Inhibition of Homologous Recombination by the Plasmid MucA′B Complex

By its functional interaction with a RecA polymer, the mutagenic UmuD′C complex possesses an antirecombination activity. We show here that MucA′B, a functional homolog of the UmuD′C complex, inhibits homologous recombination as well. In F⁻ recipients expressing MucA′B from a P_tac promoter, Hfr × F⁻ recombination decreased with increasing MucA′B concentrations down to 50-fold. In damage-induced pKM101-containing cells expressing MucA′B from the native promoter, recombination between a UV-damaged F lac plasmid and homologous chromosomal DNA decreased 10-fold. Overexpression of MucA′B together with UmuD′C resulted in a synergistic inhibition of recombination. RecA[UmuR] proteins, which are resistant to UmuD′C inhibition of recombination, are inhibited by MucA′B while promoting MucA′B-promoted mutagenesis efficiently. The data suggest that MucA′B and UmuD′C contact a RecA polymer at distinct sites. The MucA′B complex was more active than UmuD′C in promoting UV mutagenesis, yet it did not inhibit recombination more than UmuD′C does. The enhanced mutagenic potential of MucA′B may result from its inherent superior capacity to assist DNA polymerase in trans-lesion synthesis. In the course of this work, we found that the natural plasmid pKM101 expresses around 45,000 MucA and 13,000 MucB molecules per lexA(Def) cell devoid of LexA. These molecular Muc concentrations are far above those of the chromosomally encoded Umu counterparts. Plasmid pKM101 belongs to a family of broad-host-range conjugative plasmids. The elevated levels of the Muc proteins might be required for successful installation of pKM101-like plasmids into a variety of host cells.     

Effect of ischemia/reperfusion sequence on cytosolic iron status and its release in the coronary effluent in isolated rat hearts

The hypothesis that oxygen-derived free radicals play an important role in myocardial ischemic and reperfusion injury has received a lot of support. In the presence of catalytic amounts of transition metals such as iron, superoxide anions, and hydrogen peroxide can be transformed into a highly reactive hydroxyl radical °OH (Haber-Weiss reaction). In view of this, we have undertaken this study to investigate whether iron is involved in the reperfusion syndrome and therefore could aggravate free radicals injury. Coronary effluent iron concentrations and cardiac cytosolic iron levels were evaluated in rat hearts subjected to an ischemia/reperfusion sequences. In the case of total ischemia, iron concentration in coronary effluents peaked immediately in the first sample collected upon reperfusion. However, in the case of partial ischemia, iron concentration in coronary effluents peaked rather exclusively during ischemia period. Cardiac cytosolic iron level augmented significantly after 30 min of total ischemia and non significantly in the other ischemia protocols compared to perfused control hearts. It also appears that the iron released is not protein-bound, and could therefore have a marked catalytic activity. The results of the present study suggest that in the oxygen paradox, iron plays an important role in inducing alterations during reoxygenation.     

Kiwi fruit PMEI inhibits PME activity,modulates root elongation and induces pollen tube burst in Arabidopsis thaliana

Pectins are major components of primary cell wall that play a crucial role in plant development. After biosynthesis, pectins are secreted in the cell wall by Golgi-derived vesicles under a highly methylesterified form and are de-methylesterified by pectin methylesterases (PME). It is hypothesized that PME might be regulated by pectin methylesterase inhibitor (PMEI). In this paper, we show by isoelectric focalisation and subsequent zymogram that kiwi PMEI was able to inhibit Arabidopsis PME activity by forming a complex. The complexes were stable under a wide range of ionic strength and pH. Moreover, PMEI might be able to form a complex with basic PMEs including three PMEs strongly expressed in root and four PMEs expressed in pollen grains. Finally, exogenous treatment with kiwi PMEI was able to reduce the activity of cell wall resident PMEs with persistent effects such as an increase of the root growth and a dramatic effect on pollen tube stability.     

Obesity epidemic: origins and consequences

Obesity is a chronic disease with serious health consequences. Initial weight gain is related to behavioural and environmental factors acting on a biological (mainly genetic) predisposition. The evolution of the disease is characterized by the development of an inflammatory organ disease that involves the adipocytes and other adipose tissue components. These alterations lead to various clinical complications and to a progressive resistance to diet effects. The treatment of obesity must be adapted to the stage of development of the disease and to the prevalent complications.     

Cytochemical evidence of an organized microtubular cytoskeleton in Xenopus laevis oocytes: involvement in the segregation of mitochondrial populations.

An organized microtubular cytoskeleton was discovered in the cytoplasm of Xenopus laevis oocytes. The microtubules were observed in 10- to 30-micron cryostat sections by indirect immunoperoxidase labeling using an antibody to tubulin. A gradual extraction of cells with a nonionic detergent was essential for good penetration of the antibody into the cells. In the cytoplasm of all previtellogenic oocytes, a dense network of criss-crossed long microtubules was associated in a basket-like structure surrounding the mitochondrial mass. At the beginning of vitellogenesis, the network meshes enlarged, while clusters of mitochondria migrated, in close association with microtubule bundles. At the beginning of vitellogenesis, the reorganization of the microtubular network, mostly in the vegetal hemisphere, occurred during the segregation of the mitochondrial populations. Reorganization is characterized by (1) a temporary enlargement of the network and close association of mitochondrial clusters with microtubular bundles, and (2) a progressive organization of a ring-shaped microtubular structure in the crown elaboration area. It is hypothesized that these modifications of the microtubular cytoskeleton contribute to the maintenance of cell shape and the polarized organization of the cell.     

Combined Approaches for Drug Design Points the Way to Novel Proline Racemase Inhibitor Candidates to Fight Chagas’ Disease

Chagas’ disease is caused by Trypanosoma cruzi, a protozoan transmitted to humans by blood-feeding insects, blood transfusion or congenitally. Previous research led us to discover a parasite proline racemase (TcPRAC) and to establish its validity as a target for the design of new chemotherapies against the disease, including its chronic form. A known inhibitor of proline racemases, 2-pyrrolecarboxylic acid (PYC), is water-insoluble. We synthesized soluble pyrazole derivatives, but they proved weak or inactive TcPRAC inhibitors. TcPRAC catalytic site is too small and constrained when bound to PYC to allow efficient search for new inhibitors by virtual screening. Forty-nine intermediate conformations between the opened enzyme structure and the closed liganded one were built by calculating a transition path with a method we developed. A wider range of chemical compounds could dock in the partially opened intermediate active site models in silico. Four models were selected for known substrates and weak inhibitors could dock in them and were used to screen chemical libraries. Two identified soluble compounds, (E)-4-oxopent-2-enoic acid (OxoPA) and its derivative (E)-5-bromo-4-oxopent-2-enoic acid (Br-OxoPA), are irreversible competitive inhibitors that presented stronger activity than PYC on TcPRAC. We show here that increasing doses of OxoPA and Br-OxoPA hamper T. cruzi intracellular differentiation and fate in mammalian host cells. Our data confirm that through to their binding mode, these molecules are interesting and promising as lead compounds for the development of chemotherapies against diseases where active proline racemases play essential roles.     

Domain 3 of NS5A protein from the hepatitis C virus has intrinsic alpha-helical propensity and is a substrate of cyclophilin A

Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and constitutes an attractive target for antiviral drug development. Although structural data for its in-plane membrane anchor and domain D1 are available, the structure of domains 2 (D2) and 3 (D3) remain poorly defined. We report here a comparative molecular characterization of the NS5A-D3 domains of the HCV JFH-1 (genotype 2a) and Con1 (genotype 1b) strains. Combining gel filtration, CD, and NMR spectroscopy analyses, we show that NS5A-D3 is natively unfolded. However, NS5A-D3 domains from both JFH-1 and Con1 strains exhibit a propensity to partially fold into an α-helix. NMR analysis identifies two putative α-helices, for which a molecular model could be obtained. The amphipathic nature of the first helix and its conservation in all genotypes suggest that it might correspond to a molecular recognition element and, as such, promote the interaction with relevant biological partner(s). Because mutations conferring resistance to cyclophilin inhibitors have been mapped into NS5A-D3, we also investigated the functional interaction between NS5A-D3 and cyclophilin A (CypA). CypA indeed interacts with NS5A-D3, and this interaction is completely abolished by cyclosporin A. NMR heteronuclear exchange experiments demonstrate that CypA has in vitro peptidyl-prolyl cis/trans-isomerase activity toward some, but not all, of the peptidyl-prolyl bonds in NS5A-D3. These studies lead to novel insights into the structural features of NS5A-D3 and its relationships with CypA.