PARP-3 and APLF function together to accelerate nonhomologous end-joining.

PARP-3 is a member of the ADP-ribosyl transferase superfamily of unknown function. We show that PARP-3 is stimulated by DNA double-strand breaks (DSBs) in vitro and functions in the same pathway as the poly (ADP-ribose)-binding protein APLF to accelerate chromosomal DNA DSB repair. We implicate PARP-3 in the accumulation of APLF at DSBs and demonstrate that APLF promotes the retention of XRCC4/DNA ligase IV complex in chromatin, suggesting that PARP-3 and APLF accelerate DNA ligation during nonhomologous end-joining (NHEJ). Consistent with this, we show that class switch recombination in Aplf(-/-) B cells is biased toward microhomology-mediated end-joining, a pathway that operates in the absence of XRCC4/DNA ligase IV, and that the requirement for PARP-3 and APLF for NHEJ is circumvented by overexpression of XRCC4/DNA ligase IV. These data identify molecular roles for PARP-3 and APLF in chromosomal DNA double-strand break repair reactions.

Poly(ADP-ribose) polymerase 1 accelerates single-strand break repair in concert with poly(ADP-ribose) glycohydrolase.

Single-strand breaks are the commonest lesions arising in cells, and defects in their repair are implicated in neurodegenerative disease. One of the earliest events during single-strand break repair (SSBR) is the rapid synthesis of poly(ADP-ribose) (PAR) by poly(ADP-ribose) polymerase (PARP), followed by its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). While the synthesis of poly(ADP-ribose) is important for rapid rates of chromosomal SSBR, the relative importance of poly(ADP-ribose) polymerase 1 (PARP-1) and PARP-2 and of the subsequent degradation of PAR by PARG is unclear. Here we have quantified SSBR rates in human A549 cells depleted of PARP-1, PARP-2, and PARG, both separately and in combination. We report that whereas PARP-1 is critical for rapid global rates of SSBR in human A549 cells, depletion of PARP-2 has only a minor impact, even in the presence of depleted levels of PARP-1. Moreover, we identify PARG as a novel and critical component of SSBR that accelerates this process in concert with PARP-1.

Why nutraceuticals do not prevent or treat Alzheimer's disease.

A great deal of research has pointed to deleterious roles of metal ions in the development of Alzheimer's disease. These include: i) the precipitation and aggregation of amyloid beta (Abeta) peptides to form senile plaques and neurofibrillary tangles, and/or ii) the augmentation of oxidative stress by metal ion mediated production and activation of hydrogen peroxide. The growing trend in nutraceutical intake is in part a result of the belief that they postpone the development of dementias such as Alzheimer's disease. However, pathogenic events centred on metal ions are expected to be aggravated by frequent nutraceutical intake. Novel therapeutic approaches centred on chelators with specificity for copper and iron ions should be fully explored.

Therapeutic chelators for the twenty first century: new treatments for iron and copper mediated inflammatory and neurological disorders.

Superoxide, hydrogen peroxide, hydroxyl radicals and peroxynitrite are collectively termed reactive oxygen and nitrogen species (RONS). They have been ascribed an important role in oxidative stress contributing to the progression of inflammatory diseases. RONS generating systems include the inflammatory response, enzymatic pathways and as side products of catabolism. Protective enzymes exist for the regulation of RONS such as superoxide dismutase, catalase and glutathione peroxidase. Furthermore, vitamins play a secondary role in deactivating RONS. The redox active metal ions such as ferrous and cuprous ions are released from the storage proteins ferritin and caeruloplasmin by RONS. Redox active metal ions further activate/generate RONS and thus perpetuate their damaging effects. Here we report recent therapies that focus on intervening in the roles of metal ions in oxidative stress. These include: i) chelators which complex labile metal ions to form antioxidant enzyme mimetics, ii) site-specific RONS scavengers, where dual functionality co-localizes the scavenger and chelation centre to direct scavenging, and iii) redox silencing, metal complexation with concomitant stabilization of the metal ion in the oxidized form to prevent further activation of RONS. The rationale for this new therapeutic approach and recent advances will be presented in this review.

Metal ion chelating peptoids with potential as anti-oxidants: complexation studies with cupric ions.

The cupric ion binding characteristics of the chelator EDTA bis (ethyl tyrosinate) are reported. Potentiometric studies in aqueous solutions over the pH range of 2.0-12.0 allowed identification and quantification of the species in solution. The principal species CuA predominates over the physiological pH range of 4.0-8.0 pH units. The logarithm of the stability constant (log beta(pqr)) for this species is 16.43. The cupric ion binding characteristics were further assessed using electronic absorption spectroscopic investigations. These results support the use of this chelator as a metal binding anti-oxidant.

Iron supplements: the quick fix with long-term consequences.

Co-supplementation of ferrous salts with vitamin C exacerbates oxidative stress in the gastrointestinal tract leading to ulceration in healthy individuals, exacerbation of chronic gastrointestinal inflammatory diseases and can lead to cancer. Reactive oxygen and nitrogen species (RONS) have been ascribed an important role in oxidative stress. Redox-active metal ions such as Fe(II) and Cu(I) further activate RONS and thus perpetuate their damaging effects. Ascorbic acid can exert a pro-oxidant effect by its interaction with metal ions via a number of established RONS generating systems which are reviewed here. Further studies are required to examine the detrimental effects of nutraceuticals especially in chronic inflammatory conditions which co-present with anaemia.

Lipophilic ionophore complexes as superoxide dismutase mimetics.

A wide range of metal ion complexes exhibit superoxide dismutase like activities as detected by inhibition of nitroblue tetrazolium reduction. Mn(II) and Cu(II) complexes of EDTA, EHPG, and EGTA exhibit SOD like activities commensurate with many of the purpose-built SOD mimics. Here, we report analogous lipophilic chelators that localise metal ions (Cu(II), Mn(II), and Fe(III)) in the lipid membranes and lipoproteins to protect them from superoxide mediated oxidative damage. Spectroscopic titrations and Jobs method confirm that both 1:1 and 2:1 metal ion monensin complexes form. The cupric complexes are the most active exhibiting IC(50) values of 0.09 and 0.18 microM for 2Cu(II)-monensin and Cu(II)-monensin, respectively, for superoxide destruction. In addition, the IC(50) value for Mn(II)-monensin is 0.31 microM. In conclusion, Mn(II) and Cu(II) complexes of the ionophore monensin exhibit considerable superoxide scavenging activities and represent a novel class of catalytic antioxidants for the protection of lipid structures.

EDTA bis-(methyl tyrosinate): a chelating peptoid peroxynitrite scavenger.

Conjugation of ethylenediaminetetra-acetic acid (EDTA) to methyl tyrosinate generates a chelating peptoid EDTA bis-(methyl tyrosinate), (EBMT). Peroxynitrite-mediated nitration was studied for the free peptoid and its ferric and cupric complexes. The nitration products were monitored by electronic absorption spectroscopy at lambda(max) of 420 nm (mono-nitrated) and 440 nm (di-nitrated). Peak deconvolution was effected by pH manipulation as the mono-nitrated analogue of tyrosine exhibited a bathochromic shift from 365 nm (below its pK(a) of 6.8) to 420 nm. Rates of nitration were: free peptoid

Catalytic superoxide scavenging by metal complexes of the calcium chelator EGTA and contrast agent EHPG.

Metal ion chelators widely used in experimental protocols and clinical diagnosis are generally assumed to be inert. We previously reported that the ubiquitous chelator EDTA has high levels of superoxide suppressing activity. Here, we report that the common chelators calcium chelator EGTA and contrast agent EHPG have significant activities in suppressing superoxide levels depending on the nature of metal ion chelated. The most active species is Mn(II)-EGTA which exhibited an IC50 value of 0.19 microM for superoxide destruction. In addition, IC50 values for Mn(II)-EHPG and 2Cu(II)-EGTA were 0.69 and 0.60 microM, respectively. In conclusion, Mn(II) and Cu(II) complexes of the common chelators EGTA and EHPG exhibit considerable superoxide scavenging activities. Caution should be employed in their use in biological systems where superoxide has a key role and they may be useful for the development of catalytic anti-oxidants.

Superoxide and hydrogen peroxide suppression by metal ions and their EDTA complexes.

Redox-active metal ions such as Fe(II)\(III) and Cu(I)\(II) have been proposed to activate reactive oxygen and nitrogen species (RONS) and thus, perpetuate oxidative damage. Here, we show that concentrations of metal ions and EDTA complexes with superoxide-destroying activities equivalent to 1 U SOD are Fe(III) 5.1 microM, Mn(II) 0.77 microM, Cu(II)-EDTA 3.55 microM, Fe(III)-EDTA 2.34 microM, and Mn(II)-EDTA 1.38 microM. The most active being the aquated Cu(II) species which exhibited superoxide-destroying activity equivalent to 2U of SOD at 0.29 microM. Hydrogen peroxide-destroying activities were as follows Fe(III)-EDTA ca. 70 U/mg and aquated Fe(III) 141 U/mg. In contrast, DTPA prevented superoxide-destroying activity and significantly depleted hydrogen peroxide-destroying activity. In conclusion, non-protein bound transition metal ions may have significant anti-oxidant effects in biological systems. Caution should be employed in bioassays when chelating metal ions. Our results demonstrate that DTPA is preferential to EDTA for inactivating redox-active metal ions in bioassays.