PARP-1, PARP-2 and ATM in the DNA damage response: functional synergy in mouse development

Poly(ADP-ribosyl)ation is an immediate DNA damage-dependent posttranslational modification of histones and nuclear proteins that contributes to the survival of injured proliferating cells. Poly(ADP-ribose) polymerases (PARPs) now constitute a superfamily of 18 proteins, encoded by different genes and displaying a common conserved catalytic domain. PARP-1 (113kDa), the founding member, and PARP-2 (62kDa) are both involved in DNA-break sensing and signaling when single strand break repair (SSBR) or base excision repair (BER) pathways are engaged. The generation by homologous recombination of deficient mouse models have confirmed the caretaker function of PARP-1 and PARP-2 in mammalian cells under genotoxic stress. This review summarizes our present knowledge on their physiological role in the cellular response to DNA damage and on the genetic interactions between PARP-1, PARP-2, Atm that play an essential role during early embryogenesis.     

Influence of rheumatoid arthritis in the enantioselective disposition of fenoprofen

To investigate the influence of rheumatoid arthritis on the stereoselective disposition of fenoprofen administered as a racemic mixture, eight patients with rheumatoid arthritis receiving calcium rac-fenoprofen (200 mg/8 h) and 7 healthy volunteers given single oral dose (600 mg) were investigated. Serial blood samples and urine were collected from zero to 24 h after fenoprofen (FEN) administration. The following differences were observed between the (+)-(S) and (-)-(R)-FEN in the patients with rheumatoid arthritis (means 95% CI, Wilcoxon test, P < 0.05): C(max) 14.1 (12.5-15.8) versus 3.6 (2.5-4.7) microg/ml; AUC(ss) (0-8) 80.5 (67.3-93.7) versus 12.1 (8.8-15.4) microg.h/ml; Cl(T)/f 1.3 (1.0-1.5) versus 9.1 (6.5-11.8) l/h; and t(1/2) 3.1 (2.3-3.9) versus 1.2 (0.8-1.6) h. The Cl(T)/f of (-)-(R)-FEN was reduced in patients with rheumatoid arthritis when compared to healthy volunteers: 9.1 (6.5-11.8) versus 17.4 (13.9-20.9) l/h; P < 0.05 Mann-Whitney test. The administration of rac-FEN as a single dose to healthy volunteers or multiple doses to patients with rheumatoid arthritis resulted in lower Cl(T)/f for the (+)-(S)-FEN. The lower Cl(T)/f of (-)-(R)-FEN observed for patients with rheumatoid arthritis is consistent with lower clearance by inversion, although other metabolic pathways, drug interactions, and bioavailability of the individual enantiomers may also contribute to the difference.     

Is water availability really the main environmental factor controlling the phenology of woody vegetation in the central Sahel?

Rainfall distribution and the soil moisture regime have been recognized to be the key drivers of the phenological rhythms in Sahelian woody plants, although different climate triggers have been assumed to be involved in determining the date of the onset of the phenophase. However, almost no comparisons have been made of the actual relative predictive power of these environmental factors. The aim of our study was to quantify the ability of several factors to predict phenophase occurrence in the dominant woody populations of northern Mali. Canopy leafing, flowering and fruiting were monitored from May 2005 to July 2007. Multiple logistic regressions were used to test the predictive power of cumulative rainfall, soil moisture, air temperature, air humidity and day length, with time lags of up to 2 months. Artificial variables derived from time lags observed in phenophases were included as predictors to account for possible auto-correlation and cross-correlation among phenophases. Surprisingly, a decrease in temperature associated with different time lags was most often found to be the strongest predictor of both leafing and reproductive phenophases. In Sahelian shrubs, morphological and physiological adaptations strongly contribute to the relative independence of their activity from water availability, leaf phenology being a way to adjust the plant water balance to current water availability and atmospheric water content. This study provides insight towards the development of a mechanistic understanding of phenological control in the Sahel, which is becoming increasingly important in the context of expected climate changes.     

Analysis of carvedilol enantiomers in human plasma using chiral stationary phase column and liquid chromatography with tandem mass spectrometry

Carvedilol is an antihypertensive drug available as a racemic mixture. (?)‐(S)‐carvedilol is responsible for the nonselective β‐blocker activity but both enantiomers present similar activity on α₁‐adrenergic receptor. To our knowledge, this is the first study of carvedilol enantiomers in human plasma using a chiral stationary phase column and liquid chromatography with tandem mass spectrometry. The method involves plasma extraction with diisopropyl ether using metoprolol as internal standard and direct separation of the carvedilol enantiomers on a Chirobiotic T® (Teicoplanin) column. Protonated ions [M + H]⁺ and their respective ion products were monitored at transitions of 407 > 100 for the carvedilol enantiomers and 268 > 116 for the internal standard. The quantification limit was 0.2 ng ml^?1 for both enantiomers in plasma. The method was applied to study enantioselectivity in the pharmacokinetics of carvedilol administered as a single dose of 25 mg to a hypertensive patient. The results showed a higher plasma concentration of (+)‐(R)‐carvedilol (AUC^0–∞ 205.52 vs. 82.61 (ng h) ml^?1), with an enantiomer ratio of 2.48. Chirality, 2012. © 2012 Wiley Periodicals, Inc.     

Limb bud colonization by somite-derived angioblasts is a crucial step for myoblast emigration

We have combined the use of mouse genetic strains and the mouse-into-chicken chimera system to determine precisely the sequence of forelimb colonization by presomitic mesoderm (PSM)-derived myoblasts and angioblasts, and the possible role of this latter cell type in myoblast guidance. By creating a new Flk1/Pax3 double reporter mouse line, we have established the precise timetable for angioblast and myoblast delamination/migration from the somite to the limb bud. This timetable was conserved when mouse PSM was grafted into a chicken host, which further validates the experimental model. The use of Pax3(GFP/GFP) knockout mice showed that establishment of vascular endothelial and smooth muscle cells (SMCs) is not compromised by the absence of Pax3. Of note, Pax3(GFP/GFP) knockout mouse PSM-derived cells can contribute to aortic, but not to limb, SMCs that are derived from the somatopleure. Finally, using the Flk1(lacZ)(/)(lacZ) knockout mouse, we show that, in the absence of angioblast and vascular network formation, myoblasts are prevented from migrating into the limb. Taken together, our study establishes for the first time the time schedule for endothelial and skeletal muscle cell colonization in the mouse limb bud and establishes the absolute requirement of endothelial cells for myoblast delamination and migration to the limb. It also reveals that cells delaminating from the somites display marked differentiation traits, suggesting that if a common progenitor exists, its lifespan is extremely short and restricted to the somite.     

Characterization of Two Virulent Phages of Lactobacillus plantarum

We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria.     

Sequential activation of poly(ADP-ribose) polymerase 1, calpains, and Bax is essential in apoptosis-inducing factor-mediated programmed necrosis

Alkylating DNA damage induces a necrotic type of programmed cell death through the poly(ADP-ribose) polymerases (PARP) and apoptosis-inducing factor (AIF). Following PARP activation, AIF is released from mitochondria and translocates to the nucleus, where it causes chromatin condensation and DNA fragmentation. By employing a large panel of gene knockout cells, we identified and describe here two essential molecular links between PARP and AIF: calpains and Bax. Alkylating DNA damage initiated a p53-independent form of death involving PARP-1 but not PARP-2. Once activated, PARP-1 mediated mitochondrial AIF release and necrosis through a mechanism requiring calpains but not cathepsins or caspases. Importantly, single ablation of the proapoptotic Bcl-2 family member Bax, but not Bak, prevented both AIF release and alkylating DNA damage-induced death. Thus, Bax is indispensable for this type of necrosis. Our data also revealed that Bcl-2 regulates N-methyl-N'-nitro-N'-nitrosoguanidine-induced necrosis. Finally, we established the molecular ordering of PARP-1, calpains, Bax, and AIF activation, and we showed that AIF downregulation confers resistance to alkylating DNA damage-induced necrosis. Our data shed new light on the mechanisms regulating AIF-dependent necrosis and support the notion that, like apoptosis, necrosis could be a highly regulated cell death program.