Mechanism and characteristics of protein release from lactitol-based cross-linked hydrogel

Lactitol-based cross-linked hydrogel was synthesized, and model proteins (alpha-chymotrypsin, beta-lactoglobulin, bovine serum albumin (BSA), and gamma-globulin) were incorporated into the cross-linked hydrogel. The larger-molecular-weight proteins have lower diffusivity (D(e)) in the hydrogel. Increasing temperature accelerated the diffusion rate of proteins; however, the diffusion did not follow the Arrhenius equation at temperatures above 37 degrees C. The swelling ratio of the hydrogel was slightly decreased after heating for 2 h at 37 and 45 degrees C, and significantly reduced after 1 h at 60 degrees C. Therefore, diffusion of beta-lactoglobulin and BSA may be decreased by hydrogel shrinking at temperature over 37 degrees C. The model proteins have high affinities to buffer solution compared to the hydrogel network structure, resulting in high partition coefficients (K > 1) which do not affect the calculation of D(e) values. Incorporated protein release follows the theory of hindered diffusion.     

Stable RNA/DNA hybrids in the mammalian genome: inducible intermediates in immunoglobulin class switch recombination

Although it is well established that mammalian class switch recombination is responsible for altering the class of immunoglobulins, the mechanistic details of the process have remained unclear. Here, we show that stable RNA/DNA hybrids form at class switch sequences in the mouse genome upon cytokine-specific stimulation of class switch in primary splenic B cells. The RNA hybridized to the switch DNA is transcribed in the physiological orientation. Mice that constitutively express an Escherichia coli ribonuclease H transgene show a marked reduction in RNA/DNA hybrid formation, an impaired ability to generate serum immunoglobulin G antibodies, and significant inhibition of class switch recombination in their splenic B cells. These data provide evidence that stable RNA/DNA hybrids exist in the mammalian nuclear genome, can serve as intermediates for physiologic processes, and are mechanistically important for efficient class switching in vivo.     

Ralstonia solanacearum nlpD (RSc1206) contributes to host adaptation

Ralstonia solanacearum is a soil-borne xylem-inhibited pathogen and causes a deadly wilting disease on a wide range of crops. This bacterium can also colonize many weeds and native plants without necessarily causing symptoms, leading to devastating epidemiological consequences; however, little is known about the biology. A R. solanacearum mutant harbouring a transposon insertion in the annotated gene RSc1206 was previously shown to retain virulence in tomato but displayed reduced virulence in model weed host Arabidopsis. In this study, we investigated the function of RSc1206 in bacterial pathogenesis in weed hosts. RSc1206 encodes a putative protein that is homologous to E. coli NlpD, and the organization of RSc1206-related gene cluster is mostly conserved among representative gram-negative bacteria analyzed. We therefore designate it R. solanacearum gene nlpD. Microscopic data revealed that the nlpD mutant had defects in cell separation and envelope integrity. An increased sensitivity to hydrophobic toxic chemicals confirmed that the cell integrity of the nlpD mutant was damaged. In addition, the activity of the nlpD mutant in biofilm formation and motility was reduced. However, enzymatic activity and tobacco pathogenesis assays revealed that this mutant functioned normally in the Type II and III secretion systems. Trans-mutation and trans-complementation analyses further verified that disruption of nlpD function was responsible for the observed defects. Our study provides evidence that NlpD contributes to R. solanacearum cell integrity and pathogenesis in weed host Arabidopsis.     

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.     

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.     

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.     

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.     

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.     

Sandwich enzyme-linked immunosorbent assay for the detection of bovine blood in animal feed

The feeding of ruminant proteins to ruminants is prohibited in most countries because the practice is thought to be responsible for the spread of bovine spongiform encephalopathy. However, currently available methods to detect ruminant blood products in rendered feedstuffs are inadequate because they lack species specificity, tissue specificity, and are not based on a thermostable analyte. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for this study that provides reliable and sensitive (0.05-0.5% v/v) detection of bovine blood materials in animal feed. The new sandwich ELISA employs two previously developed monoclonal antibodies (MAbs), Bb6G12 as the capture antibody and biotinylated MAb Bb3D6 as the detecting antibody, and is bovine-specific and blood-specific. The assay is based on the detection of a 60 kDa thermostable protein in bovine blood and provides a useful regulatory tool for monitoring fraudulent labeling or contamination of bovine blood in both heat-processed feedstuffs and unprocessed raw materials. Keywords: Bovine; blood; monoclonal antibody; sandwich ELISA.     

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.