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Autophagy induction in cancer is involved in cancer progression and the acquisition of resistance to anticancer agents. Therefore, autophagy is considered a potential therapeutic target in cancer therapy. In this study, we found that long-chain fatty acids (LCFAs) have inhibitory effects on Atg4B, which is essential for autophagosome formation, through screening based on the pharmacophore of 21f, a recently developed Atg4B inhibitor. Among these fatty acids, docosahexaenoic acid (DHA), a polyunsaturated fatty acid, exhibited the most potent Atg4B inhibitory activity. DHA inhibited autophagy induced by androgen receptor signaling inhibitors (ARSI) in LNCaP and 22Rv1 prostate cancer cells and significantly increased apoptotic cell death. Furthermore, we investigated the effect of DHA on resistance to ARSI by establishing darolutamide-resistant prostate cancer 22Rv1 (22Rv1/Dar) cells, which had developed resistance to darolutamide, a novel ARSI. At baseline, 22Rv1/Dar cells showed a higher autophagy level than parental 22Rv1 cells. DHA significantly suppressed Dar-induced autophagy and sensitized 22Rv1/Dar cells by inducing apoptotic cell death through mitochondrial dysfunction. These results suggest that DHA supplementation may improve prostate cancer therapy with ARSI., Competing Interests: Declaration of competing interest The authors disclose any conflicts of interest related to the work., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Hemothorax caused by a right intercostal artery (ICA) injury behind the left atrium (LA) is a potentially fatal complication during pulmonary vein isolation. However, their anatomic relationship has not been fully elucidated., Objective: This study aimed to investigate the clinical anatomy of the right ICA in relation to the LA., Methods: This retrospective study included 100 patients (70.2 ± 10.6 years; 39.0% female) who underwent cardiac computed tomography. The patients were divided into sinus rhythm and atrial fibrillation groups. We focused on the distance between the LA and right ICAs and its predictive factors., Results: On average, 3.7 ± 0.7 right ICAs were found behind the LA. Of these, the eighth ICA was the closest in 54% of the cases, followed by the seventh ICA in 29% and the ninth ICA in 14%. The average closest distance between them was 3.8 ± 3.8 mm, which was significantly shorter in the atrial fibrillation group than in the sinus rhythm group (3.0 ± 3.2 mm vs 4.7 ± 4.2 mm; P = .006). Multivariate analysis revealed that a thinner chest cavity (β = -0.512; P = .002) and LA dilation (β = -0.432; P = .001) were predictors of shorter distance. The closest points distributed along the vertebral column, generally near the inferior pulmonary vein orifices., Conclusion: Right ICA-LA proximity was systematically clarified. Particularly in cases with an enlarged LA or thin chest cavity, operators should be aware of the potential risk of injuring the right ICA during pulmonary vein isolation., Competing Interests: Disclosures The authors have no conflicts of interest to disclose., (Copyright © 2024 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

α-Tocopheryl succinate (TS) is a tocopherol derivative and has multifaceted anti-cancer effects; TS not only causes cancer cell-specific apoptosis but also inhibits tumor angiogenesis. Although TS has the potential to be used as a well-tolerated anti-angiogenic drug, it is still unclear which step of the angiogenic process is inhibited by TS. Here, we show that TS inhibits the expression of angiopoietin (Ang)-2, which induces destabilization of vascular structure in the initial steps of the angiogenic process. In mouse melanoma cells, TS treatment decreased mRNA and extracellular protein levels of Ang-2; however, the mRNA level of Ang-1, which stabilizes the vascular structure, remained unchanged. Furthermore, aorta ring and Matrigel plug angiogenesis assays indicated that the conditioned medium from TS-treated cells (CM-TS) inhibited neovascularization and blood leakage from the existing blood vessels, respectively. Following immunohistochemical staining of the vessels treated with CM-TS, imaging studies showed that the vascular endothelial cells were highly packed with pericytes. In conclusion, we found that TS inhibits Ang-2 expression and, consequently, stabilizes the vascular structure during the initial step of tumor angiogenesis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: To evaluate the reproducibility of proton density fat fraction (PDFF) and T2* in a fat-water phantom on three different 3 T MRI systems using 6-point DIXON methods., Methods: A phantom which included varying fat volume percentages (true fat fraction [FF]) was scanned by three 3 T MR machines, and PDFF and T2* were measured., Results: The mean difference between true FF and PDFF was small in all vendors (-2.11% to 0.41%). However, the difference ratio for T2* values was large among vendors (1.79 to 3.36)., Conclusions: True FF and PDFF were consistent across vendors; however, T2* varied greatly., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: We evaluated the efficacy and toxicity of adding oral leucovorin (LV) to S-1 when compared with S-1 monotherapy in patients with gemcitabine-refractory pancreatic cancer (PC)., Patients and Methods: Gemcitabine-refractory PC patients were randomly assigned in a 1:1 ratio to receive S-1 at 40, 50, or 60 mg according to body surface area plus LV 25 mg, both given orally twice daily for 1 week, repeated every 2 weeks (SL group), or S-1 monotherapy at the same dose as the SL group for 4 weeks, repeated every 6 weeks (S-1 group). The primary end point was progression-free survival (PFS)., Results: Among 142 patients enrolled, 140 were eligible for efficacy assessment (SL: n = 69 and S-1: n = 71). PFS was significantly longer in the SL group than in the S-1 group [median PFS, 3.8 versus 2.7 months; hazard ratio (HR), 0.56; 95% confidence interval (CI), 0.37-0.85; P = 0.003]). The disease control rate was significantly higher in the SL group than in the S-1 group (91% versus 72%; P = 0.004). Overall survival (OS) was similar in both groups (median OS, 6.3 versus 6.1 months; HR, 0.82; 95% CI, 0.54-1.22; P = 0.463). After adjusting for patient background factors in a multivariate analysis, OS tended to be better in the SL group (HR, 0.71; 95% CI, 0.47-1.07; P = 0.099). Both treatments were well tolerated, although gastrointestinal toxicities were slightly more severe in the SL group., Conclusion: The addition of LV to S-1 significantly improved PFS in patients with gemcitabine-refractory advanced PC, and a phase III trial has been initiated in a similar setting., Clinical Trials Number: Japan Pharmaceutical Information Center: JapicCTI-111554., (© The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology.)

To elucidate the interaction of intestinal intraepithelial lymphocytes (IELs) with intestinal epithelial cells (IECs), we investigated alterations of IECs by activating or inactivating IELs. The stimulation of IELs with anti-mouse CD3 monoclonal antibody induced massive apoptosis of IECs. Changes in IECs and IELs from mice that received daily administration of FK506 for 14days were investigated. IELs, particularly TCR-γδ⁺ IELs, were reduced in cell number, and a decrease of cytotoxic activity was observed. Under this condition, loss of apoptotic cells at the tips of villi and delayed turnover of IECs were detected. The expressions of alkaline phosphatase and CD98 amino acid transporters on IECs were decreased. Furthermore, abnormal skeletal organization of villi and weakened binding of IECs to the basement membrane were shown. These results suggest that inactivated IECs, which should be led to apoptosis, remained. It was strongly suggested that IELs participated in IEC turnover., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We describe a case of a 60-year-old male with dilated phase of hypertrophic cardiomyopathy caused by Fabry disease. He was diagnosed to have a cardiac variant of Fabry disease by an enzyme assay and a right ventricular endomyocardial biopsy which revealed specific features of this disease and cardiac involvement was the sole manifestation. He has developed dilated cardiomyopathy with sustained atrial flutter and frequent non-sustained ventricular tachycardia requiring isthmus ablation and cardiac resynchronization therapy with defibrillator.

The cyclic AMP receptor protein (CRP) of Escherichia coli binds preferentially to DNA sequences possessing a T:A base pair at position 6 (at which the DNA becomes kinked), but with which it does not form any direct interactions. It has been proposed that indirect readout is involved in CRP-DNA binding, in which specificity for this base pair is primarily related to sequence effects on the energetic susceptibility of the DNA to kink formation. In the current study, the possibility of contributions to indirect readout by water-mediated hydrogen bonding of CRP with the T:A base pair was investigated. A 1.0 ns molecular dynamics simulation of the CRP-cAMP-DNA complex in explicit solvent was performed, and assessed for water-mediated CRP-DNA hydrogen bonds; results were compared to several X-ray crystal structures of comparable complexes. While several water-mediated CRP-DNA hydrogen bonds were identified, none of these involved the T:A base pair at position 6. Therefore, the sequence specificity for this base pair is not likely enhanced by water-mediated hydrogen bonding with the CRP.

The hemagglutinin (HA) protein of the influenza virus binds to the host cell receptor in the early stage of viral infection. A change in binding specificity from avian 2-3 to human 2-6 receptor is essential for optimal human-to-human transmission and pandemics. Therefore, it is important to reveal the key factors governing the binding affinity of HA-receptor complex at the molecular level for the understanding and prediction of influenza pandemics. In this work, on the basis of ab initio fragment molecular orbital (FMO) method, we have carried out the interaction energy analysis of HA-receptor complexes to quantitatively elucidate the binding specificity of HAs to avian and human receptors. To discuss the binding property of influenza HA comprehensively, a number of HAs from human H1, swine H1, avian H3 and avian H5 viruses were analyzed. We performed detailed investigations about the interaction patterns of complexes of various HAs and receptor analogues, and revealed that intra-molecular interactions between conserved residues in HA play an important role for HA-receptor binding. These results may provide a hint to understand the role of conserved acidic residues at the receptor binding site which are destabilized by the electrostatic repulsion with sialic acid. The calculated binding energies and interaction patterns between receptor and HAs are consistent with the binding specificities of each HA and thus explain the receptor binding mechanism. The calculated results in the present analysis have provided a number of viewpoints regarding the models for the HA-receptor binding specificity associated with mutated residues. Examples include the role of Glu190 and Gln226 for the binding specificity of H5 HA. Since H5 HA has not yet been adapted to human receptor and the mechanism of the specificity change is unknown, this result is helpful for the prediction of the change in receptor specificity associated with forthcoming possible pandemics.

Levels of lysophosphatidic acid (LPA), an important phospholipid mediator, in serum and ascitic fluid from ovarian cancer patients were shown to be higher than those from healthy women and from patients with other type of cancer, respectively. Although LPA in human serum seems mainly to be generated by lysophospholipase D (lysoPLD), the source and pathway for LPA in the ascitic fluid remain still obscure. In this study, we examined whether lysoPLD activity producing bioactive LPA in human peritoneal fluid was significantly elevated under pathological statuses. Lysophospholipase D activity in human peritoneal fluids was measured by quantifying choline released from exogenous lysophosphatidylcholine on their incubation at 37 degrees C. We also compared the activity of lysoPLD in sera from patients with different gynecologic diseases. We found relatively high lysoPLD activity in peritoneal fluids from patients with ovarian cancer, dermoid cyst or mucinous cystadenoma, whereas there were no significant differences in the serum lysoPLD activity among clinical groups and healthy subjects. The lysoPLD in the peritoneal fluid was found to have similar substrate specificity and metal ion requirement to those of serum lysoPLD, that has been identified as autotaxin, a tumor cell-motility stimulating protein. Our results suggest that increased lysoPLD activity in peritoneal fluid from patients with certain gynecologic tumors might be relevant to its potential of tumor progression.

Formation of a colored complex between ferric iron and xylenol orange (XO) has been used for the determination of hydroperoxides (FOX method). Original or modified FOX methods were performed on aqueous or organic solutions consisting of a single phase. However, for lipid peroxides in heterogeneous samples, such as biological materials, much of the lipid is sequestered in a separate phase. Organic solvent extraction of these lipids is often incomplete and may result in additional peroxidation during the extraction procedure. In this study, we applied the FOX assay for measurement of the membrane phosphatidylcholine hydroperoxides (PC-OOH) in separated phases. The presence of membranous egg yolk phosphatidylcholine (EYPC) in 60% MeOH shifted the broad peak at 560 nm of Fe(3+)-XO complex to 610 nm with a sharp peak associated with the increased intensity of the absorbance. The shift of the peak is useful to measure the unknown amounts of Fe(3+) because the uncomplexed XO considerably contributed to the absorbance of the peak at 560 nm but did not affect the absorbance at 610 nm. EYPC was required to form the membranes to shift the peak because the shift occurred in 60% MeOH but did not by the treatment with detergents or in 90% MeOH in which EYPC did not form the membranes. The molar absorption coefficient (epsilon 610) was 32,700 M(-1) cm(-1), which was about twice the molar absorption co efficient (epsilon 560) reported. We applied this method to the assay of PC-OOH prepared from EYPC and obtained the molar absorption coefficients (epsilon 610), which were 79,100 and 115,700 M(-1) cm(-1) in the presence and absence of BHT, respectively. This finding allows the determination of PC-OOH concentration even in chemically complex systems.

Lysophosphatidic acid (LPA) is a biologically active phospholipid that has been identified as a vasoactive principle in incubated plasma and serum of mammals. Previously, we found that mammalian plasma and serum contain a lysophospholipase D, which hydrolyzes lysophosphatidylcholines (LPCs) with different fatty acyl groups to the corresponding LPAs during its incubation at 37 degree C. In this study, we examined whether lysophospholipase D activity and levels of LPCs in rabbit serum were modulated by feeding rabbits a high cholesterol diet. Results showed that the serum levels of LPCs increased gradually in animals fed a high cholesterol diet for 12 weeks. We found that the levels of individual LPAs formed on incubation of serum for 24 h increased with an increase in the period of feeding of rabbits a high cholesterol diet. LPA with a linoleate residue was the most abundant LPA, followed in order by 16:0-, 18:1- and 18:0-LPAs. LPA was found to increase attachment of the monocytic cell line THP-1 to vascular endothelial cells pre-stimulated with tumor necrosis factor-alpha. These results indicated that increases in the levels of LPAs generated by lysophospholipase D in the blood of hypercholesterolemic rabbits may be relevant to attachment of monocytes to vascular walls, a key phenomenon observed at an early stage of atherosclerosis.

Several reports have suggested an association between lipid peroxidation and human carotid atherosclerosis, but few reports have demonstrated a link between lipid peroxidation and carotid plaques in humans. In this study, we investigated the relationship between clinical features, histopathological characteristics and lipid peroxidation in patients undergoing carotid endarterectomy (CEA). Forty-one carotid plaques were obtained. A portion of the most severe lesions was subjected to histopathologic examination, and the remainder of the plaques examined for lipid peroxidation. Thiobarbituric acid-reactive substances (TBARS) values were determined as a marker for lipid peroxidation. The lipid-rich core (LC) and macrophage infiltration (Mphi) component as a percentage of total plaque area were measured morphometrically. Based on the results, all plaques were classified into four groups. Group I (GI): LC <10%; Group IIa (GIIa): LC 10-30%, Mphi <5%; Group IIb (GIIb): LC 10-30%, Mphi < or = 5%, and Group III (GIII): LC < or =30%. The plaque TBARS values of GIII were significantly higher than those of GI, GIIa, and GIIb. The TBARS values of GIIb were one-and-a-half times higher than those of GIIa. Our results show that lipid peroxidation in carotid plaques is significantly associated with carotid atherosclerosis, especially plaque instability. These findings provide direct evidence of an association between lipid peroxidation and human atherosclerosis.

Phosphatidylcholines (PCs) with platelet-activating factor (PAF)-like biological activities are known to be generated by fragmentation of the sn-2-esterified polyunsaturated fatty acyl group. The reaction is free radical-mediated and triggered by oxidants such as metal ions, oxyhemoglobin, and organic hydroperoxides. In this study, we characterized the PAF-like phospholipids produced on reaction of PC having a linoleate group with lipoxygenase enzymes at low oxygen concentrations. When the oxidized PCs were analyzed by gas chromatography-mass spectrometry, two types of oxidatively fragmented PC were detected. One PC had an sn-2-short chain saturated or unsaturated acyl group (C(8)-C(13)) with an aldehydic terminal; the abundant species were PCs with C(9) and C(13). The other PC had a short chain saturated acyl group (C(6)-C(9)) with a methyl terminal, and the most predominant species was PC with C(8). When the extracts of oxidation products were subjected to catalytic hydrogenation, PCs having saturated acyl groups (C(6)-C(14)) were detected; the most abundant was C(12) species. The less regiospecific formation of PAF-like lipids suggests that they were generated by oxidative fragmentation of PC hydroperoxides formed by non-stereoselective oxygenation of the alkyl radical of esterified linoleate that escaped from the active centers of lipoxygenases. One of the PAF-like PC with an aldehydic terminal was found to be bioactive; it inhibited the production of nitric oxide induced by lipopolysaccharide and interferon-gamma in vascular smooth muscle cells from rat aorta.

Lysophosphatidic acid has been identified as a vasopressor principle in incubated mammalian plasma and sera, and shown to be generated extracellulary by lysophospholipase D-like activity. In this study, we monitored the time course of changes in the major phospholipid fractions during incubation of plasma, and found that polyunsaturated lysophosphatidic acids accumulate more rapidly than saturated lysophosphatidic acids at expense of the corresponding lysophosphatidylcholines. We compared the phospholipase activities for producing bioactive LPA in age-matched spontaneously hypertensive rats and Wistar Kyoto rats. The lysophospholipase D activity in rat plasma was found to be independent of strain and age. We suggest that lysophospholipase D functions in rat for persistent production of bioactive LPA in the circulation throughout life. However, our finding that production of LPA in spontaneously hypertensive rats was not greater than that in Wistar Kyoto rats does not seem to support the idea that increased production of LPA is involved in the pathogenesis of hypertension.

The roles of tumor necrosis factor (TNF)-alpha and the facilitative glucose transporter (GLUT) 4 on the induction of insulin resistance in peripheral tissues of cancer patients was examined by quantitative competitive PCR on biopsies of abdominal rectal muscle from patients with gastrointestinal cancer. The degree of insulin resistance in these patients was measured by the euglycemic hyperinsulinemic glucose clamp using a high physiologic insulin concentration (100 microU/ml). Quantitative competitive PCR was carried out using DNA competitors constructed by deleting 20-30 bp between the two primer annealing sites. Decreased glucose uptake (M value) in peripheral tissues was accompanied by a significantly increased TNF-alpha mRNA in skeletal muscle (r=0.867, p=0.0025). GLUT4 mRNA, however, was positively correlated with M values (r=0.739, p=0.015). The amounts of mRNAs for TNF-alpha and GLUT4 in skeletal muscle were not correlated. Serum TNF-alpha concentrations remained below the limit of detection. These findings suggest that the insulin resistance in peripheral tissues of cancer patients is in part due to the induction TNF-alpha mRNA and the down regulation of GLUT4 mRNA in peripheral tissues.

Superoxide (O2-)-dependent lipid peroxidation on addition of xanthine oxidase (XO) and Fe(3+)-ADP was induced in egg phosphatidylcholine (PC) liposomes containing dicetylphosphate (DCP), which are negatively charged like biological membranes, but not in uncharged egg PC liposomes. Positively charged Fe(3+)-ADP interacted more with negatively charged egg PC-DCP liposomes than with uncharged egg PC liposomes. The activities of Fe(3+)-chelates for initiating O(2-)-dependent lipid peroxidation were in the order Fe(3+)-ADP > Fe(3+)-citrate > Fe(3+)-oxalate = Fe(3+)-malonate > Fe(3+)-EDTA = 0. This order was the same as that for the reduction rates of these Fe(3+)-chelates to Fe(2+)-chelates by O(2-)-generated by XO. Lineweaver-Burk plots showed that the chelators inhibited XO by different mechanisms: uncompetitively by ADP and adenosine and non-competitively by organic acid chelators (citrate and oxalate) and EDTA. These results suggest that ADP interacts with XO in a manner different from the other chelators. Lipid peroxidation by XO-xanthine and Fe(3+)-ADP was induced in egg PC liposomes containing a trace (0.31-0.35 mol%) of peroxidized egg PC (PC-OOH), but not in PC-OOH-free liposomes of egg PC obtained by their pretreatment with triphenylphosphine. PC-OOH incorporated into dimyristoyl phosphatidylcholine (DMPC) liposomes was degraded on addition of both XO-xanthine and Fe(3+)-chelate, but not of either one alone. alpha-Tocopherol in DMPC liposomes was oxidized on addition of XO-xanthine and Fe(3+)-chelates in the presence, but not in the absence of PC-OOH. Furthermore, PC-OOH was required for decrease of the ESR spectrum of the spin probe 12-(N-oxyl-4,4'-dimethyloxazolidin-2-yl)stearic acid, which labels the hydrophobic region of egg PC liposome membranes, on addition of XO-xanthine and Fe(3+)-chelates. These results indicate that the "induction message of lipid peroxidation," which is associated with reduction of Fe(3+)-ADP by O2- and concurrent degradation of PC-OOH, must be transferred from the membrane surface to the inner hydrophobic region of the membranes.

The effect of lysophosphatidic acid (LPA) on Ca2+ efflux from cultured bovine adrenal chromaffin cells was examined. LPA stimulated the efflux of 45Ca2+ from the cells in a concentration dependent manner (10(-7)-10(-5)M). Other lysophospholipids (lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol and lysophosphatidylserine), phosphatidic acid, monoolein and oleic acid had not significant effect on 45Ca2+ efflux from the cells. The LPA-stimulated 45Ca2+ efflux was dependent on the extracellular Na+ concentration and was inhibited by amiloride, inhibitor of Na+/Ca2+ exchange. LPA increased 22Na+ uptake into the cells and this effect was also sensitive to amiloride. These results indicate that LPA stimulates extracellular Na(+)-dependent 45Ca2+ efflux from cultured bovine adrenal chromaffin cells, probably through its stimulatory effect on membrane Na+/Ca2+ exchange.

To elucidate the molecular orientation and mobility of alpha-tocopherol (vitamin E) in membranes, 19F nuclear magnetic resonance (NMR) was applied. In bilayer phosphatidylcholine (PC) liposomes, 19F NMR spin-spin relaxation times (T2) of 19F-labeled methyl groups on either the chromanol moiety or the isoprenoid side chain of alpha-tocopherol were significantly decreased compared to those for alpha-tocopherol in solution (CDCl3), suggesting that the vitamin E molecule exists in the lipid core of membranes and that its motional freedom is significantly restricted. The difference in T2 values between the solution state and PC liposomes was greatest for the 5a-CF3 on the chromanol moiety compared with other methyl groups on the isoprenoid side chain. Hence, it is thought that the chromanol moiety of alpha-tocopherol may fit tightly near the surface of the membrane because of its hydrophilicity. When Pr3+ was added to the liposomal suspension, the intensity of the NMR signal for 5a-CF3 changed faster than those of the other methyl groups. When the surface of the liposomal membranes was positively charged using stearyl amine, the mobility of the 5a-methyl group, expressed as the correlation time (tau c), was quite similar to that in negatively charged membranes. These results indicate that the chromanol moiety of alpha-tocopherol may orient near the membrane surface, and that the hydroxyl group may be hydrogen bonded to the ester carbonyl of PC. For further confirmation of this conclusion, time-dependent changes in the fluidity of liposomes caused by the addition of Pr3+ were measured at the surface and inner region of liposomal membranes using a fluorescence polarization technique. The fluidity increased in the surface region and decreased in the inner region of liposomes containing alpha-tocopherol. These results show that when praseodymium ions may pass through the liposomal membrane, the hydrogen bonds between alpha-tocopherol and the ester carbonyl of PC may be broken, leading to the formation of an alpha-tocopherol-Pr3+ complex, thus causing the fluidity to increase. Based on the findings obtained, mobility and molecular orientation of vitamin E in membranes are proposed.

The effect of protein kinase C (C-kinase) on the Ca(2+)-activated K+ channel (KCa-channel) was studied in cultured smooth muscle cells from porcine coronary artery by the patch-clamp technique. In cell-attached patches, bath application of phorbol 12-myristate 13-acetate (PMA, 1 microM), a C-kinase activator, significantly decreased the open probability of the activated KCa-channel in the presence of the calcium ionophore A23187 (20 microM), which increases intracellular Ca2+. This decrease in the open probability was reversed by subsequent application of staurosporine (1 nM), a C-kinase inhibitor. Application of 1-oleoyl-2-acetylglycerol (OAG, 30 microM) or 1,2-dioctanoylglycerol (DG8; 30 microM), activators of C-kinase, also inhibited KCa-channel activation by A23187, and these inhibitions were also reversed by staurosporine. PMA (1 microM) also inhibited KCa-channel activation by dibutylyl cyclic AMP (db-cAMP, 2 mM) or caffeine (30 mM). In inside-out patches, bath application of the C-kinase fraction from rat brain in the presence of ATP (1 mM) and PMA (1 microM) markedly inhibited the KCa-channel. These results indicate that activation of C-kinase inhibits the KCa-channel and may cause membrane depolarization and vascular contraction.

Activation of the Ca(2+)-activated K+ channel (KCa-channel) by adenosine 3', 5'-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase (A-kinase) in cultured smooth muscle cells from porcine coronary artery was investigated using the patch-clamp technique. In cell-attached patches, the KCa-channel was activated when forskolin (10 microM) was applied to the bath. In excised inside-out patches, application of 50 microM cAMP to the bath activated the KCa-channel in the presence of A-kinase (10 units/ml) and ATP (1 mM). In addition, the KCa-channel was activated directly by application of cAMP to the cytoplasmic side of the membrane in the absence of A-kinase. The activation by cAMP or by A-kinase required intracellular Ca2+, and was enhanced by increase of intracellular Ca2+. At a low concentration (3 x 10(-7) M) of Ca2+, more than 2 mM cAMP was required for activation of the KCa-channel, but with 10(-6) M Ca2+, 100 microM cAMP was sufficient for activation. These results suggest that there are two mechanisms of activation of the KCa-channel by cAMP: direct activation, and indirect activation via phosphorylation of the channel by A-kinase.

Oxidation of linoleic acid (LA) in tetradecyltrimethylammonium bromide micelles was induced by ferrous- and ferric-chelates in the presence of linoleic acid hydroperoxide (LOOH). Ferrous-chelates also induced lipid peroxidation in the presence of H2O2, but ferric-chelates did not, thought they could generate OH-radicals in the presence of H2O2, resulting in deoxyribose degradation. Of the chelators tested, nitrilotriacetic acid (NTA) chelated with iron showed the highest activity for induction of H2O2- and LOOH-dependent lipid peroxidations and H2O2-dependent deoxyribose degradation. NTA with ferrous ion, but not with ferric ion, also initiated oxidation of LA after a short lag period in the absence of peroxides such as H2O2 and LOOH, but other chelators with ferrous ion did not. The peroxide-independent lipid peroxidation and associated oxidation of ferrous-NTA to ferric-NTA progressed in two steps: an induction step in a lag period and then a propagation step. Ferrous ion complexed with NTA was autoxidized pH-dependently and synchronously with oxygen uptake. The rates of both reactions increased with increase of pH, but were not related to the length of the lag period, which was also dependent on pH, and was shortest at pH 4.2. The EPR spectrum of the ferric-NTA complex prepared directly from ferric salt was different from that of the complex prepared from ferrous salt, confirming that some ferric-type active oxygen participated in induction of peroxide-independent lipid peroxidation. From these results, we propose a possible mechanism of lipid peroxidation induced by ferrous-NTA without peroxides. The finding that iron-NTA had the highest activity for induction of the oxidations of LA and deoxyribose is discussed in relation to the carcinogenic and nephrotoxic effects of this chelating agent.

To obtain information on the role of iron-catalyzed lipid peroxidation in the presence of the small amount of lipid peroxide in deterioration of biological membranes, we examined factors affecting peroxidation of fatty acids in charged micelles. Peroxidation of linoleic acid (LA) was catalyzed by Fe2+ via reductive cleavage of linoleic acid hydroperoxide (LOOH) in negatively charged sodium dodecyl sulfate micelles, but not in positively charged tetradecyltrimethylammonium bromide (TTAB) micelles. However, this Fe2(+)-induced, LOOH-dependent lipid peroxidation could be induced in TTAB micelles in the presence of a negatively charged iron chelator, nitrilotriacetic acid (NTA). The linoleic acid alkoxy radical (LO.) generated by the LOOH-dependent Fenton reaction was also trapped by N-t-butyl-alpha-phenylnitrone at the surface of TTAB micelles in the presence of NTA, but not in its absence. The degradation rates of two spin probes, N-oxyl-4,4'-dimethyloxazolidine derivatives of stearic acid (5-NS and 16-NS), were investigated to determine the site of production of radicals formed during LOOH-dependent lipid peroxidation. The rate of consumption of 16-NS during the LOOH-dependent Fenton-like reaction was higher in TTAB micelles containing LA than in those containing lauric acid (LauA), although the rates of formation of LO. in the two types of fatty acid micelles were similar. The rates of 5-NS consumption in LA and LauA micelles were almost the same and were as low as that of 16-NS consumption in LauA micelles. 16-NS was more inhibitory than 5-NS of LOOH-dependent lipid peroxidation, and this inhibition was associated with its higher consumption of 16-NS than of 5-NS. alpha-Tocopherol inhibited NTA-Fe2(+)-induced LOOH-dependent lipid peroxidation in TTAB micelles, and was oxidized during this inhibition process. The rate and amount of alpha-tocopherol oxidized by the LOOH-dependent Fenton reaction were higher in LA micelles than in LauA micelles. alpha-Tocopherol inhibited the consumption of 16-NS during NTA-Fe2(+)-induced LOOH-dependent lipid peroxidation more effectively than that of 5-NS. The distribution of the chromanol moiety of alpha-tocopherol was studied by the fluorescence quenching method. There was no difference between Stern-Volmer plots of the quenchings of alpha-tocopherol fluorescence by 5-NS and 16-NS. From these results, we discuss the mechanism of induction of LOOH-dependent peroxidation of LA and the mechanism of the antioxidant effects of alpha-tocopherol on it from the viewpoint of site-specific reaction.

Rates of oxidation of alpha-tocopherol by the hydroxyl- and superoxide free radicals were measured. The radicals were produced in known yields by radiolysis of aqueous solutions with gamma rays. Two main systems were used to dissolve the tocopherol; micelles, made up from charged and uncharged amphiphiles, and membranes made from dimyristyl phosphatidylcholine which could be charged by addition of stearyl amine or dicetyl phosphate. The HO. radicals were efficient oxidants of alpha-tocopherol in all systems, with up to 83% of radicals generated in micelle and 32% in membrane suspensions initiating the oxidation. The HO.(2) radical was an even more effective oxidant, but when most of it was in the O(2) form at neutral or alkaline pH, the oxidation rates became low. Tocopherol held in positively charged micelles or membranes was oxidized at a higher rate by the O(2) than in uncharged or negative particles. Possible biological significance of these results is discussed.

This research examined the structure of internal representation and the conceptual operation of color in two pure alexic cases (Case I and Case II) with color naming defects. Experiment I investigated the structure of the internal representation of different kinds of colors using a similarity judgment task. Experiment II examined categorical judgments of perceived colors using a two-alternative-forced choice task. Experiment III tested the classification of perceived colors using a color sorting task. The performance of Case I essentially fell within the normal range while the results of Case II showed some impairment in the conceptual operation of color. Analysis of the responses obtained from these experiments indicated that the color naming defects in Case I can be explained in terms of visual-verbal disconnection. However, the naming defects in Case II reflect disfunction in some other higher cortical processes coupled with visual-verbal disconnection.

alpha-Tocopherol inhibited H2O2-Fe2+-induced lipid peroxidation of linoleic acid (LA) by scavenging OH radicals in tetradecyltrimethylammonium bromide (TTAB) micelles. The inhibiting ability of alpha-tocopherol was much greater than that of OH-radical scavengers mannitol and t-butanol. In contrast, alpha-tocopherol enhanced linoleic acid hydroperoxide (LOOH)-Fe2+-induced lipid peroxidation through regeneration of Fe2+ in sodium dodecyl sulfate (SDS) micelles containing LA. alpha-Tocopherol was oxidized by Fenton's reagent (FeSO4 + H2O2) at a higher rate in SDS micelles than in TTAB micelles. The likely oxidants were OH radicals in the former and Fe3+ in the latter. Both reagents formed in the Fenton reaction. Ferrous ion catalyzed in a dose-dependent manner the decomposition of LOOH and conjugated diene compounds in SDS but not in TTAB micelles. alpha-Tocopherol and Fe3+ individually had no effect on the decomposition of LOOH, but together were quite effective. The rate of the decomposition was a function of the concentration of alpha-tocopherol. The mechanism of "site-specific" antioxidant action of alpha-tocopherol in charged micelles is discussed.

Egg yolk phosphatidylcholine liposomes were rapidly oxidized in the presence of chelated iron and a superoxide-generating system. alpha-Tocopherol incorporated in the bilayer was oxidized at the same time. No lipid or alpha-tocopherol oxidation occurred in liposomes composed of dimyristoyl phosphatidylcholine. The antioxidant did not inhibit lipid peroxidation until its concentration reached a critical level, which depended on the effectiveness of the oxidative stress. Beyond this level, peroxidation was inhibited completely and, simultaneously, the rate of oxidation of tocopherol was lowered. The results suggest that the antioxidant efficiency of alpha-tocopherol depends on its ability to react mainly with the chain-initiating or chain-propagating lipid radicals. This, in turn, is closely tied to the tocopherol content of the membrane. Ascorbate inhibited the consumption of alpha-tocopherol, possibly by regenerating its reduced form.

Generation of hydroxyl radicals by the Fenton reaction resulted in lipid peroxidation of linoleic acid (LA) (H2O2-Fe2+-induced lipid peroxidation) in positively charged tetradecyltrimethylammonium bromide (TTAB) micelles, but not in negatively charged sodium dodecyl sulfate (SDS) micelles. However, more OH radicals formed via the Fenton reaction were trapped by N-t-butyl-alpha-phenylnitrone (PBN) in SDS micelles than in TTAB micelles. When detergent-dispersed LA was contaminated with linoleic acid hydroperoxide (LOOH), lipid peroxidation was catalyzed by Fe2+ via reductive cleavage of LOOH (LOOH-Fe2+-induced lipid peroxidation), and Fe2+ was oxidized simultaneously in SDS micelles, even when H2O2 was not present. In contrast, LOOH-Fe2+-induced lipid peroxidation and simultaneous oxidation of Fe2+ were not observed in TTAB micelles. An ESR spectrum presumed to be due to an alkoxy radical trapped by PBN was also detected in SDS micelles, but not in TTAB micelles in the LOOH-Fe2+-induced lipid peroxidation system. The results are discussed in the light of the localization of iron, the unsaturated bonding moiety of LA, the OOH-group of LOOH, and the trapping site of PBN in different charged micelles.