Molecular basis of diseases caused by the mtDNA mutation m.8969G>A in the subunit a of ATP synthase

The ATP synthase which provides aerobic eukaryotes with ATP, organizes into a membrane-extrinsic catalytic domain, where ATP is generated, and a membrane-embedded F_O domain that shuttles protons across the membrane. We previously identified a mutation in the mitochondrial MT-ATP6 gene (m.8969G>A) in a 14-year-old Chinese female who developed an isolated nephropathy followed by brain and muscle problems. This mutation replaces a highly conserved serine residue into asparagine at amino acid position 148 of the membrane-embedded subunit a of ATP synthase. We showed that an equivalent of this mutation in yeast (aS₁₇₅N) prevents F_O-mediated proton translocation. Herein we identified four first-site intragenic suppressors (aN₁₇₅D, aN₁₇₅K, aN₁₇₅I, and aN₁₇₅T), which, in light of a recently published atomic structure of yeast F_O indicates that the detrimental consequences of the original mutation result from the establishment of hydrogen bonds between aN₁₇₅ and a nearby glutamate residue (aE₁₇₂) that was proposed to be critical for the exit of protons from the ATP synthase towards the mitochondrial matrix. Interestingly also, we found that the aS₁₇₅N mutation can be suppressed by second-site suppressors (aP₁₂S, aI₁₇₁F, aI₁₇₁N, aI₂₃₉F, and aI₂₀₀M), of which some are very distantly located (by 20–30?Å) from the original mutation. The possibility to compensate through long-range effects the aS₁₇₅N mutation is an interesting observation that holds promise for the development of therapeutic molecules.     

Changes in peroxidases in the suspension culture of Rubus fruticosus during growth

The growth parameters of a cell suspension culture of Rubus fruticosus L. were determined over a culture period including exponential growth, stationary phase and a glucose starvation period at the end of the normal culture cycle. Peroxidase activities were measured in the cytoplasm, in the cell wall, and in the culture medium by the guaiacol assay. There is a relationship between the activity found in the spent medium and the dry matter mass of the cells during the exponential growth. In the three compartments a bimodal repartition of peroxidase activities was observed, with the two peaks at day 4 and day 26, respectively. This suggests that the first peak corresponds to actively dividing cells whereas the second is associated with senescence, or stress due to starvation. Fractionation of the peroxidases from the culture mediuim revealed the presence of two sets of cationic isoenzymes, with minor amount of anionic peroxidases. Interestingly, the second peak of cationic enzymes which was of weak intensity at day 10 of the culture, becameprevalent at day 26. This indicates that not only the total amount of peroxidases varies as a function of culture time, but also that the nature of the peroxidases secreted into the medium changes during growth.Abbreviations DW dry weight - FW fresh weight - MV medium volume - SV suspension volume - BSA bovine serum albumin     

Actin Depolymerizing Factor (ADF/Cofilin) Enhances the Rate of Filament Turnover: Implication in Actin-based Motility

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.     

Contribution a l'étude desAphelinidae aphidiphages et de leurs hotes en France [Hym. Chalcidoidea]

Résumé L'auteur donne un nouvel exemple de variabilité de la pigmentation chez un Aphelinide. L'inventaire des Aphelinides de France est dressé; 26 h?tes nouveaux sont précisés. L'observation du comportement de plusieurs espèces d'Aphelinides dans la nature a révélé l'importance du facteur de l'humidité ambiante pour l'apparition de taux de parasitisme élevés.

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Summary New data on the pigmentation variability byA. chaonia Wlk. (Aphelinidae) under natural conditions are given. The provisional list of the 11 species ofAphelinidae collected in France till now includesAphelinus flavipes Foerster andA. varipes Foerster, two species not recorded before in this country; 26 new hosts are recorded as parasites of 6 species ofAphelinus. Field observations on several Aphelinids emphasize the importance of the high level of surrounding humidity to determine high parasitism rates.

     

Première expérimentation écologique sur les Entomophthorales (Phycomycètes) parasites de pucerons en vergers de pêchers

Résumé Un essai infructueux d'implantation de l'entomophthorose dans une population deMyzus persicae en verger de Pêcher a révélé les principales difficultés inhérentes à ce genre d'expérimentation écologique où l'apport d'inoculum est accompagné d'une tentative de modification du milieu par des aspersions contr?lées. L'épizootiologie de l'entomophthorose dans les vergers de la vallée du Rh?ne est discutée en fonction de la fréquence des jours pendant lesquels l'humidité relative demeure élevée pendant plus de 12 heures et la température maximale excède 20°C.

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Summary A preliminary experiment has been undertaken in May 1970 in the Rhone Valley in order to implant focuses ofEntomophthora aphidis Hoffm. andE. thaxteriana Petch. in colonies ofMyzus persicae Sulz. and to study the effect of sprinkling. The aim of the sprinkling is to provide the presence of a continuous film of water on the leaves of the trees during the delay (8 to 14 hours) required by the critical phase of the cycle of the pathogen (sporulation-infection). Four plots were considered: The check-plot without inoculum or sprinkling, a plot with inoculum but without sprinkling, a plot with sprinkling but without inoculum and the fourth plot with inoculum and sprinkling. By an extra infestation, an homogenous population has been established on the 6 trees of each plot. The inoculum consists partly in cultures of the fungous in Petri dishes, partly in dead aphids in the phase of projection of conidia. The failure of the experiment is attributed to the very quick destruction of the inoculum by sorching winds, to the working-failure of the sprinkling system and specially to the adversity of the climatic conditions during the last decade of May (no diurnal rains, high frequency of windy days). The epizootiology of Entomophthorales in orchards of the Rhone Valley is discussed at light of the frequence of the days with high relative humidity during more than 12 hours and that of the days with maximal temperatures exceeding 20°C.

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Communication présentée à la 2^e réunion du groupe de travail PBI ?Biological Control of Aphids?, Paris, 16–18 septembre 1970.     

An alternative approach for gene transfer in trees using wild-typeAgrobacterium strains

Micropropagated shoots of three forest tree species, poplar (Populus tremula × P.alba), wild cherry (Prunus avium L.) and walnut (Juglans nigra × J. regia), were inoculated each with six different wild-typeAgrobacterium strains. Poplar and wild cherry developed tumors that grew hormone-independently, whereas on walnut, gall formation was weak. On poplar and wild cherry, tumors induced by nopaline strains developed spontaneously shoots that had a normal phenotype and did not carry oncogenic T-DNA. From these observations, we have established a co-inoculation method to transform plants, using poplar as an experimental model. The method is based on inoculation of stem internodes with anAgrobacterium suspension containing both an oncogenic strain that induces shoot differentiation and a disarmed strain that provides the suitable genes in a binary vector. We used the vector pBI121 carryingneo (kanamycin resistance) anduidA (-glucuronidase) genes to facilitate early selection and screening. Poplar plants derived from kanamycin-resistant shoots that did not carry oncogenic T-DNA, were shown to contain and to expressneo anduidA genes. These results suggest that wild-typeAgrobacterium strains that induce shoot formation directly from tumors can be used as a general tool for gene transfer, avoiding difficult regeneration procedures.This work is dedicated to the late Marie-France Michel who initiated the poplar biotechnology project at INRA.     

Metabolic network visualization eliminating node redundance and preserving metabolic pathways

Background  

The tools that are available to draw and to manipulate the representations of metabolism are usually restricted to metabolic pathways. This limitation becomes problematic when studying processes that span several pathways. The various attempts that have been made to draw genome-scale metabolic networks are confronted with two shortcomings: 1- they do not use contextual information which leads to dense, hard to interpret drawings, 2- they impose to fit to very constrained standards, which implies, in particular, duplicating nodes making topological analysis considerably more difficult.