Glucose Oxidase Treatment Research Articles

Spatiotemporal expression patterns of ZBP1 in the brain of mouse experimental stroke model

Z-DNA binding protein 1 (ZBP1) is a cytosolic nucleic acid sensor, functioning as a critical mediator of inflammation and cell death pathways. Since neuroinflammation could occur in response to damage-associated molecular patterns (DAMPs), ZBP1 might be involved in neuroinflammation after stroke. However, the spatiotemporal expression profile of ZBP1 in the post-stroke brain remains to be elucidated. The aim of this study is to demonstrate the spatiotemporal expression patterns of ZBP1 in the post-stroke brain using a mouse photothrombotic stroke model. Real-time PCR assays showed that ZBP1 is induced on days 3–14 post stroke. ZBP1 immunoreactivity was observed in Iba1-positive microglia/macrophages in peri-infarct regions by immunohistochemistry. ZBP1-positive cells were spread in layers surrounding the infarct core by 7–14 days post stroke. Interestingly, ZBP1 immunoreactivity was also detected in CD206-positive border-associated macrophages (BAMs) in the meninges. Furthermore, ZBP1-expressing cells were positive for antibodies against inflammatory mediators such as Toll-like receptor 4 (TLR4), Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF), and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Morphological analysis with confocal microscopy showed that the co-localization signals of ZBP1 and its adaptor, TRIF, are increased by glucose oxidase (GOx) treatment, which has been reported to induce mitochondrial DNA (mtDNA) release. These results suggest that ZBP1 is induced in peri-infarct microglia/macrophages and may be involved in DAMPs-mediated neuroinflammation involving mtDNA in the post-infarct brain.

Impact of glucose oxidase treatment in high sugar and pH musts on volatile composition of white wines

Climate change is modifying the composition of the grapes, increasing sugar and pH levels, which produces unbalanced wines with high alcohol degree, low acidity and poor aromatic notes. In this study, glucose oxidase (GOX) and catalase (CAT) were applied in white must with high sugar content and pH (>3.8) to simultaneously decrease glucose concentration and pH. The effect of enzyme treatment on volatile composition of wine was investigated. A concentration of 0.17 mkat/L for both GOX and CAT was sufficient to produce a remarkable reduction of glucose concentration in the must (61.5 g/L), achieving similar results within the pH range of 3–4. The musts subjected to enzymatic treatment yielded more balanced wines, lowering their alcohol content by 10–27 mL/L and pH by 0.3–0.5, while leaving their chromatic characteristics unchanged compared to the control wines. As positive effects, enzyme treatment reduced the concentrations of C6-alcohols with green-herbaceous notes and high-chain ethyl esters with soapy notes in wines, and did not modify the concentrations of short-chain ethyl esters, acids and higher alcohols. However, the concentrations of heptyl acetate and 2-phenylethanol with floral notes, and some ketones with floral and fruit notes, were lower in wines from treated musts.

Effect of glucose oxidase treatment on the aroma qualities and release of cooked off-odor components from heat-treated Hami melon juice

Melon juice produces strong cooked off-odors during heat processing, leading to serious deterioration of aroma quality. In this work, the aroma quality of melon juice, the changes in GOD reaction products, and the interactions of reaction products and cooked off-odor components were analyzed by sensory evaluation, gas chromatography–mass spectrometry/olfactory, ultraperformance liquid chromatography–triple quadrupole mass spectrometry, and isothermal titration calorimetry to study the effect mechanism of glucose oxidase (GOD) on the release of cooked off-odor components from heat-treated melon juice. The results showed that GOD treatment improved the aroma quality mainly by controlling off-odor attributes and maintaining characteristic odor attributes. This was because the reaction products (hydrogen peroxide and gluconic acid) of GOD treatment inhibited the release of cooked off-odor components from heat-treated melon juice through oxidation and hydrophobic effects. Furthermore, these products reduced the loss of characteristic odor compounds by restraining Maillard, degradation, and oxidation reactions during heat processing.

Regulation of carboxyl groups and structural defects of graphitic carbon nitride via environmental-friendly glucose oxidase ring-opening modulation

Structural defects and surface functional groups defects engineering in the graphitic carbon nitride (g-C3N4) polymer semiconductor usually has great benefit in optimizing its electron structure and photocatalytic performance. In this study, one type of carboxyl group defects-grafting g-C3N4 was prepared through glucose oxidase treatment on the initial O sites in the g-C3N4, which played multi-functional roles in the generation of carboxyl groups and melon structure defect. FT-IR, XPS and 13C NMR analysis verified the presence of highly favorable carboxyl groups, which was beneficial to the fast separation and migration of photogenerated carriers. The singularity of enzyme mediated defects control of carbon nitride materials was greatly boosted on O-enriched g-C3N4 (OCN) further supporting the overall O sites ring-opening hypothesis. The glucose oxidase (GOD) treated reconstructed g-C3N4 photocatalysts showed narrowed band gaps and fast charge separation ability as verified by UV–vis DRS and electrochemical tests. As a result, the optimized photocatalyst (GOD-OCN-3d) showed a high Cr(VI) photocatalytic detoxification rate of 99 %, which was 6.6 times higher than that of raw g-C3N4 (15 %) in 120 min. These findings open the understanding on precise molecular defects modulation of g-C3N4 via an environmental-friendly enzyme treatment approach, as well as providing a new avenue for understanding the function of structural defects of photocatalysts on prompting charge separation.

Composition and foam properties of whole wheat dough liquor as affected by xylanase and glucose oxidase

Xylanase (XYL) and glucose oxidase (GOX) have been applied to improve the crumb structure of whole wheat buns. However, their effects on the constituents dissolved in the dough liquor (DL), which supposedly contributed to gas cell stabilization and crumb structure, have not been elucidated. In this study, we aimed to unravel the effects of these enzymes on the protein and arabinoxylan contents, DL yields, bulk viscosity, as well as foaming properties. DL was first separated through ultracentrifugation from whole wheat dough prior treated by XYL and GOX. The results indicated that XYL and GOX treatment increased the DL yields. Moreover, while XYL increased the water extractable arabinoxylan content, GOX increased the protein level in the DL. The free sulfhydryl groups of soluble proteins declined significantly in the presence of GOX. Moreover, GOX might have catalyzed the formation of large protein aggregates and protein-polysaccharide complexes. Furthermore, while the foaming capacity of DL increased only in the presence of XYL, the foam stability was increased by both XYL and GOX, especially under heat treatment. The present research might help to understand the mechanism of crumb structure improvement of whole wheat buns by XYL/GOX treatment from the view of their effects on the DL.

Probing the Performance of Glucose Oxidase Treated Graphene-Based Field Effect Transistors.

Graphene due to its unique properties of biocompatibility has received considerable attention for biosensing applications. Here, we report the effect of glucose oxidase treatment on the graphene devices. Raman spectroscopy and the electrical transport measurements are performed to study the graphene intrinsic characteristics before glucose oxidase treatment. The absence of the defect peak in the Raman spectrum shows high-quality graphene. The modulation in the electrical properties is further investigated by different period of glucose oxidase treatment. Our results illustrate that the mobility of the graphene-based field effect transistor is gradually enhanced with the glucose oxidase treatment time. The enhancement in the electrical performance of graphene FET with the biomolecules could be the suitable route for the bioelectronic devices.

Glucose oxidase promotes gallic acid-myofibrillar protein interaction and thermal gelation

The effect of glucose oxidase (GOx) catalytic oxidation on the efficacy of gallic acid (GA) to modify the chemical structure and gelling behavior of myofibrillar protein (MP) was investigated. In contrast to non-oxidized MP samples where GA induced very little changes, GA (0, 6, 30, and 60 µmol/g MP) under GOx treatment promoted sulfhydryl and amine loss (up to 58% and 49%, respectively). The attenuation of intrinsic tryptophan fluorescence in the GA/GOx-treated MP corroborated the finding. The gelling capacity of MP, corresponding to disulfide and non-disulfide bond formation in protein aggregates, was markedly enhanced by 60 µmol GA under GOx, up to 86% in gel storage modulus G' and 53% in gel strength. The GOx-aided GA modification of MP could be a potential ingredient strategy in meat processing to promote textural attributes of cooked products.

Intracellular Thiol Oxidation Is Linked with Loss of Δψm and Disease Progression in Acute Promyelocytic Leukaemia

Acute promyelocytic leukaemia (APL) is a type of myeloid malignancy defined by the chromosomal translocation t(15;17) and subsequent expression of the PML-RARα fusion protein. Long term remission in APL is achieved through a combination of high dose all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), the latter of which has potential to induce mitochondrial derived oxidative stress and initiate the intrinsic apoptosis pathway. ATO is particularly effective in the treatment of APL when compared with acute myeloid leukaemia (AML), suggesting that the mitochondrial membrane potential (Δψm) of APL cells is compromised. Here we show that loss of Δψm is associated with intracellular thiol oxidation and disease progression in APL.The mitochondrial probe 3,3'-diethyloxacrbocyanine iodide (DiOC2(3)) was used to evaluate MRP8-PML/RARA transgenic mice Δψm in bone marrow mononuclear cells (BMMC) at three disease stages: 1) pre-APL, 2) APL and 3) ATRA treated APL. Our data show that MRP8-PML/RARA BMMC cells during the APL stage accumulate significantly less DiOC2(3) compared with pre-APL cells (0.05±0.02 vs 0.24±0.05, p<0.05; data are normalised FL1/FL3 MFI ratio). The DiOC2(3) accumulation in BMMC from ATRA treated APL mice was equivalent to that of pre-APL cells (p>0.05). This finding indicates a loss of Δψm in APL. This may result in free radical leakage from the mitochondria, which in turn could oxidise intracellular thiols.To evaluate intracellular thiol oxidation, a previously optimised flow cytometric assay that incorporates the thiol reactive probe fluorescein-5 maleimide (F5M) was used. Our data show a significant decrease in F5M MFI for MRP8-PML/RARA BMMC cells during the APL stage, compared with pre-APL cells (219.01±47.67 vs 672.66±131.04, p<0.05; data are F5M MFI relative to unstained controls). This finding signifies that there is more thiol oxidation at the APL stage. ATRA treated APL mice showed an equivalent F5M MFI to that of pre-APL cells. Confocal microscopy confirmed that the F5M signal was intracellular. This observation shows that there is an increase in intracellular thiol oxidation in APL, which may be tracked during disease progression and treatment course. This finding may explain why APL cells are more sensitive to pro-oxidant treatments i.e. ATO.To understand these observations further, the APL cell line (NB4) was subjected to glucose oxidase (GOX) mediated oxidative stress in cell culture over a 4 hour time-course. F5M MFI signal was compared with the THP1 and Kasumi-1 AML cell lines subjected to the same treatment. All cell lines showed similar significant increases in F5M MFI after 1 hour GOX treatment relative to control (p<0.05), indicating an increase in ‘reduced’ cellular thiol i.e. reductive stress. However, after 4 hours NB4 cells showed a significant decrease in F5M MFI relative to control (p<0.05), indicating an increase in ‘oxidised’ cellular thiol i.e. oxidative stress. In contrast, THP1 cells showed no difference in MFI after 4 hours (p>0.05), whereas F5M MFI Kasumi-1 remained significantly increased after 4 hours (p<0.05) relative to control. This finding indicates differences in the sensitivity to oxidative stress between APL and AML cells.Taken together, this study shows that APL cells are more sensitive to oxidative stress compared with AML cells. Our F5M flow cytometric assay illustrates that an increase in cellular thiol oxidation is linked to disease progression in APL, with loss of Δψm associated with this process. Our F5M flow cytometric assay may therefore have clinical utility in monitoring treatment efficacy in APL. DisclosuresNo relevant conflicts of interest to declare.

Technical Feasibility of Glucose Oxidase as a Prefermentation Treatment for Lowering the Alcoholic Degree of Red Wine

In the present work, the use of the glucose oxidase/catalase enzymatic system was evaluated as an alternative to decrease glucose concentration and eventually produce a reduced-alcohol wine. The effects of glucose oxidase, catalase, and aeration on glucose concentration were evaluated after 24 and 48 hr of treatment of 27°Brix Carmenere must. The results showed that the effect of aeration and glucose oxidase was not significant compared with the effect produced by glucose oxidase itself. In addition, the use of catalase combined with glucose oxidase provided the best result, decreasing the glucose concentration by 51 and 78% after 24 and 48 hr, respectively, when 200 U/mL of both enzymes was used. The alcoholic degree obtained after three and five days under this treatment and subsequent fermentations were 15% (v/v) ± 0.8 and 14% (v/v) ± 0.8, respectively. A major drawback of this treatment was the color change of Carmenere must because H2O2 was produced during the glucose oxidase treatment, despite the presence of catalase. The technical feasibility of using this prefermentative process led to a divided conclusion; obtaining a lower alcoholic degree using the glucose oxidase/catalase system was possible, but if the goal is the industrial application of this technique, the color change should be investigated further. An evaluation of the glucose oxidase/catalase ratio was projected to show an improvement of the H2O2 elimination and, subsequently, decrease the effect on color change.

Synergistic effects of ascorbate and sorafenib in hepatocellular carcinoma: New insights into ascorbate cytotoxicity

We investigated the mechanism of selective ascorbate-induced cytotoxicity in tumor cells, including Hep G2 cells, compared to primary hepatocytes. H2O2 formation was required for ascorbate cytotoxicity, as extracellular catalase treatment protected tumor cells. H2O2 generated by glucose oxidase treatment also caused cell killing, but treatment with a pharmacologic dose (5–20mM) of ascorbate was significantly more cytotoxic at comparable rates of H2O2 production, suggesting that ascorbate enhanced H2O2 cytotoxicity. This was further supported by the finding that ascorbate at a non-cytotoxic dose (1mM) enhanced cell killing caused by glucose oxidase. Consistent with this conclusion, ascorbate treatment caused deregulation of cellular calcium homeostasis, resulting in massive mitochondrial calcium accumulation. Ascorbate acted synergistically with the chemotherapeutic sorafenib in killing Hep G2 cells, but not primary hepatocytes, suggesting adjuvant ascorbate treatment can broaden sorafenib’s therapeutic range. Sorafenib caused mitochondrial depolarization and prevented mitochondrial calcium sequestration. Subsequent ascorbate addition further deregulated cellular calcium homeostasis promoting cell death. Additionally, we present the case of a patient with hepatocellular carcinoma (HCC) who had prolonged regression of a rib metastasis upon combination treatment with ascorbate and sorafenib, indicating that these studies have direct clinical relevance.

Stimulus-dependent neuronal cell responses in SH-SY5Y neuroblastoma cells.

The aim of the present study was to elucidate the intracellular mechanisms that cause neuronal cell death following exposure to excitatory neurotransmitter‑induced neurotoxicity, neurotoxins and oxidative stress. Human SH‑SY5Y neuroblastoma cells were exposed to various stimuli, including glutamate, 6‑hydroxydopamine (6‑OHDA), and glucose oxidase, and cell viability was determined by MTT assay. Early apoptosis and necrosis were examined by Annexin V/propidium iodide double staining and flow cytometric analysis. Intracellular calcium ion concentration and mitochondrial membrane potential were assessed by Fluo‑3a and JC‑1 staining, respectively. In addition, protein expression of receptor‑interacting protein (RIP) kinase 1 and RIP kinase 3 were evaluated by western blotting. Glutamate, 6‑OHDA and glucose oxidase treatment decreased cell viability. Glutamate induced apoptosis and necrosis, whereas, 6‑OHDA induced cell necrosis and glucose oxidase induced apoptosis. Furthermore, glutamate, 6‑OHDA or glucose oxidase treatment significantly increased intracellular calcium concentrations (P<0.05). The effect of glutamate on mitochondrial membrane potential varied with high and low concentrations, whereas 6‑OHDA and glucose oxidase significantly increased the mitochondrial membrane potential in the SH‑SY5Y cells (P<0.05). Glutamate significantly upregulated expression levels of RIP kinase 1 (P<0.05), but not RIP kinase 3. These findings demonstrate that the response of SH‑SY5Y cells varies with the stimuli. Furthermore, RIP kinase 1 may specifically regulate programmed necrosis in glutamate‑mediated excitatory toxicity, but not in cell damage induced by either 6-OHDA or glucose oxidase.

C-myb Regulates Autophagy for Pulp Vitality in Glucose Oxidative Stress

Diabetes mellitus is closely related to oral-complicated diseases by oxidative stress. This study investigates whether cellular myeloblastosis (c-myb) could protect human dental pulp cells against glucose oxidative stress and regulate autophagy activity for pulp vitality. Diabetes mellitus was induced by streptozotocin in Sprague-Dawley rats, and their pulp tissue in teeth was analyzed in terms of pulp cavity and molecules by hematoxylin and eosin and immunohistochemistry staining. Human dental pulp cells were serially subcultured and treated with glucose oxidase in the presence of elevated glucose to generate glucose oxidative stress. The replication-deficient adenovirus c-myb and small interfering RNA c-myb were introduced for c-myb expression. The pulp tissue from the diabetic rats was structurally different from normal tissue in terms of narrow pulp capacity, reduced c-myb, and dentinogenesis molecules. Glucose oxidase treatment decreased c-myb and dentinogenesis molecules (bone morphogenetic protein 2 and 7, dentin matrix protein 1, and dentin sialophosphoprotein) in human dental pulp cells. However, overexpression of c-myb by adenovirus c-myb increased dentinogenesis, autophagy molecules (autophagy protein 5, microtubule-associated protein 1A/1B–light chain 3, and Beclin-1), and cell survival via p-AMPK/AKT signaling even with glucose oxidative stress. In contrast, the lack of c-myb decreased the above molecules and cell survival by downregulating p-AMPK/AKT signaling. The results indicate that diabetes leads to irreversible damage to dental pulp, which is related to downexpression of autophagy via the p-AMPK/AKT pathway by decline of c-myb. The findings of this study provide a new insight that c-myb could ameliorate autophagy activity and that it is applicable for monitoring complicated diseases of dental pulp. The involvement of c-myb in pulp pathology could serve a therapeutic target in oral-complicated diseases.

Gelation and In Vitro Digestibility of Soybean Protein Isolate Treated by a Ternary System Containing Horseradish Peroxidase, Glucose Oxidase, and Glucose

The aim of the present study was to investigate the potential application of a ternary system containing horseradish peroxidase, glucose oxidase, and glucose for the oxidative cross-linking of soybean protein isolate. Protein content, reaction temperature, and original pH were fixed at 30 g/L, 37°C, and 7.0, while suitable glucose, glucose oxidase, horseradish peroxidase addition, and reaction time selected from single factor trials for the one-step treatment were 0.1 mmol, 4.0 U, 200 U/g protein, and 3 h, respectively. In the two-step treatment, glucose, glucose oxidase, and horseradish peroxidase addition were the same, but a reaction time of 1 or 2 h was used for the glucose oxidase or horseradish peroxidase treatment. Two modified soybean protein isolates obtained by the one- and two-step treatment contained some protein polymers, had relative dityrosine content of 414 and 381, respectively, and showed lower in vitro digestibility and less free sulfhydryl than the control soybean protein isolate. During acidification induced by glucono-δ-lactone, the modified soybean protein isolates showed shorter gelation time (57 and 61 versus 72 min), lower loss tangent and gelation point temperature (52.9 and 53.2 versus 62.7°C) but higher final storage modulus than the control soybean protein isolate. The acidified gels prepared from the modified soybean protein isolates also exhibited enhanced hardness, springiness, cohesiveness, and chewiness, and finer microstructural features. The ternary system was capable of treating protein ingredients to improve their gelation properties.

P21: Cytoprotective effects of AP39, a mitochondrially targeted hydrogen sulfide donor in oxidatively stressed endothelial cells: Protection against the loss of cell viability and bioenergetics and attenuation of mitochondrial DNA injury

The purpose of the current study was to investigate the effect of the recently synthesized mitochondrially-targeted hydrogen sulfide (H2S) donor, AP39, on bioenergetics, viability and mitochondrial DNA integrity of the murine microvascular endothelial cell line bEnd.3 in vitro, under normal conditions, and during oxidative stress induced by glucose oxidase. Administration of AP39 (30–300 nM) to bEnd.3 cells increased intracellular H2S levels, with preferential response seen in the mitochondrial regions. Consistently with the bioenergetic effect of authentic H2S, AP39 exerted a concentration-dependent effect on mitochondrial activity, which consisted from a stimulation of mitochondrial electron transport and cellular bioenergetic function at lower concentrations (30–100 nM), followed by an inhibitory effect at a higher concentration (300 nM). AP39 did not affect mitochondrial or nuclear DNA integrity under basal conditions. Under oxidative stress conditions induced by glucose oxidase, there was a suppression of cell viability, and an inhibition of cellular bioenergetic function. AP39 pretreatment protected the cells against these responses. In accordance with our recent studies, glucose oxidase treatment induced a preferential damage to the mitochondrial DNA and AP39 (100 nM) pretreatment protected against this loss of mitochondrial DNA integrity. In conclusion, AP39 induced antioxidant and cytoprotective effects under oxidative stress conditions, which includes a protection against the loss of mitochondrial DNA integrity. Given the recent data indicating a direct causative effect of mitochondrial DNA damage in cell death, we speculate that maintenance of mitochondrial DNA integrity may be a novel mechanisms through which H2S can exert cytoprotective effects.

Ets-1 regulates intracellular glutathione levels: key target for resistant ovarian cancer

BackgroundOvarian cancer is characterized by high rates of metastasis and therapeutic resistance. Many chemotherapeutic agents rely on the induction of oxidative stress to cause cancer cell death, thus targeting redox regulation is a promising strategy to overcome drug resistance.MethodsWe have used a tetracycline-inducible Ets-1 overexpression model derived from 2008 ovarian cancer cells in the present study. To examine the role of Ets-1 in glutathione regulation we have measured intracellular reactive oxygen species and glutathione levels, as well as glutathione peroxidase enzyme activity. Glutathione synthesis was limited using transsulfuration or Sxc- pathway blocking agents, and glutamate release was measured to confirm Sxc- blockade. Cell viability following drug treatment was assessed via crystal violet assay. Oxidative stress was induced through glucose oxidase treatment, which produces hydrogen peroxide by glucose oxidation. The protein expressions of redox-related factors were measured through western blotting.ResultsOverexpression of Ets-1 was associated with decreased intracellular ROS, concomitantly with increased intracellular GSH, GPX antioxidant activity, and Sxc- transporter activity. Under basal conditions, inhibition of the transsulfuration pathway resulted in decreased GSH levels and GPX activity in all cell lines, whereas inhibition of Sxc- by sulfasalazine decreased GPX activity in Ets-1-expressing cells only. However, under oxidative stress the intracellular GSH levels decreased significantly in correlation with increased Ets-1 expression following sulfasalazine treatment.ConclusionsIn this study we have identified a role for proto-oncogene Ets-1 in the regulation of intracellular glutathione levels, and examined the effects of the anti-inflammatory drug sulfasalazine on glutathione depletion using an ovarian cancer cell model. The findings from this study show that Ets-1 mediates enhanced Sxc- activity to increase glutathione levels under oxidative stress, suggesting that Ets-1 could be a promising putative target to enhance conventional therapeutic strategies.

Identification of proteins containing redox-sensitive thiols after PRDX1, PRDX3 and GCLC silencing and/or glucose oxidase treatment in Hepa 1–6 cells

The oxidation and reduction of cysteine thiols are thought to be a major mechanism for redox regulation. The aim of this study was to identify proteins with reactive thiols and determine their oxidation profiles under oxidative stress induced by simultaneous silencing of antioxidant defences (peroxiredoxin-1, peroxiredoxin-3, and the catalytic subunit of the glutamate-cysteine ligase), and/or treatment with glucose oxidase (GO). Using an approach that combined the labelling of reversibly oxidised cysteines, 2-DE protein separation and MS analysis, we identified 26 proteins with cysteines prone to reversible oxidation belonging to different functional classes. Among these proteins are those that have not been previously recognised as reversible oxidation targets, including cytoplasmic aspartate aminotransferase, proteasome subunit alpha type-6, heterogeneous nuclear ribonucleoproteins isoA2/B1 and A/B, and histidine triad nucleotide-binding protein 1. We provide the first evidence of reversible oxidation for specific cysteines, including Cys112 and Cys146 in glutamate dehydrogenase 1, Cys17 in actins, Cys5 in protein disulfide-isomerase A3, and Cys267 in the heat shock cognate 71 kDa protein. Silencing induced lower oxidative stress than GO treatment. Nevertheless, we detected some proteins particularly sensitive to oxidation by silencing. We hypothesised that these proteins may play a role in regulatory mechanisms by redox stress.

Impaired dephosphorylation renders G6PD-knockdown HepG2 cells more susceptible to H 2O 2-induced apoptosis

Glucose-6-phosphate dehydrogenase (G6PD) plays a key role in the regeneration of NADPH and maintenance of cellular redox balance. In the present study, we investigate the effect of G6PD deficiency on H 2O 2-elicited signaling in HepG2 cells. H 2O 2 was found to inhibit cellular protein tyrosine phosphatase (PTP) activity, resulting in activation of MAPKs. MKP-1 expression increased in the late phase of H 2O 2 signaling. Using RNAi technology, we found that G6PD knockdown enhanced the inhibitory effect of H 2O 2 on PTPs and led to sustained MAPK activation. This was accompanied by delayed expression and inhibition of MKP-1. Using a pharmacological inhibitor and siRNA, we demonstrate that MKP-1 acts as a regulator of MAPK activation in H 2O 2 signaling. The prolonged MAPK activation in G6PD-knockdown cells was associated with an increased susceptibility to H 2O 2-induced apoptosis and growth retardation. Treatment with p38 and JNK inhibitors or N-acetylcysteine ameliorated such cellular effect, while triptolide and MKP-1-siRNA did the opposite. Glucose oxidase treatment had similar effects as addition of H 2O 2. Taken together, these findings suggest that G6PD knockdown enhances the magnitude and duration of H 2O 2-induced MAPK signaling through inhibition of cellular PTPs, and the resultant anomalous signaling may lead to cell demise.

Regulatory regions responsive to oxidative stress in the promoter of the human DNA glycosylase gene NEIL2

Reactive oxygen species (ROS) generated endogenously or from exogenous sources produce mutagenic DNA lesions. If not repaired, these lesions could lead to genomic instability and, potentially, to cancer development. NEIL2 (EC 4.2.99.18), a mammalian base excision repair (BER) protein and ortholog of the bacterial Fpg/Nei, excises oxidized DNA lesions from bubble or single-stranded structures, suggesting its involvement in transcription-coupled DNA repair. Perturbation in NEIL2 expression may, therefore, significantly impact BER capacity and promote genomic instability. To characterize the genetic and environmental factors regulating NEIL2 gene expression, we mapped the human NEIL2 transcriptional start site and partially characterized the promoter region of the gene using a luciferase reporter assay. We identified a strong positive regulatory region from nucleotide -206 to +90 and found that expression from this region was contingent on its being isolated from an adjacent strong negative regulatory region located downstream (+49 to +710 bp), suggesting that NEIL2 transcription is influenced by both these regions. We also found that oxidative stress, induced by glucose oxidase treatment, reduced the positive regulatory region expression levels, suggesting that ROS may play a significant role in regulating NEIL2 transcription. In an initial attempt to characterize the underlying mechanisms, we used in silico analysis to identify putative cis-acting binding sites for ROS-responsive transcription factors within this region and then used site-directed mutagenesis to investigate their role. A single-base change in the region encompassing nucleotides -206 to +90 abolished the effect of oxidative stress that was observed in the absence of the mutation. Our study is the first to provide an initial partial characterization of the NEIL2 promoter and opens the door for future research aimed at understanding the role of genetic and environmental factors in regulating NEIL2 expression.

Sustained Hydrogen Peroxide Induces Iron Uptake by Transferrin Receptor-1 Independent of the Iron Regulatory Protein/Iron-responsive Element Network

Local and systemic inflammatory conditions are characterized by the intracellular deposition of excess iron, which may promote tissue damage via Fenton chemistry. Because the Fenton reactant H(2)O(2) is continuously released by inflammatory cells, a tight regulation of iron homeostasis is required. Here, we show that exposure of cultured cells to sustained low levels of H(2)O(2) that mimic its release by inflammatory cells leads to up-regulation of transferrin receptor 1 (TfR1), the major iron uptake protein. The increase in TfR1 results in increased transferrin-mediated iron uptake and cellular accumulation of the metal. Although iron regulatory protein 1 is transiently activated by H(2)O(2), this response is not sufficient to stabilize TfR1 mRNA and to repress the synthesis of the iron storage protein ferritin. The induction of TfR1 is also independent of transcriptional activation via hypoxia-inducible factor 1alpha or significant protein stabilization. In contrast, pulse experiments with (35)S-labeled methionine/cysteine revealed an increased rate of TfR1 synthesis in cells exposed to sustained low H(2)O(2) levels. Our results suggest a novel mechanism of iron accumulation by sustained H(2)O(2), based on the translational activation of TfR1, which could provide an important (patho) physiological link between iron metabolism and inflammation.

Rebuilding gluten network of damaged wheat by means of glucose oxidase treatment

AbstractThe disrupted gluten structure of infested wheat flours leads to low‐quality doughs unusable in bread‐making processes. Enzymes are replacing chemical treatments in the food industry as a tool to treat weak flours. Glucose oxidase is one of the most promising oxidative enzymes, although its efficiency compared with the alcohol‐soluble fraction of gluten proteins has not yet been demonstrated. If this enzyme could restore the broken covalent bonds between glutenin subunits, the gluten network of damaged wheat flour would recover its native structure and functionality. This treatment would allow bakers to use damaged flour, reducing the economic losses caused by this plague around Europe and North Africa. Electrophoretic studies demonstrated the formation of high‐molecular‐weight aggregates in the glutenin fraction, which had a characteristic thermal stability depending on enzyme dosage. Those molecular studies agreed with the bread‐making assays made with maximum enzyme dosage and microstructure determination. Overall results showed that glucose oxidase is a real alternative to traditionally used chemical oxidants. It acted specifically on the high‐molecular‐weight glutenin subunits of damaged wheat, forming dityrosine crosslinks between the wheat proteins, which reinforced the gluten network and gave away the dough functionality. Copyright © 2007 Society of Chemical Industry