Biochemical characterization of a Caspase-3 far-red fluorescent probe for non-invasive optical imaging of neuronal apoptosis.

Apoptosis 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 cerebellar 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 H2O2 and PACAP, respectively, both in cell lysates and in cultured neurons. Finally, intravenous injection of the probe after cerebral 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.

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.

Effects of curcumin and curcumin derivatives on mitochondrial permeability transition pore.

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a natural compound with antiproliferative properties. Recent studies suggest that these properties might be due to the ability of curcumin to induce apoptosis in tumor cells by increasing the permeability of the mitochondrial membrane. In the present study, we confirm these observations and provide a molecular mechanism for the action of curcumin in rat liver mitochondria. Curcumin induced mitochondrial swelling, the collapse of Deltapsi, and the release of cytochrome C, events associated with the opening of the permeability transition pore (PTP). Experiments were performed with chemically substituted curcumin derivatives. Some derivatives were obtained by modification of groups on the terminal aromatic rings, and others were obtained by substitution of the diketone function with the cyclohexanone function. They demonstrated that phenol and methoxy groups were essential to promote PTP opening. Curcumin and curcumin derivatives that open the PTP were able to oxidize thiol groups. In addition, PTP opening was abolished in medium devoid of O2 and decreased in the presence of catalase, ferrozine, o-phenanthroline, mannitol, or N-ethylmaleimide. These data suggest that the mechanism by which curcumin promotes PTP opening involves the reduction of Fe3+ to Fe2+, inducing hydroxyl radical (HO*) production and oxidation of thiol groups in the membrane, leading to pore opening.