Effect of stress on the antioxidant enzymes and gastric ulceration

The effect of cold-restraint stress on the antioxidant enzymes of the rat gastric mucosa was studied with a view to finding out their role in stress induced gastric ulceration. Histological examination revealed stress induced extensive damage of the surface epithelial cell with lesions extending upto submucosa in some cases. Stress causes time-dependent increase in histamine and pepsin content but decrease in acid content of the gastric fluid with the progress of ulceration (ulcer index) for two hours. The tissue lipid peroxidation was significantly increased as evidenced by accumulation of malondialdehyde. Since lipid peroxidation results from the generation of reactive oxygen species, stress effect was studied on some antioxidant enzymes such as superoxide dismutase, peroxidases and prostaglandin synthetase as a function of time. The time dependent increase in stress ulcer correlates well with the concomitant increase in superoxide dismutase activity and decrease in peroxidase and prostaglandin synthetase activity. This creates a favourable condition for accumulation of endogenous H₂O₂ and more reactive hydroxyl radical (OH·). Administration of antioxidants such as reduced glutathione or sodium benzoate prior to stress causes significant decrease in ulcer index and lipid peroxidation and protection of gastric peroxidase activity suggesting the involvement of reactive oxygen species in stress induced gastric ulceration. This is supported by thein vitro observation that OH· can also inactivate peroxidase and induce lipid peroxidation. As prostaglandin is known to offer cytoprotection, stress-induced loss of prostaglandin synthetase activity appears to aggravate the oxidative damage caused by reactive oxygen species.Abbreviations ROS reactive oxygen species - GPO gastric peroxidase - SOD superoxide dismutase - MDA malondialdehyde - GSH reduced glutathione - TCA trichloroacetic acid     

Oxidative dechlorination of halogenated phenols catalyzed by two distinct enzymes: Horseradish peroxidase and dehaloperoxidase

The mechanism of the dehalogenation step catalyzed by dehaloperoxidase (DHP) from Amphitrite ornata, an unusual heme-containing protein with a globin fold and peroxidase activity, has remarkable similarity with that of the classical heme peroxidase, horseradish peroxidase (HRP). Based on quantum mechanical/molecular mechanical (QM/MM) modeling and experimentally determined chlorine kinetic isotope effects, we have concluded that two sequential one electron oxidations of the halogenated phenol substrate leads to a cationic intermediate that strongly resembles a Meisenheimer intermediate – a commonly formed reactive complex during nucleophilic aromatic substitution reactions especially in the case of arenes carrying electron withdrawing groups.     

Immune and proliferative cellular responses to Helicobacter pylori infection in the gastric mucosa of Mexican children

BACKGROUND: Helicobacter pylori infection occurs mostly during childhood, but few studies on this age group have addressed the innate immune and the proliferative response to this infection. Mexico has a high H. pylori prevalence in children, but a low risk of gastric cancer. The aim of this work was to study the cellular responses of the gastric mucosa to this infection in Mexican children. METHODS: Antral and corpus gastric biopsies were obtained from 44 H. pylori-infected children (mean age 12 +/- 3.2 years) and 44 uninfected children (mean age 10 +/- 3 years). Mucosal cellular responses were studied by immunohistochemistry, using anti-Ki67 antibodies for proliferation studies, antihuman tryptase for mast cells, and antihuman CD68 for macrophages. T and B lymphocytes were stained with a commercial integrated system. The intensity of cellular responses was estimated histologically using the software KS300. RESULTS: Epithelium proliferation and infiltration of macrophages and T and B lymphocytes were significantly higher in H. pylori-infected than in uninfected children. A balanced increase of CD4, CD8, and CD20 lymphocytes was observed in infected children. However, activated mast cells were decreased, and infiltration of neutrophil and mononuclear cells was low. Epithelial proliferation was associated with polymorphonuclear infiltration but not with infiltration of macrophages or lymphocytes. Inflammation and proliferation was higher in CagA (+)-infected children. CONCLUSIONS: Mexican children respond to H. pylori infection with a low inflammatory response, a balanced increase of T and B lymphocytes, and a high regenerative activity.     

Limited utilization of exogenous arachidonic acid by the prostaglandin cyclooxygenase in gastric mucosa: The role of protein binding, glutathione peroxidase, and hydrogen peroxides

We investigated the utilization of exogenous ¹⁴C-labelled arachidonic acid by the cyclooxygenase system of the gastric mucosa and its alteration by cytosolic factors, protein binding, glutathione peroxidase (GSH-Px), and hydrogen peroxides.Total prostaglandin (PG) synthesis from gastric microsomes was reduced in a dose- dependent manner to 12% and 68% of controls by increasing amounts of the 105,000g supernatant or albumin (8mg protein/ml), respectively (p<0.01). The inhibitory cytosolic factor was heat labile, protease sensitive, and was retained by a 300,000 Dalton ultrafiltration membrane. Thus, it was likely a protein. Other possible inhibitory mechanisms like protease- or heme-induced destabilization of the cyclooxygenase, haptoglobin-mediated inhibition, or self-inactivation by endogenous substrate were excluded.N-ethylmaleimide (NEM), an agent that alkylates sulfhydryl-groups thereby inhibiting GSH-Px, abolished the inhibitory effect of cytosol in a dose-dependent fashion. In contrast to their inhibition of prostaglandin synthesis, the binding of arachidonic acid by albumin or cytosolic proteins accounted to 75% and 19% under comparable conditions, respectively, however, cytosolic fatty acid binding was unaffected by NEM. Thus, it was concluded that the inhibitory effect of cytosol, in contrast to albumin, was mediated by a sulfhydryl-depending process, probably a GSH-Px. This conclusion was supported by a qualitatively comparable inhibition by a purified GSH-Px from bovine erythrocytes.The inhibitory action of cytosolic proteins was reduced significantly by increasing concentrations or repeated application of arachidonic acid; therefore, cytosolic GSH-Px was likely to affect substrate utilization by the microsomal PGH synthase through reduction of activating substrate peroxides.Similarly, the in vitro formation of cyclooxygenase products by mucosal homogenate or gastric microsomes in the absence of cytosol was limited at substrate concentrations below 80μM, despite sufficient nonesterified arachidonic acid remaining in the incubate. This limitation was mediated only partially by self-inactivation of the prostaglandin cyclooxygenase. Neither N-ethylmaleimide nor repeated application of hydrogen peroxides increased substrate utilization by isolated microsomes, excluding contamination by GSH-Px or simply a lack of hydrogen peroxides as possible mechanisms for the limited utilization. From these results, a special role of substrate-linked lipid peroxides in the activation of mucosal prostaglandin synthesis is proposed. The reduction of these peroxides by glutathione dependent or independent peroxidases, e.g. the PGH synthase-linked hydroperoxidase activity itself, could explain the reduced utilization of nonesterified arachidonic acid by the gastric mucosa.     

Limited utilization of exogenous arachidonic acid by the prostaglandin cyclooxygenase in gastric mucosa: the role of protein binding, glutathione peroxidase, and hydrogen peroxides.

We investigated the utilization of exogenous 14C-labelled arachidonic acid by the cyclooxygenase system of the gastric mucosa and its alteration by cytosolic factors, protein binding, glutathione peroxidase (GSH-Px), and hydrogen peroxides. Total prostaglandin (PG) synthesis from gastric microsomes was reduced in a dose- dependent manner to 12% and 68% of controls by increasing amounts of the 105,000g supernatant or albumin (8mg protein/ml), respectively (p less than 0.01). The inhibitory cytosolic factor was heat labile, protease sensitive, and was retained by a 300,000 Dalton ultrafiltration membrane. Thus, it was likely a protein. Other possible inhibitory mechanisms like protease- or heme-induced destabilization of the cyclooxygenase, haptoglobin-mediated inhibition, or self-inactivation by endogenous substrate were excluded. N-ethylmaleimide (NEM), an agent that alkylates sulfhydryl-groups thereby inhibiting GSH-Px, abolished the inhibitory effect of cytosol in a dose-dependent fashion. In contrast to their inhibition of prostaglandin synthesis, the binding of arachidonic acid by albumin or cytosolic proteins accounted to 75% and 19% under comparable conditions, respectively, however, cytosolic fatty acid binding was unaffected by NEM. Thus, it was concluded that the inhibitory effect of cytosol, in contrast to albumin, was mediated by a sulfhydryl-depending process, probably a GSH-Px. This conclusion was supported by a qualitatively comparable inhibition by a purified GSH-Px from bovine erythrocytes. The inhibitory action of cytosolic proteins was reduced significantly by increasing concentrations or repeated application of arachidonic acid; therefore, cytosolic GSH-Px was likely to affect substrate utilization by the microsomal PGH synthase through reduction of activating substrate peroxides. Similarly, the in vitro formation of cyclooxygenase products by mucosal homogenate or gastric microsomes in the absence of cytosol was limited at substrate concentrations below 80 microM, despite sufficient nonesterified arachidonic acid remaining in the incubate. This limitation was mediated only partially by self-inactivation of the prostaglandin cyclooxygenase. Neither N-ethylmaleimide nor repeated application of hydrogen peroxides increased substrate utilization by isolated microsomes, excluding contamination by GSH-Px or simply a lack of hydrogen peroxides as possible mechanisms for the limited utilization. From these results, a special role of substrate-linked lipid peroxides in the activation of mucosal prostaglandin synthesis is proposed. The reduction of these peroxides by glutathione dependent or independent peroxidases, e.g. the PGH synthase-linked hydroperoxidase activity itself, could explain the reduced utilization of nonesterified arachidonic acid by the gastric mucosa.     

Matrine involves in the progression of gastric cancer through inhibiting miR-93-5p and upregulating the expression of target gene AHNAK

Matrine, also known as oxymatrine, is an important active ingredient of traditional Chinese herb Sophora flavescens. Recent studies have found that matrine may inhibit multiple tumors through inhibiting the tumor cell proliferation, inducing cell apoptosis, blocking cell cycle, suppressing cell invasion and migration and assisting in the synergy, and attenuation of radiotherapy and chemotherapy. This study mainly investigated the role of matrine in gastric cancer and its possible mechanism. The real-time fluorescence quantitative polymerase chain reaction technique showed that matrine inhibited the proliferation and migration of gastric tumor cells and significantly suppressed the expression of miR-93-5p. The dual-luciferase reporter gene assay indicated that AHNAK was a target gene of miR-93-5p and regulated by miR-93-5p and matrine. The torsion test demonstrated that matrine exerted its role via miR-93-5p while miR-93-5p played a role by targeting AHNAK. Thus, this study found that matrine affected the progression of gastric cancer by inhibiting the function of gastric cancer cells through the possible mechanism of inhibiting miR-93-5p expression to increase the expression level of the downstream target gene AHNAK.     

Inactivation of red cell glutathione peroxidase by divicine and its relation to the hemolysis of favism

A significant inactivation of red blood cell glutathione peroxidase (25% less than the physiological value) was observed after exposure of intact erythrocytes to 2 mM divicine (an autoxidizable aminophenol from Vicia faba seeds) and 2 mM ascorbate for 3 h at 37°C. Addition of catalase and conversion of Hb to the carbomonoxy derivative resulted in protection against enzyme inactivation. Oxidation of Hb was a concurrent phenomenon, and augmented the inactivating effect. In hemolysates, much stronger effects were observed at shorter times (2 h); divicine was effective also without ascorbate, and the presence of reductants (ascorbate or glutathione or NADPH) enhanced its inactivating power. Of the other antioxidant enzymes, superoxide dismutase was unaffected under the same experimental conditions. Catalase was found to be much less sensitive to the inactivation; it was almost unaffected in experiments with intact erythrocytes and specifically protected by NADPH in experiments with hemolysates. This specific damage of glutathione peroxidase, apparently involving interaction of H₂O₂ and HbO₂, may be related to the pathogenesis of hemolysis in favism.     

Up- and down-regulation of membrane receptors as possible mechanisms related to the antiulcer actions of milk in rat gastric mucosa

The effects of a cow's milk diet on receptor activity and histamine metabolism in gastric glands and mucosa isolated from adult rats were examined. The milk diet was associated with (1) a decreased mobilization of H₂ receptors by histamine and (2) an increased mobilization of PGE₂ (prostaglandin E₂) receptors in mucous cells (cytoprotective effect) and parietal cells (antiacid effect). These changes are not observed for the receptors reducing pentagastrin- and histamine-induced gastric acid secretion (pancreatic/enteroglucagons, somatostatin) and stimulating mucus, bicarbonate and pepsin secretions in the rat (secretin). Cimetidine produced a parallel displacement of the histamine dose-response curve, suggesting competitive inhibition between this classical H₂ receptor antagonist and histamine in the two experimental groups. Prostaglandins and other components in milk such as EGF (epidermal growth factor) and somatostatin might therefore protect gastric mucosa by a differential control of PGE₂ and histamine H₂ receptor activity eitherdirectly (PGE₂ in milk) orindirectly (inhibition of endogeneous histamine synthesis/release and stimulation of PGE-I synthesis/release).     

Dampening HOTAIR sensitizes the gastric cancer cells to oxaliplatin through miR-195-5p and ABCG2 pathway

Long non-coding RNAs (lncRNA) have an extensive role in the progression and chemoresistance of gastric cancer (GC). Deeply study the regulatory role of lncRNAs could provide potential therapeutic targets. The aim of this study is to explore the regulatory role of HOTAIR in the progression and oxaliplatin resistance of GC. The expression of HOTAIR in GC and cell lines were detected by using qRT-PCR. Cell proliferation and apoptosis were analysed by CCK-8, EdU incorporation and flow cytometry. Luciferase reporter assay was used to identify the interaction between HOTAIR and ABCG2 (ATP-binding cassette (ABC) superfamily G member 2, ABCG2) via miR-195-5p. The regulatory functions were verified by using molecular biology experiments. HOTAIR was significantly overexpressed in GC and associated with poor prognosis. Knock-down of HOTAIR inhibited the GC cells proliferation and oxaliplatin resistance, while overexpression of HOTAIR showed opposite functions. Further studies found that HOTAIR acted as a competing endogenous RNA (ceRNA) to absorb miR-195-5p and elevated the expression of ABCG2, which leads to resistance of GC cells to oxaliplatin. Taken together, our findings demonstrated that HOTAIR regulates ABCG2 induced resistance of GC to oxaliplatin through miR-195-5p signalling and illustrate the great potential of developing new therapeutic targets for GC patients.     

Effect of culture conditions on the production of ligninolytic enzymes by white rot fungi Phanerochaete chrysosporium (ATCC 20696) and separation of its lignin peroxidase

The present work was carried out to determine the optimum culture conditions of Phanerochaete chrysosporium (ATCC 20696) for maximizing ligninolytic enzyme production. Additionally, separation of its lignin peroxidase was conducted. After experiments, an optimized culture medium/condition was constructed (per liter of Kirk’s medium): dextrose 10 g, ammonium tartrate 0.11 g, Tween-80 0.5 g, MnSO₄ 7 mg, and veratryl alcohol 0.3 g in 10 mM acetic acid buffer pH 4.5. Under the optimized experimental condition, both lignin peroxidase (LiP) and manganese peroxidase (MnP) were detected and reach the highest yield at 30°C on the 8th day culture. Salt precipitation methods was used in the extraction and purification processes. Results show that salt precipitation with 60% (NH₄)₂SO₄ yielded the best result, especially toward LiP. Enzyme separation was conducted and two fractions with LiP activity. LiP1 and LiP2 were produced using three columns sequentially: desalting column, Q FF ion exchange column and Sepharyl S-300 HR gel filtration. LiP1 and LiP2 had been purified by 9.6- and 7.6-fold with a yield of 22.9% and 18.6%, respectively. According to the data of sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE), the molecular weights of the enzymes are 38 kDa and 40 kDa, respectively.     

CircRNA circPDSS1 promotes the gastric cancer progression by sponging miR-186-5p and modulating NEK2

The aim of this study is to investigate the regulatory mechanism of circPDSS1/miR-186-5p/NEK2 axis on the viability and proliferation in gastric cancer (GC) cell line. Differentially expressed circRNAs, miRNAs, and mRNAs in GC tissues and paracarcinoma tissues were analyzed using gene chips GSE83521, GSE89143, and GSE93415. Then, the expression of circPDSS1, miR-186-5p, and NEK2 was analyzed via quantitative real-time polymerase chain reaction (qRT-PCR). Survival analysis was adopted to explore the association between the circPDSS1 expression and the prognosis of GC. The effect of circPDSS1 on GC cell cycle and apoptosis was verified with the flow cytometry. Targeting relationships among circPDSS1, miR-186-5p, and NEK2 were predicted via bioinformatics analysis and demonstrated by the dual-luciferase reporter assay. Our results showed that circPDSS1 and NEK2 were high-expressed whereas miR-186-5p was low-expressed in GC tissues and cells. CircPDSS1 promoted GC cell cycle and inhibited apoptosis by sponging miR-186-5p, while miR-186-5p inhibited cell cycle and promoted apoptosis by targeting NEK2. Thus, circPDSS1 acts as a tumor promoter by regulating miR-186-5p and NEK2, which could be a potential biomarker and therapeutic target for the management of GC.     

LncRNA SNHG20 contributes to cell proliferation and invasion by upregulating ZFX expression sponging miR-495-3p in gastric cancer

Long noncoding RNAs (lncRNAs) exert key regulators in cancer development and progression. The functional significance of lncRNA small nucleolar RNA host gene 20 (SNHG20) was reported in gastric cancer (GC); however, the underlying molecular mechanism in GC development is largely unknown. Here, our results showed that the lncRNA SNHG20 expression was significantly higher in GC tissues compared with adjacent normal tissues by quantitative real-time PCR (qRT-PCR) analysis. Higher lncRNA SNHG20 expression was highly associated with tumor size and lymphatic metastasis of patients. Patients with higher lncRNA SNHG20 expression predicted a short disease-free survival (DFS) and overall survival (OS). Furthermore, lncRNA SNHG20 expression negatively associated with miR-495-3p expression and regulated miR-495-3p expression. Function assays confirmed that lncRNA SNHG20 knockdown using RNA interference suppressed cell proliferation and invasion of GC by negatively regulating miR-495-3p expression. Moreover, we demonstrated that lncRNA SNHG20 inhibited zinc finger protein X-linked (ZFX) expression by negatively miR-495-3p expression in GC cells. In vivo, the current study also indicated that lncRNA SNHG20 knockdown reduced the tumor growth by downregulating ZFX expression. Thus, our results implied that inhibition of SNHG20/miR-495-3p/ZFX axis may provide valuable target for GC treatment.     

Production of lignin peroxidase by Phanerochaete chrysosporium immobilized on porous poly(styrene-divinylbenzene) carrier and its application to the degrading of 2-chlorophenol

Porous poly(styrene-divinylbenzene) carriers, for the immobilization of white rot fungus Phanerochaete chrysosporium have been prepared by the concentrated emulsion polymerization method. The concentrated emulsion consists of a mixture of styrene and divinylbenzene containing a suitable surfactant and an initiator as the continuous phase, and water as the dispersed phase. The polymerization of the monomers of the continuous phase generated the polymer carrier with a porcus structure. The white rot fungus Phanerochaete chrysosporium has been immobilized on porous poly(styrene-divinylbenzene) carriers and used for the batch production and the repeated batch production of lignin peroxidase in shake cultures based on a carbon-limited medium containing veratryl alcohol. The best results were achieved when a spore inoculum was used for immobilization instead of 1-day-old mycelial pellets, for both the batch production and the repeated batch production. The porous poly(styrene-divinylbenzene) immobilized Phanerochaete chrysosporium and freely suspended mycelial pellets were used as biocatalysts for the degradation of 2-chilorophenol in a 2-L bioreactor. The porous poly(styrene-divinylbenzene) particle (diameter congruent with 0.2 cm) immobilized spores exhibited a much higher activity in the degradation of 2-chlorophenol than the freely suspended mycelial pellets. (c) 1994 John Wiley & Sons, Inc.     

Effect of a high fructose diet on the activities of enzymes implicated in the conversion of fructose to glucose by the liver and the intestinal mucosa of the rat

The activity of enzymes implicated in the metabolic pathway of fructose to glucose conversion was shown in rat liver and intestine. In rats on normal diet, the specific activity of glucose-6-phosphatase, fructokinase, fructose-1,6-diphosphatase and triokinase was low in the intestine confirming that sugar conversion is not operative in this organ. In rats on a fructose diet, all the specific enzymatic activities tested were increased except for the hepatic triokinase and triose phosphate isomerase and for the intestinal triose phosphate isomerase. The intestine acquires the possibility to transform fructose to glucose by modifying the activities of enzymes implicated in the same metabolic pathway as that intervening in the liver.