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1.
The influence of thyroid hormone (L-3, 3', 5-triiodothyronine, T 3) on Kupffer cell function was studied in the isolated perfused rat liver by colloidal carbon infusion. Rates of carbon uptake were determined from the influent minus effluent concentration difference and the flow rate, and the respective carbon-induced respiratory activity was calculated by integration of the area under the O 2 curves during carbon infusion. In the concentration range of 0.2 to 2.0 mg of carbon/ml, livers from euthyroid rats exhibited a sigmoidal-type kinetics of carbon uptake, with a Vmax of 4.8 mg/g liver/min and a concentration of 0.82 mg/ml for half-maximal rate; carbon-induced O 2 uptake presented a hyperbolic-type kinetics, with a Vmax of 4.57 μmol of O 2/g liver and a Km of 0.74 mg of carbon/ml, which significantly correlates with the carbon uptake rates. Light-microscopy showed that carbon was taken up exclusively by non-parenchymal cells, predominantly by Kupffer cells. Thyroid calorigenesis was found in parallel with increased rates of hepatic O 2 consumption and thiobarbituric acid reactive substances (TBARS) formation, glutathione (GSH) depletion, and higher sinusoidal lactate dehydrogenase (LDH) efflux compared to control values. In the concentration range of 0.25 to 0.75 mg/ml, carbon infusion did not modify liver LDH efflux in control rats, while it was significantly enhanced in T 3-treated animals. In this latter group, higher carbon concentrations (1 and 1.3 mg/ml) led to loss of viability of the liver. At 0.25 to 0.75 mg of carbon/ml, both the rates of carbon uptake and the associated carbon-induced respiratory activities were significantly increased by T 3 treatment, effects that were abolished by pretreatment of the rats with gadolinium chloride (GdCl 3). In addition, GdCl 3 decreased by 50% the changes induced by T 3 in hepatic GSH content and TBARS formation. It is concluded that hyperthyroidism enhances Kupffer cell function, correlated with the increased number of liver macrophages observed histologically, which may represent an alternate source of reactive O 2 species to that induced in parenchymal cells, thus contributing to the enhanced oxidative stress status developed. 相似文献
2.
Thyroglobulin (Tg) was subjected to metal-catalyzed oxidation, and the oxidative degradation was analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions. In contrast to no effect of hydrogen peroxide (H 2O 2) alone on the Tg degradation, the inclusion of Cu 2+ (30 μM), in combination with 2 mM H 2O 2, caused a remarkable degradation of Tg, time- and concentration-dependent. The action of Cu 2+ was not mimicked by Fe 2+, suggesting that Tg may interact selectively with Cu 2+. A similar degradation of Tg was also observed with Cu 2+corbate system, and the concentration of Cu 2+ (5-10 μM), in combination with ascorbate, required for the effective degradation was smaller than that of Cu 2+ (10-30 μM) in combination with H 2O 2. In support of involvement of H 2O 2 in the Cu 2+ corbate action, catalase expressed a complete protection. However, hydroxyl radical scavengers such as dimethylsulfoxide or mannitol failed to prevent the oxidation of Tg whereas phenolic compounds, which can interact with Cu 2+, diminished the oxidative degradation, presumably consistent with the mechanism for Cu 2+-catalyzed oxidation of protein. Moreover, the amount of carbonyl groups in Tg was increased as the concentration (3-100 μM) of Cu 2+ was enhanced, while the formation of acid-soluble peptides was not remarkable in the presence of Cu 2+ up to 200 μM. In further studies, Tg pretreated with heat or trichloroacetic acid seemed to be somewhat resistant to Cu 2+-catalyzed oxidation, implying a possible involvement of protein conformation in the susceptibility to the oxidation. Based on these observations, it is proposed that Tg could be degraded non-enzymatically by Cu 2+-catalyzed oxidation. 相似文献
3.
The rates of respiratory O 2 uptake have been studied in leaves, stems and whole shoots of several freshwater plants: 6 angiosperms, 2 bryophytes and one alga. For angiosperm leaves, rates varied widely with species (30–142 μmol O 2 (gDW) −1 h −1), were correlated with chlorophyll content and were higher than those of the stems (13–71 μmol O 2 (gDQ) −1 h −1). The rates for the shoots of bryophytes (53–66 μmol O 2 (gDW) −1 h −1) and for the alga Cladophora glomerata (L.) Kütz. (96 μmol O 2 (gDW) −1 h −1) were slightly higher than those of most angiosperm stems, but lower than those for most leaves. These plants had a significant cyanide-resistant respiration, suggesting the existence of an alternative pathway to the “classic” cytochrome system. This pathway was found to be active in all the species studied, as judged by responses to a specific inhibitor, SHAM (salicylhydroxamic acid). Measurement of electron-transport system (ETS) activity showed that there is a large electron-transport capacity which is not normally used by respiration in vivo. 相似文献
4.
We have previously shown that crystals of calcium oxalate (COM) elicit a superoxide (O 2−) response from mitochondria. We have now investigated: (i) if other microparticles can elicit the same response, (ii) if processing of crystals is involved, and (iii) at what level of mitochondrial function oxalate acts. O 2− was measured in digitonin-permeabilized MDCK cells by lucigenin (10 μM) chemiluminescence. [ 14C]-COM dissociation was examined with or without EDTA and employing alternative chelators. Whereas mitochondrial O 2− in COM-treated cells was three- to fourfold enhanced compared to controls, other particulates (uric acid, zymosan, and latex beads) either did not increase O 2− or were much less effective (hydroxyapatite +50%, p < 0.01), with all at 28 μg/cm 2. Free oxalate (750 μM), at the level released from COM with EDTA (1 mM), increased O 2− (+50%, p < 0.01). Omitting EDTA abrogated this signal, which was restored completely by EGTA and partially by ascorbate, but not by desferrioxamine or citrate. Omission of phosphate abrogated O 2−, implicating phosphate-dependent mitochondrial dicarboxylate transport. COM caused a time-related increase in the mitochondrial membrane potential (Δψ m) measured using TMRM fluorescence and confocal microscopy. Application of COM to Fura 2-loaded cells induced rapid, large-amplitude cytosolic Ca 2+ transients, which were inhibited by thapsigargin, indicating that COM induces release of Ca 2+ from internal stores. Thus, COM-induced mitochondrial O 2− requires the release of free oxalate and contributes to a synergistic response. Intracellular dissociation of COM and the mitochondrial dicarboxylate transporter are important in O 2− production, which is probably regulated by Δψ m. 相似文献
5.
Formation of reactive O 2 species in biological systems can be accomplished by copper-(II) (Cu 2+) catalysis, with the consequent cytotoxic response. We have evaluated the influence of Cu 2+ on the respiratory activity of Kupffer cells in the perfused liver after colloidal carbon infusion. Studies were carried out in untreated rats and in animals pretreated with the Kupffer cell inactivator gadolinium chloride (GdCl 3) or with the metallothionein (MT) inducing agent zinc sulphate, and results were correlated with changes in liver sinusoidal efflux of lactate dehydrogenase (LDH) as an index of hepatotoxicity. In the concentration range of 0.1–1 μM, Cu 2+ did not modify carbon phagocytosis by Kupffer cells, whereas the carbon-induced liver O 2 uptake showed a sigmoidal-type kinetics with a half-maximal concentration of 0.23 μM. Carbon-induced O 2 uptake occurred concomitantly with an increased LDH efflux, effects that were significantly correlated and abolished by GdCl 3 pretreatment or by MT induction. It is hypothesized that Cu 2+ increases Kupffer cell-dependent O 2 utilization by promotion of the free radical processes related to the respiratory burst of activated liver macrophages, which may contribute to the concomitant development of hepatocellular injury. 相似文献
6.
Paraoxonase1 (PON1), one of HDL-asssociated antioxidant proteins, is known to be sensitive to oxidative stress. Here, the effect of endogenous reducing compounds on Cu 2+-mediated inactivation of PON1 was examined. Cu 2+-mediated inactivation of PON1 was enhanced remarkably by catecholamines, but not by uric acid or homocysteine. Furthermore, catecholamines such as 3,4-dihydroxyphenylalanine (DOPA), dopamine or norepinephrine were more effective than caffeic acid or pyrocatechol in promoting Cu 2+-mediated inactivation of PON1, suggesting the importance of dihydroxybenzene group as well as amino group. DOPA at relatively low concentrations showed a concentration-dependent inactivation of PON1 in a concert with Cu 2+, but not Fe 2+. The DOPA/Cu 2+-induced inactivation of PON1 was prevented by catalase, but not hydroxyl radical scavengers, consistent with Cu 2+-catalyzed oxidation. A similar result was also observed when HDL-associated PON1 (HDL-PON1) was exposed to DOPA/Cu 2+. Separately, it was found that DOPA at low concentrations (1-6 μM) acted as a pro-oxidant by enhancing Cu 2+-induced oxidation of HDL, while it exhibited an antioxidant action at ≥10 μM. In addition, Cu 2+-oxidized HDL lost the antioxidant action against LDL oxidation. Meanwhile, the role of DOPA/Cu 2+-oxidized HDL differed according to DOPA concentration; HDL oxidized with Cu 2+ in the presence of DOPA (60 or 120 μM) maintained antioxidant activity of native HDL, in contrast to an adverse effect of DOPA at 3 or 6 μM. These data indicate that DOPA at micromolar level may act as a pro-oxidant in Cu 2+-induced inactivation of PON1 as well as oxidation of HDL. Also, it is proposed that the oxidative inactivation of HDL-PON1 is independent of HDL oxidation. 相似文献
7.
Poly(ethylene glycol dimethacrylate- n-vinyl imidazole) [poly(EGDMA–VIM)] hydrogel (average diameter 150–200 μm) was prepared copolymerizing ethylene glycol dimethacrylate (EGDMA) with n-vinyl imidazole (VIM). Poly(EGDMA–VIM) beads had a specific surface area of 59.8 m 2/g. Poly(EGDMA–VIM) beads were characterized by swelling studies and scanning electron microscope (SEM). Cu 2+ ions were chelated on the poly(EGDMA–VIM) beads (452 μmol Cu 2+/g), then the metal-chelated beads were used in the adsorption of yeast invertase in a batch system. The maximum invertase adsorption capacity of the poly(EGDMA–VIM)–Cu 2+ beads was observed as 35.2 mg/g at pH 4.5. The adsorption isotherm of the poly(EGDMA–VIM)–Cu 2+ beads can be well fitted to the Langmuir model. Adsorption kinetics data were tested using pseudo-first- and -second-order models. Kinetic studies showed that the adsorption followed a pseudo-second-order reaction. The value of the Michaelis constant Km of invertase was significantly larger upon adsorption, indicating decreased affinity by the enzyme for its substrate, whereas Vmax was smaller for the adsorbed invertase. The optimum temperature for the adsorbed preparation of poly(EGDMA–VIM)–Cu 2+-invertase at 50 °C, 10 °C higher than that of the free enzyme at 40 °C. Storage stability was found to increase with adsorption. Adsorbed invertase retains an activity of 82% after 10 batch successive reactions, demonstrating the usefulness of the enzyme-loaded beads in biocatalytic applications. 相似文献
8.
1. By using dibromothymoquinone as the electron acceptor, it is possible to isolate functionally that segment of the chloroplast electron transport chain which includes only Photosystem II and only one of the two energy conservation sites coupled to the complete chain (Coupling Site II, observed P/e 2 = 0.3–0.4). A light-dependent, reversible proton translocation reaction is associated with the electron transport pathway: H 2O → Photosystem II → dibromothymoquinone. We have studied the characteristics of this proton uptake reaction and its relationship to the electron transport and ATP formation associated with Coupling Site II. 2. The initial phase of H+ uptake, analyzed by a flash-yield technique, exhibits linear kinetics (0–3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush”) encountered in the overall Hill reaction with methylviologen. Thus the initial rate of H+ uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH change tracing obtained under continuous illumination. 3. Dibromothymoquinone reduction, observed as O2 evolution by a similar flash-yield technique, is also linear for at least the first 5 s, the rate of O2 evolution agreeing well with the steady-state rate observed under continuous illumination. 4. Such measurements of the initial rates of O2 evolution and H+ uptake yield an H+/e− ratio close to 0.5 for the Photosystem II partial reaction regardless of pH from 6 to 8. (Parallel experiments for the methylviologen Hill reaction yield an H+/e− ratio of 1.7 at pH 7.6.) 5. When dibromothymoquinone is being reduced, concurrent phosphorylation (or arsenylation) markedly lowers the extent of H+ uptake (by 40–60%). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to an unequivocal interpretation since phosphorylation does not alter the rate of electron transport in the Photosystem II partial reaction. ADP, Pi and hexokinase, when added individually, have no effect on proton uptake in this system. 6. The involvement of a proton uptake reaction with an H+/e− ratio of 0.5 in the Photosystem II partial reaction H2O → Photosystem II → dibromothymoquinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/e2) and proton uptake (H+/e−) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of energy coupling. 相似文献
9.
1. 1. The mechanism of the photooxidation of ascorbate and of Mn2+ by isolated chloroplasts was reinvestigated. 2. 2. Our results suggest that ascorbate or Mn2+ oxidation is the result of the Photosystem I-mediated production of the radical superoxide, and that neither ascorbate nor Mn2+ compete with water as electron donors to Photosystem II nor affect the rate of electron transport through the two photosystems: The radical superoxide is formed as a result of the autooxidation of the reduced forms of low potential electron acceptors, such as methylviologen, diquat, napthaquinone, or ferredoxin. 3. 3. In the absence of ascorbate or Mn2+ the superoxide formed dismutases either spontaneously or enzymatically producing O2 and H2O2. In the presence of ascorbate or Mn2+, however, the superoxide is reduced to H2O2 with no formation of O2. Consequently, in the absence of reducing compounds, in the reaction H2O to low potential acceptor one O2 (net) is taken up per four electrons transported where as in the presence of ascorbate, Mn2+ or other suitable reductants up to three molecules O2 can be taken up per four electrons transported. 4. 4. This interpretation is supported by the following observations: (a) in a chloroplast-free model system containing NADPH and ferredoxin-NADP reductase, methylviologen can be reduced to a free radical which is autooxidizable in the presence of O2; the addition of ascorbate or Mn2+ to this system results in a two fold stimulation of O2 uptake, with no stimulation of NADPH oxidation. The stimulation of O2 uptake is inhibited by the enzyme superoxide dismutase; (b) the stimulation of light-dependent O2 uptake in the system H2O → methylviologen in chloroplasts is likewise inhibited by the enzyme superoxide dismutase. 5. 5. In Class II chloroplasts in the system H2O → NADP upon the addition of ascorbate or Mn2+ an apparent inhibition of O2 evolution is observed. This is explained by the interaction of these reductants with the superoxide formed by the autooxidation of ferredoxin, a reaction which proceeds simultaneously with the photoreduction of NADP. Such an effect usually does not occur in Class I chloroplasts in which the enzyme superoxide dismutase is presumably more active than in Class II chloroplasts. 6. 6. It is proposed that since in the Photosystem I-mediated reaction from reduced 2,4-dichlorophenolindophenol to such low potential electron acceptor as methylviologen, superoxide is formed and results in the oxidation of the ascorbate present in the system, the ratio ATP/2e in this system (when the rate of electron flow is based on the rate of O2 uptake) should be revised in the upward direction.
Abbreviations: DCMU, 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea; HEPES, hydroxyethyl-piperazineethanesulfonic acid; MES, (N-morpholino)ethanesulfonic acid; DCIP, 2,4-dichlorophenol-indophenol 相似文献
10.
The effect ot Cu 2+ and Ca 2+ ions, on the ultraviolet differential (UVD) spectra of single-stranded poly I was studied and the coordination (Δε b) and conformation (Δε c) conponents of the spectra calculated The comparison of Δε b and the UVD spectrum of protonated IMP leads to the conclusion that N(7) ot inosine-5'-monophosphate (IMP) is a coordinating site tor Ca 2+ and Cu 2+ ions on the polymer bases. The binding ot Ca 2+ and Cu 2+ ions causes differently directed displacements of the four absorption bands of poly I, which are observed in the wavenumber range (50-34) × 10 3 cm −1 The calculation of concentration dependencies tor the association constants ( K“) ot Ca 2+ and Cu 2+ ions binding to poly I bases shows that the binding is cooperative The K“ values for the poly I + Ca 2+ complex are two orders of magnitude lower than those for the poly 1 + Cu 2+ complex At low ion concentrations, binding to the poly I phosphates predominates and increases the degree of the polynucleotide helicity. At higher concentrations the spectra are mainly affected by the ion binding to bases, which results in melting of the helical parts of poly I At Ca 2+ concentrations exceeding 10 −3 M light-scattering aggregates are formed. The degree of monomer order in them is close to that observed in multistranded helices of poly I 相似文献
11.
1. 1. The nature and mechanism of the reduction of fungal laccase (p-diphenol: O2 oxidoreductase, EC 1.10.3.2) obtained on an increase in pH have been studied by optical and electron paramagnetic resonance (EPR) spectroscopy and by measurements of O2 concentration. 2. 2. The decreases in the optical absorption and the EPR signal of the “blue” Type 1 Cu2+ at high pH indicate that this ion is reduced. This is confirmed by oxidation with hexachloroiridate(IV) which restores the blue color. The “nonblue” Type 2 Cu2+ remains divalent over the pH range studied, as seen from the EPR spectra. 3. 3. Approximately one equivalent of hexachloroiridate(IV) is sufficient to restore the color of a pH-bleached protein which suggests that the reduction involves a single electron. A comparison between the optical spectra at pH 5 and 8 shows that the two-electron accepting unit, which at pH 5.5 is reduced concomitantly with the Type 1 Cu2+, remains oxidized in the protein brought to high pH. This unit can be reduced at pH 8.3 by octacyanotungstate(IV), as shown by the fact that this reductant in anaerobic titrations is found to add about two electrons (and no more) to a protein already having the Type 1 copper reduced. Thus, an increase in pH introduces a difference in the reduction behavior of the electron acceptors in fungal laccase. 4. 4. Oxygraph experiments show that there is no production of O2 with an increase in pH, as would occur if water was oxidized by laccase. On the contrary, there is a continuous consumption of O2 at both pH 5 and 8, indicating that the protein preparation contains a reducing substance which is responsible for the pH-dependent reduction.
Abbreviations: EPR; electron paramagnetic resonance 相似文献
12.
The effects of N-ethylmaleimide (NEM) on mouse platelet serotonin (5-HT) and 86Rb + uptake were studied. The 5-HT transport system showed a biphasic response to increasing concentrations of NEM, with low concentrations (25–50 μM) stimulating and high concentrations (200–400 μM) inhibiting 5-HT transport. Fluoxetine, an inhibitor of the platelet 5-HT transporter, blocked NEM-induced stimulation of 5-HT transport. The kinetics of 5-HT uptake indicated that NEM (50 μM) markedly increased the maximal rate of 5-HT transport ( Vmax control = 28.4±1.4 pmol/10 8 platelets/4 min vs Vmax NEM = 64.5±9.5 pmol/10 8 platelets/4 min but had no significant effect on the Km value. Platelet Na + K + ATPase activity was determined by measuring 86Rb + uptake. Platelet 86Rb + uptake showed a biphasic response to NEM, with low concentrations (25–100 μM) significantly stimulating and high concentrations (400 μM) inhibiting uptake. These changes in platelet 86Rb + uptake paralleled the biphasic changes in 5-HT transport. In the presence of fluoxetine, 5-HT transport was markedly inhibited but no change in the ability of NEM to stimulate 86Rb + uptake was observed. These data suggest that low concentrations of NEM activate plasma membrane Na + K + ATPase which results in a marked stimulation of platelet 5-HT transport. 相似文献
13.
The cationic monoalkylated derivatives of the well-known metalloligand [Pt 2(μ-S) 2(PPh 3) 4], viz. [Pt 2(μ-S)(μ-SR)(PPh 3) 4] + (R = n-Bu, CH 2Ph) are themselves able to act as metalloligands towards the Ph 3PAu + and R′Hg + (R′ = Ph or ferrocenyl) fragments, by reaction with Ph 3PAuCl or R′HgCl, respectively. The resulting dicationic products [Pt 2(μ-SR)(μ-SAuPPh 3)(PPh 3) 4] 2+ and [Pt 2(μ-SR)(μ-SHgR′)(PPh 3) 4] 2+ are readily isolated as their hexafluorophosphate salts, and have been fully characterised by spectroscopic techniques and an X-ray structure determination on [Pt 2(μ-SR)(μ-SHgFc)(PPh 3) 4](PF 6) 2. 相似文献
14.
Isolated hepatocytes incubated with selenite (30–100 μM) exhibited changes in the glutathione redox system as shown by an increase in O 2 consumption, oxidation of glutathione and loss of NADPH. Selenite (50 μM) raised O 2 consumption within the 1 h and induced an partial depletion of thiols with a concomitant increase in oxidized glutathione, as well as a decrease in NADPH levels within 2 h. With 100 μM selenite more pronounced effects were obtained such as a total depletion of thiols. This concentration of selenite also lysed cells within 3 h. Arsenite, HgCl 2 and KCN prevented the increase in O 2 uptake, counteracted loss of thiols and delayed selenite induced lysis. p-Tert-butylbenzoic acid, an inhibitor of gluconeogenesis, decreased selenite dependent O 2 consumption and potentiated the effect on NADPH levels as well as the toxic effect. Finally, methionine further enhanced O 2 consumption by selenite and also delayed loss of thiols and potentiated selenite toxicity. These results indicated that selenite catalyzed a reduction of O 2 in glutathione dependent redox cycles with NADPH as an electron donor. With subtoxic concentrations of selenite (50 μM) there were indications that O 2 reduction was terminated by selenite biotransformation to methylated metabolites. With toxic concentrations of selenite (100 μM) it appeared that O 2 reduction was eventually limited by the capacity of the cell to regenerate NADPH. It is suggested that a depletion of NADPH mediated the observed cytotoxicity of selenite. 相似文献
15.
Microsensor measurements of CO 2, O 2, pH and Ca 2+ in the vicinity of the symbiont-bearing planktonic foraminifer Orbulina universa showed major light-modulated changes in the chemical microenvironment due to symbiont photosynthesis, respiration of the holobiont, and precipitation of the calcite shell. Under saturating light conditions, microprofiles measured towards the shell surface showed an O 2 increase of up to 220% air saturation, a decrease in CO 2 concentration to 4.9 μM, and a pH increase to 8.8 due to symbiont photosynthesis. The Ca 2+ concentration decreased to ∼9.6 mM in two specimens, while it increased to 10.2-10.8 mM in three other specimens kept in light. In darkness, the respiration of the community decreased the O 2 concentration to 82% of air saturation, CO 2 increased up to 15 μM, the pH decreased to 8.0, and the Ca 2+ concentration increased up to 10.4 mM. These data, and derived calculations of the distribution of HCO 3- and CO 32- near the shell, showed that the carbonate system in the vicinity of O. universa was significantly different from conditions in the surrounding seawater, both in light and darkness, due to the metabolism of the foraminifer and its associated algae. Experimental light-dark cycles indicated a sufficient CO 2 supply sustaining high carbon fixation rates of the symbiotic algae via conversion of HCO 3- or via CO 2 release from calcification and host respiration. Our findings on irradiance-dependent CO 2 and pH changes in the vicinity of symbiont-bearing planktonic foraminifera give direct experimental evidence for the predictions of isotope fractionation models used in palaeoclimatology stating that metabolic processes affect the isotopic carbon signal (δ 13C) in foraminifera. 相似文献
16.
The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca 2+ levels ([Ca 2+] i) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca 2+ indicator. Clomiphene at concentrations between 10-50 μM increased [Ca 2+] i in a concentration-dependent manner. The [Ca 2+] i signal was biphasic with an initial rise and a slow decay. Ca 2+ removal inhibited the Ca 2+ signal by 41%. Adding 3 mM Ca 2+ increased [Ca 2+] i in cells pretreated with clomiphene in Ca 2+-free medium, confirming that clomiphene induced Ca 2+ entry. In Ca 2+-free medium, pretreatment with 50 μM brefeldin A (to permeabilize the Golgi complex), 1 μM thapsigargin (to inhibit the endoplasmic reticulum Ca 2+ pump), and 2 μM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 μM clomiphene-induced store Ca 2+ release. Conversely, pretreatment with 50 μM clomiphene in Ca 2+-free medium abolished the [Ca 2+] i increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 μM clomiphene-induced Ca 2+release was unaltered by inhibiting phospholipase C with 2 μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 μM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca 2+] i increases in PC3 cells by releasing store Ca 2+ from multiple stores in an phospholipase C-independent manner, and by activating Ca 2+ influx; and clomiphene was of mild cytotoxicity. 相似文献
17.
Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 μM (270 μg l −1). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml −1 LDH and 0.8 U ml −1 LOD in reactions containing 246 μM pyruvate and 7.5 μM NADPH. PCP detection limits were an EC10 of 800 nM (213 μg l −1) and a minimum inhibition detectable ( MID) limit of 650 nM (173 μg l −1). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 μM. PCP detection limits were obtained for an assay containing 5 U ml −1 LDH, 0.8 U ml −1 LOD and 0.1 U ml −1 GDH with 246 μM pyruvate, 400 mM glucose and 2 μM NADPH. The EC10 limit was 150 nM (39.9 μg l −1) and the MID was 100 nM (26.6 μg l −1). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring. 相似文献
18.
We investigated the restoration of [Ca 2+] i in fura-2-loaded human platelets following discharge of internal Ca 2+ stores in the absence of external Ca 2+. After stimulation by thrombin [Ca 2+] i returned from a peak level of 0.6 μM to resting levels within 4 min. When ionomycin discharged the internal stores the recovery was slower with [Ca 2+] i still elevated at around 0.5 μM after 5 min. Thrombin added shortly after ionomycin could accelerate the recovery of [Ca 2+] i and restore resting levels within 5 min, an effect that was mimicked by phorbol-12-myristate-13-acetate (PMA). Since the continued presence of ionomycin precluded reuptake into the internal stores we conclude that thrombin and PMA stimulate Ca 2+ efflux, perhaps via protein kinase C actions on a plasma membrane Ca 2+ pump. 相似文献
19.
Norathyriol, aglycone of a xanthone C-glycoside mangiferin isolated from Tripterospermum lanceolatum, concentration dependently inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O 2˙−) generation and O 2 consumption in rat neutrophils. In cell-free oxygen radical generating system, norathyriol inhibited the O 2˙− generation during dihydroxyfumaric acid (DHF) autoxidation and in hypoxanthine-xanthine oxidase system. fMLP-induced transient elevation of [Ca 2+] i and the formation of inositol trisphosphate (IP 3) were significantly inhibited by norathyriol (30 μM) (about 30 and 46% inhibition, respectively). Norathyriol concentration dependently suppressed the neutrophil cytosolic phospholipase C (PLC). In contrast with the marked attenuation of fMLP-induced protein tyrosine phosphorylation (about 70% inhibition at 10 μM norathyriol), norathyriol only slightly modulated the phospholipase D (PLD) activity as determined by the formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt). Norathyriol did not modulate the intracellular cyclic AMP level. In the presence of NADPH, the phorbol 12-myristate 13-acetate (PMA)-activated particulate NADPH oxidase activity was suppressed by norathyriol in a concentration-dependent manner and the inhibition was noncompetitive with respect to NADPH. Norathyriol inhibited the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free NADPH oxidase system at the same concentration range as those used in the suppression of PMA-activated particulate NADPH oxidase activity. Taken together, these results suggest that the scavenging ability of norathyriol contributes to the reduction of generated O 2˙−, however, the inhibition of O 2˙− generation from neutrophils by norathyriol is attributed to the blockade of PLC pathway, the attenuation of protein tyrosine phosphorylation, and to the suppression of NADPH oxidase through the interruption of electrons transport. 相似文献
20.
The trinuclear clusters [Pd 3(μ-dppm) 3(CO)] 2+ and [PtPdCo(μ-dppm) 2(CO) 3(CN tBu)] + exhibit a large and a small cavity, respectively, formed by the phenyl rings of the bridging diphosphine ligands. Their binding constants ( K11) with halide ions (X −) were obtained by UV-Vis spectroscopy. The binding ability varies as I − > Br − > Cl −, and [Pd 3(μ-dppm) 3(CO)] 2+ > [ptPdCo(μ-dppm) 2-(CO) 3(CN tBu)] +. The MO diagram for the related cluster [Pd 2Co(μ-dppm) 2(CO) 4] + has been addressed theoretically in order to predict the nature of the lowest energy electronic bands. For this class of compounds, the lowest energy bands are assigned to charge transfers from the Co center to the Pd 2 centers. 相似文献
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