C-terminal periplasmic domain of Escherichia coli quinoprotein glucose dehydrogenase transfers electrons to ubiquinone.
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Membrane-bound quinoprotein glucose dehydrogenase (GDH) in Escherichia coli donates electrons directly to ubiquinone during the oxidation of d-glucose as a substrate, and these electrons are subsequently transferred to ubiquinol oxidase in the respiratory chain. To determine whether the specific ubiquinone-reacting site of GDH resides in the N-terminal transmembrane domain or in the large C-terminal periplasmic catalytic domain (cGDH), we constructed a fusion protein between the signal sequence of beta-lactamase and cGDH. This truncated GDH was found to complement a GDH gene-disrupted strain in vivo. The signal sequence of the fused protein was shown to be cleaved off, and the remaining cGDH was shown to be recovered in the membrane fraction, suggesting that cGDH has a membrane-interacting site that is responsible for binding to membrane, like peripheral proteins. Kinetic analysis and reconstitution experiments revealed that cGDH has ubiquinone reductase activity nearly equivalent to that of the wild-type GDH. Thus, it is likely that the C-terminal periplasmic domain of GDH possesses a ubiquinone-reacting site and transfers electrons directly to ubiquinone. (+info)
Reconstitution of bactericidal activity in chronic granulomatous disease cells by glucose-oxidase-containing liposomes.
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Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency characterized by phagocytes devoid of a functioning nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The failure of CGD phagocytes to produce reactive oxygen species (ROS) results in a marked increase in the susceptibility of affected patients to life-threatening bacterial and fungal infections. This study investigated whether loading of CGD phagocytes with glucose oxidase (GO)-containing liposomes (GOLs) could restore cellular production of bactericidal ROS (eg, H2O2 and HOCl) in vitro. Results indicate that GO encapsulated in liposomes enabled NADPH oxidase-deficient phagocytes to use H2O2 for the production of highly bactericidal HOCl. The intracellular colocalization of bacteria and liposomes (or liposome-derived ferritin) was demonstrated by confocal laser microscopy and electron microscopy. After uptake of GOLs (approximately 0.2 U/mL at 1 mM total lipid concentration, size approximately 180 nm), CGD granulocytes produced HOCl levels comparable to those of normal phagocytes. Remarkably, after treatment with GOLs, CGD phagocytes killed Staphylococcus aureus as efficiently as normal granulocytes. Moreover, treated cells retained sufficient motility toward chemotactic stimuli as measured by chemotaxis assay. Side effects were evaluated by measuring the H2O2 concentrations and the production of methemoglobin in whole blood. These studies revealed that H2O2 produced by GOLs was degraded immediately by the antioxidative capacity of whole blood. Elevated methemoglobin levels were observed only after application of extremely high amounts of GOLs (2 U/mL). In summary, the application of negatively charged GOLs might provide a novel effective approach in the treatment of patients with CGD at high risk for life-threatening infections. (+info)
Glucose sensing based on interdigitated array microelectrode.
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A micro glucose sensor consisting of an interdigitated array gold microelectrode was developed. The interdigitated array structure, which has 10 microns band width and 10 microns band gap, was fabricated in a small region (2.5 x 5 mm2) on a quartz substrate. Glucose oxidase was chemically fixed onto the electrode surface through self-assembled monolayer of 11-mercaptoundecanoic acid; ferroceneacetic acid was used as electron mediator. Electrochemical properties of the glucose oxidase-immobilized microelectrode were investigated by cyclic voltammogram measurements. Results confirmed that the reductive ferroceneacetic acid generated at counter electrode diffuses through a narrow band gap (10 microns) and can reach the working electrode surface. (+info)
Redox cycling of diaspirin cross-linked hemoglobin induces G2/M arrest and apoptosis in cultured endothelial cells.
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It is hypothesized that oxidative reactions of hemoglobin driven by reactive oxygen species in the vasculature lead to endothelial cell injury or death. Bovine aortic endothelial cells were incubated with diaspirin cross-linked hemoglobin (DBBF-Hb), developed as a hemoglobin-based oxygen carrier, and hydrogen peroxide (H(2)O(2)), generated by the glucose oxidase system. The low steady flux of H(2)O(2) oxidizes the ferrous form of DBBF-Hb and drives the redox cycling of ferric and ferryl DBBF-Hb. Cells underwent rounding, swelling and detachment, and accumulated in the G2/M phase of the cell cycle. G2/M arrest preceded the onset of apoptosis as determined by increases in phosphatidylserine (PS) externalization and sub-G1 events. Redox cycling of unmodified hemoglobin also led to G2/M arrest and apoptosis. The rate and extent of DBBF-Hb oxidation correlated with the onset and extent of G2/M arrest and apoptosis and induced significant decreases in soluble reduced thiols. Earlier depletion of glutathione by pretreatment with buthionine sulfoximine rendered cells more susceptible to G2/M arrest and apoptosis. The caspase inhibitor, z-VAD-fmk, had no effect on the induction of G2/M arrest but completely inhibited the subsequent increases in PS externalization and sub-G1 events. Catalase inhibited DBBF-Hb oxidation, the loss of thiols, and the onset of G2/M arrest and apoptosis. These data support a causative role for the ferric-ferryl redox cycle in the development of endothelial cell injury. (+info)
Hemolysis and iodination of erythrocyte components by a myeloperoxidase-mediated system.
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Erythrocytes are hemolyzed by myeloperoxidase, an H2O2-generating system (glucose + glucose oxidase; hypoxanthine + xanthine oxidase) and an oxidizable cofactor (chloride, iodide, thyroxine, triiodothyronine). The combined effect of chloride and either iodide or the thyroid hormones is greater than additive. Myeloperoxidase can be replaced by lactoperoxidase in the iodide-, thyroxine and triiodothyronine-dependent, but not in the chloride-dependent, systems. Hemolysis is is inhibited by the peroxidase inhibitors, azide and cyanide, and by catalase and is stimulated by superoxide dismutase when the xanthine oxidase system is employed as the source of H2O2. Hemolysis by the iodide-dependent system is associated with the iodination of erythrocyte components. (+info)
Size-dependent intracellular immunotargeting of therapeutic cargoes into endothelial cells.
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Cell-selective intracellular targeting is a key element of more specific and safe enzyme, toxin, and gene therapies. Endothelium poorly internalizes certain candidate carriers for vascular immunotargeting, such as antibodies to platelet endothelial cell adhesion molecule 1 (PECAM-1). Conjugation of poorly internalizable antibodies with streptavidin (SA) facilitates the intracellular uptake. Although both small and large (100-nm versus 1000-nm diameter) anti-PECAM/SA-beta galactosidase (SA-beta-gal) conjugates bound selectively to PECAM-expressing cells, only small conjugates showed intracellular accumulation of active beta-gal. To study whether size of the conjugates controls the uptake, a series of anti-PECAM/SA and anti-PECAM/bead conjugates ranging from 80 nm to 5 microm in diameter were produced. Human umbilical vein endothelial cells and PECAM-transfected mesothelioma cells internalized 80- to 350-nm anti-PECAM conjugates, but not conjugates larger than 500 nm. Further, size controls intracellular targeting of active therapeutic cargoes in vitro and in vivo. Small anti-PECAM/DNA conjugates transfected target cells in culture 5-fold more effectively than their large counterpart (350- versus 4200-nm diameter). To evaluate the practical significance of the size-controlled subcellular addressing, we coupled glucose oxidase (GOX) to anti-PECAM and antithrombomodulin. Both types of conjugates had equally high pulmonary uptake after intravenous injection in mice, yet only small (200- to 250-nm), not large (600- to 700-nm), GOX conjugates caused profound oxidative vascular injury in the lungs, presumably owing to intracellular generation of H(2)O(2). Thus, engineering of affinity carriers of specific size permits intracellular delivery of active cargoes to endothelium in vitro and in vivo, a paradigm useful for the targeting of drugs, genes, and toxins. (+info)
A novel two-enzyme amperometric electrode for lactose determination.
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Coimmobilization of beta-galactosidase and glucose oxidase in a redox polymer, polyvinylferrocenium perchlorate (PVF+ ClO4-), led to the development of an enzyme electrode for the determination of lactose. The amperometric response of the electrode was measured at +0.70 V vs. SCE, which was due to the electrooxidation of enzymatically produced H2O2. The effects of the substrate and buffer concentrations as well as the pH on the electrode response were elucidated. (+info)
Vascular immunotargeting of glucose oxidase to the endothelial antigens induces distinct forms of oxidant acute lung injury: targeting to thrombomodulin, but not to PECAM-1, causes pulmonary thrombosis and neutrophil transmigration.
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Oxidative endothelial stress, leukocyte transmigration, and pulmonary thrombosis are important pathological factors in acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Vascular immunotargeting of the H(2)O(2)-generating enzyme glucose oxidase (GOX) to the pulmonary endothelium causes an acute oxidative lung injury in mice.(1) In the present study we compared the pulmonary thrombosis and leukocyte transmigration caused by GOX targeting to the endothelial antigens platelet-endothelial cell adhesion molecule (PECAM) and thrombomodulin (TM). Both anti-PECAM and anti-TM delivered similar amounts of (125)I-GOX to the lungs and caused a dose-dependent, tissue-selective lung injury manifested within 2 to 4 hours by high lethality, vascular congestion, polymorphonuclear neutrophil (PMN) sequestration in the pulmonary vasculature, severe pulmonary edema, and tissue oxidation, yet at an equal dose, anti-TM/GOX inflicted more severe lung injury than anti-PECAM/GOX. Moreover, anti-TM/GOX-induced injury was accompanied by PMN transmigration in the alveolar space, whereas anti-PECAM/GOX-induced injury was accompanied by PMN degranulation within vascular lumen without PMN transmigration, likely because of PECAM blockage. Anti-TM/GOX caused markedly more severe pulmonary thrombosis than anti-PECAM/GOX, likely because of TM inhibition. These results indicate that blocking of specific endothelial antigens by GOX immunotargeting modulates important pathological features of the lung injury initiated by local generation of H(2)O(2) and that this approach provides specific and robust models of diverse variants of human ALI/ARDS in mice. In particular, anti-TM/GOX causes lung injury combining oxidative, prothrombotic, and inflammatory components characteristic of the complex pathological picture seen in human ALI/ARDS. (+info)