Diallyl disulfide and diallyl trisulfide up-regulate the expression of the pi class of glutathione S-transferase via an AP-1-dependent pathway

Garlic organosulfur compounds are recognized as potential chemopreventive compounds. This protection is related to the induction of phase II detoxification enzymes. We previously reported that diallyl disulfide (DADS) and diallyl trisulfide (DATS) up-regulate the gene expression of the pi class of glutathione S-transferase (GSTP) and that an enhancer element named GPE I is required for this induction. In the present study, we further investigated the signal pathway involved in DADS and DATS up-regulation of this detoxification enzyme in Clone 9 cells. Cells were cultured with 25-200 micromol/L of DADS or DATS for 24 h. Western and Northern blots showed that both garlic allyl sulfides concentration dependently induced GSTP protein and mRNA expression, respectively. Changes in GST activity toward ethacrynic acid were consistent with the increase in GSTP expression (P < 0.05). Electromobility gel shift assay showed that the DNA binding activity of nuclear activator protein-1 (AP-1) is concentration-dependently increased in the presence of DADS and DATS as compared with that of the control cells. The phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), but not of p38, was stimulated in the presence of both garlic allyl sulfides. Pretreatment with SP600125 and PD98059, which are JNK and ERK inhibitors, respectively, abolished the increase in AP-1-DNA binding activity and also the induction of GSTP protein by either allyl sulfide. Our results indicate that the effectiveness of DADS and DATS on GSTP expression is likely related to the JNK-AP-1 and ERK-AP-1 signaling pathways and, thus, that DADS and DATS enhance the binding of AP-1 to GPE I.     

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.     

The wheat germ agglutinin binding sites and development of the mitochondria-rich cells in gills of tilapia (Oreochromis mossambicus)

By labelling with gold-colloid-conjugated wheat germ agglutinin (WGA), the WGA-binding sites were identified in the apical region of certain mitochondria-rich (MR) cells in gills of freshwater-adapted tilapia (Oreochromis mossambicus). Bromo-deoxyuridine (BrdU) injection was used to discern the differentiation of the WGA binding sites during development of MR cells. Double labeling of gill sections of fish 2, 4, 6, and 8 days after a single BrdU injection showed that the BrdU labeled MR cells 2 and 4 days after injection were mostly WGA negative, whereas 6 and 8 days after injection, WGA and BrdU-labeled MR cell increased gradually in number. Furthermore, the ratio of the WGA-positive to total MR-cells was higher in gills of tilapia adapted to low calcium ([Ca] = 0.015 mm) freshwater than in hard freshwater ([Ca] = 1.898 mm). The results indicate that WGA binding site may be a marker expressed late during differentiation of MR cells, and physiologically functional in assisting uptake of calcium when environmental calcium is low. According to their WGA binding and also characteristics their likely function in calcium absorption, the WGA positive MR cells shown in this study are considered to be similar to the cells described by Pisam et al. (1995).     

Characterization of the pattern of cytokeratin proteins in the epidermal cells of loach,Misgurnus anguillicaudatus (Cyprinformes)

The pattern of cytokeratin proteins in the epidermal cells of loach was studied by immunotechniques and partial separation of the epidermal cells. Two monoclonal antibodies, namely 8F7 and 1C45, against the cytokeratin proteins of the loach epidermis were prepared. these two monoclonal antibodies exhibit distinctive results in immunohistochemical staining. The 8F7 monoclonal antibody stains mainly with the epithelial cells, while the 1C45 monoclonal antibody stains specifically with the club cells. The pattern of cytokeratin proteins in the club cells and the epithelial cells of various epidermal layers was further determined by partial separation of these cells. Immunoblotting analysis of the cell fractions confirms the cytokeratin proteins to be differentially expressed in the club cells and the epithelial cells. However, the cytokeratin proteins expressed in the epithelial cells of the basal, middle and outer layers are same. The results indicate that differentiation of the epithelial cells seems limited during their translocation from basal to upper layers, but in those cells that do differentiate into club cells, the cytokeratin pattern changes.     

Integration of independent component analysis with near infrared spectroscopy for evaluation of rice freshness

The storage time and conditions of rice has an enormous effect on its appearance, flavor, and quality of the nutrients; and the acidity of rice usually increases with prolonged storage. Therefore, evaluation of freshness is an important issue for rice quality. In this study, the NIR (near infrared) spectra combined with independent component analysis (ICA) technique was used to evaluate the rice freshness. A total of 180 white rice samples were collected from 6 crop seasons for the purpose of developing an ICA-NIR based procedure for rice freshness as quantified by pH values. Values of pH were determined by a BTB-MR (bromothymol blue – methyl red) method. The best calibration model of white rice was developed using the smoothed first derivative spectra, five ICs and cross-validation; the results indicated that r² (coefficient of determination) = 0.924, and in units of pH, SEC (standard error of calibration) = 0.145, SEP (standard error of prediction) = 0.146, bias = 0.001, and RPD (residual predictive deviation) = 3.65. Freshness of white rice could be distinguished either visually by a 3-dimensional diagram composed from ICs 2, 3 and 4, or statistically by a calibration model. The results show that ICA with NIR has the potential to be adopted as an effective method for evaluating rice freshness.     

Effects of whey protein concentrate (WPC) on the distributions of lymphocyte subpopulations in rats with excessive alcohol intake

To investigate the effects of whey protein concentrate (WPC) on antioxidant statuses and the lymphocyte subpopulations in the rats with alcohol intake, the antioxidant statuses in the peripheral blood (PB) and the lymphocyte subpopulations in the PB, spleen, and bone marrow (BM) of the rats fed with WPC (0.334 g/kg) and alcohol (6 g/kg) for 3 months were analyzed. Results showed that the effects of WPC on the glutathione peroxidase and glutathione in the PB, the T and B cells in the spleen, and the B cells in the BM were more apparent in the rats with alcohol intake; however, they are not apparent in the controls. Taken together, our results indicated that the immunity of rats might be enhanced by the increased antioxidant ability after WPC supplementation and the effects of WPC on the lymphocyte subpopulations were mainly in the spleen and BM and not in the PB.     

Detection of antibodies against Bordetella avium in turkeys by avidin-biotin enhancement of the enzyme-linked immunosorbent assay and the dot-immunobinding assay.

An avidin-biotin-enhanced enzyme-linked immunosorbent assay (AB-ELISA) and an avidin-biotin-enhanced dot-immunobinding (AB-DIB) assay for detecting antibody to Bordetella avium in turkey sera were developed and compared with the microagglutination (MA) test. Whole-cell antigen, biotin-labeled goat anti-turkey IgG conjugate, and horseradish-peroxidase-labeled streptavidin were used in the AB-ELISA and AB-DIB assay. The AB-ELISA and AB-DIB assay were sensitive, specific, and reproducible. These assays were superior to the MA test for measuring acquired and maternal antibodies against B. avium. All MA-positive sera were positive by two assays, but some sera negative by MA test had titers in the AB-ELISA and AB-DIB assay. AB-ELISA and AB-DIB titers showed a positive correlation (r = 0.866), and AB-ELISA was more sensitive than the AB-DIB assay.