Baicalein induction of hydroxyl radical formation via 12-lipoxygenase in human platelets: an ESR study

The pro-oxidant activities of baicalein, morin, myricetin, quercetin, and rutin were examined in various cell-containing systems including human platelets, rat vascular smooth muscle cells, human umbilical vein endothelial cells (HUVECs), human THP-1 cells, and fibroblast cells. Electron spin resonance (ESR) results showed that only baicalein generated hydroxyl radicals in a resting human platelet suspension, whereas the other flavonoids showed no effects on any of the resting cell systems. A low concentration of arachidonic acid (AA) increased the intensity of hydroxyl radicals, but a high concentration inhibited it. Collagen and thrombin, platelet aggregatory agents that can cause the release of AA by platelets, enhanced baicalein-induced hydroxyl radical formation, whereas ADP and U44619 showed no significant effects. Quinacrine and 5,8,11,14-eicosatetraenoic trifluoromethyl ketone, both PLA2 inhibitors, significantly attenuated baicalein-induced hydroxyl radical formation. These results suggest that baicalein-induced hydroxyl radical formation is associated with AA metabolite enzymes in human platelets. The formation of hydroxyl radicals was significantly inhibited by lipoxygenase inhibitors including nordihydroguaiaretic acid, (-)-epicatechin, (-)-epicatechin gallate, and hinokitiol, but was not affected by desferroxamine or the heme protein inhibitors KCN and NaN3. On the other hand, semiquinone free radicals were generated when baicalein was incubated with horseradish peroxidase/H2O2 or platelets/AA. The semiquinone radicals formed in the platelets/AA system could be extensively inhibited by desferroxamine, diethylenetriaminepentaacetic acid, KCN, and NaN3, indicating that prostaglandin H synthase (PGHS)-peroxidase may be involved. The results of this study led to the proposal that baicalein induces hydroxyl radical formation via 12-lipoxygenase and induces semiquinone radical formation via PGHS-peroxidase in human platelets.     

Monoclonal antibodies specific to thermostable proteins in animal blood

Bovine plasma proteins are used as high-quality protein supplements in animal feed and as binders or colorants in food for human consumption. Religious observance, as well as recent fears of epidemic bovine spongiform encephalopathy, highlights the need for methods to detect bovine blood in processed food and animal feed for regulatory purposes, as the currently available methods are neither species-specific, blood-specific, nor valid for excessively heat-processed samples. This paper reports the development of monoclonal antibodies (MAbs) raised against bovine thermostable plasma proteins that display a unique species specificity pattern for plasma proteins. Immunoblotting revealed several thermostable antigenic proteins (10, 25, 40, and 60 kDa) in bovine plasma sterilized at 121 degrees C for 15 min. These MAbs can be employed individually or combined in immunoassays for analytical purposes and investigations of the chemical and biological properties of the thermostable plasma proteins identified here.     

Molecular action mechanism against apoptosis by aqueous extract from guava budding leaves elucidated with human umbilical vein endothelial cell (HUVEC) model

Chronic cardiovascular and neurodegenerative complications induced by hyperglycemia have been considered to be associated most relevantly with endothelial cell damages (ECD). The protective effects of the aqueous extract of Psidium guajava L. budding leaves (PE) on the ECD in human umbilical vein endothelial cell (HUVEC) model were investigated. Results revealed that glyoxal (GO) and methylglyoxal (MGO) resulting from the glycative and autoxidative reactions of the high blood sugar glucose (G) evoked a huge production of ROS and NO, which in turn increased the production of peroxynitrite, combined with the activation of the nuclear factor kappaB (NFkappaB), leading to cell apoptosis. High plasma glucose activated p38-MAPK, and high GO increased the expressions of p38-MAPK and JNK-MAPK, whereas high MGO levels induced the activity of ERK-MAPK. Glucose and dicarbonyl compounds were all found to be good inducers of intracellular PKCs, which together with MAPK acted as the upstream triggering factor to activate NFkappaB. Conclusively, high plasma glucose together with dicarbonyl compounds can trigger the signaling pathways of MAPK and PKC and induce cell apoptosis through ROS and peroxynitrite stimulation and finally by NFkappaB activation. Such effects of PE were ascribed to its high plant polyphenolic (PPP) contents, the latter being potent ROS inhibitors capable of blocking the glycation of proteins, which otherwise could have brought forth severe detrimental effects to the cells.     

Hibiscus protocatechuic acid or esculetin can inhibit oxidative LDL induced by either copper ion or nitric oxide donor

Oxidation of low-density lipoprotein (LDL) could increase the incidence of atherosclerosis. Previous studies have shown that copper and sodium nitroprusside (SNP) possess the ability to oxidize LDL in a dose-dependent condition. They increase the existing negative charge in LDL and increase the electrophoretic mobility. In this study, we used protocatechuic acid (PCA) and/or esculetin (ECT) to define the antioxidative activity in oxidative LDL by relative electrophoretic mobility (REM) and thiobarbituric acid-relative substances (TBARS). The data showed that ECT and PCA possessed stronger antioxidative activity than vitamin E in oxidative LDL. A previous study showed that the level of oxidative LDL can be determined by the cholesterol degradation and fragmentation of Apo B. Our results showed that Cu(2+)-mediated oxidative LDL can induce 31% cholesterol degradation and significant fragmentation of Apo B. Both PCA and ECT exhibited remarkable ability to rescue the cholesterol degradation and Apo B fragmentation. Taken together, both PCA and ECT showed strong potency to inhibit oxidative LDL induced by copper or an NO donor. Additionally, their nontoxic characteristics elevated the possibility for their use in the daily diet; and should further prevent atherosclerosis effectively.     

Antcin B and its ester derivative from Antrodia camphorata induce apoptosis in hepatocellular carcinoma cells involves enhancing oxidative stress coincident with activation of intrinsic and extrinsic apoptotic pathway

The triterpenoids methylantcinate B (MAB) and antcin B (AB), isolated from the medicinal mushroom Antrodia camphorata , have been identified as strong cytotoxic agents against various type of cancer cells; however, the mechanisms of MAB- and AB-induced cytotoxicity have not been adequately explored. This study investigated the roles of caspase cascades, reactive oxygen species (ROS), DNA damage, mitochondrial disruption, and Bax and Bcl-2 proteins in MAB- and AB-induced apoptosis of hepatocellular carcinoma (HCC) HepG2 cells. Here, we showed that MAB and AB induced apoptosis in HepG2 cells, as characterized by increased DNA fragmentation, cleavage of PARP, sub-G1 population, chromatin condensation, loss of mitochondrial membrane potential, and release of cytochrome c. Increasing the levels of caspase-2, -3, -8, and -9 activities was involved in MAB- and AB-induced apoptosis, and they could be attenuated by inhibitors of specific caspases, indicating that MAB and AB triggered the caspase-dependent apoptotic pathway. Additionally, the enhanced apoptotic effect correlates with high expression of Fas, Fas ligand, as well as Bax and decreased protein levels of Bcl-(XL) and Bcl-2, suggesting that both the extrinsic and intrinsic apoptosis pathways were involved in the apoptotic processes. Incubation of HepG2 cells with antioxidant enzymes superoxide dismutase and catalase and antioxidants N-acetylcysteine and ascorbic acid attenuated the ROS generation and apoptosis induced by MAB and AB, which indicate that ROS plays a pivotal role in cell death. NADPH oxidase activation was observed in MAB- and AB-stimulated HepG2 cells; however, inhibition of such activation by diphenylamine significantly blocked MAB- and AB-induced ROS production and increased cell viability. Taken together, our results provide the first evidence that triterpenoids MAB and AB induced a NADPH oxidase-provoked oxidative stress and extrinsic and intrinsic apoptosis as a critical mechanism of cause cell death in HCC cells.     

Anti-inflammatory effects of resveratrol and oligostilbenes from Vitis thunbergii var. taiwaniana against lipopolysaccharide-induced arthritis

Vitis thunbergii Sieb. and Zucc. var. taiwaniana Lu is an endemic plant in Taiwan used as a dietary supplement for bone health. In this study, human chondrocytes were induced to produce COX-2, MMP-3, -13, and PGE(2) by LPS. An (18)F-FDG microPET imaging system was used to evaluate the anti-inflammatory arthritic effects in vivo. Six stilbenes, resveratrol (1), (+)-ε-viniferin (2), ampelopsin C (3), ampelopsin A (4), (-)-vitisin B (5), and (+)-vitisin A (6), were isolated from the stem part of V. thunbergii, which displayed the strongest PGE(2) inhibition. Among these compounds, 1 significantly decreased COX-2 activity, PGE(2), MMP-3, and -13 production in vitro, and (18)F-FDG uptake and serum PGE(2) in rabbits in vivo. Anti-inflammatory effects were enhanced through the combined usage of 1 and other oligostilbenes. Taken together, the synergistic effects of 1 and oligostilbenes resulted in stem part extracts with lower 1 content displaying the better anti-inflammatory arthritis effects.     

Protein-protein interactions during high-moisture extrusion for fibrous meat analogues and comparison of protein solubility methods using different solvent systems

Soy protein, mixed with gluten and starch, was extruded into fibrous meat analogues under high-moisture and high-temperature conditions. The protein solubility of samples collected at different extruder zones and extrudates made with different moistures was determined by 11 extraction solutions consisting of 6 selective reagents and their combinations: phosphate salts, urea, DTT, thiourea, Triton X-100, and CHAPS. Protein solubility by most extractants showed decreasing patterns as the material passed through the extruder, but the solution containing all 6 reagents, known as isoelectric focus (IEF) buffer, solubilized the highest levels and equal amounts of proteins in all samples, indicating that there are no other covalent bonds involved besides disulfide bonds. With regard to relative importance between disulfide bonds and non-covalent interactions, different conclusions could be made from protein solubility patterns, depending on the type of extracting systems and a baseline used for comparison. The observation points out pitfalls and limitation of current protein solubility methodology and explains why controversy exists in the literature. Using the IEF buffer system with omission of one or more selective reagents is considered to be the right methodology to conduct protein solubility study and thus recommended. Results obtained with this system indicate that disulfide bonding plays a more important role than non-covalent bonds in not only holding the rigid structure of extrudates but also forming fibrous texture. The sharpest decrease in protein solubility occurred when the mix passed through the intermediate section of the extruder barrel, indicating formation of new disulfide bonds during the stage of dramatic increase in both temperature and moisture. After this stage, although the physical form of the product might undergo change and fiber formation might occur as it passed through the cooling die, the chemical nature of the product did not change significantly.     

Molecular phylogenetic relationships of puffer fish inferred from partial sequences of cytochrome b gene and restriction fragment length polymorphism analysis

Phylogenetic relationships among puffer fish were investigated by comparing cytochrome b gene sequences and restriction endonuclease assays of 16 species from Taiwan. DNA was prepared for sequencing by PCR. No variation in sequences was detected among individuals within each species. Direct estimates of mitochondrial cytochrome b gene sequence divergence among 16 puffer fish were from 3.41 to 31.78%. Different restriction patterns were found among 16 puffer fish with 10 restriction endonucleases, whereas no variation in patterns was detected among individuals within each species. The polymorphisms obtained by RFLP have provided a new set of genetic markers for the accurate identification of sibling puffer species. It is the first molecularly based study of puffer diversity and sheds light on the evolution and taxonomy of this major puffer fish family.     

Mosquito-killing water molds isolated from soil samples collected in Taiwan

Mosquito-killing water molds were isolated from soils collected from various parts in Taiwan. The first-instar larvae of Aedes aegypti and Aedes albopictus were used as baits. A total of 453 soil samples were collected from Taipei, Ilan, Hualien, Taoyuan, Hsinchu, Miaoli, Taichung, Tainan, Nantou, Chiayi, Yunlin and Pintung. Four mosquito-killing Pythium spp. and one Saprolegnia sp. were isolated from the soil samples. Using zoospores of the Pythium spp. as inoculum to infect the first-instar larvae, scanning electron micrographs showed that most zoospores attached in the anal gill and a few attached between head and thorax. Thus, Pythium spp. is a potential biocontrol agent against first-instar larvae of A. aegypti and A. albopictus.