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PubMed2002

Enzyme-based sensor arrays for rapid characterization of complex disaccharide solutions.

An enzyme-based sensor array has been developed to detect multiple disaccharides in aqueous solutions. Porous agarose beads, derivatized with enzymes for assaying disaccharides, are localized within wells etched into a silicon chip in a regular 5 x 7 array. Each well is individually addressable and acts as a microanalysis chamber where sample solution passes through the agarose matrix and is exposed to the enzymes. Detection is achieved by observing the increase in absorbance of a quinoneimine dye produced during the reaction. This technique is used to quantify the disaccharides lactose, sucrose, and maltose and the monosaccharide glucose. Preexisting glucose in the sample complicates multicomponent sensing but can be accounted for by including a glucose sensor in the array. This detection strategy is applied to the simultaneous analysis of these sugars in several beverages.

Beverages↗
PubMed1998

Role of algae in fate of carcinogenic polycyclic aromatic hydrocarbons in the aquatic environment.

Polycyclic aromatic hydrocarbons (PAHs) represent an ecotoxicologically relevant, combustion-related substance group. The bioconcentration and transformation of a priority PAH, benzo[a]pyrene (BaP), by brown (Fucus vesiculosus and Chorda filum), red (Furcellaria lumbricalis), green (Enteromorpha intestinalis, and Cladophora glomerata), and chara (Chara aspera) algae have been studied. A flux budget was made of the amounts of BaP that are accumulated and metabolized by different algae during an estimated time. The results indicated that of all the BaP consumed, 89-99% was found in the biomass of Fucus, an insignificant part was in the solution, and the remainder (up to 4%) was not recovered, i.e., was considered to have been metabolized. For green and chara algae the proportion of transformed PAHs was more essential, 42-49%. The transformation of BaP in marine and freshwater algae is species specific and depends on the presence and activity of enzymes localized in the plant cells. The most important enzyme systems for detoxification of BaP are o-diphenol oxidase, cytochrome P450, and peroxidase. The data obtained indicate the important role of marine and freshwater algae in the fate of carcinogenic PAHs in the environment.

Benzo(a)pyrene↗
PubMed1980

Oxygen utilization by Lactobacillus plantarum. I. Oxygen consuming reactions.

Lactobacillus plantarum (ATCC 8014) cells, grown aerobically on glucose medium, consumed molecular oxygen when incubated with either glucose, D/L-lactate or pyruvate as substrate. Cell extracts catalyzed the oxidation of NADH, D/L-lactate of pyruvate with O2. Per mol O2 2mol of NADH were consumed indicating that O2 was reduced to H2O; reduction proceeded via H2O2 involving a NADH oxidase and a NADH peroxidase. Catalase activity was absent. Pyruvate oxidation with O2 led to the formation of H2O2, lactate oxidation to the formation of H2O. Thus in L. plantarum different mechanisms are available by which molecular oxygen can be used as electron acceptor for oxidation reactions.

Glucose↗
PubMed1982

Selective enrichment of NADPH oxidase activity in phagosomes from guinea pig polymorphonuclear leukocytes.

Recent studies have demonstrated that the activated NADPH oxidase, the enzyme responsible for the stimulation of O2 consumption with O2 formation during phagocytosis, is located in the plasma membrane of leukocytes. The present work deals with whether the activation induced by phagocytosis involves the enzyme of the entire membrane or only that of the portion of the membrane that interacts with the phagocytosable particle and forms the phagosome. The results presented show that the activity of the NADPH oxidase of phagosomal membrane, isolated by centrifugation of homogenates on discontinuous sucrose gradients, is increased 12.6-fold with respect that of homogenate. In contrast, the activities of 5'-nucleotidase and of acid p-nitrophenyl phosphatase, enzyme markers of the plasma membrane not activated during phagocytosis and uniformly distributed on the entire membrane, are increased only about three-fold with respect to that of homogenate. These results indicate that during phagocytosis and activation of NADPH oxidase is a segmentary response that involves only the enzyme that forms the phagocytic vacuole. This fact is relevant for the function of toxic intermediates of oxygen reduction that are discharged in direct contact with the engulfed agent.

4-Nitrophenylphosphatase↗
PubMed2006

Earlier onset of neutrophil-mediated inflammation in the ultraviolet-exposed skin of mice deficient in myeloperoxidase and NADPH oxidase.

OBJECTIVE AND DESIGN: This study examined the role of neutrophil-derived reactive oxygen species (ROS) in neutrophil recruitment into ultraviolet B (UVB)-exposed skin of mice. METHODS: Mouse dorsal skin was irradiated with UVB (600 mJ/cm2). Accumulation of neutrophils within the inflammatory sites was observed histochemically. Keratinocyte-derived chemokine (KC) and macrophage inflammatory protein 2 (MIP-2) were quantified, and in vivo chemotaxis of neutrophils toward KC and MIP-2 was examined. RESULTS: UVB exposure of mice deficient in myeloperoxidase (MPO), NADPH oxidase, or both, caused skin neutrophil infiltration peaking at 60, 48, and 48 h, respectively, which was earlier than the 72-h peak in wild-type mice. MIP-2 level was higher in mutant than wild-type mice. Mutant neutrophils produced more MIP-2 in vitro. Neutrophil migration toward a localized source of KC was higher in mutant than wild type mice. NADPH oxidase deficiency had a greater effect on migration than MPO deficiency. CONCLUSIONS: These results suggest that ROS produced by neutrophils regulate expression of MIP-2 and migration of neutrophils toward KC. This may explain the earlier infiltration of mutant neutrophils in response to UVB.

Animals↗
PubMed2005

Effects of crude oil and diesel exposures on biochemical activities of polymorphonuclear leukocytes in cattle.

Cattle exposed to low doses of an Alberta crude oil, Pembina Cardium crude oil (PCCO), or a winter diesel oil no. 2 (WDO-2) were assessed for their biochemical activities in polymorphonuclear leukocyte (PMNL) cells (mainly neutrophils). The study used a randomized block design containing five treatment groups (8 animals/group). The animals were dosed per gavage with the test substance on study days 0, 14, 28, and 42. The dosages given (on per kg body weight) were: Group 1 (control), 10 mL/kg of potable water; Group 2, 5 mL/kg WDO-2; Group 3, 2.5 mL/kg PCCO; Group 4, 5 mL/kg PCCO; and Group 5, 10 mL/kg PCCO. Blood was collected at the specified intervals during the pre- and post-exposure periods, and the biochemical activities of isolated PMNL were analyzed. Cattle groups exposed to WDO-2 and PCCO showed moderate and statistically significant reductions (p < 0.01) in the activities of (1) phorbol myristate acetate (PMA) stimulated cellular respiration (respiratory burst), (2) NADPH-oxidase (PMA-stimulated production of superoxide anion), (3) myeloperoxidase, and (4) n-acetylglucosidase as compared to the control group. These biochemical parameters also showed statistically significant (p < 0.01) dose-related periodic (study day) trends. In general, these biochemical activities were decreased after each dosing; however, they subsequently recovered to near the pre-dosing levels. Such a biochemical response in PMNL provides a valuable biological tool to follow exposure effects in cattle accidentally exposed to low doses of petroleum hydrocarbons.

Animals↗
PubMed1998

Confocal Raman microspectroscopy of the activation of single neutrophilic granulocytes.

Confocal Raman micro-spectroscopy has been applied to investigate the activation process of single, living neutrophilic granulocytes. Both resting cells as well as activated cells were measured. The activation of cells was performed with phorbol-12-myristate-13-acetate activator and Escherichia Coli bacteria. Raman microspectroscopy combines a high spatial resolution inside a single, living cell with detailed material information. Using this approach it can be concluded that activation of the cells with phorbol-12-myristate-13-acetate causes a change in the redox state of cytochrome b558. This protein is a part of the NADPH-oxidase complex that neutrophilic granulocytes employ to generate O-2, superoxide anion. Additionally a change in the redox state of myeloperoxidase can be observed. Myeloperoxidase is known to react with O-2. Activation of the cells with bacteria gives rise to corresponding changes in the Raman spectra. From this single cell study it can be concluded that the enzymes cytochrome b558 and myeloperoxidase are present inside the cytoplasm of the living cell, while participating in the redox processes. Activation causes an intra-cellular release of oxygen metabolites. Activation with bacteria of neutrophilic granulocytes from a patient with chronic granulomatous disease, that contain no cytochrome b558, led to typical changes in the redox state of myeloperoxidase. This indicates that in the bacterium/neutrophilic granulocyte system oxygen metabolites are generated that are capable of reacting with MPO.

Granulomatous Disease, Chronic↗
PubMed2002

Biotechnological applications and potential of wood-degrading mushrooms of the genus Pleurotus.

The genus Pleurotus comprises a group of edible ligninolytic mushrooms with medicinal properties and important biotechnological and environmental applications. The cultivation of Pleurotus spp is an economically important food industry worldwide which has expanded in the past few years. P. ostreatus is the third most important cultivated mushroom for food purposes. Nutritionally, it has unique flavor and aromatic properties; and it is considered to be rich in protein, fiber, carbohydrates, vitamins and minerals. Pleurotus spp are promising as medicinal mushrooms, exhibiting hematological, antiviral, antitumor, antibiotic, antibacterial, hypocholesterolic and immunomodulation activities. The bioactive molecules isolated from the different fungi are polysaccharides. One of the most important aspects of Pleurotus spp is related to the use of their ligninolytic system for a variety of applications, such as the bioconversion of agricultural wastes into valuable products for animal feed and other food products and the use of their ligninolytic enzymes for the biodegradation of organopollutants, xenobiotics and industrial contaminants. In this Mini-Review, we describe the properties of Pleurotus spp in relation to their biotechnological applications and potential.

Biodegradation, Environmental↗
PubMed2006

Regulation of antioxidant and oxidant enzymes in vascular cells and implications for vascular disease.

Data from numerous studies demonstrate that oxidative stress plays an important role in the pathogenesis of vascular disease. Oxidative stress leads to many pathologic events, such as inactivation of nitric oxide, lipid oxidation, enhanced mitogenicity and apoptosis of vascular cells, and increased expression and activation of redox-sensitive genes, which contribute to atherogenesis at all stages of the disease. Multiple enzymes are expressed in vascular cells that are involved in the elimination and production of reactive oxygen species, including the superoxide dismutases, catalase, thioredoxin reductase, glutathione peroxidase, NAD(P)H oxidase, xanthine oxidase, myeloperoxidase, and endothelial nitric oxide synthase. Several agonists and pathologic conditions that predispose to vascular disease induce changes in the expression and activity levels of these antioxidant and oxidant enzyme systems, leading to modulation of vascular oxygen radical load. Identification of key enzymes and mechanisms of vascular oxidative stress is important for the development of novel, specific pharmacologic interventions.

Animals↗
PubMed1987

One-step enzyme immunochromatographic assay for theophylline.

The convenience of the previously described enzyme immunochromatography method for visually quantifying theophylline in whole blood has been improved with the development of a one-step protocol. The capillary migration and color generation in the two-step enzyme immunochromatographic assay have been combined into a single step. Ascorbic acid is used as a signal inhibitor to delay enzymatic color product formation until the inhibitor itself is consumed. The concept of internal delay reaction is presented and the mechanism of ascorbate's action as an inhibitor to temporarily delay color generation is described. The internal delay reaction has been applied to a practical one-step quantitative visual enzyme immunochromatographic assay for theophylline in whole blood.

Ascorbic Acid↗