Homologous recombination-mediated repair of DNA double-strand breaks operates in mammalian mitochondria

Mitochondrial DNA is frequently exposed to oxidative damage, as compared to nuclear DNA. Previously, we have shown that while microhomology-mediated end joining can account for DNA deletions in mitochondria, classical nonhomologous DNA end joining, the predominant double-strand break (DSB) repair pathway in nucleus, is undetectable. In the present study, we investigated the presence of homologous recombination (HR) in mitochondria to maintain its genomic integrity. Biochemical studies revealed that HR-mediated repair of DSBs is more efficient in the mitochondria of testes as compared to that of brain, kidney and spleen. Interestingly, a significant increase in the efficiency of HR was observed when a DSB was introduced. Analyses of the clones suggest that most of the recombinants were generated through reciprocal exchange, while ~ 30% of recombinants were due to gene conversion in testicular extracts. Colocalization and immunoblotting studies showed the presence of RAD51 and MRN complex proteins in the mitochondria and immunodepletion of MRE11, RAD51 or NIBRIN suppressed the HR-mediated repair. Thus, our results reveal importance of homologous recombination in the maintenance of mitochondrial genome stability.     

The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells

Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design.     

Dynamics of proteins in Golgi membranes: comparisons between mammalian and plant cells highlighted by photobleaching techniques

In less than a decade the green fluorescent protein (GFP) has become one of the most popular tools for cell biologists for the study of dynamic processes in vivo. GFP has revolutionised the scientific approach for the study of vital organelles, such as the Golgi apparatus. As Golgi proteins can be tagged with GFP, in most cases without altering their targeting and function, it is a great substitute to conventional dyes used in the past to highlight this compartment. In this review, we cover the application of GFP and its spectral derivatives in the study of Golgi dynamics in mammalian and plant cells. In particular, we focus on the technique of selective photobleaching known as fluorescence recovery after photobleaching, which has successfully shed light on essential differences in the biology of the Golgi apparatus in mammalian and plant cells.     

Induction of neovascularization in vivo by glycerol

Glycerol, injected into a site between the femoral vessels of the rat, induced neovascularization, both from the preexisting microcirculation and from the side of the femoral vein facing the artery-vein interstitium where the glycerol was administered. The use of glycerol together with a known angiogenic substance (PGE₂) did not modify the neocapillary density (NCD) obtained with glycerol alone. In contrast, the lower level of NCD achieved with an acylglycerol (triacetylglycerol) was increased when the latter was associated with PGE₂. Values reached were similar to, but never higher than, those for glycerol alone, or combined with PGE₂. The results suggest that glycerol and some substances containing glycerol, amongst which 1-butyrylglycerol has been previously considered¹, may stimulate angiogenesis by a direct or indirect mechanism of action.     

Induction of the formation of a coli-bacterio-phage and colicin by hydroperoxide

Zusammenfassung Die Arbeit gibt die Resultate der Induktion der Lysogenie und der Colicinogenie im selben StammeE. coli (L II-18) mit Wasserstoffperoxid an. Der Phagentiter wurde 35mal, der Colicintiter 7mal erhöht. Die optimale Konzentration des H₂O₂ für Phagenbildung warM/500, die für ColicinbildungM/2000. Im Vergleich mit der Induktion desselben Stammes mit UV-Licht ist die Wirkung des H₂O₂ verhältnismässig schwach.     

Uptake by mammalian cells of nucleic acids combined with a basic protein

Résumé Les acides nucléiques se combinent avec l'albumine méthylée et forment des complexes qui ne sont pas hydrolysés par les nucléases, mais dissociés par des ions inorganiques. Ces nucléoprotéines pénètrent dans les cellules en quantité beaucoup plus élevée que les acides nucléiques correspondants, et contrairement à ceux-ci, elles ne sont pas catabolisées. La combinaison avec l'albumine méthylée rend inactif l'acide ribonucléique viral: les ions inorganiques, et non les cellules vivantes, sont capables de dissocier ce complexe et de libérer l'ARN infectieux.     

Induction of morphological changes in bacteria by optochin

Zusammenfassung Es wird die Wirkung des Optochins auf die Zellmorphologie desBacterium anitratum beschrieben und aus Versuchsergebnissen geschlossen, dass Optochin zur Gruppe chemischer Substanzen gehört, die zu hochgradigen morphologischen Veränderungen der Bakterienzelle führen.     

Inhibition of mammalian acetylcholinesterase by phenylmethanesulfonyl fluoride

Resumen El fenilmentano sulfonil fluoro (FMSF) 4×10^–5 M inhibe a la acetilcolinesterasa (E. C. 3.1.1.7) de cerebro de rata en más de 50%, y 9×10^–4 M más de 90%. La constante de reacción inhibidor enzima es de 4.7×10² (1 mol^–1 min^–1). La colinesterasa (E. C. 3.1.1.8) de cerebro de rata es insensible a FMSF.

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Supported by NIMH Grant No. 1 RO3 MH 19125, by Comisión Nacional de Investigación Cientiffica Proyecto No. 80, and by Comisión de Investigación Cientiffica Universidad de Chile, Proyecto No. 95.     

Induction of uncoiled chromosomes by vibration

Summary Chromatin condensation during metaphase can be removed by simple vibration of metaphase cells prior to fixation. Uncoiled chromosome arms consist of long threads with dense regions at irregular distances each from the other.Acknowledgment. This work is supported by the Hellenic Anticancer Institute.