-phenylmethylsilicone oil) in the presence of polyethylenimine. Electrodes prepared from the enzyme-modified carbon paste were optimized with respect to their sensitivity and selectivity. Different membranes were cast or electropolymerized directly on the surface of the electrode to increase the long-term stability of the biosensor. The compatibility with the reversed-phase chromatographic system was established. A PLRP-S polymer-based separation column was used with phosphate buffer as the mobile phase. The selectivity of the enzyme electrode was also determined by injecting some easily oxidizable and possibly interfering species normally present in biological samples. The enzyme electrode was also used in an on-line system, consisting of a microdialysis probe as the sampling unit, the CLC system and the biosensor detection device, for the selective following of the ethanol produced when a paper pulp industrial waste water was fermented with Saccharomyces cerevisiae.

Biosensing Techniques↗

TerELISA: the ELISA test performed in Terasaki plates.

The use of Terasaki (10 microliter samples) and microtitration (100 microliter samples) plates as the solid phase in enzyme immunoassays was compared. Various antigens were used for coating the plates and antibodies present in human sera were evaluated using the same anti-human Ig antibody labelled with either beta -galactosidase, alkaline phosphatase, peroxidase or glucose oxidase. The results obtained, either by scoring with the naked eye or by absorbance reading with appropriate densitometers, showed that both plates were equally suitable and that the 4 enzymes were equally effective in detecting the same lowest quantity of antibody. A comparative evaluation using either Terasaki or microtitration plates for the quantitation of human anti-Echinococcus granulosus antibody ii 50 sera demonstrated that there was a good correlation between the two procedures (r = 0.8097). Finally, the use of glucose oxidase as the enzyme marked allowed a clear-cut distinction to be made between positive and negative samples with the naked eye alone.

Antigen-Antibody Reactions↗

Fluorescence and chemiluminescence enzyme immunoassays of 17 alpha-hydroxyprogesterone in dried blood spotted on filter paper.

Enzyme immunoassays for 17 alpha-hydroxyprogesterone (17-OHP) were developed. Horseradish peroxidase (HRP), glucose oxidase (GOD), invertase (INV) and glucose-6-phosphate dehydrogenase (G6PDH) were used as label enzymes. Double antibody coated beads or tubes were used for separating the bound and free fractions. Antisera used were prepared by using 4-carboxyethylthio-17-OHP and 3-carboxymethyl oxime-17-OHP-bovine serum albumin as immunogens. The bridge heterologous system was more sensitive than other site heterologous and homologous systems. The minimum amounts of 17-OHP detected were 0.25 and 1.0 pg/tube for fluorescence EIAs using HRP and GOD, and 0.1, 10 and 0.1 pg/tube for chemiluminescence EIAs using GOD, INV and G6PDH, respectively. The reproducibility and correlation with RIA were also studied. The present study demonstrates the feasibility of a neonatal screening for congenital adrenal hyperplasia.

17-alpha-Hydroxyprogesterone↗

Electrocatalytic oxidation of ascorbate by heme-FeIII/heme-FeII redox couple of the HRP and its effect on the electrochemical behaviour of an L-lactate biosensor.

The measurements of L-lactate using the carbon paste electrode modified with lactate oxidase (LOD), horseradish peroxidase (HRP) and ferrocene (FcH) operating at low working potential in flow injection mode showed that the intensity as well as the shape of peaks were dependent on the concentration of the reducing species present in samples (e.g. ascorbate) even at low operating potentials (-200 to 0 mV vs. Ag/AgCl). The mechanism of the electrochemical contribution of ascorbate to the L-lactate response was examined by using cyclic voltammetry, hydrodynamic voltammetry and FIA results. Comparative studies showed that HRP was catalytically active for the oxidation of ascorbate leading to a decrease in the cathodic electrochemical signal of L-lactate. The results of our investigation postulated that the direct electron transfer from the HRP-Fe(III)/HRP-Fe(II) redox couple to the electrode surface was involved in the electrocatalytic oxidation of ascorbate at the electrode surface.

Ascorbic Acid↗

Simultaneous detection of the release of glutamate and nitric oxide from adherently growing cells using an array of glutamate and nitric oxide selective electrodes.

The simultaneous detection of nitric oxide and glutamate using an array of individually addressable electrodes, in which the individual electrodes in the array were suitably modified with a highly sensitive nitric oxide sensing chemistry or a glutamate oxidase/redox hydrogel-based glutamate biosensor is presented. In a sequence of modification steps one of the electrodes was covered first with a positively charged Ni porphyrin entrapped into a negatively charged electrodeposition paint followed by the manual modification of the second working electrode by a bienzyme sensor architecture based on crosslinked redox hydrogels with entrapped peroxidase and glutamate oxidase. Adherently growing C6-glioma cells were grown on membrane inserts and placed in close distance to the modified sensor surfaces. The current responses recorded at each electrode after stimulation of glutamate and NO release by means of K+ and bradykinin clearly demonstrate the ability of the individual electrode in the array to detect the analyte towards which its sensitivity and selectivity was targeted without interference from the neighbouring electrode or other analytes present in the test mixture.

Animals↗

On-line monitoring of methanol in n-hexane by an organic-phase alcohol biosensor.

An organic-phase alcohol biosensor has been developed by co-entrapping alcohol oxidase and horseradish peroxidase within an ionotropy polymer hydrogel matrix fabricated from silica gel particles, hydroxyethyl carboxymethylcellulose, an adduct of 3-methoxy-4-ethoxybenzaldehyde and 4-tert-butylpyridinium acetohydrazone, and octadecylsilica particles. The viability of the immobilised enzymes for the biocatalytic reaction of methanol in n-hexane was comparatively studied by using a bulk cell or a volume-changeable flow-through cell coupled with an oxygen optical transducer. It was found that the microenvironment around the enzyme, the deterioration property of the enzyme, the substrate throughput and the mass transfer process of the reactant in the bioreactor were the crucial parameters affecting the performance of the alcohol organic-phase biosensor. Our optimal biosensor was constructed from a flow-through cell packed with small particles of immobilised enzymes and it could maintain the biocatalytic reaction at high and stable rate for on-line detection of methanol in n-hexane under flow operation mode. The biosensor had an analytical working range of 2.3-90 mM methanol in n-hexane. The response times (t95) were 4.5 and 7.5 min for 60 and 10 mM methanol, respectively. The operational lifetime of the biosensor was more than 45 assays and the shelf lifetime was longer than 2 weeks. The biosensor has been successfully applied to determine the methanol content in a commercial gasoline-methanol blend sample with good recovery.

Alcohol Oxidoreductases↗

Extracellular oxidative systems of the lignin-degrading Basidiomycete Phanerochaete chrysosporium.

The US Department of Energy has assembled a high quality draft genome of Phanerochaete chrysosporium, a white rot Basidiomycete capable of completely degrading all major components of plant cell walls including cellulose, hemicellulose and lignin. Hundreds of sequences are predicted to encode extracellular enzymes including an impressive number of oxidative enzymes potentially involved in lignocellulose degradation. Herein, we summarize the number, organization, and expression of genes encoding peroxidases, copper radical oxidases, FAD-dependent oxidases, and multicopper oxidases. Possibly relevant to extracellular oxidative systems are genes involved in posttranslational processes and a large number of hypothetical proteins.

Lignin↗

Machine learning-assisted Mn-N-C nanozyme colorimetric sensor array for trace-level detection of biogenic amines in meat.

Accurate detection of biogenic amines (BAs) in meat remains challenging due to their high structural similarity and co-occurrence. Herein, an Mn-N-C nanozyme was synthesized via a metal-organic framework confined pyrolysis strategy, possessing excellent oxidase (OXD)- and peroxidase (POD)-like activities. The dual enzyme-like activity showed Km values of 0.1584 mM (OXD) and 0.1498 mM (POD), respectively, in detection system. Leveraging these properties, a colorimetric sensor array was constructed, enabling the detection of four representative BAs within a concentration range of 2-10 ppm with 100% classification accuracy. In addition, a concentration independent recognition model based on an artificial neural network was developed to address signal nonlinearity interference in meat. The integrated system achieved accurate trace-level identification of BAs in perishable fish, pork, and chicken, demonstrating its applicability for early-stage BAs monitoring and quality deterioration warning during storage and transportation.

Biogenic Amines↗

Studies on the physiology of pollen and pollen tube growth. 1. pinus roxburghii.

Pollen grains and pollen tubes of Pinus roxburghii were subjected to histochemical technique with a view to determining the distributional pattern of reserve substances (ascorbic acid, proteins starch, polysaccharides, nucleic acids) and some enzymes (acid phosphatase, glucose-6-phosphatase, peroxidase, esterase, cytochrome oxidase, succinate dehydrogenase). An attempt is made to correlate the activity of different enzymes in relation to pollen tube growth.

Enzymes↗

Resonance Raman spectroscopy as a probe of heme protein structure and dynamics.

Our understanding of metalloporphyrin resonance Raman spectra has advanced to the point where it is possible to obtain detailed information about the structure of the heme group in situ in heme proteins. The porphyrin skeletal mode frequencies can be analyzed in terms of the ligation and spin state of the heme and may provide information about protein-induced stresses. The high-frequency region of the spectrum also contains bands due to vibrations of the porphyrin peripheral substituents, which are potentially monitors of the protein contacts. In the low-frequency region, it is possible to locate bands, at least in some states of the heme protein, which are associated with vibrations of the axial ligands. They give direct information about the nature of the bonding to exogenous ligands or to the proximal protein residue. Thus, a variety of evidence is potentially available in the resonance Raman spectra from which a fairly complete picture of the heme site can be assembled for a particular protein in its various functional states. Detailed studies have been pursued for paradigmatic heme proteins, including myoglobin, hemoglobin, cytochrome c, horseradish peroxidase, and cytochrome oxidase. These studies provide a substantial data base from which the exploration of lesser known systems can be launched. Another extension of current knowledge to new frontiers is in the time domain, since pulsed lasers now make it feasible to carry out time-resolved resonance Raman studies on heme protein reactions. Time-resolved resonance Raman spectroscopy is capable of elucidating the temporal evolution of heme structure and provides a link between heme chemistry and protein dynamics. This link is being elucidated for hemoglobin and cytochrome c, where specific heme intermediates have been identified following ligand photodissociation or electron transfer.

Carbon Monoxide↗

Immunohistochemical demonstration of epidermal growth factor (EGF) receptors in normal human placental villi.

With an avidin-biotin-peroxidase (or glucose oxidase) complex method using anti-epidermal growth factor receptor monoclonal antibody (528 IgG), the tissue and cellular distribution of the receptors for epidermal growth factors (EGF) in normal human placental villi, from 6 to 42 weeks of gestation, were studied. EGF receptors were mainly localized on the free surface of the syncytiotrophoblast that directly faced to intervillous space of the maternal circulation. The cell surface of cytotrophoblasts, except for the region that was adjacent to the basal lamina, was also positive for EGF receptors. The receptors were in close contact to the fetal vessels in the villous stroma. The EGF receptors on the syncytiotrophoblast were thought to be involved in the production and secretion of human chorionic gonadotropin and placental lactogen, probably under the control of maternal EGF. The receptors on cytotrophoblasts may play a role in trophoblastic proliferation, possibly mediated by EGF in the fetal circulatory system.

Chorionic Villi↗

Carbonate anions; effects on the oxidation of luminol, oxidative hemolysis, gamma-irradiation and the reaction of activated oxygen species with enzymes containing various active centres.

Presence of carbonate anions increases the oxidation of luminol in different chemical systems. Lysis of human erythrocytes due to the action of dihydroxyfumaric acid or of perborate is also stimulated by carbonate ions. These anions also change considerably the loss of activity of different enzymes treated with superoxide, hydroxyl or formate radicals and can increase or decrease the effect as a function of the nature of the active centre of the enzyme. The relative effects of superoxide, hydroxyl, formate and carbonate radicals for the inactivation of various enzymes (superoxide dismutases, catalase, ribonuclease, glucose oxidase and glutathione peroxidase) have been examined. Three systems were used: gamma-irradiation under different conditions, photoproduction of radicals and sonication. Inactivation of the enzymes is a function not only of the radical used but also of the nature of the active site. Thus glutathione peroxidase is remarkably resistant to hydroxyl radicals while the superoxide dismutases are rapidly inactivated by carbonate radicals. All of the results combine to show that the presence or absence of carbonate anions must be considered in all studies of oxygen containing free radicals whether chemical, biochemical or biological or high energy irradiation.

Animals↗

An electrochemical multienzymatic biosensor for determination of cholesterol.

This paper describes an electrochemical biosensor for free cholesterol monitoring. The sensor is a multienzymatic electrodic system in which horseradish peroxidase and cholesterol oxidase are simultaneously immobilized within a polymeric film, on the surface of a pyrolitic graphite electrode. From voltammetric and amperometric (flow-injection) data obtained, the efficiency, reproducibility and stability of the system are discussed. Results obtained, of interest for basic and applied biochemistry, represent a first step for construction of a mediator-free biosensor with potentialities for a successful application in the biosensor area.

Biosensing Techniques↗

Fluorometric determination of hypoxanthine and xanthine in biological fluids by high-performance liquid chromatography using enzyme reactors.

A selective and sensitive assay of hypoxanthine and xanthine in biological fluids by high-performance liquid chromatography coupled with immobilized-enzyme reactors was developed. The separations were achieved by reversed-phase liquid chromatography. Hydrogen peroxide produced from hypoxanthine and xanthine by immobilized xanthine oxidase was determined fluorometrically using immobilized peroxidase and p-hydroxyphenylacetic acid. Immobilized enzymes were prepared by intermolecular cross-linking to controlled-pore glass. Assay of allopurinol was also possible by the present method. The method was applied to serum and urine. The detection limits of hypoxanthine and xanthine were approximately 50 and 120 pg per injection, respectively.

Chromatography, High Pressure Liquid↗

Graphite-Teflon composite bienzyme electrodes for the determination of L-lactate: application to food samples.

A bienzyme amperometric graphite-Teflon composite biosensor, in which lactate oxidase (LOD) and peroxidase, together with the mediator ferrocene, are incorporated into the electrode matrix, was developed for the determination of L-lactate in food samples such as wine and yogurt by using both batch- and flow-injection modes. This bienzyme electrode was fabricated by simple physical inclusion of the enzymes and the mediator in the bulk of the graphite-Teflon matrix. A Teflon content of 70%, an applied potential of 0.00 V, and a pH of 7.4 were employed as working conditions. The composite bioelectrode exhibited long-term operation because of the renewability of its surface by polishing. Reproducible amperometric responses were achieved with different electrodes fabricated from different composite matrices, and no significant loss of the enzyme activity occurred after 6 months of storage at 4 degrees C. Detection limits for L-lactate of 1.4 and 0.9 microM were obtained by batch amperometry in stirred solutions and flow-injection with amperometric detection, respectively. An interferences study with different substances which may be present in wine and yogurt together with L-lactic acid demonstrated very good selectivity for the determination of this analyte. The bienzyme composite electrode was applied to the determination of L-lactic acid in red wine and shaken yogurt, and the methods were validated by comparing these results with those obtained by applying a recommended reference method.

Biosensing Techniques↗

Graphite-teflon composite bienzyme electrodes for the determination of cholesterol in reversed micelles. Application to food samples.

A bienzyme amperometric composite biosensor for the determination of free and total cholesterol in food samples is reported. Cholesterol oxidase and horseradish peroxidase, together with potassium ferrocyanide as a mediator, are incorporated into a graphite-70% Teflon matrix. The compatibility of this biosensor design with predominantly nonaqueous media allows the use of reversed micelles as working medium. The reversed micelles are formed with ethyl acetate as continuous phase (in which cholesterol is soluble), a 4% final concentration of 0.05 mol L(-1) phosphate buffer solution, pH 7.4, as dispersed phase, and 0.1 mol L(-1) AOT as emulsifying agent. Studies on the repeatability of the amperometric response obtained at +0.10 V, with and without regeneration of the electrode surface by polishing, on the useful lifetime of one single biosensor and on the reproducibility in the fabrication of different pellets illustrate the robustness of the biosensor design. Determination of free and total cholesterol in food samples such as butter, lard, and egg yoke was carried out, and the obtained results were advantageously compared with those provided by using a commercial Boehringer test kit.

Biosensing Techniques↗

Electroanalysis of metabolic flux from single cells in simple picoliter-volume microsystems.

A picoliter-volume electrochemical analytical chamber has been developed for detecting the metabolic flux resulting from the stress responses of a single plant cell. Electrochemical cells, with volumes as small as 100 pL, were fabricated by controlled electrochemical dissolution of a gold wire sealed in glass (the back-etching of the metal realizing an ultralow-volume titer chamber). In the first instance, the electrode contained within the chamber was characterized by the microinjection of standard aliquots of either ascorbic acid or hydrogen peroxide. In all cases, experimental currents obtained correlated well with theoretical calculations. Subsequently, single plant cells were micromanipulated into the chambers and were exposed to amounts of the detergent SDS (which permeabilized the cell membrane and released the intracellular contents). The flux of metabolite released from a single cell was estimated by using electrochemical-linked assays based upon the enzymes catalase, ascorbate oxidase, and horseradish peroxidase (in each case), in the presence of a mediator. In so doing, we investigated the activity of the cellular protection mechanisms through the determination of peroxides, while the individual cell was "stressed". The technique was found to provide a reliable and reproducible method for making single-cell measurements, using fabrication procedures that are both simple and do not require photolithographic methods.

Journal Article↗