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Gentamicin is an aminoglucoside antibiotic used in clinics to cure severe infections due to Gram negative bacteria. After glomerular filtration, gentamicin is reabsorbed by endocytosis in the proximal tubular cells and accumulates principally inside lysosomes ( De Broe and al., 1984; Silverblatt et al., 1978; Tulkens et al;, 1978). At a therapeutic dose, gentamicin induces apoptosis in vivo in rat kidney (El Mouedden and al., 2000a). This process can be reproduced in cultured cells (LLC-PK1, MDCK) (El Mouedden and al., 2000b) and Servais et al have characterized the mechanism involved. They showed on LLC-PK1 cells that’s gentamicin induces a destabilization of the lysosomal membrane detected after 2 hours, a drop of the mitochondrial membrane potential after 10 hours, and an activation of caspace-3 in parallel with nuclear fragmentation after 24 hours (Servais and al., submitted).
In the present work, we tried to understand the cellular mechanisms involved in apoptosis induced by gentamicin 2mM and especially the roles of the lysosomes mitochondria.
The perméabilisation of the lysosomal membrane induced by gentamicin could be responsible for the release of lysosomal proteases which could be able in turn to part recruitment and activation of Bcl-2 prtoein family (like Bax) and to activate the mitochondrial pathway and release of cytochrome c e.g.
By western blot and RT-PCR, we showed an increase of Bax protein in the cells rtreated for 8 hours with 2mM gentamicin but, we did not notice any difference at the level of mRNA
Then, by cellular fractionation and western blot, we evidenced a translocation of Bax from cytosol to mitochondri. In parallel, cytochrome c underwent a translocation from mitochondri to cytosol. Lastly, using western blot, we observed a delocalisation of cathepsin D (a lysosomal protease that could be able to activate Bax) from MLP fraction to thee nuclear fraction and the cytosol and by DAPI technic we showed a decrease of the number of apoptosis cells if cells are pretreated with pepstatinA, an inhibitor of cathepsin D.
By these observations, we suggest that apoptsis induced by gentamicin in LLc-PK cells could be initiated by a permeabilisation of the lysosomal membranes after 2 hours, involving a release of proteases (including cathepsin D) and gentamicin which would be responsible for activation of mitochondria and release of cytochrom c from mitochondria to cytosol Résumé : La gentamicine, un antibiotique de la famille des aminoglycides, est utilisé en clinique pour traiter des infections sévères à bactéries Gram négatif. A dose thérapeutiques, la gentamicine induit de l’apoptose in vivo dans les reins de rats traités (el Mouedden et al., 2000a.). Ce processus a pu être reproduit sur plusieurs types cellulaires dont les fibroblastes d’embryons de rats et des cellules rénales de type LLC-PK1 et MDCK (El Mouedden et al, 2000b).Des travaux récents ont montré que, sur des cellules de type LLC-PK1, la gentamicine induit une perméabilité de la membrane lysosomiale détectée après 2 heurs. Cette perméabilisation lysososmiale serait suivie d’une pertubation du potentiel membranaire mitochondriale après 10 heures entrainant l’activation des caspases et la fragmentation nucléaire après 24 heures (Servais et al., soumis).
Notre travail a tenté de comprendre les mécanismes cellulaires impliqués dans l’apoptose induite par la gentamicine et plus particulièrement les rôles du lysosome et de la mitochondrie.
Dans un premier temps, nous nous sommes plus particulièrement intéressés à la cathepsine D et nous avons cherché à montrer par microscopie confocale et western blot, sur des cellules LLC-PK1 incubées en présence de gentamicine 2mM, une éventuelle délocalisation de cette protéase. Le western blot a permis d’observer une délocalisation de cette cathepsine de la fraction MLP vers la fraction nucléaire et le surnageant. Nous avons également testé l’effet de la pepstatine A, un inhibiteur de la cathepsine D et montré par la technique DAPI une diminution du nombre de cellules en apoptose en présence de cet inhibiteur.
Dans un second temps, nous avons tenté de mettre en évidence par western blot et par RT-PCR l’implication de la protéine Bax. Les résultats obtenus montrent qu’il y a bien une augmentation de la protéine Bax dans les cellules traitées à la gentamicine 2mM (pour des temps égaux ou supérieur à 8 heures d’incubation) mais, nous ne constatons aucune différence au niveau de l’ARNm indiquant qu’il ne s’agit pas d’une augmentation de la transcription. Enfin, par fractionnement cellulaire et western blot, nous avons cherché à montrer, comme pour la cathepsine D, des changements de localisation de la protéine Bax et du cytochrome c. Nous montrons qu’au cours de l’apoptose induite par la gentamicine, la protéine Bax est transloquée du cytosol à la mitochondrie tandis que le cytochrome c subit une translocation de la mitochondrie au cytosol.
Sur base de nos travaux, nous suggérons que l’apoptose induite par la gentamicine dans les cellules LLC-PK1 serait initiée par une perméabilisation de la membrane lysosomiale après 2 heures, entraînant un relargage de protéases lysosomiales (dont la cathepsine D) qui seraient responsables de l’activation de la mitochondrie. Au cours de cette cascade, il y aurait une activation de la protéine Bax participant à la perméabilisation mitochondriale et au relargage du cytochrome c

Gentamicins --- Cytochromes c --- Cathepsin D --- Apoptosis

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This book will describe the nuclear encoded genes and their expressed proteins of mitochondrial oxidative phosphorylation. Most of these genes occur in eukaryotic cells, but not in bacteria or archaea. The main function of mitochondria, the synthesis of ATP, is performed at subunits of proton pumps (complexes I, III, IV and V), which are encoded on mitochondrial DNA. The nuclear encoded subunits have mostly a regulatory function. However, the specific physiological functions of the nuclear encoded subunits of complexes I, III, IV, and V are mostly unknown. New data indicates that they are essential for life of higher organisms, which is characterized by an adult life without cell division (postmeiotic stage) in most tissues, after the juvenile growth. For complex IV (cytochrome c oxidase) some of these subunits occur in tissue-specific (subunits IV, VIa, VIb, VIIa, VIII), developmental-specific (subunits IV, VIa, and VIIa) as well as species-specific isoforms. Defective genes of some subunits were shown to induce mitochondrial diseases. Mitochondrial genes and human diseases will also be covered.

DNA, Mitochrondrial. --- Eukaryotic cells. --- Mitochondrial DNA. --- Mitochondrial DNA --- Mitochondrial pathology --- Eukaryotic cells --- Cytochrome c Group --- Subcellular Fractions --- Multienzyme Complexes --- Phosphorylation --- Metabolic Phenomena --- Energy Metabolism --- Organelles --- Phenomena and Processes --- Cytoplasmic Structures --- Biochemical Processes --- Cytochromes --- Cellular Structures --- Enzymes --- Chemical Processes --- Hemeproteins --- Enzymes and Coenzymes --- Biochemical Phenomena --- Cytoplasm --- Cells --- Proteins --- Chemicals and Drugs --- Intracellular Space --- Chemical Phenomena --- Anatomy --- Amino Acids, Peptides, and Proteins --- Oxidative Phosphorylation --- Electron Transport Chain Complex Proteins --- Metabolism --- Mitochondria --- Cytochromes c --- Biology --- Medicine --- Human Anatomy & Physiology --- Health & Biological Sciences --- Pathology --- Animal Biochemistry --- Genetics --- Mitochondria. --- Mitochondrial pathology. --- Phosphorylation. --- Mitochondrial disorders --- Chondriosomes --- Diseases --- Medicine. --- Human genetics. --- Gene expression. --- Cell biology. --- Biomedicine. --- Human Genetics. --- Gene Expression. --- Cell Biology. --- Cell biology --- Cellular biology --- Cytologists --- Genes --- Genetic regulation --- Heredity, Human --- Human biology --- Physical anthropology --- Clinical sciences --- Medical profession --- Life sciences --- Medical sciences --- Physicians --- Expression --- Chemical reactions --- Pathology, Cellular --- Cell organelles --- Protoplasm --- Disorders --- Cytology.