ESR study on the structure-antioxidant activity relationship of tea catechins and their epimers.
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The purpose of this study is to examine the relationship between the free radical scavenging activities and the chemical structures of tea catechins ((-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC) and (-)-epicatechin (EC)) and their corresponding epimers ((-)-gallocatechin gallate (GCG), (-)-gallocatechin (GC) and (+)-catechin ((+)-C)). With electron spin resonance (ESR) we investigated their scavenging effects on superoxide anions (O-.2) generated in the irradiated riboflavin system, singlet oxygen(1O2) generated in the photoradiation-hemoporphyrin system, the free radicals generated from 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical. The results showed that the scavenging effects of galloylated catechins (EGCG and GCG) on the four free radicals were stronger than those of nongalloylated catechins (EGC, GC, EC, (+)-C), and the scavenging effects of EGC and GC were stronger than those of EC and (+)-C. Thus, it is suggested that the presence of the gallate group at the 3 position plays the most important role in their free radical-scavenging abilities and an additional insertion of the hydroxyl group at the 5' position in the B ring also contributes to their scavenging activities. Moreover, the corresponding phenoxyl radicals formed after the reaction with O-.2 were trapped by DMPO and the ESR spectra of DMPO/phenoxyl radical adducts were observed (aN=15.6 G and aHbeta=21.5 G). No significant differences were found between the scavenging effects of the catechins and their epimers when their concentrations were high. However, significant differences were observed at relatively low concentrations, and the lower their concentrations, the higher the differences. The scavenging abilities of GCG, GC and (+)-C were stronger than those of their corresponding epimers (EGCG, EGC and EC). The differences between their sterical structures played a more important role in their abilities to scavenge large free radicals, such as the free radicals generated from AAPH and the DPPH radical, than to scavenge small free radicals, such as O-.2 and 1O2, especially in the case with EGCG and GCG with more bulky steric hindrance. (+info)
Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast cancer cells.
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We investigated the effects of polyamine biosynthesis inhibition on the estrogenic signaling pathway of MCF-7 breast cancer cells using a protein-protein interaction system. Estrogen receptor (ER) linked to glutathione-S-transferase (GST) was used to examine the effects of two polyamine biosynthesis inhibitors, difluoromethylornithine (DFMO) and CGP 48664. ER was specifically associated with a 45 kDa protein in control cells. In cells treated with estradiol, nine proteins were associated with ER. Cells treated with polyamine biosynthesis inhibitors in the absence of estradiol retained the binding of their ER with a 45 kDa protein and the ER also showed low-affinity interactions with a number of cellular proteins; however, these associations were decreased by the presence of estradiol and the inhibitors. When samples from the estradiol+DFMO treatment group were incubated with spermidine prior to GST-ER pull down assay, an increased association of several proteins with ER was detected. The intensity of the ER-associated 45 kDa protein increased by 10-fold in the presence of 1000 microM spermidine. These results indicate a specific role for spermidine in ER association of proteins. Western blot analysis of samples eluted from GST-ER showed the presence of chicken ovalbumin upstream promoter-transcription factor, an orphan nuclear receptor, and the endogenous full-length ER. These results show that multiple proteins associate with ER and that the binding of some of these proteins is highly sensitive to intracellular polyamine concentrations. Overall, our results indicate the importance of the polyamine pathway in the gene regulatory function of estradiol in breast cancer cells. (+info)
When and why a water-soluble antioxidant becomes pro-oxidant during copper-induced low-density lipoprotein oxidation: a study using uric acid.
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The inclusion of uric acid in the incubation medium during copper-induced low-density lipoprotein (LDL) oxidation exerted either an antioxidant or pro-oxidant effect. The pro-oxidant effect, as mirrored by an enhanced formation of conjugated dienes, lipid peroxides, thiobarbituric acid-reactive substances and increase in negative charge, occurred when uric acid was added late during the inhibitory or lag phase and during the subsequent extensive propagation phase of copper-stimulated LDL oxidation. The pro-oxidant effect of uric acid was specific for copper-induced LDL oxidation and required the presence of copper as either Cu(I) or Cu(II). In addition, it became much more evident when the copper to LDL molar ratio was below a threshold value of approx. 50. In native LDL, the shift between the antioxidant and the pro-oxidant activities was related to the availability of lipid hydroperoxides formed during the early phases of copper-promoted LDL oxidation. The artificial enrichment of isolated LDL with alpha-tocopherol delayed the onset of the pro-oxidant activity of uric acid and also decreased the rate of stimulated lipid peroxidation. However, previous depletion of alpha-tocopherol was not a prerequisite for unmasking the pro-oxidant activity of uric acid, since this became apparent even when alpha-tocopherol was still present in significant amounts (more than 50% of the original values) in LDL. These results suggest, irrespective of the levels of endogenous alpha-tocopherol, that uric acid may enhance LDL oxidation by reducing Cu(II) to Cu(I), thus making more Cu(I) available for subsequent radical decomposition of lipid peroxides and propagation reactions. (+info)
Pharmacokinetics and pharmacodynamics of Ro 44-3888 after single ascending oral doses of sibrafiban, an oral platelet aggregation inhibitor, in healthy male volunteers.
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AIMS: This study constituted the first administration of the oral platelet inhibitor, sibrafiban, to humans. The aim was to investigate the pharmacokinetics and pharmacodynamics of Ro 44-3888, the active principle of sibrafiban, after single ascending oral doses of sibrafiban. Particular emphasis was placed on intersubject variability of the pharmacokinetic and pharmacodynamic parameters of Ro 44-3888. METHODS: The study consisted of three parts. Part I was an open ascending-dose study to determine target effect ranges of sibrafiban. Part II, a double-blind, placebo-controlled, parallel-group study, addressed the intersubject variability of pharmacokinetic and pharmacodynamic parameters of the active principle at a sibrafiban dose achieving an intermediate effect. Part III was a double-blind, placebo-controlled, ascending-dose design covering the complete plasma concentration vs pharmacodynamic response curve of sibrafiban. RESULTS: At sibrafiban doses between 5 mg and 12 mg, the pharmacokinetics of free Ro 44-3888 in plasma were linear whereas those of total Ro 44-3888 were non-linear because of the saturable binding to the glycoprotein IIb-IIIa receptor. Saturation of the GP IIb-IIIa receptor was reached at plasma concentrations of 15.9 ng ml-1. At sibrafiban doses up to 2 mg, ADP-induced platelet aggregation was inhibited by 50%, whereas the inhibition of TRAP-induced platelet aggregation was about 20-30%. At the higher doses, ADP-induced platelet aggregation was almost completely inhibited while a clear dose-response could be observed with TRAP-induced inhibition of platelet aggregation at sibrafiban doses of 5 to 12 mg. Ivy bleeding time increased very steeply with dose with a significant prolongation observed at doses of 5 to 7 mg of sibrafiban (5-7 min, >30 min in one case). At a sibrafiban dose of 12 mg, the stopping criterion for dose escalation (prolongation of the Ivy bleeding time >30 min in three out of four subjects per dose group) was reached. The interindividual coefficients of variation of the integrated pharmacokinetic and pharmacodynamic parameters (AUC and AUE) were below 20%, thus lying well within the pre-set level of acceptance. CONCLUSIONS: With a low intersubject variability of its pharmacokinetic and pharmacodynamic parameters, linear pharmacokinetics and pharmacodynamic effects closely related to its plasma concentrations, Ro 44-3888 has good pharmacological prerequisites for a well controllable therapy of secondary prevention of arterial thrombosis in patients with acute coronary syndrome. (+info)
Morphological classification of ganglion cells in the central retina of chicks.
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Classification of retinal ganglion cells (RGCs) in the chick central retina was studied by retrograde labeling of carbocyanine dye (DiI) and intracellular filling with Lucifer Yellow. Ganglion cells were divided into 4 groups, Group Ic/Is, Group IIc/IIs, Group IIIs, Group IVc, according to sizes of somal area and dendritic field and dendritic branching pattern. Group I cells had small somal area and small dendritic field. They were further divided into 2 subgroups by complexity (subgroup Ic) and simplicity (subgroup Is) of the dendritic arborization. Group II cells had medium-sized soma and dendritic field. They were also divided into subgroup IIc and IIs by the same definitions as those of subgroup Ic and Is. Group IIIs had medium-sized soma, large and simple dendritic arborization. Group IVc in which all cells had large soma, showed large and complex dendritic arborization. Cell populations of each group were 51.8% (subgroup Ic), 21.1% (subgroup Is), 6.2% (subgroup IIc), 14.6% (subgroup IIs), 4.2% (Group IIIs), and 2.1% (Group IVc). Subgroup Ic cells, which were very similar to beta-cells in the mammalian central area, represented about a half of the ganglion cell population. Cells in subgroup Is and IIs, which were not reported in the mammalian retina, were found in the chick central retina in relatively high population (35.7%). Morphological features of chick RGCs in the central retina were considered in comparison with those of other vertebrates. (+info)
1. The aim of this study was to investigate the effect of N-(3-(aminomethyl)benzyl)acetamidine (1400W), a selective inhibitor of inducible calcium-independent nitric oxide synthase (iNOS), on the functional and histopathological outcomes of experimental transient focal cerebral ischaemia in rats. 2. Transient ischaemia was produced by the occlusion for 2 h of both the left middle cerebral artery and common carotid artery. Treatments with 1400W (20 mg kg(-1)) or vehicle were started 18 h after occlusion of the arteries and consisted in seven subcutaneous injections at 8 h interval. Ischaemic outcomes and NOS activities (constitutive and calcium-independent NOS) were evaluated 3 days after ischaemia. 3. 1400W significantly reduced ischaemic lesion volume by 31%, and attenuated weight loss and neurological dysfunction. 4. 1400W attenuated the calcium-independent NOS activity in the infarct by 36% without affecting the constitutive NOS activity. 5. These findings suggest that iNOS activation contributes to tissue damage and that selective inhibitors of this isoform may be of interest for the treatment of stroke. (+info)
Formation of the aldehydic choline glycerophospholipids in human red blood cell membrane peroxidized with an azo initiator.
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The production of phospholipid hydroperoxide and aldehydic phospholipid was examined in human red blood cell (RBC) membranes after peroxidation with 2,2-azobis(2-amidinopropane)dihydrochloride (AAPH) or xanthine/xanthine oxidase (XO/XOD/Fe3+). Both radical-generation systems caused a profound decrease in the amount of polyunsaturated fatty acid (PUFA) in choline glycerophospholipid (CGP) and induced formation of peroxidized CGP in RBC membranes to different extents. No consistent generation of peroxidized lipids from CGP was evident after peroxidation with XO/XOD/Fe3+, which caused the apparent decomposition of phospholipids and the formation of large amounts of thiobarbituric acid-reactive substance (TBARS). On the other hand, CGP hydroperoxide was formed as a primary product of peroxidation with AAPH. Aldehydic CGP was also detected as a secondary product of hydroperoxide decomposition in AAPH-peroxidized RBC membranes. Aldehydic CGP was preferentially generated from arachidonoyl CGP rather than from linoleoyl CGP in AAPH-peroxidized membranes. AAPH mainly oxidized CGP to hydroperoxide and aldehydic phospholipids. The sum of hydroperoxide and aldehyde of CGP corresponded to the loss of CGP due to peroxidation by AAPH. This result indicates that CGP was mainly converted into these two oxidized phospholipids in AAPH-peroxidized RBC membranes. (+info)
Salt-induced hypertension in Dahl salt-resistant and salt-sensitive rats with NOS II inhibition.
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Although recent evidence suggests that reduced nitric oxide (NO) production may be involved in salt-induced hypertension, the specific NO synthase (NOS) responsible for the conveyance of salt sensitivity remains unknown. To determine the role of inducible NOS (NOS II) in salt-induced hypertension, we treated Dahl salt-resistant (DR) rats with the selective NOS II inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) for 12 days. Tail-cuff systolic blood pressures rose 29 +/- 6 and 42 +/- 8 mmHg in DR rats given 150 and 300 nmol AMT/h, respectively (P < 0.01, 2-way ANOVA) after 7 days of 8% NaCl diet. We observed similar results with two other potent selective NOS II inhibitors, S-ethylisourea (EIT) and N-[3-(aminomethyl)benzyl]acetamidine hydrochloride (1400W). Additionally, AMT effects were independent of alterations in endothelial function as assessed by diameter change of mesenteric arterioles in response to methacholine using videomicroscopy. We, therefore, conclude from these data that NOS II is important in salt-induced hypertension. (+info)