γ-Oryzanol from Rice Bran Antagonizes Glutamate-Induced Excitotoxicity in an In Vitro Model of Differentiated HT-22 Cells.

The excessive activation of glutamate in the brain is a factor in the development of vascular dementia. γ-Oryzanol is a natural compound that has been shown to enhance brain function, but more research is needed to determine its potential as a treatment for vascular dementia. This study investigated if γ-oryzanol can delay or improve glutamate neurotoxicity in an in vitro model of differentiated HT-22 cells and explored its neuroprotective mechanisms. The differentiated HT-22 cells were treated with 0.1 mmol/L glutamate for 24 h then given γ-oryzanol at appropriate concentrations or memantine (10 µmol/L) for another 24 h. Glutamate produced reactive oxygen species and depleted glutathione in the cells, which reduced their viability. Mitochondrial dysfunction was also observed, including the inhibition of mitochondrial respiratory chain complex I activity, the collapse of mitochondrial transmembrane potential, and the reduction of intracellular ATP levels in the HT-22 cells. Calcium influx triggered by glutamate subsequently activated type II calcium/calmodulin-dependent protein kinase (CaMKII) in the HT-22 cells. The activation of CaMKII-ASK1-JNK MAP kinase cascade, decreased Bcl-2/Bax ratio, and increased Apaf-1-dependent caspase-9 activation were also observed due to glutamate induction, which were associated with increased DNA fragmentation. These events were attenuated when the cells were treated with γ-oryzanol (0.4 mmol/L) or the N-methyl-D-aspartate receptor antagonist memantine. The results suggest that γ-oryzanol has potent neuroprotective properties against glutamate excitotoxicity in differentiated HT-22 cells. Therefore, γ-oryzanol could be a promising candidate for the development of therapies for glutamate excitotoxicity-associated neurodegenerative diseases, including vascular dementia.

Gigantol has Protective Effects against High Glucose-Evoked Nephrotoxicity in Mouse Glomerulus Mesangial Cells by Suppressing ROS/MAPK/NF-κB Signaling Pathways.

Gigantol is a bibenzyl compound derived from several medicinal orchids. This biologically active compound has shown promising therapeutic potential against diabetic cataracts, but whether this compound exerts beneficial effects on the other diabetic microvascular complications remains unclear. This study was carried out to examine effects of gigantol on high glucose-induced renal cell injury in cultured mouse kidney mesangial cells (MES-13). MES-13 cells were pretreated with gigantol (1, 5, 10 or 20 µmol/L) for 1 h followed by further exposure to high (33.3 mmol/L) glucose for 48 h. Gigantol concentration dependently enhanced cell viability followed by high glucose treatment in MES-13 cells. High glucose induced reactive oxygen species (ROS) generation, malondialdehyde production and glutathione deficiency were recoved in MES-13 cells pretreated with gigantol. High glucose triggered cell apoptosis via the the loss of mitochondrial membrane potential, depletion of adenosine triphosphate, upregulation of caspases 9 and 3, enhancement of cytochrome c release, and subsequent interruption of the Bax/Bcl-2 balance. These detrimental effects were ameliorated by gigantol. High glucose also induced activation of JNK, p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) in MES-13 cells, which were blocked by gigantol. The results suggest that treatment MES-13 cells with gigantol halts high glucose-induced renal dysfunction through the suppression of the ROS/MAPK/NF-κB signaling pathways. Our data are of value to the understanding the mechanism for gigantol, and would benefit the study of drug development or food supplement for diabetes and nephropathy.

Hesperidin Prevents High Glucose-Induced Damage of Retinal Pigment Epithelial Cells.

The present study aimed to determine whether hesperidin, a plant-based active flavanone found in citrus fruits, can prevent high glucose-induced retinal pigment epithelial (RPE) cell impairment. Cultured human RPE cells (ARPE-19) were exposed to a normal glucose concentration (5.5 mM) for 4 d and then soaked in either normal (5.5 mM) or high (33.3 mM) concentrations of D-glucose with or without different concentrations of hesperidin (10, 20, or 40 µM) for another 48 h. The survival rates of the cells were measured using a 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction assay. With the help of a fluorescent probe, the intracellular production of reactive oxygen species (ROS) was evaluated. Colorimetric assay kits were used to assess the antioxidant enzyme activities, and western blotting was used to measure the expression of apoptosis-related protein. Hesperidin was effective in inhibiting high glucose-induced ROS production, preventing loss of cell viability, and promoting the endogenous antioxidant defense components, including glutathione peroxidase, superoxide dismutase, catalase, and glutathione, in a concentration-dependent manner. Furthermore, high glucose triggered cell apoptosis via the upregulation of caspase-9/3, enhancement of cytochrome c release into the cytosol, and subsequent interruption of the Bax/Bcl-2 balance. These detrimental effects were ameliorated by hesperidin in a concentration-dependent manner. We conclude that through the scavenging of ROS and modulation of the mitochondria-mediated apoptotic pathway, hesperidin may protect RPE cells from high glucose-induced injury and thus may be a candidate in preventing the visual impairment caused by diabetic retinopathy.

Protective Effects of Hesperidin (Citrus Flavonone) on High Glucose Induced Oxidative Stress and Apoptosis in a Cellular Model for Diabetic Retinopathy.

The aim of this study was to investigate the protective effects and mechanisms of hesperidin, a plant based active flavanone found in citrus fruits, under the oxidative stress and apoptosis induced by high levels of glucose in retinal ganglial cells (RGCs). RGC-5 cells were pretreated with hesperidin (12.5, 25, or 50 µmol/L) for 6 h followed by exposure to high (33.3 mmol/L) d-glucose for 48 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was adopted to evaluate cell viability. Mitochondrial function was estimated by measuring the mitochondrial membrane potential (ΔΨm). A fluorescent probe was employed to evaluate the intercellular production of reactive oxygen species (ROS). Colorimetric assay kits were used to evaluate lipid peroxidation, antioxidant enzyme activities, and protein carbonyls formation. The expression of apoptosis-related proteins and mitogen-activated protein kinase (MAPK) were measured with Western blotting. Hesperidin inhibited high glucose-mediated cell loss and restored mitochondrial function including a reversion of ΔΨm loss and cytochrome c release. Treated with hesperidin, high glucose-induced increase in ROS, malondialdehyde, and protein carbonyl levels were blocked in RGC-5 cells. Hesperidin was found to elevate the activities of superoxide dismutase, catalase, glutathione peroxidase, and to recover glutathione levels. Hesperidin inhibited high glucose-induced cell apoptosis by attenuating the downregulation of caspase-9, caspase-3, and Bax/Bcl-2. Furthermore, the phosphorylation of c-Jun N-terminal kinases (JNK) and p38 MAPK triggered by high glucose were attenuated in RGC-5 cells after their incubation with hesperdin. We concluded that hesperidin may protect RGC-5 cells from high glucose-induced injury since it owns the properties of antioxidant action and blocks mitochondria-mediated apoptosis.

The Benefits of the Citrus Flavonoid Diosmin on Human Retinal Pigment Epithelial Cells under High-Glucose Conditions.

We investigate diosmin for its effect on the ARPE-19 human retinal pigment epithelial cells exposed to high glucose, a model of diabetic retinopathy (DR). After incubation for 4 days with a normal (5 mmol/L) concentration of D-glucose, ARPE-19 cells were exposed separately to normal or high concentrations of D-glucose (30 mmol/L) with or without diosmin at different concentrations (0.1, 1, 10 µg/mL) for another 48 h. Next, we assessed cell viability, reactive oxygen species (ROS) generation and antioxidant enzyme activities. In order to examine the underlying molecular mechanisms, we meanwhile analyzed the expressions of Bax, Bcl-2, total and phosphorylated JNK and p38 mitogen-activated protein kinase (MAPK). Diosmin dose dependently enhanced cell viability following high glucose treatment in ARPE-19 cells. The activities of superoxide dismutase and glutathione peroxidase, as well as the levels of reduced glutathione were decreased, while it was observed that levels of ROS in high glucose cultured ARPE-19 cells increased. High glucose also disturbed Bax and Bcl-2 expression, interrupted Bcl-2/Bax balance, and triggered subsequent cytochrome c release into the cytosol and activation of caspase-3. These detrimental effects were ameliorated dose dependently by diosmin. Furthermore, diosmin could abrogate high glucose-induced apoptosis as well as JNK and P38 MAPK phosphorylation in ARPE-19 cells. Our results suggest that treatment ARPE-19 cells with diosmin halts hyperglycemia-mediated oxidative damage and thus this compound may be a candidate for preventing the visual impairment caused by DR.

Antioxidant-Rich Extract from Plantaginis Semen Ameliorates Diabetic Retinal Injury in a Streptozotocin-Induced Diabetic Rat Model.

Plantaginis semen, the dried ripe seed of Plantago asiatica L. or Plantago depressa Willd. (Plantaginaceae), has been traditionally used to treat blurred vision in Asia. The aim of this work was to investigate the effect of plantaginis semen ethanol extract (PSEE) on the amelioration of diabetic retinopathy (DR) in streptozotocin (STZ)-diabetic rats. PSEE has abundant polyphenols with strong antioxidant activity. PSEE (100, 200 or 300 mg/kg) was oral administrated to the diabetic rats once daily consecutively for 8 weeks. Oral administration of PSEE resulted in significant reduction of hyperglycemia, the diameter of the retinal vessels, and retinal vascular permeability and leukostasis in diabetic rats. In addition, PSEE administration increased the activities of superoxidase dismutase (SOD) and catalase (CAT), and glutathione peroxidase (GSH) level in diabetic retinae. PSEE treatment inhibited the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) and the phosphorylation of Akt without altering the Akt protein expression in diabetic retinae. PSEE not only down-regulated the gene expression of hypoxia-inducible factor-1α (TNF-α) and interleukin-1ß (IL-1ß), but also reduced ICAM-1 and VCAM-1 expression in diabetic retinae. Moreover, PSEE reduced the nuclear factor-κB (NF-κB) activation and corrected imbalance between histone deacetylases (HDAC) and histone acetyltransferases (HAT) activities in diabetic retinae. In conclusion, phenolic antioxidants extract from plantaginis semen has potential benefits in the prevention and/or progression of DR.

The ethanol extract of Zingiber zerumbet rhizomes mitigates vascular lesions in the diabetic retina.

Diabetic retinopathy (DR) is a common diabetic eye disease which is well-known as the result of microvascular retinal changes. Although the ethanol extract from Zingiber zerumbet (L.) Smith rhizome (EEZZR) has been indicated to ameliorate hyperglycemia in diabetes, its protective effect on DR remains unclear. The aim of this study was to determine the effects of EEZZR on DR in streptozotocin (STZ) diabetic rats. Diabetic rats were treated orally with EEZZR (200, 300 mg/kg per day) or calcium dobesilate (CD; 500 mg/kg per day) for 12 weeks. EEZZR displayed similar characteristics to CD in reducing blood-retinal barrier permeability in diabetic rats. Retinal histopathological observation showed that retinal vessels were decreased in EEZZR-treated diabetic rats. EEZZR decreased the increased retinal expression of vascular endothelial growth factor (VEGF) and upregulate the expressions of renal pigment epithelium-derived factor (PEDF) in diabetic rats. Retinal mRNA expression of tumor necrosis factor-α, interleukin (IL)-1, IL-6, monocyte chemotactic proteins-1, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 were all decreased in EEZZR-treated diabetic rats. Moreover, EEZZR could attenuate phosphorylation of nuclear factor Kappa B (NF-κB) p65 and extracellular signal-regulated kinase (ERK)1/2 as well as inhibit the nuclear translocation of pNF-κB p65 induced by diabetes. In conclusion, restoring the balance between stimulators and inhibitors of angiogenesis may be associated with the protective effect of EEZZR on DR. In addition, EEZZR can ameliorate retinal inflammation via transrepression of NF-κB and inhibition of ERK1/2 signaling pathway.

Consumption of Polyphenol-Rich Zingiber Zerumbet Rhizome Extracts Protects against the Breakdown of the Blood-Retinal Barrier and Retinal Inflammation Induced by Diabetes.

The present study investigates the amelioration of diabetic retinopathy (DR) by Zingiber zerumbet rhizome ethanol extracts (ZZRext) in streptozotocin-induced diabetic rats (STZ-diabetic rats). ZZRext contains high phenolic and flavonoid contents. STZ-diabetic rats were treated orally with ZZRext (200, 300 mg/kg per day) for three months. Blood-retinal barrier (BRB) breakdown and increased vascular permeability were found in diabetic rats, with downregulation of occludin, and claudin-5. ZZRext treatment effectively preserved the expression of occludin, and claudin-5, leading to less BRB breakdown and less vascular permeability. Retinal histopathological observation showed that the disarrangement and reduction in thickness of retinal layers were reversed in ZZRext-treated diabetic rats. Retinal gene expression of tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, vascular endothelial growth factor, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 were all decreased in ZZRext-treated diabetic rats. Moreover, ZZRext treatment not only inhibited the nuclear factor κB (NF-κB) activation, but also downregulated the protein expression of p38 mitogen-activated protein kinase (MAPK) in diabetic retina. In conclusion, the results suggest that the retinal protective effects of ZZRext occur through improved retinal structural change and inhibiting retinal inflammation. The antiretinopathy property of ZZRext might be related to the downregulation of p38 MAPK and NF-κB signal transduction induced by diabetes.

Biogenic amine content, histamine-forming bacteria, and adulteration of pork in tuna sausage products.

Twenty-five tuna sausage products were purchased from retail markets in Taiwan. The rates of occurrence of biogenic amines, histamine-forming bacteria, and adulteration by pork and poultry were determined. The average content of various biogenic amines in all tested samples was less than 2.0 mg/100 g (<0.05 to 1.85 mg/100 g). Thirteen histamine-producing bacterial strains isolated from tested samples produced 12.1 to 1,261 ppm of histamine in Trypticase soy broth supplemented with 1.0% L-histidine. Among them, Raoultella ornithinolytica (one strain), Enterobacter aerogenes (one strain), and Staphylococcus pasteuri (two strains) were identified as prolific histamine formers. PCR assay revealed that the adulteration rates were 80% (20 of 25) and 4% (1 of 25) for pork and poultry, respectively, in tuna sausage. The fish species in the tuna sausage samples were identified as Thunnus albacares for 22 samples (88%), Thunnus alalunga for 1 sample (4%), and Thunnus thynnus for 1 sample (4%), whereas the remaining sample was identified as Makaira nigricans (blue marlin).

Cloning and functional characterization of a complex endo-beta-1,3-glucanase from Paenibacillus sp.

A beta-1,3-glucanase gene, encoding a protein of 1,793 amino acids, was cloned from a strain of Paenibacillus sp. in this study. This large protein, designated as LamA, consists of many putative functional units, which include, from N to C terminus, a leader peptide, three repeats of the S-layer homologous module, a catalytic module of glycoside hydrolase family 16, four repeats of the carbohydrate-binding module of family CBM_4_9, and an analogue of coagulation factor Fa5/8C. Several truncated proteins, composed of the catalytic module with various organizations of the appended modules, were successfully expressed and characterized in this study. Data indicated that the catalytic module specifically hydrolyze beta-1,3- and beta-1,3-1,4-glucans. Also, laminaritriose was the major product upon endolytic hydrolysis of laminarin. The CBM repeats and Fa5/8C analogue substantially enhanced the hydrolyzing activity of the catalytic module, particularly toward insoluble complex substrates, suggesting their modulating functions in the enzymatic activity of LamA. Carbohydrate-binding assay confirmed the binding capabilities of the CBM repeats and Fa5/8C analogue to beta-1,3-, beta-1,3-1,4-, and even beta-1,4-glucans. These appended modules also enhanced the inhibition effect of the catalytic module on the growth of Candida albicans and Rhizoctonia solani.

The 1.5 A structure of endo-1,3-beta-glucanase from Streptomyces sioyaensis: evolution of the active-site structure for 1,3-beta-glucan-binding specificity and hydrolysis.

The catalytic domain structure of Streptomyces sioyaensis 1,3-beta-glucanase (278 amino acids), a member of glycosyl hydrolase family 16 (GHF16), was determined to 1.5 A resolution in space group P2(1)2(1)2(1). The enzyme specifically hydrolyzes the glycosidic bond of the 1,3-beta-linked glucan substrate. The overall structure contains two antiparallel six-and seven-stranded beta-sheets stacked in a beta-sandwich jelly-roll motif similar to the fold of GHF16 1,3-1,4-beta-glucanases. The active-site cleft of the enzyme is distinct, with the closure of one end primarily caused by two protruding loop insertions and two key residues, Tyr38 and Tyr134. The current known structures of 1,3-1,4-beta-glucanases and 1,3-beta-glucanase from Nocardiopsis sp., on the other hand, have open-channel active-site clefts that can accommodate six beta-D-glucopyranosyl units. The active-site structure of 1,3-beta-glucanase was compared with those of other homologous structures in order to address the binding and enzymatic specificity for 1,3-beta-linked glucans in Streptomyces. This information could be helpful in the development of specific antifungal agents.

Isolation and biochemical characterization of an endo-1,3-beta-glucanase from Streptomyces sioyaensis containing a C-terminal family 6 carbohydrate-binding module that binds to 1,3-beta-glucan.

A gene encoding 1,3-beta-glucanase was isolated from Streptomyces sioyaensis based on an activity plate assay. Analysis of the deduced amino acid sequence of the gene revealed that the matured 1,3-beta-glucanase has two functional domains separated by a stretch of nine glycine residues. The N-terminal domain shares sequence similarity with bacterial endo-1,3-beta-glucanases classified in glycosyl hydrolase family 16 (GHF 16), while the C-terminal domain is a putative carbohydrate-binding module (CBM) grouped into CBM family 6. To characterize the function of each domain, both the full-length and the CBM-truncated versions of the protein were expressed in Escherichia coli and purified to homogeneity. Biochemical data suggest that the glycosyl hydrolase domain preferentially catalyses the hydrolysis of glucans with 1,3-beta linkage, and has an endolytic mode of action. Binding assay indicated that the C-terminal CBM binds to various insoluble beta-glucans (1,3-, 1,3-1,4- and 1,4- linkages) but not to xylan, a primary binding target for most members of CBM family 6. The full-length and the CBM-truncated proteins had similar specific activity (units per mol of hydrolase domain) on soluble 1,3-beta-glucan, whereas the former had much stronger specific activity on insoluble 1,3-beta-glucans, suggesting that the C-terminal CBM enhances the activity of the S. sioyaensis 1,3-beta-glucanase against insoluble substrates, presumably by increasing the frequency of encounter events between the hydrolase domain and the substrate.