Multiple apoptotic pathways induced by p53-dependent acidification in benzo[a]pyrene-exposed hepatic F258 cells

Polycyclic aromatic hydrocarbons (PAH), such as benzo[a]pyrene (B[a]P), are ubiquitous genotoxic environmental pollutants. Their DNA-damaging effects lead to apoptosis induction, through similar pathways to those identified after exposure to other DNA-damaging stimuli with activation of p53-related genes and the involvement of the intrinsic apoptotic pathway. However, at a low concentration of B[a]P (50 nM), our previous results pointed to the involvement of intracellular pH (pHi) variations during B[a]P-induced apoptosis in a rat liver epithelial cell line (F258). In the present work, we identified the mitochondrial F0F1-ATPase activity reversal as possibly responsible for pHi decrease. This acidification not only promoted executive caspase activation, but also activated leucocyte elastase inhibitor/leucocyte-derived DNase II (LEI/L-DNase II) pathway. p53 appeared to regulate mitochondria homeostasis, by initiating F0F1-ATPase reversal and endonuclease G (Endo G) release. In conclusion, a low dose of B[a]P induced apoptosis by recruiting a large panel of executioners apparently depending on p53 phosphorylation and, for some of them, on acidification.     

Oleic acid is elevated in cell membranes during rapid cold-hardening and pupal diapause in the flesh fly, Sarcophaga crassipalpis

The integrity of cellular membranes is critical to the survival of insects at low temperatures, thus an advantage is conferred to insects that can adjust their composition of membrane fatty acids (FAs). Such changes contribute to homeoviscous adaption, a process that allows cellular membranes to maintain a liquid-crystalline state at temperatures that are potentially low enough to cause the membrane to enter the gel state and thereby lose its ability to maintain homeostasis. Flesh flies (Sarcophaga crassipalpis) were subjected to two experimental conditions that elicit low temperature tolerance: rapid cold-hardening and diapause. FAs were isolated and analyzed using gas chromatography-mass spectrometry. FAs changed in response to both rapid cold-hardening and diapause. In response to rapid cold-hardening (8 h at 4 degrees C), the proportion of oleic acid (18:1n-9) in pharate adults increased from 30% to 47% of the total FA pool. The proportion of almost every other FA was reduced. By entering diapause, pupae experienced an even greater increase in oleic acid proportion, to 58% of the total FA pool. Oleic acid not only promotes membrane fluidity at low temperature but also allows the cell membrane to maintain a liquid crystalline state if temperatures increase.     

Aminopeptidase N during the ontogeny of the chick

Little is known about the production and function of metallopeptidases in embryonic development. One such enzyme, aminopeptidase N (APN), is present in several epithelia, the brain and angiogenic vessels in adults. APN promotes vascular growth and endothelial cell proliferation in physiological and pathological models of angiogenesis. However, its possible role in embryonic angiogenesis or other developmental processes is unknown. Its expression profile in the early phase of embryonic development has not been reported. We report here the expression of this enzyme during the early development of the chick embryo, using complementary techniques for monitoring APN mRNA, protein, and enzymatic activity. We detected APN in the embryo as early as gastrulation. In addition to the known sites of APN production identified in both adults and rat fetuses toward the end of gestation, APN was found in unexpected sites, such as the primitive streak, the dorsal folds of the neural tube, the somites, and the primordia of several organs. APN was present mostly in the cardiovascular compartment during the first 13 days of incubation, and in the hematopoietic compartment (yolk sac and aorta-gonad-mesonephros region) early in development. This study provides clues as to the possible role of APN in embryonic development.     

Therapeutic peptides: Targeting the mitochondrion to modulate apoptosis

For many years, medical drug discovery has extensively exploited peptides as lead compounds. Currently, novel structures of therapeutic peptides are derived from active pre-existing peptides or from high-throughput screening, and optimized following a rational drug design approach. Molecules of interest may prove their ability to influence the disease outcome in animal models and must respond to a set of criteria based on toxicity studies, ease of administration, the cost of their synthesis, and logistic for clinical use to validate it as a good candidate in a therapeutic perspective. This applies to the potential use of peptides to target one central intracellular organelle, the mitochondrion, to modulate (i.e. activate or prevent) apoptosis. Putative mitochondrial protein targets and the strategies already elaborated to correct the defects linked to these proteins (overexpression, inactivation, mutation..., etc.) are described, and recent advances that led or may lead to the conception of therapeutic peptides via a specific action on these mitochondrial targets in the future are discussed.     

Normal acute and chronic inflammatory responses in sphingosine kinase 1 knockout mice

Sphingosine-1-phosophate, generated from the phosphorylation of sphingosine by sphingosine kinase enzymes, is suggested to function as an intracellular second messenger for inflammatory mediators, including formyl peptide, C5a, and Fc. More recently, a role for sphingosine kinases during inflammation has also been proposed. Here we show that sphingosine kinase 1 knockout mice exhibit normal inflammatory cell recruitment during thioglycollate-induced peritonitis and that sphingosine kinase 1-null neutrophils respond normally to formyl peptide. In the collagen-induced arthritis model of rheumatoid arthritis, sphingosine kinase 1 knockout mice developed arthritis with normal incidence and severity. Our findings show that sphingosine kinase 1 is dispensable for inflammatory responses and support the need for more extensive studies of sphingosine kinases in inflammation.     

Synthesis and primary cytotoxicity evaluation of arylmethylenenaphthofuranones derivatives

New series of 2(or 3)-arylmethylenenaphtho[2,1-b]furan-3(or 2)-ones were synthesized, characterized and tested for anticancer properties in vitro. The target compounds were prepared by Knoevenagel coupling between the naphthofuranones 3, 28-30 and formyl derivatives. 2-(4-Oxo-1-benzopyran-3-ylmethylene)naphtho[2,1-b]furan-3-one 36 was the most active compound (IC50 (L1210) = 1.6 microM). These compounds were also evaluated, in an independent manner, as inhibitors of Src protein tyrosine kinase, but only minor activity was observed.     

Sponge-associated microbial Antarctic communities exhibiting antimicrobial activity against Burkholderia cepacia complex bacteria

The aerobic heterotrophic bacterial communities isolated from three different Antarctic sponge species were analyzed for their ability to produce antimicrobial compounds active toward Cystic Fibrosis opportunistic pathogens belonging to the Burkholderia cepacia complex (Bcc). The phylogenetic analysis performed on the 16S rRNA genes affiliated the 140 bacterial strains analyzed to 15 genera. Just three of them (Psychrobacter, Pseudoalteromonas and Arthrobacter) were shared by the three sponges. The further Random Amplified Polymorphic DNA analysis allowed to demonstrate that microbial communities are highly sponge-specific and a very low degree of genus/species/strain sharing was detected. Data obtained revealed that most of these sponge-associated Antarctic bacteria and belonging to different genera were able to completely inhibit the growth of bacteria belonging to the Bcc. On the other hand, the same Antarctic strains did not have any effect on the growth of other pathogenic bacteria, strongly suggesting that the inhibition is specific for Bcc bacteria. Moreover, the antimicrobial compounds synthesized by the most active Antarctic bacteria are very likely Volatile Organic Compounds (VOCs), a finding that was confirmed by the SPME-GC-MS technique, which revealed the production of a large set of VOCs by a representative set of Antarctic bacteria. The synthesis of these VOCs appeared to be related neither to the presence of pks genes nor the presence of plasmid molecules. The whole body of data obtained in this work indicates that sponge-associated bacteria represent an untapped source for the identification of new antimicrobial compounds and are paving the way for the discovery of new drugs that can be efficiently and successfully used for the treatment of CF infections.     

Voltage-dependent-anion-channels (VDACs) in Arabidopsis have a dual localization in the cell but show a distinct role in mitochondria

In mammals, the Voltage-dependent anion channels (VDACs) are predominant proteins of the outer mitochondrial membrane (OMM) where they contribute to the exchange of small metabolites essential for respiration. They were shown to be as well associated with the plasma membrane (PM) and act as redox enzyme or are involved in ATP release for example. In Arabidopsis, we show that four out of six genomic sequences encode AtVDAC proteins. All four AtVDACs are ubiquitously expressed in the plant but each of them displays a specific expression pattern in root cell types. Using two complementary approaches, we demonstrate conclusively that the four expressed AtVDACs are targeted to both mitochondria and plasma membrane but in differential abundance, AtVDAC3 being the most abundant in PM, and conversely, AtVDAC4 almost exclusively associated with mitochondria. These are the first plant proteins to be shown to reside in both these two membranes. To investigate a putative function of AtVDACs, we analyzed T-DNA insertion lines in each of the corresponding genes. Knock-out mutants for AtVDAC1, AtVDAC2 and AtVDAC4 present slow growth, reduced fertility and yellow spots in leaves when atvdac3 does not show any visible difference compared to wildtype plants. Analyses of atvdac1 and atvdac4 reveal that yellow areas correspond to necrosis and the mitochondria are swollen in these two mutants. All these results suggest that, in spite of a localization in plasma membrane for three of them, AtVDAC1, AtVDAC2 and AtVDAC4 have a main function in mitochondria.     

An alternating GluN1-2-1-2 subunit arrangement in mature NMDA receptors

NMDA receptors (NMDARs) form glutamate-gated ion channels that play a critical role in CNS physiology and pathology. Together with AMPA and kainate receptors, NMDARs are known to operate as tetrameric complexes with four membrane-embedded subunits associating to form a single central ion-conducting pore. While AMPA and some kainate receptors can function as homomers, NMDARs are obligatory heteromers composed of homologous but distinct subunits, most usually of the GluN1 and GluN2 types. A fundamental structural feature of NMDARs, that of the subunit arrangement around the ion pore, is still controversial. Thus, in a typical NMDAR associating two GluN1 and two GluN2 subunits, there is evidence for both alternating 1/2/1/2 and non-alternating 1/1/2/2 arrangements. Here, using a combination of electrophysiological and cross-linking experiments, we provide evidence that functional GluN1/GluN2A receptors adopt the 1/2/1/2 arrangement in which like subunits are diagonal to one another. Moreover, based on the recent crystal structure of an AMPA receptor, we show that in the agonist-binding and pore regions, the GluN1 subunits occupy a "proximal" position, closer to the central axis of the channel pore than that of GluN2 subunits. Finally, results obtained with reducing agents that differ in their membrane permeability indicate that immature (intracellular) and functional (plasma-membrane inserted) pools of NMDARs can adopt different subunit arrangements, thus stressing the importance of discriminating between the two receptor pools in assembly studies. Elucidating the quaternary arrangement of NMDARs helps to define the interface between the subunits and to understand the mechanism and pharmacology of these key signaling receptors.