Human Rad51 Protein Requires Higher Concentrations of Calcium Ions for D-Loop Formation than for Oligonucleotide Strand Exchange.

Human Rad51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. Both the activator protein Swi5/Sfr1 and Ca2+ ions stimulate different reaction steps and induce perpendicular DNA base alignment in the presynaptic complex. To investigate the role of base orientation in the strand exchange reaction, we examined the Ca2+ concentration dependence of strand exchange activities and structural changes in the presynaptic complex. Our results show that optimal D-loop formation (strand exchange with closed circular DNA) required Ca2+ concentrations greater than 5 mM, whereas 1 mM Ca2+ was sufficient for strand exchange between two oligonucleotides. Structural changes indicated by increased fluorescence intensity of poly(dεA) (a poly(dA) analog) reached a plateau at 1 mM Ca2+. Ca2+ > 2 mM was required for saturation of linear dichroism signal intensity at 260 nm, associated with rigid perpendicular DNA base orientation, suggesting a correlation with the stimulation of D-loop formation. Therefore, Ca2+ exerts two different effects. Thermal stability measurements suggest that HsRad51 binds two Ca2+ ions with KD values of 0.2 and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. Our results indicate parallels between the Mg2+ activation of RecA and the Ca2+ activation of HsRad51.

Well-Known Mediators of Selective Oxidation with Unknown Electronic Structure: Metal-Free Generation and EPR Study of Imide-N-oxyl Radicals.

Nitroxyl radicals are widely used in chemistry, materials sciences, and biology. Imide-N-oxyl radicals are subclass of unique nitroxyl radicals that proved to be useful catalysts and mediators of selective oxidation and CH-functionalization. An efficient metal-free method was developed for the generation of imide-N-oxyl radicals from N-hydroxyimides at room temperature by the reaction with (diacetoxyiodo)benzene. The method allows for the production of high concentrations of free radicals and provides high resolution of their EPR spectra exhibiting the superhyperfine structure from benzene ring protons distant from the radical center. An analysis of the spectra shows that, regardless of the electronic effects of the substituents in the benzene ring, the superhyperfine coupling constant of an unpaired electron with the distant protons at positions 4 and 5 of the aromatic system is substantially greater than that with the protons at positions 3 and 6 that are closer to the N-oxyl radical center. This is indicative of an unusual character of the spin density distribution of the unpaired electron in substituted phthalimide-N-oxyl radicals. Understanding of the nature of the electron density distribution in imide-N-oxyl radicals may be useful for the development of commercial mediators of oxidation based on N-hydroxyimides.

Anti-breast Cancer Agents Derived from Plants.

Upon emergence of modern anticancer therapy, medical community is divided into two opposite camps, one of them claiming absolute necessity of using isolated or synthesized chemical compounds for efficient patient treatment and another one advocating alternative cancer therapies, in particular those based on natural sources, including extracts from plants. It seems, in reality, that the two camps are reconcilable: while natural sources, plant extracts or juices play both curative and protective role, drugs represent the ultimate possibility to inhibit or reverse tumor development. In this paper we tried to analyze anti-breast cancer potencies of quite a few extracts from different plant sources and to compare their anti-proliferative efficiency of crude extracts with actions of their purified ingredients.

Approach for the preparation of various classes of peroxides based on the reaction of triketones with H2O2: first examples of ozonide rearrangements.

The reaction of ß,δ-triketones with an ethereal solution of H2O2 catalyzed by heteropoly acids in the presence of a polar aprotic co-solvent proceeds via three pathways to form three classes of peroxides: tricyclic monoperoxides, bridged tetraoxanes, and a pair of stereoisomeric ozonides. The reaction is unusual in that produces bridged tetraoxanes and ozonides with one of the three carbonyl groups remaining intact. In the synthesis of bridged tetraoxanes, the peroxide ring is formed by the reaction of hydrogen peroxide with two carbonyl groups at the ß positions. The synthesis of ozonides from ketones and hydrogen peroxide is a unique process in which the ozonide ring is formed with the participation of two carbonyl groups at the δ positions. Rearrangements of ozonides were found for the first time after more than one century of their active investigation. Ozonides are interconverted with each other and rearranged into tricyclic monoperoxides, whereas ozonides and tricyclic monoperoxides are transformed into bridged tetraoxanes. The individual reaction products were isolated by column chromatography and characterized by NMR spectroscopy, mass spectrometry, and elemental analysis. One representative of each class of peroxides was characterized by X-ray diffraction.

Phosphomolybdic and phosphotungstic acids as efficient catalysts for the synthesis of bridged 1,2,4,5-tetraoxanes from ß-diketones and hydrogen peroxide.

Phosphomolybdic acid (PMA) and phosphotungstic acid (PTA) efficiently catalyze the addition of H2O2 to ß-diketones to form bridged 1,2,4,5-tetraoxanes. These reactions are not accompanied by the formation of monocyclic peroxides containing hydroxy and hydroperoxide groups or polymeric peroxides. The use of these catalysts made it possible to obtain bridged tetraoxanes from easily oxidizable benzoylacetone derivatives and α-unsubstituted ß-diketones. The syntheses are scaled up to ten grams. The resulting peroxides can be easily isolated from the reaction mixture by column chromatography. The yield of tetraoxanes depends on the structure of ß-diketone and varies from 12 to 83%. NMR monitoring of two bridged 1,2,4,5-tetraoxanes synthesis was carried out.

Interplay of Ca(2+) and Mg (2+) in sodium-calcium exchanger and in other Ca(2+)-binding proteins: magnesium, watchdog that blocks each turn if able.

Sodium-calcium exchange across plasma membrane is regulated by intracellular calcium ions. The sodium-calcium exchanger (NCX1) is activated by successive saturation of numerous Ca(2+)-binding sites located in the intracellular loop of the protein. The progressive saturation of the binding domain CBD12 by Ca(2+) results in a series of conformational changes of CBD12 as well as of entire NCX1 molecule. Like other soluble and membrane Ca(2+)-binding proteins, NCX1 can also be regulated by Mg(2+) that antagonises Ca(2+) at the level of divalent cation-binding sites. This chapter summarises data on Mg(2+) impacts in the cells. Regulatory action of Mg(2+) on intracellular Ca(2+)-dependent processes can be achieved due to changes of its cytoplasmic level, which take place in the range of [Mg(2+)](i) from 0.5 to 3 mM. Under normal conditions, these changes are ensured by activation of plasmalemmal Mg(2+) transport systems and by variations in ATP level in cytoplasm. In heart and in brain, some pathological conditions, such as hypoxia, ischemia and ischemia followed by reperfusion, are associated with an important increase in intracellular Ca(2+). The tissue damage due to Ca(2+) overload may be prevented by Mg(2+). The protective actions of Mg(2+) can be achieved due to its ability to compete with Ca(2+) for the binding sites in a number of proteins responsible for the rise in intracellular free Ca(2+), including NCX1, in case when the reverse mode of Na(+)/Ca(2+) exchange becomes predominant. Saturation of CBD12 by Mg(2+) results in important changes of NCX1 conformation. Modulating actions of Mg(2+) on the conformation of NCX1 were detected at a narrow range of Mg(2+) concentration, from 0.5 to 1 mM. These data support an idea that variations of intracellular Mg(2+) could modify transmembrane Ca(2+) movements ensured by NCX1.

A quantitative evaluation of redox-active compounds in human blood lipids.

Endogenous low molecular weight redox active compounds (RACs) comprise antioxidants, pro-oxidants, transition metal cations and metal chelators. Traditional electrochemical methods of measuring RACs are limited to aqueous solutions, thus providing information of only hydrophilic RAC pools. In a large number of diseases associated with oxidative stress and/or with metal toxicity, redox states of hydrophilic as well as hydrophobic compartments are modified, and therefore development of methods for their detection is both necessary and important. The pools of lipid soluble RACs in reduced and oxidized forms in n-hexane extracts obtained from blood plasma, erythrocytes and whole blood of healthy donors were determined by spectrophotometric detection of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, which stoichiometrically interacts with hydrogen donors in non polar solutions. Measurements of RACs in extracts before and after treatment with NaBH(4) provided information about the levels of both reduced and oxidized RACs. Vitamin E was also determined using a fluorescence method. The results have shown that vitamin E is the major RAC in blood plasma lipids but not in blood cell lipids, where other phenols and quinones appear to predominate.

Aziridine alkaloids as potential therapeutic agents.

The present review describes research on natural aziridine alkaloids isolated from both terrestrial and marine species, as well as their lipophilic semi-synthetic, and/or synthetic analogs. Over 130 biologically active aziridine-containing compounds demonstrate confirmed pharmacological activity including antitumor, antimicrobial, antibacterial effects. The structures, origin, and biological activities of aziridine alkaloids are reviewed. Consequently this review emphasizes the role of aziridine alkaloids as an important source of drug prototypes and leads for drug discovery.

Iron chelators and free radical scavengers in naturally occurring polyhydroxylated 1,4-naphthoquinones.

The cardioprotective effect of polyhydroxylated 1,4-naphthoquinones on the experimental model of myocardial ischemia-reperfusion has been demonstrated previously. In this study, using different models, such as bulk organic phase, liposomes and sarcoplasmic reticulum (SR) vesicles, we have shown the ability of naturally occurring polyhydroxynaphthoquinones, echinochrome (Ech), spinochromes C, D and E (SpC, SpD and SpE) to inhibit free-radical oxidation induced by heme iron (hemin) or by free iron ions (in ferrous/ascorbate system). The polyhydroxy-1,4-naphthoquinones (PHNQs) were more effective in inhibiting the phosphatidyl choline liposome peroxidation induced by ferrous/ascorbate than that induced by hemin. The iron chelating ability of PHNQs was determined spectrophotometrically. Prevention of the ferrous/ascorbate-induced leakage of calcium by Ech was demonstrated in isolated SR vesicles from rabbit skeletal muscle. The PHNQs displayed high scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. We concluded that iron chelation predominates in the overall antioxidant potential of the polyhydroxynaphthoquinones.

Three types of muscles express three sodium-calcium exchanger isoforms.

The sodium-calcium exchanger (NCX) of plasma membrane is expressed in any animal cell. The specific role of its three isoforms (NCX1-3) is not yet established. Their levels vary considerably during murine postnatal development. In particular, in skeletal muscle, NCX1 expression decreases gradually upon aging while reciprocal changes take place for NCX3. NCX2 expression is restricted to brain and smooth muscles. The data on SDS-gel mobility shifts indicate that all three isoforms undergo Ca2+-dependent conformational changes, and that an exchanger regulatory Ca2+-binding domain interacts directly with mutually exclusive exons A and B inducing two different NCX1 conformations.

Fetal muscle-derived cells can repair dystrophic muscles in mdx mice.

We have previously reported that CD34(+) cells purified from mouse fetal muscles can differentiate into skeletal muscle in vitro and in vivo when injected into muscle tissue of dystrophic mdx mice. In this study, we investigate the ability of such donor cells to restore dystrophin expression, and to improve the functional muscle capacity of the extensor digitorum longus muscle (EDL) of mdx mice. For this purpose green fluorescent-positive fetal GFP(+)/CD34(+) cells or desmin(+)/(-)LacZ/CD34(+) cells were transplanted into irradiated or non-irradiated mdx EDL muscle. Donor fetal muscle-derived cells predominantly fused with existing fibers. Indeed more than 50% of the myofibers of the host EDL contained donor nuclei delivering dystrophin along 80-90% of the length of their sarcolemma. The presence of significant amounts of dystrophin (about 60-70% of that found in a control wild-type mouse muscle) was confirmed by Western blot analyses. Dystrophin expression also outcompeted that of utrophin, as revealed by a spatial shift in the distribution of utrophin. At 1 month post-transplant, the recipient muscle appeared to have greater resistance to fatigue than control mdx EDL muscle during repeated maximal contractions.

Echinochrome, a naturally occurring iron chelator and free radical scavenger in artificial and natural membrane systems.

Echinochrome, or 6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone, possesses cardioprotective activity, and diminishes the myocardial ischemia/reperfusion injury that is known to be accompanied by free-radical oxidative damage and calcium overload. In this study, we investigated the lipophilicity of echinochrome, its ability to inhibit free-radical oxidation both in the bulk organic phase and in an artificial membrane system (liposomes), and to prevent the ferrous/ascorbate-induced leakage of calcium from the isolated sarcoplasmic reticulum (SR) of rabbit skeletal muscle. The experimentally-determined octanol/water partition coefficient (LogP) of echinochrome was +3.11, and the distribution coefficient (LogD) was +2.58 at pH 6.0 and -0.15 at pH 8.0. Echinochrome displayed high scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals with a stoichiometry of about 1:7. Echinochrome was more effective in inhibiting the phosphatidyl choline liposome peroxidation induced by Fe2+/ascorbate than that induced by hemin. The iron chelating ability of echinochrome was estimated spectrophotometrically. In isolated SR, echinochrome protected the ATP-dependent Ca2+-pump system from damage by Fe2+/ascorbate. It was concluded that iron chelation predominates in the overall antioxidant potential of echinochrome.

Endothelial cells within embryonic skeletal muscles: a potential source of myogenic progenitors.

We investigated whether the vessel-associated or endothelial cells within mouse embryo muscles can be a source of myogenic progenitors. Immunodetection of the stem cell surface markers, CD34 and Flk1, which are known to characterize the endothelial lineage, was done throughout the course of embryo muscle development. Both markers appeared to be restricted to the vessel-associated cells. On the basis of CD34 labeling, the reactive cells were purified by magnetic-bead selection from the limb muscles of 17-dpc desmin+/-LacZ mouse embryos and characterized by fluorescence-activated cell sorting. The cells in the selected CD34(+) population appeared to be approximately 95% positive for Flk1, but usually negative for CD45. We demonstrated that in vitro the CD34(+)/Flk1(+) population differentiated into endothelial cells and skeletal myofibers. When transplanted into mdx mouse muscle, this population displayed a high propensity to disperse within the recipient muscle, fuse with the host myofibers, and restore dystrophin expression. The marked ability of the embryonic muscle endothelial cells to activate myogenic program could be related to their somitic origin.

Na(+)/Ca(2+) exchange in human myotubes: intracellular calcium rises in response to external sodium depletion are enhanced in DMD.

This study aims to investigate the sodium/calcium exchanger expression in human co-cultured skeletal muscle cells and to compare the effects of Na(+)/Ca(2+) exchange activity in normal and dystrophic (Duchenne's muscular dystrophy) human co-cultured myotubes. For this purpose, variations of intracellular calcium concentration ([Ca(2+)](int)) were monitored, as the variations of the fluorescence ratio of indo-1 probe, in response to external sodium depletion. External sodium withdrawal induced [Ca(2+)](int) rises within several seconds in both normal and Duchenne's muscular dystrophy myotubes. These Na(+)-free-induced [Ca(2+)](int) elevations were attributed to the reverse mode of the Na(+)/Ca(2+) exchange mechanism since the phenomenon was dependent on extracellular calcium concentration ([Ca(2+)](ext)), and since it was sensitive to external Ni(2+) ions. Amplitudes of Na(+)-free-induced [Ca(2+)](int) rises were significantly greater in Duchenne's muscular dystrophy cells than in normal ones. Such a difference disappeared when the sarcoplasmic reticulum was pharmacologically blocked, suggesting that the reverse mode of the Na(+)/Ca(2+) exchange mechanism was able to generate enhanced calcium-induced calcium-release in Duchenne's muscular dystrophy myotubes. Immunostaining images of Na(+)/Ca(2+) exchanger (NCX) isoforms, obtained by confocal microscopy, revealed the presence of NCX1 and NCX3 at the sarcolemmal level of both normal and Duchenne's muscular dystrophy myotubes. No differences were observed in the location of NCX isoforms expression between normal and Duchenne's muscular dystrophy co-cultured myotubes.