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81.
Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (1O2) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of 1O2 with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (•OH) adduct of DMPO (DMPO-OH) resulting from 1O2-dependent generation of •OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its •OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of •OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of •OH from 1O2, and that spin trap-mediated •OH generation hardly occurs with DEPMPO. 相似文献
82.
In Saccharomyces cerevisiae, there are two isoenzymes of fumarate reductase (FRDS1 and FRDS2), encoded by the FRDS and OSM1 genes, respectively. Simultaneous disruption of these two genes results in a growth defect of the yeast under anaerobic conditions, while disruption of the OSM1 gene causes slow growth. However, the metabolic role of these isoenzymes has been unclear until now. In the present study, we found that the anaerobic growth of the strain disrupted for both the FRDS and OSM1 genes was fully restored by adding the oxidized form of methylene blue or phenazine methosulfate, which non-enzymatically oxidize cellular NADH to NAD(+). When methylene blue was added at growth-limiting concentrations, growth was completely arrested after exhaustion of oxidized methylene blue. In the double-disrupted strain, the accumulation of succinate in the supernatant was markedly decreased during anaerobic growth in the presence of methylene blue. These results suggest that fumarate reductase isoenzymes are required for the reoxidation of intracellular NADH under anaerobic conditions, but not aerobic conditions. 相似文献
83.
Selective recovery of lactate dehydrogenase using affinity foam 总被引:3,自引:0,他引:3
Selective isolation of lactate dehydrogenase (LDH) from porcine muscle extract was studied using foam generated from the vigorous stirring of a non-ionic surfactant, Triton X-114 derivatized with Cibacron blue. The cloud point of the surfactant-dye conjugate was higher than that of the native Triton X-114, and also the foam prepared from the affinity surfactant was more rigid taking a longer time to collapse. The equilibrium dissociation constant between pure LDH and surfactant-dye conjugate was 5.0 microM as compared to the value of 2.2 microM for the enzyme and free dye as measured by differential spectroscopy. The isolation procedure involved mixing of the porcine muscle extract with the affinity foam, separating and collapsing the foam, and warming the solution formed to 37 degrees C to yield the surfactant-dye phase and an aqueous phase containing the enzyme. The effect of surfactant concentration and protein load on enzyme recovery and purification was investigated. Under optimal conditions, LDH was quantitatively recovered with high purification factor in a very short time. Both recovery and purification were higher when foam prepared from an equivalent mixture of surfactant-dye conjugate and unmodified surfactant was used. The selectivity of interaction between LDH and detergent-dye conjugate was confirmed by lowered recovery when NADH was included during the binding step. 相似文献
84.
Heuberger EH Veenhoff LM Duurkens RH Friesen RH Poolman B 《Journal of molecular biology》2002,317(4):591-600
Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins have not been studied. By comparison with analytical ultracentrifugation, we have evaluated whether the oligomeric state of membrane transport proteins is reflected reliably with blue native electrophoresis. For the analysis we have used two well-characterized transporters, that is, the major facilitator superfamily protein LacS and the phosphotransferase system EII(Mtl). For another member of the major facilitator superfamily, the xyloside transporter XylP from Lactobacillus pentosus, the complete analysis of the quaternary structure determined by analytical ultracentrifugation and freeze-fracture electron microscopy is presented.Our experiments show that during blue native electrophoresis the detergent bound to the proteins is replaced by the amphipathic Coomassie brilliant blue (CBB) dye. The mass of the bound CBB dye was quantified. Provided this additional mass of bound CBB dye is accounted for and care is taken in the choice and concentration of the detergent used, the mass of LacS, XylP and EII(Mtl) and four other membrane (transport) proteins could be deduced within 10 % error. Our data underscore the fact that the oligomeric state of many membrane transport proteins is dimeric. 相似文献
85.
An improved method for the isolation of rubella virus-like particles (RVLP) from cell culture supernatant of transfected Chinese hamster ovary (CHO24S) cells is described. It employs a combination of membrane filtration with sucrose gradient ultracentrifugation. It was found that staining the RVLP band with Coomassie brilliant blue G (CBB) resulted in the CBB crystals adsorbing RVLP. After ultracentrifugation (25,000 rpm, 3h, 4 degrees C) a sharp blue band with crystals (diameter 30-40 microm) was observed (at a density of 1.250 g/ml at 25 degrees C) in a 30-60% sucrose gradient. Using a combination of SDS-PAGE and Western blotting techniques, E1 rubella virus structural protein was detected only in the solutions derived from the sharp blue band. A decrease in crystal concentration a few millimeters above or below the main band was associated with a decrease in protein concentration. By dilution with a saturated ice-cold 30% sucrose solution it was possible to pellet the crystals by centrifugation (15,000 rpm, 10 min). SDS-PAGE showed a much higher concentration of RVLP structural protein in the pellet than in the supernatant. This RVLP-containing material is especially suitable for the preparation of rubella virus immunoblot stripes. 相似文献
86.
Summary. The possibility that nitric oxide synthase (NOS) inhibitors influence dynorphin immunoreactivity following hyperthermia was
examined in a rat model using a pharmacological approach. Previous reports from our laboratory show that hyperthermia induces
an upregulation of NOS in several brain regions that seems to be instrumental in causing cell injury. Recent reports suggest
that nitric oxide (NO) can influence dynorphin neurotransmission in the normal brain as well as in several pathological states.
Since dynorphin is neurotoxic in different animal models of brain or spinal cord injury, it may be that the peptide will contribute
to the cell injury in hyperthermia. The present investigation was carried out to determine whether hyperthermia can influence
dynorphin immunoreactivity in the brain, and if so, whether inhibition of NOS will influence the peptide distribution in the
brain following heat stress. Rats subjected to hyperthermia at 38°C for 4 h in a biological oxygen demand incubator (BOD)
resulted in a marked upregulation of dynorphin immunoreactivity in several brain regions e.g., cerebral cortex, hippocampus,
cerebellum and brain stem. Pretreatment of rats with two potent NOS inhibitors, L-NAME (30 mg/kg/day, i.p. for 7 days) or
L-NMMA (35 mg/kg/day, i.p. for 7 days) significantly attenuated the dynorphin immunoreactivity in the brain. These drugs were
also able to reduce hyperthermia induced blood-brain barrier (BBB) permeability, brain edema formation and cell injury. Taken
together, our results suggest that (i) hyperthermia has the capacity to upregulate dynorphin immunoreactivity in the brain,
(ii) inhibition of NOS considerably attenuates the dynorphin immunoreaction following heat stress and (iii) upregulation of
dynorphin is somehow contributing to hyperthermia induced brain damage, not reported earlier.
Received July 3, 2001 Accepted August 6, 2001 Published online July 31, 2002 相似文献
87.
The study was conducted to identify the self-incompatibility mechanism in Eucalyptus globulus ssp. globulus. Controlled self- and cross-pollinations were conducted on individual flowers from three mature trees that had self-incompatibility levels of 76, 99.6 and 100%. Flowers were harvested at 4, 6 and 8 weeks after pollination. Embryology was investigated by bright field microscopy on material harvested at 4 and 6 weeks after pollination. Fertilization had taken place at 4 weeks after pollination with zygotes and free nuclear endosperm visible. There was a greater proportion of healthy, fertilized ovules in the cross- compared with the self-pollination treatment, and approx. half the ovules examined from both pollen treatments were not fertilized or were degenerating. By 6 weeks after pollination a few zygotes were starting to divide. The number of healthy, fertilized ovules was still greater in the cross-pollination treatment, but the number of healthy fertilized ovules was lower in both treatments compared with 4 weeks after pollination, and many ovules were degenerating. Fertilized ovules were significantly larger than non-fertilized or degenerating ovules and this difference was detectable by eye at 6 and 8 weeks after pollination. The mechanism of self-incompatibility appears to have both late pre- and post-zygotic components. 相似文献
88.
89.
90.
The ovoid cells of the branchial heart complex of Sepia officinalis L. were investigated with respect to their role in detoxification processes. The electron microscopical localization of in situ injected ferritin in the endocytotic-lysosomal system, and the fluorescence microscopical localization of protein-bound Evans blue in the ovoid cells of in vivo incubated animals, indicate that foreign materials are eliminated from the hemolymph by the branchial heart tissue. In addition to the non-circulating ovoid cells of the branchial heart, hemocytes in the circulating blood and in the wall of the branchial heart are also involved in the incorporation of allogeneic substances and bacteria, or their debris. Based on these observations, we propose that the ovoid cells, together with circulating and adhesive hemocytes in the branchial hearts, are an important component of a more comprehensive defence and detoxification system in dibranchiate cephalopods that prevents contamination of the whole organism by endocytotic removal of noxious substances from the hemolymph. 相似文献