Three histidine residues of amyloid-beta peptide control the redox activity of copper and iron.

Zinc, iron and copper are concentrated in senile plaques of Alzheimer disease. Copper and iron catalyze the Fenton-Haber-Weiss reaction, which likely contributes to oxidative stress in neuronal cells. In this study, we found that ascorbate oxidase activity and the intensity of ascorbate radicals measured using ESR spectroscopy, generated by free Cu(II), was decreased in the presence of amyloid-beta (Abeta), the major component of senile plaques. Specifically, the ascorbate oxidase activity was strongly inhibited (85% decrease) in the presence of Abeta1-16 or Abeta1-42, whereas it was only slightly inhibited in the presence of Abeta1-12 or Abeta25-35 (<20% inhibition). Ascorbate-dependent hydroxyl radical generation by free Cu(II) decreased in the presence of Abeta in the identical order of Abeta1-42, Abeta1-16 > Abeta1-12 and was abolished in the presence of 2-fold molar excess glycylhystidyllysine (GHK). Ascorbate oxidase activity and ascorbate-dependent hydroxyl radical generation by free Fe(III) were inhibited by Abeta1-42, Abeta1-16, and Abeta1-12. Although Cu(II)-Abeta shows a significant SOD-like activity, the rate constant for the reaction of superoxide with Cu(II)-Abeta was much slower than that with SOD. Overall, our results suggest that His6, His13, and His14 residues of Abeta1-42 control the redox activity of transition metals present in senile plaques.

Effects of MCI-186 upon neutrophil-derived active oxygens.

Reactions of 3-methyl-1-phenyl-2-pyrazoline-5-one (MCI-186) with hypochlorous acid and superoxide were analysed by spectrophotometry and mass spectrometry. The results were applied to the neutrophil system to evaluate the scavenging activity of neutrophil-derived active oxygen species by MCI-186. MCI-186 reacted rapidly with hypochlorous acid (1 x 10(6) M(-1)s(-1)) to form a chlorinated intermediate, followed by a slow conversion to a new spectrum. MCI-186 consumed 3 moles of hypochlorous acid and did not react with superoxide. The newly synthesized fluorescence probes, 2-[6-(4'-amino)-phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) and 2-[6-(4'-hydroxy)phenoxy-3H-anthen-3-on-9-yl]benzoic acid (HPF) successfully detected neutrophil-derived active oxygens (Setsukinai K, Urano Y, Kakinuma K, Majima HJ, Nagano T. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J Biol Chem 2003; 278: 3170-3175). The rate constants for the reaction of hypochlorous acid with MCI-186 and fluorescence probes was in the order of MCI-186 > APF > HPF. Fluorescence due to the oxidation of APF and HPF was observed with the stimulated neutrophils. The result that the intensity from APF oxidation was higher than that from HPF oxidation is compatible with reports that APF selectively reacts with hypochlorous acid. Fluorescence due to oxidation of both APF and HPF decreased when the reactions were carried out in the presence of a fluorescence probe and MCI-186 in a dose-dependent manner. These results indicate that MCI-186 effectively scavenges neutrophil-derived hypochlorous acid and other active oxygens.

Cu-metallothioneins (Cu(I)8-MTs) in LEC rat livers 13 weeks after birth still act as antioxidants.

Redox properties of metallothioneins (MTs) and Cu in the cytosol from Long-Evans Cinnamon (LEC) rat livers 13 weeks after birth were investigated. MTs from LEC rat livers contain 8 g atoms of Cu and 1 g atom of Zn per mole of protein (Cu(I)8-MTs). Titration of Cu(I)8-MTs with CuCl2 indicates that Cu(I)8-MTs were able to reduce further 2-g atoms of cupric ions per mole MTs as bound form. Hg2+-induced hydroxyl radical generation from Cu(I)8-MTs was demonstrated by ESR using the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The intensity of DMPO-OH signal from Cu-loaded MTs was increased with the increasing number of Cu in MTs. The used cytosol fraction contained 1.37 mM total Cu and 5 mM DTNB titrable-SH groups has a potential to reduce 2 mM CuCl2. No ESR signal due to Cu2+ was also detected with LEC rat liver cytosol, whereas strong Cu2+ signal appeared by the addition of HgCl2. The rate constants for the reaction of Cu(I)8-MTs with superoxide and hydroxyl radicals were estimated to be 2 x 10(6) and > or = 10(12) M(-1)s(-1), respectively, from competition kinetics. Cu2+-catalyzed oxidation of DNA was strongly inhibited both in the presence of Cu-unsaturated MTs and GSH. The results suggest that Cu(I)8-MTs from LEC rat livers just before hepatitis still act as antioxidants.

Dissociation of DNA double strand by hypohalous acids.

Calf thymus DNA was treated with authentic HOCl, and hypohalous acid-generating systems. This caused a decrease in fluorescence of ethidium-DNA complexes when ethidium bromide was subsequently added to the DNA. The fluorescence continued to decrease up to 30 min after adding HOCl. Loss in fluorescence was proportional to the concentration of HOCl and was complete when a 3-fold excess of HOCl was added to the DNA. No significant decrease in the fluorescence was observed when the chlorination was carried out in the presence of a concentration of monochlorodimedone (MCD) equivalent to that of HOCl. MCD is known to react stoichiometrically with HOCl. The decrease in fluorescence was completely inhibited by H2O2, ascorbate and glutathione (GSH). We have estimated the rate constant for the reaction of HOCl with H2O, to be 1-2 x 10(5) M(-1)s(-1). When compared with authentic HOCl, HOCl-generating systems (Cl + H2O2 + MPO or chloroperoxidase) were found to be inefficient in damaging DNA. This result most likely arises because the rate constant for reaction of HOCl with H2O2 is about 1000-fold faster than that for the reaction with DNA. HOBr and HOI generating systems also had a limited ability to damage DNA. We conclude that good chlorine acceptors and antioxidants protect DNA from hypohalous acid-induced oxidative damage.

Effects of CP-060S, a novel Ca(2+) channel blocker, on oxidative stress in cultured cardiac myocytes.

The effect of (-)-(S)-2-[3,5-bis(1, 1-dimethylethyl)-4-hydroxyphenyl]-3-[3-[N-methyl-N-[2-(3, 4-methylenedioxyphenoxy)ethyl]amino]propyl]-1,3-thiazolidin- 4-one hydrogen fumarate (CP-060S), a novel Ca(2+) channel blocker, on hydrogen peroxide (H(2)O(2))-induced cytotoxicity was studied in cultured rat cardiac myocytes. The CP-060S effect was compared with that of CP-060R, an optical isomer of CP-060S with a less potent Ca(2+) channel blocking action than CP-060S. H(2)O(2) increased the release of lactate dehydrogenase from cardiac myocytes and decreased the formation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) formazan in cardiac myocytes (i.e., cytotoxic action). Both CP-060S (1 microM) and CP-060R (1 microM) attenuated to a similar extent the foregoing alterations induced by H(2)O(2). On the other hand, 1,3-dimethyl-2-thiourea (10 mM), a scavenger of both H(2)O(2) and hydroxyl radical, also attenuated the H(2)O(2)-induced cytotoxicity whereas diltiazem (10 microM) did not. In an experiment using electron spin resonance (ESR) with 5, 5-dimethyl-1-pyrroline N-oxide (DMPO), a spin-trapping agent, both CP-060S and CP-060R decreased the intensity of DMPO-hydroxyl radical signal concentration dependently. These results suggest that CP-060S protects cardiac myocytes from oxidative stress through its radical scavenging action.

Computed tomography of the retropancreatic peritoneal recess: a case of gastric leiomyosarcoma.

We report computed tomography of the retropancreatic peritoneal recess in a case in which gastric leiomyosarcoma developed. Because of its location behind the pancreas, an abnormal finding in the retropancreatic peritoneal recess may be radiologically mistaken for a retroperitoneal abnormality. Knowledge of anatomy of posterior peritoneal recesses and understanding of fetal development are important in the radiological diagnosis of the lesions behind the pancreas.

Metal-induced hydroxyl radical generation by Cu(+)-metallothioneins from LEC rat liver.

Reactions of LEC (Long-Evans rats with a cinnamonlike coat color) rat liver Cu(I)-metallothioneins (MTs) with HgCl2 or K3Fe(CN)6 were investigated by ESR spectroscopy and generation of hydroxyl radicals was demonstrated using the ESR spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). When Cu(I)-MTs were incubated with more than one equivalent mole HgCl2 or K3Fe(CN)6 to Cu+ bound to MTs, strong signals due to Cu2+ appeared. ESR spectra, which were a combination of the DMPO-OH adduct signal and a six-line signal, were observed in the reaction of Cu(I)-MTs with HgCl2, whereas no oxygen radical signal was seen with K3Fe(CN)6. The DMPO-OH signal intensity was greater in the presence of SOD while the signal disappeared in the presence of catalase. The results suggest that addition of HgCl2 causes the liberation of cuprous ions from MTs followed by a reaction with oxygen, leading to hydroxyl radical formation through a Fenton-type Haber-Weiss reaction.

Mice lacking p27(Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors.

SUMMARY: Mice lacking p27(Kip1) have been created by gene targeting in embryonic stem cells. These mice are larger than the control animals, with thymus, pituitary, and adrenal glands and gonadal organs exhibiting striking enlargement. CDK2 activity is elevated about 10-fold in p27(-/-) thymocytes. Development of ovarian follicles seems to be impaired, resulting in female sterility. Similar to mice with the Rb mutation, the p27(-/-) mice often develop pituitary tumors spontaneously. The retinas of the mutant mice show a disturbed organization of the normal cellular layer pattern. These findings indicate that p27(Kip1) acts to regulate the growth of a variety of cells. Unexpectedly, the cell cycle arrest mediated by TGFbeta, rapamycin, or contact inhibition remained intact in p27(-/-) cells, suggesting that p27(Kip1) is not required in these pathways.

2-(2-Pyridyl)ethyl group: new type protecting group in the synthesis of DNA via phosphoramidite intermediates.

2-(2-Pyridyl)ethyl group is a new type P-O protecting group for the synthesis of oligodeoxyribonucleotides by the phosphite triester method. This group is stable to alkali and acid conditions, and to be removed from internucleotidic bonds under mild conditions via two step procedures without any side reactions. Further we have found that bis(diisopropylamino)chlorophosphine is much more effective for the preparation of bis(diisopropylamino)alkoxyphosphines than various dichlorophosphines.