DNA damage by peroxynitrite characterized with DNA repair enzymes.

The DNA damage induced by peroxynitrite in isolated bacteriophage PM2 DNA was characterized by means of several repair enzymes with defined substrate specificities. Similar results were obtained with peroxynitrite itself and with 3-morpholinosydnonimine (SIN-1), a compound generating the precursors of peroxynitrite, nitric oxide and superoxide. A high number of base modifications sensitive to Fpg protein which, according to HPLC analysis, were mostly 8-hydroxyguanine residues, and half as many single-strand breaks were observed, while the numbers of oxidized pyrimidines (sensitive to endonuclease III) and of sites of base loss (sensitive to exonuclease III or T4 endonuclease V) were relatively low. This DNA damage profile caused by peroxynitrite is significantly different from that obtained with hydroxyl radicals or with singlet molecular oxygen. The effects of various radical scavengers and other additives (t-butanol, selenomethionine, selenocystine, desferrioxamine) were the same for single-strand breaks and Fpg-sensitive modifications and indicate that a single reactive intermediate but not peroxynitrite itself is responsible for the damage.     

Peroxynitrite diminishes gap junctional communication: protection by selenite supplementation

Loss of intercellular communication via gap junctions has been correlated with progression of cells to a malignant phenotype. Here, we show that peroxynitrite, a mediator of toxicity in inflammatory processes, diminishes gap junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells, assayed by the scrapeloading dye-transfer technique as well as by microinjection of a fluorescent dye into single cells. Exposure of cultured cells to a steady-state concentration of peroxynitrite of 1.6 microM for 4 min or to 3-morpholinosydnonimine (SIN-1) at 0.5 mM strongly diminished GJIC. These concentrations of peroxynitrite or SIN-1 were not cytotoxic. When cells were grown in a medium supplemented with sodium selenite (0.1-1 microM) for 72 h, substantial protection was afforded against the decrease in GJIC by peroxynitrite. Thus, peroxynitrite can disrupt GJIC, and selenium-containing proteins protect.     

Activation pattern of mitogen-activated protein kinases elicited by peroxynitrite: attenuation by selenite supplementation

Peroxynitrite is a mediator of toxicity in pathological processes in vivo and causes damage by oxidation and nitration reactions. Here, we report a differential induction of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells by peroxynitrite. For the exposure of cultured cells with peroxynitrite, we employed a newly developed infusion method. At 6.5 microM steady-state concentration, the activation of p38 MAPK was immediate, while JNK1/2 and ERK1/2 were activated 60 min and 15 min subsequent to 3 min of exposure to peroxynitrite, respectively. Protein-bound 3-nitrotyrosine was detected. When cells were grown in a medium supplemented with sodium selenite (1 microM) for 48 h, complete protection was afforded against the activation of p38 and against nitration of tyrosine residues. These data suggest a new role for peroxynitrite in activating signal transduction pathways capable of modulating gene expression. Further, the abolition of the effects of peroxynitrite by selenite supplementation suggests a protective role of selenium-containing proteins.     

A Novel 1,4-Dihydropyridine Derivative Improves Spatial Learning and Memory and Modifies Brain Protein Expression in Wild Type and Transgenic APPSweDI Mice

Ca²⁺ blockers, particularly those capable of crossing the blood-brain barrier (BBB), have been suggested as a possible treatment or disease modifying agents for neurodegenerative disorders, e.g., Alzheimer’s disease. The present study investigated the effects of a novel 4-(N-dodecyl) pyridinium group-containing 1,4-dihydropyridine derivative (AP-12) on cognition and synaptic protein expression in the brain. Treatment of AP-12 was investigated in wild type C57BL/6J mice and transgenic Alzheimer’s disease model mice (Tg APP_SweDI) using behavioral tests and immunohistochemistry, as well as mass spectrometry to assess the blood-brain barrier (BBB) penetration. The data demonstrated the ability of AP-12 to cross the BBB, improve spatial learning and memory in both mice strains, induce anxiolytic action in transgenic mice, and increase expression of hippocampal and cortical proteins (GAD67, Homer-1) related to synaptic plasticity. The compound AP-12 can be seen as a prototype molecule for use in the design of novel drugs useful to halt progression of clinical symptoms (more specifically, anxiety and decline in memory) of neurodegenerative diseases, particularly Alzheimer’s disease.     

Pristine Early Eocene Wood Buried Deeply in Kimberlite from Northern Canada

We report exceptional preservation of fossil wood buried deeply in a kimberlite pipe that intruded northwestern Canada’s Slave Province 53.3±0.6 million years ago (Ma), revealed during excavation of diamond source rock. The wood originated from forest surrounding the eruption zone and collapsed into the diatreme before resettling in volcaniclastic kimberlite to depths >300 m, where it was mummified in a sterile environment. Anatomy of the unpermineralized wood permits conclusive identification to the genus Metasequoia (Cupressaceae). The wood yields genuine cellulose and occluded amber, both of which have been characterized spectroscopically and isotopically. From cellulose δ¹⁸O and δ²H measurements, we infer that Early Eocene paleoclimates in the western Canadian subarctic were 12–17°C warmer and four times wetter than present. Canadian kimberlites offer Lagerstätte-quality preservation of wood from a region with limited alternate sources of paleobotanical information.     

Mitogen-activated protein kinase (p38-, JNK-, ERK-) activation pattern induced by extracellular and intracellular singlet oxygen and UVA.

Ultraviolet A (UVA; 320-400 nm) radiation in human skin fibroblasts induces a pattern of mitogen-activated protein kinase (MAPK) activation consisting of a rapid and transient induction of p38 and c-Jun-N-terminal kinase (JNK) activity but not extracellular signal-regulated kinases (ERK). UVA activation of p38 can be inhibited by the singlet oxygen (1O2) quenchers azide and imidazole, but not by the hydroxyl radical scavengers mannitol or dimethylsulfoxide, pointing to the involvement of 1O2. The same effect has been shown for JNK. Like UVA, 1O2 generated intracellularly upon photoexcitation of Rose Bengal activates p38 and JNK but not ERK. p38 and JNK activation was also elicited by chemiexcitation for the intracellular generation of 1O2 by the lipophilic 1,4-endoperoxide of N,N'-di(2,3-dihydroxypropyl)-1, 4-naphthalene dipropionamide. In contrast, extracellular generation of 1O2, by irradiation of Rose Bengal immobilized on agarose beads or by chemiexcitation employing the hydrophilic 1,4-endoperoxide of disodium 3,3'-(1,4-naphthylidene) dipropionate, was ineffective in activating p38 or JNK. These data suggest that the activation of p38 and JNK by 1O2 occurs only when the electronically excited molecule is generated intracellularly.     

Zur Kenntnis der Zell- und Kernteilung in dem primären Meristem

Ohne Zusammenfassung     

Carotenoids in petals of perennial Medicago species

The carotenoid content in the petals of fourteen Medicago species was examined, together with eight species studied previously, caratenoids in all the known perennials of the genus are reported. The species can be arranged in relationship groups on the basis of their interfertility. No major carotenoid was species- or groupspecific; a few minor pigments, however, were group- or species-specific. The amount of carotenoids ranged from 7 μg/g dry matter in violet-flowered M. sativa to 2120 μg/g in brownish-yellow M. platycarpos. Xanthophylls constituted 76–99% of the total, with lutein as the major component. β-Carotene, lutein and flavoxanthin were ubiquitous in petals. In M sativa leaves β-carotene, lutein, violaxanthin and neoxanthin constituted 88% of the total. The xanthophylls were esterified in petals but not in leaves.     

Protection against peroxynitrite

Peroxynitrite formed in vivo from superoxide and nitric oxide can mediate oxidation, nitration, or nitrosation reactions, leading to impaired function, toxicity, and alterations in signaling pathways. Protection against peroxynitrite is important for defense of normal tissue, especially during inflammation. Biological protection against peroxynitrite is organized in three categories: prevention, interception, and repair. Prevention is the control of the formation of peroxynitrite precursors, nitric oxide and superoxide. Interception is by direct reaction with peroxynitrite, leading to non-toxic products. In this regard, organoselenium compounds, metalloporphyrin derivatives, and peroxidases (e.g. glutathione peroxidase and myeloperoxidase) exhibit high second-order rate constants with peroxynitrite. Ebselen, like glutathione peroxidase, protects in a catalytic fashion utilizing glutathione as reductant in the peroxynitrite reductase reaction. Protection by metalloporphyrins can be maintained through glutathione or ascorbate. Repair processes remove damaged products and restitute intact biomolecules.     

A half-marathon and a marathon run induce oxidative DNA damage, reduce antioxidant capacity to protect DNA against damage and modify immune function in hobby runners

This study investigated whether a 21.1 km (half-marathon) or a 42.195 km (marathon) run modulates DNA damage, antioxidant capacity in lymphocytes and plasma, and the immune system in healthy hobby runners. Ten and 12 volunteers who completed the Baden-Marathon race in Karlsruhe with a running distance of 21.1 km and 41.195 km, respectively, were assessed 10 days before and immediately after the finish. There was no increase in the levels of endogenous DNA strand breaks immediately after half-marathon or marathon races. A statistically significant increase in the levels of oxidative DNA damage in lymphocytes was found using endonuclease III but not formamidopyrimidine glycolase (Fpg). The resistance of DNA to oxidative damage induced by hydrogen peroxide in isolated lymphocytes was significantly decreased after both races. The levels of plasma antioxidants such as alpha-tocopherol, beta-carotene and lycopene were close to, or higher than, those considered optimal for reducing the risk of cardiovascular diseases and there were no significant changes after the races in antioxidant capacity of LDL (lag-time test) or plasma in ORAC, TEAC or paraoxonase assays. The number and percentage of granulocytes and monocytes able to generate oxidative burst were significantly increased after both races, but the lytic activity of NK cells was significantly increased at the end of the half-marathon; no effect was observed in the marathon runners. Thus, oxidative DNA damage in lymphocytes, decreased the antioxidant capacity to protect lymphocytes against DNA strand breaks and increased the formation of reactive species by phagocytes in well-nourished hobby runners indicating moderate oxidative damage during such high-intensity exercise.