Adding Glucose Oxidase Research Articles

Glucose-Responsive Silk Fibroin Microneedles for Transdermal Delivery of Insulin.

Microneedles (MNs) have attracted great interest as a drug delivery alternative to subcutaneous injections for treating diabetes mellitus. We report MNs prepared from polylysine-modified cationized silk fibroin (SF) for responsive transdermal insulin delivery. Scanning electron microscopy analysis of MNs' appearance and morphology revealed that the MNs were well arranged and formed an array with 0.5 mm pitch, and the length of single MNs is approximately 430 μm. The average breaking force of an MN is above 1.25 N, which guarantees that it can pierce the skin quickly and reach the dermis. Cationized SF MNs are pH-responsive. MNs dissolution rate increases as pH decreases and the rate of insulin release are accelerated. The swelling rate reached 223% at pH = 4, while only 172% at pH = 9. After adding glucose oxidase, cationized SF MNs are glucose-responsive. As the glucose concentration increases, the pH inside the MNs decreases, the MNs' pore size increases, and the insulin release rate accelerates. In vivo experiments demonstrated that in normal Sprague Dawley (SD) rats, the amount of insulin released within the SF MNs was significantly smaller than that in diabetic rats. Before feeding, the blood glucose (BG) of diabetic rats in the injection group decreased rapidly to 6.9 mmol/L, and the diabetic rats in the patch group gradually reduced to 11.7 mmol/L. After feeding, the BG of diabetic rats in the injection group increased rapidly to 33.1 mmol/L and decreased slowly, while the diabetic rats in the patch group increased first to 21.7 mmol/L and then decreased to 15.3 mmol/L at 6 h. This demonstrated that the insulin inside the microneedle was released as the blood glucose concentration increased. Cationized SF MNs are expected to replace subcutaneous injections of insulin as a new modality for diabetes treatment.

Metal-doped carbon dots as peroxidase mimic for hydrogen peroxide and glucose detection.

Carbon dots (CDs) have several superior characteristics including sufficient carbon sources, easy preparation, no toxicity, and high catalytic efficiency as a new kind of nanozyme. Herein, Ce-doped carbon dots (Ce-CDs), Cr-doped carbon dots (Cr-CDs), Cu-doped carbon dots (Cu-CDs), Fe-doped carbon dots (Fe-CDs), Mn-doped carbon dots (Mn-CDs), and non-metal-doped carbon dots (0-CDs) were synthesized to explore the detection of hydrogen peroxide (H2O2) and glucose as peroxidase mimic. The prepared CDs could efficiently oxidize the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxTMB in the presence of H2O2. After adding glucose oxidase (GOD) to the CDs/TMB system, a colorimetric method for glucose detection was developed. The results show that Fe-CDs possess the highest catalytic activity. When using Fe-CDs as peroxidase mimetics, the detection limit of this assay for glucose was 0.029mmol L-1. This successfully provides a sensitive and selective colorimetric method for hydrogen peroxide and glucose determination.

A regression-based machine learning approach for pH and glucose detection with redox-sensitive colorimetric paper sensors.

Colorimetric paper sensors are used in various fields due to their convenience and intuitive manner. However, these sensors present low accuracy in practical use because it is difficult to distinguish color changes for a minute amount of analyte with the naked eye. Herein, we demonstrate that a machine learning (ML)-based paper sensor platform accurately determines the color changes. We fabricated a colorimetric paper sensor by adsorbing polyaniline nanoparticles (PAni-NPs), whose color changes from blue to green when the ambient pH decreases. Adding glucose oxidase (GOx) to the paper sensor enables colorimetric glucose detection. Target analytes (10 μL) were aliquoted onto the paper sensors, and their images were taken with a smartphone under the same conditions in a darkroom. The red-green-blue (RGB) data from the images were extracted and used to train and test three regression models: support vector regression (SVR), decision tree regression (DTR), and random forest regression (RFR). Of the three regression models, RFR performed the best at estimating pH levels (R2 = 0.957) ranging from pH 2 to 10 and glucose concentrations (R2 = 0.922) ranging from 0 to 10 mg mL-1.

Evaluation of Oxidation Process by Ozonation and Glucose Oxidase Enzyme on the Degradation of Benzoquinone in Wheat Flour

Background: Wheat flour is an important food ingredient for humans, which is the basic ingredient of bread and other bakery products. Objective: This study aimed to assess the effect of adding Glucose Oxidase (GOX), and exposure to ozone gas on methyl-1, 4-benzoquinone (MBQ), and ethyl-1, 4-benzoquinone (EBQ) secreted by Tribolium castaneum in flour. Methods: The flour contaminated by MBQ and EBQ was treated with ozone gas at (10, 20, and 40 ppm) with exposure times (15, 30, and 45 min). Similarly, GOX was added to flour at (10, 15, and 20 ppm), leaving the dough for periods between 10 and 45 min after treatments. The MBQ and EBQ determined by HPLC, and the UV-Visible Spectrophotometer and Fourier Transform Infrared Spectroscopy (FTIR) were used to describe the changes that occurred in the main structure of EBQ after ozonation at 40 ppm for 45 min. Results: The results indicated that adding GOX enzyme to the flour at level 20 ppm degrade the MBQ to 13.7, 20.23, and 39.6 after 15, 30, and 45 min from mixing time, respectively. On the other hnad, the EBQ degrades to 13.6, 18.9, and 35.9%. In contrast, the percentages of degradation of MBQ and EBQ increases after ozonation at 40 ppm for 45min were 84.1 and 78.8%, respectively. The results obtained by UV–vis spectroscopy and FTIR reflect that many oxidation products formed as aldehydes, ketones, and carboxylic acids. Conclusion: In general, ozonation was a reliable treatment for the degradation of benzoquinone in flour.

Effect of glucose oxidase addition on the textural characteristics of wheat-maize dough and bread

Abstract The aim of this study was to assess the effect of adding glucose oxidase (GOX) on wheat-maize dough and bread textural characteristics. It strengthens the importance and need of replacing wheat flour with maize flour in regions where extensive cultivation of wheat is not possible. For improving the rheological properties of bread, GOX was added to wheat and wheat-maize flours. Wheat and maize flours were used in several ratios, but the best results were obtained for (on flour basis): 80:20, 75:25, 70:30, where the highest values correspond to the wheat flour. Wheat-maize dough and bread volume were slightly influenced by GOX addition. Dough strength was higher in the case of dough without GOX addition than the samples enriched by GOX, while crumb strength of GOX enriched maize bread was higher than the samples without GOX. The obtained data might be useful for future studies, while it was demonstrated here the effect of GOX addition on wheat-maize dough and bread.

Enhancing the Rheological Performance of Wheat Flour Dough with Glucose Oxidase, Transglutaminase or Supplementary Gluten

The enzymes glucose oxidase and transglutaminase are frequently used to improve the breadmaking performance of wheat flours, as they have the ability to considerably alter the viscoelastic nature of the gluten network. To evaluate a flour’s breadmaking performance, rheological tests offer an attractive framework. In this study, the rheological impact of adding glucose oxidase or transglutaminase to wheat flour dough is investigated by means of linear oscillatory shear tests, creep-recovery shear tests and startup extensional tests. The former tests reveal that the enzymes render the dough stiffer and enhance its elastic character, until saturation is reached. In the breadmaking process, the use of excessive amounts of enzyme is known to be counterproductive. The strain-hardening index clearly reveals this overcross-linking effect. Besides enzymes, the gluten network can also be reinforced by adding supplementary gluten, which was indeed found to enhance the extent of strain-hardening.

Effects of Wheat Flour Dough’s Viscoelastic Level by Adding Glucose Oxidase on its Dynamic Shear Properties whatever the Strain Modes

The objective of this work was to study the effects of wheat flour dough’s viscoelastic level by adding glucose oxidase (Gox) on its rheological properties at dynamic shear strain mode to predict the final product quality. Dough does display a linear viscoelastic domain. Glucose oxidase (Gox) was added to dough in order to enhance its viscoelasticity and to take into account the possible effects of this viscoelasticity on the results. Whatever the types of dough strain used G’ increased, tan δ decreased and led to less sticky dough. Wheat flour dough, an increase in G’ with extension may be associated to a strain-hardening phenomenon but the role of dough viscoelasticity is discussed.

P47phox-deficient immune microenvironment signals dysregulate naive T-cell apoptosis

The phagocyte NADPH oxidase is a multicomponent enzyme complex mediating microbial killing. We find that NADPH oxidase p47(phox)-deficient (p47(phox-/-)) chronic granulomatous disease (CGD) mice develop lymph node hyperplasia even without obvious infection, where increased number of T and B lymphocytes is associated with increased percent of naïve cells and a lower T : B cell ratio than wild type. Paradoxically, despite lymphoid hyperplasia in vivo, when lymphocytes are placed in culture, p47(phox-/-) CD8(+) lymphocytes progress more rapidly to apoptosis than wild type. This is associated in cultured p47(phox-/-) CD8(+) lymphocytes with the induction of proapoptotic Bim and Puma expression, increased mitochondrial outer membrane permeabilization and depressed Bcl-2 expression. Addition of IL-7 to the culture partially corrects Bcl-2 levels in cultured p47(phox-/-) CD8(+) lymphocytes and improves the survival. Adding glucose oxidase to the culture to generate hydrogen peroxide along with IL-7 further improves p47(phox-/-) CD8(+) lymphocyte survival, but only to 30% of wild type. We conclude that p47(phox-/-) CD8(+) lymphocytes have an intrinsic survival defect likely in part related to the oxidase deficiency, but in vivo in lymph nodes of CGD mice, there are microenvironmental factors yet to be delineated that suppress the progression of apoptosis and allow the accumulation of lymphocytes leading to lymphoid hyperplasia.

Cyanidin 3-glucoside protects 3T3-L1 adipocytes against H2O2- or TNF-α-induced insulin resistance by inhibiting c-Jun NH2-terminal kinase activation

Anthocyanins are naturally occurring plant pigments and exhibit an array of pharmacological properties. Our previous study showed that black rice pigment extract rich in anthocyanin prevents and ameliorates high-fructose-induced insulin resistance in rats. In present study, cyanidin 3-glucoside (Cy-3-G), a typical anthocyanin most abundant in black rice was used to examine its protective effect on insulin sensitivity in 3T3-L1 adipocytes exposed to H2O2 (generated by adding glucose oxidase to the medium) or tumor necrosis factor α (TNF-α). Twelve-hour exposure of 3T3-L1 adipocytes to H2O2 or TNF-α resulted in the increase of c-Jun NH2-terminal kinase (JNK) activation and insulin receptor substrate 1 (IRS1) serine 307 phosphorylation, concomitantly with the decrease in insulin-stimulated IRS1 tyrosine phosphorylation and cellular glucose uptake. Blocking JNK expression using RNA interference efficiently prevented the H2O2- or TNF-α-induced defects in insulin action. Pretreatment of cells with Cy-3-G reduced the intracellular production of reactive oxygen species, the activation of JNK, and attenuated H2O2- or TNF-α-induced insulin resistance in a dose-dependent manner. In parallel, N-acetyl-cysteine, an antioxidant compound, did not exhibit an attenuation of TNF-α-induced insulin resistance. Taken together, these results indicated that Cy-3-G exerts a protective role against H2O2- or TNF-α-induced insulin resistance in 3T3-L1 adipocytes by inhibiting the JNK signal pathway.

Redox regulation of proliferation of lens epithelial cells in culture

Both oxidants and antioxidants have been shown to modulate cell proliferation. We studied the effects of hydrogen peroxide and two antioxidants on the rate of proliferation of lens epithelial cells in culture. Hydrogen peroxide at concentrations higher than 32 microM caused a significant inhibition of proliferation. However, in the concentration range of 0.01-0.5 microM, hydrogen peroxide stimulated the rate of proliferation. The effect of hydrogen peroxide was dependent on the amount of cells in an individual culture well, indicating decomposition of hydrogen peroxide by cellular enzymes. In order to eliminate the possibility of decomposition of the dose of hydrogen peroxide given as a bolus, we induced continual production of hydrogen peroxide by adding glucose oxidase to the incubation medium. We found that hydrogen peroxide, generated by 1-50 microU x ml(-1) of glucose oxidase significantly increased the rate of cell proliferation. This effect was most apparent at the beginning of the exponential phase of cellular growth. Glucose oxidase alone (100-500 microU x ml(-1)) did not produce any effect. The effects of pro-oxidative hydrogen peroxide were compared with the effects of two biologically important antioxidants, alpha-tocopherol and retinol. Both antioxidants completely inhibited proliferation at concentrations of 30 microM and higher. In contrast to retinol, the effect of alpha-tocopherol was dependent on the amount of cells, indicating cellular decomposition of alpha-tocopherol. The results document the possibility of redox regulation of cellular proliferation at physiologically relevant reactant concentrations.

IRS1 degradation and increased serine phosphorylation cannot predict the degree of metabolic insulin resistance induced by oxidative stress.

Oxidative stress was shown to selectively induce impaired metabolic response to insulin, raising the possible involvement of alterations in Insulin-Receptor-Substrate (IRS) proteins. This study was conducted to assess whether oxidative stress induced IRS protein degradation and enhanced serine phosphorylation, and to assess their functional importance. 3T3-L1 adipocytes and rat hepatoma cells (FAO) were exposed to micro-molar H(2)O(2) by adding glucose oxidase to the culture medium, and IRS1 content, its serine phosphorylation and downstream metabolic insulin effects were measured. Cells exposed to oxidative stress exhibited decreased IRS1 (but not IRS2) content, and increased serine phosphorylation of both proteins. Total protein ubiquitination was increased in oxidized cells, but not in cells exposed to prolonged insulin treatment. Yet, lactacystin and MG132, two unrelated proteasome inhibitors, prevented IRS1 degradation induced by prolonged insulin but not by oxidative stress. The PI 3-kinase inhibitor LY294002 and the mTOR inhibitor rapamycin, but not the MEK1 inhibitor PD98059, could prevent IRS1 changes in oxidized cells. Rapamycin, which protected against IRS1 degradation and serine phosphorylation was not associated with improved response to acute insulin stimulation. Moreover, the antioxidant alpha lipoic acid, while protecting against oxidative stress-induced insulin resistance in 3T3-L1 adipocytes, could not prevent IRS1 degradation and serine phosphorylation. Oxidative stress induces serine phosphorylation of IRS1 and increases its degradation by a proteasome-independent pathway; yet, these changes do not correlate with the induction of impaired metabolic response to insulin.

Synthesis and Purification of 3-Hydroxykynurenine- O-β-glucoside, a Primate Lens Ultraviolet Filter, and Its Application in a Two-Step Assay for β-Glucosidase Activity

3-Hydroxykynurenine-3- O-β-glucoside (3-HKG) functions in the primate lens as a filter of 295- 400-nm light, thereby protecting the retina from damaging UV radiation. Although extensive studies have been conducted to determine the functional role of 3-HKG in the primate lens, an efficient method for its synthesis and purification has yet to be developed. Several procedures have been reported for the synthesis of 3-HKG; however, these procedures either result in low yields or require numerous sequential reactions and purification steps. In this study, we report a two-step synthesis of 3-HKG with a one-step purification and a two- to eightfold increase in yield over previously reported methods. Additionally, an assay was developed to confirm the presence of a β-glycosidic linkage in the purified reaction product and we propose a method by which 3-HKG can be used as a general probe of β-glucosidase activity. The assay consists of adding glucose oxidase to the 3-HKG/glucosidase solution and then allowing the hydrogen peroxide, generated from the interaction of glucose with glucose oxidase, to oxidize 3-hydroxykynurenine to xanthomattin (XAN) and 4,6-dihydroxyquinolinequinone carboxylic acid (DHQCA). Both XAN and DHQCA absorb strongly between 400 and 500 nm and the color change of the solution can be seen by eye. In addition, XAN fluoresces in the visible region with λ max = 527 nm.

An oxidative stress-responsive biosensor: responses to hydrogen peroxide generated by an extracellular enzyme

The promoter region of the katG gene of Escherichia coli was fused to the bacterial luciferase reporter gene luxCDABE from Photorhadbus luminescens. In E. coli, this system is limited to compounds that produce oxidative stress. Consequently we designed a system that would detect compounds that do not generate such stress. As proof of concept we choose a commonly used oxidase enzyme, for example, glucose oxidase that oxidises glucose to hydrogen peroxide and gluconolactone. The resultant hydrogen peroxide puts stress onto the whole sensor and results in an increase in luminescence over time. We found that the system was specific for glucose and had a range of sensitivity from 2 to 12 mM glucose. We propose that by adding glucose oxidase to the oxidative stress whole cell biosensor the specificity of the oxidative stress response can be increased.

Lipoic acid protects against oxidative stress induced impairment in insulin stimulation of protein kinase B and glucose transport in 3T3-L1 adipocytes.

Oxidative stress has been shown to impair insulin-stimulated glucose transporter 4 translocation in 3T3-L1 adipocytes. This study explores the potential of the antioxidant lipoic acid to protect the cells against the induction of insulin resistance when given before exposure to oxidative stress. 3T3-L1 were exposed for 16 h to lipoic acid after which cells were exposed for 2 h to continuous production of H2O2 by adding glucose oxidase to the culture medium. These conditions resulted in a 50-70% reduction in insulin-stimulated glucose transport activity associated with a decrease in reduced glutathione content from 37.4 +/- 3.1 to 26.4 +/- 4.9 nmol/mg protein, (p < 0.005). Lipoic acid pretreatment increased insulin-stimulated glucose transport following oxidative stress, reaching 84.8 +/- 4.4% of the control, associated with an increase in reduced glutathione content. Oxidation impaired the 4.89 +/- 0.36-fold insulin-stimulated increase in glucose transporter 4 content in plasma membrane lawns of control cells. Lipoic acid pretreatment was, however, associated with preserved insulin-induced glucose transporter 4 translocation in cells exposed to oxidation, yielding 80% of its content in controls. Although tyrosine phosphorylation patterns were not affected by lipoic acid pretreatment, insulin-stimulated protein kinase B/Akt serine 473 phosphorylation and activity were considerably impaired by oxidation but protected by lipoic acid pretreatment. A protective effect was not observed with either troglitazone, its isolated vitamin E moiety, or with vitamin C. This study shows the ability of lipoic acid to provide partial protection against the impaired insulin-stimulated glucose transporter 4 translocation and protein kinase B/Akt activation induced by oxidative stress, potentially by its capacity to maintain intracellular redox state.

Oxidative Stress Disrupts Insulin-induced Cellular Redistribution of Insulin Receptor Substrate-1 and Phosphatidylinositol 3-Kinase in 3T3-L1 Adipocytes: A PUTATIVE CELLULAR MECHANISM FOR IMPAIRED PROTEIN KINASE B ACTIVATION AND GLUT4 TRANSLOCATION

In a recent study we have demonstrated that 3T3-L1 adipocytes exposed to low micromolar H2O2 concentrations display impaired insulin stimulated GLUT4 translocation from internal membrane pools to the plasma membrane (Rudich, A., Tirosh, A., Potashnik, R., Hemi, R., Kannety, H., and Bashan, N. (1998) Diabetes 47, 1562-1569). In this study we further characterize the cellular mechanisms responsible for this observation. Two-hour exposure to approximately 25 microM H2O2 (generated by adding glucose oxidase to the medium) resulted in disruption of the normal insulin stimulated insulin receptor substrate (IRS)-1 and phosphatidylinositol (PI) 3-kinase cellular redistribution between the cytosol and an internal membrane pool (low density microsomal fraction (LDM)). This was associated with reduced insulin-stimulated IRS-1 and p85-associated PI 3-kinase activities in the LDM (84 and 96% inhibition, respectively). The effect of this finding on the downstream insulin signal was demonstrated by a 90% reduction in insulin stimulated protein kinase B (PKB) serine 473 phosphorylation and impaired activation of PKBalpha and PKBgamma. Both control and oxidized cells exposed to heat shock displayed a wortmannin insensitive PKB serine phosphorylation and activity. These data suggest that activation of PKB and GLUT4 translocation are insulin signaling events dependent upon a normal insulin induced cellular compartmentalization of PI 3-kinase and IRS-1, which is oxidative stress-sensitive. These findings represent a novel cellular mechanism for the induction of insulin resistance in response to changes in the extracellular environment.

Lung edema due to hydrogen peroxide is independent of cyclooxygenase products.

Active oxygen species can cause lung injury. Although a direct action on endothelial cells is proposed, the possibility exists that they might cause injury via mediators. We considered that active oxygen species would stimulate the generation of cyclooxygenase metabolites, which then alter pulmonary vasoreactivity and cause edema. We chemically produced hydrogen peroxide by adding glucose oxidase to a plasma- and cell-free, but beta-D-glucose-containing, solution, which perfused isolated rat lungs. Addition of glucose oxidase to the perfusate caused a marked decrease in pulmonary vasoreactivity, accompanied by an increase in the concentrations of prostacyclin, thromboxane A2, and prostaglandin F2 alpha. Pretreatment with catalase, a specific scavenger of hydrogen peroxide, preserved pulmonary vasoreactivity, inhibited the increase of the concentration of the measured prostaglandins, and prevented edema formation. Indomethacin effectively blocked lung prostaglandin production but neither prevented the decrease in vasoreactivity nor inhibited edema formation. From these data we conclude that hydrogen peroxide impaired pulmonary vasoreactivity and subsequently caused edema. Despite the fact that hydrogen peroxide stimulated lung prostaglandin production, cyclooxygenase-derived products neither caused the decrease in vasoreactivity nor the development of edema.