Détermination des effets mitochondriaux de la curcumine et de quelques dérivés (proposition d'un mécanisme d'action impliquant le pore de transition de perméabilité)

La mitochondrie joue un rôle clé dans la production des radicaux libres oxygénés (RLO) et dans le processus apoptotique. La première partie de ce travail a consisté à étudier les effets mitochondriaux de la curcumine et a révélé qu'elle réduisait les productions de RLO et induisait l'ouverture du pore de transition de perméabilité (PTP). Pour comprendre son mécanisme d'action, notre travail a, ensuite, consisté à tester les effets mitochondriaux de 22 dérivés de la curcumine obtenus par substitution de certaines fonctions chimiques. Cette étude révéla que la curcumine et certains dérivés induisaient l'ouverture du PTP en réduisant l'ion Fe entraînant la production du radical H0 qui oxyde certaines protéines membranaires au niveau de leurs fonctions thiols. D'autre part, cette propriété semble conférée par le groupe phénol associé au groupe méthoxy en ortho. Enfin, cette étude a mis en évidence des molécules fortement anti-oxydantes et d'autres potentiellement anti-cancéreuses.Mitochondria play an important role in free radical oxygen species (ROS) generation and in apoptotic process. The first part of tins work was the study of the mitochondrial effects of curcumin: it decreased ROS productions and induced permeability transition pore (PTP) opening. To understand its mechanism of action, we study, in the second part of this work, the mitochondrial effects of 22 curcumin-derivatives obtained after chemical substitutions. We showed that curcuniin and some derivatives iriduced Fe reduction leading to H0 generation. Then, tins radical promoted membrane thiol groups oxidation leading to PTP opening. The cheniical functions that are needed to promote PTP opening were the hydroxy and the methoxy groups located at the phenolic cycle. Furthermore, this study indicated that some curcumin-derivatives, like curcumin, could constitute a pool of antitumoral compounds whereas other compounds could be protective agents during oxidant stress.PARIS12-CRETEIL BU Multidisc. (940282102) / SudocSudocFranceF

Molecular analysis of resistance to streptogramin A compounds conferred by the Vga proteins of staphylococci.

The Vga and Msr resistance determinants, encoded by mobile genetic elements in various staphylococcal strains, belong to a family of ATP-binding cassette (ABC) proteins whose functions and structures are ill defined. Their amino acid sequences are similar to those of proteins involved in the immunity of streptomycetes to the macrolide-lincosamide-streptogramin antibiotics that they produce. Sequence analysis of the genomes of the gram-positive bacteria with low G+C contents revealed that Lmo0919 from Listeria monocytogenes is more closely related to Vga variants than to Msr variants. In the present study we compared the antibiotic resistance profiles conferred by the Vga-like proteins in two staphylococcal hosts. It was shown that Vga(A), the Vga(A) variant [Vga(A)v], and Lmo0919 can confer resistance to lincosamides and streptogramin A compounds, while only Vga(B) is able to increase the level of resistance to pristinamycin, a mixture of streptogramin A and streptogramin B compounds. By using polyclonal antibodies, we found that the Vga(A) protein colocalized with the beta subunit of the F(1)-F(0) ATPase in the membrane fractions of staphylococcal cells. In order to identify functional units in these atypical ABC proteins, such as regions that might be involved in substrate specificity and/or membrane targeting, we analyzed the resistance phenotypes conferred by various plasmids carrying parts or modified versions of the vga(A) gene and we determined the subcellular localization of the gene products. Only polypeptides composed of two ABC domains were detected in the cell membranes. No region of drug specificity was identified. Resistance properties were dependent on the integrities of both Walker B motifs

In Vitro and Ex Vivo Evaluation of Smart Infra-Red Fluorescent Caspase-3 Probes for Molecular Imaging of Cardiovascular Apoptosis

International audiencePURPOSE:The aim of this paper is to develop new optical bioprobes for the imaging of apoptosis.PROCEDURE:We developed quenched near-infrared probes which become fluorescent upon cleavage by caspase-3, the key regulatory enzyme of apoptosis.RESULTS:Probes were shown to be selectively cleaved by recombinant caspase-3. Apoptosis of cultured endothelial cells was associated with an increased fluorescent signal for the cleaved probes, which colocalized with caspase-3 and was reduced by the addition of a caspase-3 inhibitor. Flow cytometry demonstrated a similar profile between the cleaved probes and annexin V. Ex vivo experiments showed that sections of hearts obtained from mice treated with the proapoptotic drug doxorubicin displayed an increase in the fluorescent signal for the cleaved probes, which was reduced by a caspase-3 inhibitor.CONCLUSION:We demonstrated the capacity of these novel probes to detect apoptosis by optical imaging in vitro and ex vivo

Biochemical Characterization of a Caspase-3 Far-red Fluorescent Probe for Non-invasive Optical Imaging of Neuronal Apoptosis

International audienceApoptosis is a regulated process, leading to cell death, which is involved in several pathologies including neurodegenerative diseases and stroke. Caspase-3 is a key enzyme of the apoptotic pathway and is considered as a major target for the treatment of abnormal cell death. Sensitive and non-invasive methods to monitor caspase-3 activity in cells and in the brain of living animals are needed to test the efficiency of novel therapeutic strategies. In the present study, we have biochemically characterized a caspase-3 far-red fluorescent probe, QCASP3.2, that can be used to detect apoptosis in vivo. The specificity of cleavage of QCASP3.2 was demonstrated using recombinant caspases and protease inhibitors. The functionality of the probe was also established in cere-bellar neurons cultured in apoptotic conditions. QCASP3.2 did not exhibit any toxicity and appeared to accurately reflect the induction and inhibition of caspase activity by H 2 O 2 and PACAP, respectively, both in cell lysates and in cultured neurons. Finally, intravenous injection of the probe after cere-bral ischemia revealed activation of caspase-3 in the infarcted hemisphere. Thus, the present study demonstrates that QCASP3.2 is a suitable probe to monitor apoptosis both in vitro and in vivo and illustrates some of the possible applications of this caspase-3 fluorescent probe