Secreted proteins produced by fungi associated with Botryosphaeria dieback trigger distinct defense responses in Vitis vinifera and Vitis rupestris cells.

Grapevine trunk diseases (Eutypa dieback, esca and Botryosphaeria dieback) are caused by a complex of xylem-inhabiting fungi, which severely reduce yields in vineyards. Botryosphaeria dieback is associated with Botryosphaeriaceae. In order to develop effective strategies against Botryosphaeria dieback, we investigated the molecular basis of grapevine interactions with a virulent species, Neofusicoccum parvum, and a weak pathogen, Diplodia seriata. We investigated defenses induced by purified secreted fungal proteins within suspension cells of Vitis (Vitis rupestris and Vitis vinifera cv. Gewurztraminer) with putative different susceptibility to Botryosphaeria dieback. Our results show that Vitis cells are able to detect secreted proteins produced by Botryosphaeriaceae, resulting in a rapid alkalinization of the extracellular medium and the production of reactive oxygen species. Concerning early defense responses, N. parvum proteins induced a more intense response compared to D. seriata. Early and late defense responses, i.e., extracellular medium alkalinization, cell death, and expression of PR defense genes were stronger in V. rupestris compared to V. vinifera, except for stilbene production. Secreted Botryosphaeriaceae proteins triggered a high accumulation of δ-viniferin in V. vinifera suspension cells. Artificial inoculation assays on detached canes with N. parvum and D. seriata showed that the development of necrosis is reduced in V. rupestris compared to V. vinifera cv. Gewurztraminer. This may be related to a more efficient induction of defense responses in V. rupestris, although not sufficient to completely inhibit fungal colonization. Overall, our work shows a specific signature of defense responses depending on the grapevine genotype and the fungal species.

Changes in morphology and in polyphosphoinositide turnover of human erythrocytes after cholesterol depletion.

Human erythrocytes were cholesterol-depleted (5-25%) by incubation with phosphatidylcholine vesicles in media containing Ca2+ at different concentrations (0, 28 nM, 5 microM or 1 mM). After removal of the vesicles, the cells were reincubated with [32P]phosphate in the same media. Control (incubated in buffer alone) and cholesterol-maintained erythrocytes (incubated with cholesterol/phosphatidylcholine vesicles) were treated similarly. Cholesterol depletion induced the conversion of the cells into stomatocytes III and spherostomatocytes and decreased the turnover rate of phosphatidylinositol phosphate and of phosphatidylinositol bisphosphate. None of these effects were observed in cholesterol-maintained cells. In cholesterol-depleted cells, they occurred without changes in the ATP specific activity or in the polyphosphoinositide concentrations. Moreover, these modifications of shape and of lipid metabolism were proportional to the extent of the cholesterol depletion and were independent of the external Ca2+ concentration. In contrast, other effects of cholesterol depletion, a decrease in the turnover rate of phosphatidic acid, a decrease in diacylglycerol and in phosphatidic acid concentrations were dependent on the external Ca2+ concentration. Thus it appears that the shape change was not correlated with a change in the concentrations of these phospholipids or of diacylglycerol and therefore cannot be explained by a bilayer couple mechanism involving these phospholipids. However, the spherostomatocytic transformation was correlated with the decrease in the turnover rate of the polyphosphoinositides, but not with the turnover rate of phosphatidic acid, suggesting a role for the turnover of the polyphosphoinositides in the maintenance of the erythrocyte shape.

A high-density immunoblotting methodology for quantification of total protein levels and phosphorylation modifications.

The components of many signaling pathways have been identified and there is now a need to conduct quantitative data-rich temporal experiments for systems biology and modeling approaches to better understand pathway dynamics and regulation. Here we present a modified Western blotting method that allows the rapid and reproducible quantification and analysis of hundreds of data points per day on proteins and their phosphorylation state at individual sites. The approach is of particular use where samples show a high degree of sample-to-sample variability such as primary cells from multiple donors. We present a case study on the analysis of >800 phosphorylation data points from three phosphorylation sites in three signaling proteins over multiple time points from platelets isolated from ten donors, demonstrating the technique's potential to determine kinetic and regulatory information from limited cell numbers and to investigate signaling variation within a population. We envisage the approach being of use in the analysis of many cellular processes such as signaling pathway dynamics to identify regulatory feedback loops and the investigation of potential drug/inhibitor responses, using primary cells and tissues, to generate information about how a cell's physiological state changes over time.

Changes in the respiratory chain complexes activities and in the mitochondrial DNA content during ageing in D. subobscura.

The time course (age 0-8 weeks) of the enzyme activities of respiratory chain complexes I, III and IV and of citrate synthase, and the cell mitochondrial/nuclear DNA content ratio were studied in Drosophila subobscura. The activities of the three respiratory complexes decreased with age, but with different kinetics. The activities of complexes I and III remained nearly stable between weeks 0 and 3 (falling by 6% and 15%, respectively), and then gradually decreased; after 8 weeks residual activities were about 50% of the initial value for complexes I and III. The activity of complex IV fell in the first week, decreasing continually to week 8, where residual activity was 30% of the initial value. No significant age-related change in citrate synthase activity was observed. Mitochondrial DNA (measured by mitDNA/nucDNA) increased linearly up to week 5 (2.6-fold) and then dropped by 40% in week 6 though it remained higher than initial values.

Three-dimensional modelling of the motion range of axial rotation of the upper arm.

Computer simulation of human movements and postures suffers generally from the lack of reliable data, such as data on joint limits. In this paper, the motion range of axial rotation of the upper arm was investigated. An original surface regression fitting method using an orthogonal homogeneous polynomial basis has been presented. The method was used to fit both maximum internal and external upper arm axial rotation surfaces based on the experimental data from seven male subjects of ages 23-34. Under the assumption of normal distribution, the sample mean and the confidence limits for population mean of both internal and external upper arm axial rotation limits were derived. It has been shown that the axial motion range of the upper arm depends strongly on the position of the upper arm in the shoulder sinus cone and varies on average from 94 to 157 degrees. The present work can be considered as an extension of the shoulder kinematic data base established by Engin and Chen (1986), Journal of Biomechanical Engineering 108, 215-221.

[Snake bite by European vipers. A multicenter study of tolerance to Viperfav, a new intravenous antivenom]. / Envenimations par vipères européennes. Etude multicentrique de tolérance du Viperfav, nouvel antivenin par voie intraveineuse.

OBJECTIVES: To evaluate the tolerance and the effectiveness of i.v. Viperfav, a new antivenom containing F(ab')2 fragments of equine antibodies, for the treatment of European viper envenomed patients. STUDY DESIGN: Open, multicentre field trial, associated with a cohort study. PATIENTS: The study included 46 patients of either gender, nine aged less then 10 years, eight between 10 and 15 years, and 28 adults, who sustained a moderate or severe viper envenomation (Grade 2 or 3). METHOD: At the inclusion, a single infusion of Viperfav was given. Depending on the clinical course, up to four additional infusions were to have been administered at 4-hour intervals. To evaluate tolerance, all symptoms were recorded. There were three effectiveness evaluation criterion (duration of hospitalisation, course of the severity grade, recovery (sequelae)) and one subjective criteria (value of the antivenom as ascertained by investigators). RESULTS: In the 46 included patients, 79 infusions were administrated. Concerning tolerance, six mild symptoms were associated to the antivenom infusions. No severe reaction occurred. The mean duration of hospitalisation was 4 days 19 hours +/- 13 hours. A severity grade decrease by at least one point was observed in 35 patients, and all were discharged without sequelae. For the investigators the antivenom was inefficient in only two patients (grade 3 with tissue lesions). CONCLUSIONS: In comparison with literature data (5 to 10% of severe reactions attributable to the antivenom), the tolerance of Viperfav can be considered as satisfactory. As all criteria were in favour of a positive benefit to risk ratio, the authors recommend the use of Viperfav i.v. for the grade 2 and 3 envenomations instead of the current less purified antivenom, which can only be administered by the intramuscular route.

Filipin and digitonin studies of membrane cholesterol in frog atrial fibers with unusual gap junction configurations.

The gap junctions of frog myocardium present, in freeze-fracture, an atypical organisation of their junctional particles. Freeze-fracture cytochemistry with the cholesterol probes filipin and digitonin has been used to investigate whether the particular arrangement of the particles involves a lipid segregation in the plane of the membrane. Both probes labeled uniformly the non-junctional membrane, but no deformations were ever found inside the smooth membrane area circumscribed by the circle of junctional particles. At the level of junction formation zones almost no sterol probe complexes were found in the intramembranous particle free area which surrounded small clusters of junctional particles. These results suggest a regional variation in cholesterol related to the necessity of membrane fluidity during junction morphogenesis.

[The junctions of the sinoatrial fascicle in frogs]. / Etude des jonctions des faisceaux sino-auriculaires de grenouille

The demonstration of gap-junctions on frog atria muscle similar to those described on mammalian myocardium has been controversed. En bloc staining with uranyl acetate allows us to show such gap junctions on this tissue.

[Disorganization of the structure of gap junctions isolated from rat liver and treated by the action of a mixture of 2 detergents]. / Désorganisation de la structure des jonctions gap isolées de foie de rat et soumises à l'action d'un mélange de deux détergents.

Isolated rat liver gap junctions were treated with two non denaturant detergents, digitonin and octyl glucoside. The structural changes of the gap junctions were observed by electron microscopy (thin sections and freeze-fracture). Octyl glucoside alone was inefficient in modifying gap junction structure. Digitonin induced a partial spacing of the two adjacent junctional membranes. The combined action of the two detergents resulted in a complete disorganization of the particle network normally seen in freeze-fracture and of the pentalaminar sheet seen in thin sections. This disappearance of the junction structure suggests that the inter-subunits bonds have been disrupted.

Freeze-fracture studies of frog atrial fibres.

The freeze-fracturing technique was used to characterize the junctional devices involved in the electrical coupling of frog atrial fibres. These fibres are connected by a type of junction which can be interpreted as a morphological variant of the "gap junction" or "nexus". The most characteristic features are rows of 9-nm junctional particles forming single or anastomosed circular profiles on the inner membrane face, and corresponding pits on the outer membrane face. Very seldom aggregates consisting of few geometrically disposed 9-nm particles are found. The significance of the junctional structures in the atrial fibres is discussed, with respect to present knowledge about junctional features of gap junctions in various tissues, including embryonic ones.

[Physiological importance of gap junctions]. / Importance physiologique des jonctions communicantes.

Some structural features of the different types of intercellular junctions which occur in vertebrate tissues (desmosomes, tight and gap junctions, Table 1) are first mentioned. Then, this review is exclusively concerned with gap junctions. The ubiquitous occurrence of these junctions throughout the phylogenetic scale up to man points to a major functional role. Cells of most organized tissues make cell-to-cell channels, 1-2 nm diameter, that provide a structural hydrophilic pathway for free diffusion of inorganic ions and small molecules. Ionic coupling and metabolic cooperation have been shown to be functional expressions of the direct intercellular communication. The role of gap junctions in nonexcitable tissues is not well established (Chap. III). While these junctions are clearly involved in the regulation of some enzymatic activities and exocrine and endocrine secretions, the cell-to-cell transmission of signal molecules necessary for growth control remains largely hypothetical.

Restoration of gap junction-like structure after detergent solubilization of the proteins from liver gap junctions.

Gap junctions isolated from rat liver were partially solubilized with a mixture of digitonin and octyl glucoside. After supplementation with lecithin and cholesterol, the octyl glucoside was removed from the soluble fraction by dialysis. The membranes of the reconstituted vesicles, observed in freeze-fracture, contained particles ranging from 7 to 12 nm diameter, more or less aggregated depending on the protein-to-lipid ratio. At every protein concentration, the arrangement of particles in contact areas between adjacent membranes closely resembles the organization of intact gap junctions. We conclude that the mixture of digitonin and octyl glucoside is able to solubilize the proteins of the liver gap junctions while preserving their property of restoring a gap junction-like structure.

Voltage dependence of liver gap-junction channels reconstituted into liposomes and incorporated into planar bilayers.

The voltage dependence of rat liver gap junctions was investigated using non-denaturing solubilization and reconstitution of gap-junction protein into proteoliposomes in controlled conditions of connexon aggregation. The presence of liver connexin 32 in reconstituted proteoliposomes was checked with specific antibodies. The proteoliposomes were inserted into planar lipid bilayers by fusion. The single-channel conductance was voltage independent, and its magnitude was 700-1900 pS in 1 M NaCl, as expected from other reports, assuming that conductance is linear with ion activity. The channels were open at zero voltage and completely closed above 40 mV in either direction. This steep voltage dependence corresponded to an open/closed-state voltage difference of 19 mV and to 3.5 gating charges moving through the field. When several channels were inserted into the bilayer, a large fraction of the membrane conductance became voltage insensitive. These results show that the isolated channel units are highly voltage dependent and are consistent with the assumption that aggregated connexons interact through links which prevent voltage-sensitive conformational changes.

Fate of intercellular junctions in isolated adult rat cardiac cells.

Freeze fracture and thin section techniques have revealed morphological changes in gap junctions and intercalated discs of adult rat myocytes following enzymatic dissociation. Cell separation leaves behind small vesicular remnants of formerly adjacent cells connected to the intact cell by gap junctions; in contrast, desmosomes cleave at the region of intercellular contact. Apparently, the next step in gap junction breakdown is internalization of the remnants. In thin section, lanthanum penetration reveals that the cleft of some apparently internalized gap junctions is in contact with the sarcolemma, while that of others is not. In freeze fracture replicas, cytoplasmic gap junctions frequently possess hexagonally packed domains of E-face pits separated by smooth regions that may correspond to separations of membranes of internalized junctions found in thin section. Study of material maintained overnight at 37 degrees C showed no surface junctional remnants; topologies of cytoplasmic gap junctions were generally complex, and concentric membrane vesicles were common. These observations suggest that enzymatic dissociation initiates a progressive, defined sequence of junctional internalization that begins with attached cell remnants and may have as the last determinable step the separation into single membranes inside the intact cell.

Ultrastructural changes in gap junctions associated with CO2 uncoupling in frog atrial fibres.

The correlation between gap junction morphology and the state of electrical coupling was investigated in the frog auricle, which presents an atypical gap-junction organization. Electrical uncoupling of the tissue was achieved by perfusion with CO2-saturated Ringer medium. The tissue was fixed with glutaraldehyde and freeze-fractured before and during the application of CO2-saturated Ringer medium and after returning to the initial medium. The electrical tissue coupling was assayed by microelectrode recording just before fixation. At least 97% uncoupling was induced by CO2-saturated Ringer medium, as estimated from double sucrose gap experiments on several single atrial trabeculae. Several modifications were induced by CO2-saturated Ringer medium. A decrease in the number of particles per junctional assembly and dispersion of these assemblies in the plane of the membrane, indicate a decrease in the organization of the gap junctions. In parallel, loose clusters of particles became evident on the P-fracture face of the membrane. The size of the particles in these clusters was larger than the size of the background particles, and of the gap junction particles. They never corresponded with complementary pits on the E-fracture face. On return to the initial Ringer perfusion medium the cell coupling was reversed and the gap junction dispersion was also reversed. However, the gap junctions remained small. The relationship between these morphological modifications and the conducting state of the tissue is discussed.

Evolution of Antp-class genes and differential expression of Hydra Hox/paraHox genes in anterior patterning.

The conservation of developmental functions exerted by Antp-class homeoproteins in protostomes and deuterostomes suggested that homologs with related functions are present in diploblastic animals. Our phylogenetic analyses showed that Antp-class homeodomains belong either to non-Hox or to Hox/paraHox families. Among the 13 non-Hox families, 9 have diploblastic homologs, Msx, Emx, Barx, Evx, Tlx, NK-2, and Prh/Hex, Not, and Dlx, reported here. Among the Hox/paraHox, poriferan sequences were not found, and the cnidarian sequences formed at least five distinct cnox families. Two are significantly related to the paraHox Gsx (cnox-2) and the mox (cnox-5) sequences, whereas three display some relatedness to the Hox paralog groups 1 (cnox-1), 9/10 (cnox-3) and the paraHox cdx (cnox-4). Intermediate Hox/paraHox genes (PG 3 to 8 and lox) did not have clear cnidarian counterparts. In Hydra, cnox-1, cnox-2, and cnox-3 were not found chromosomally linked within a 150-kb range and displayed specific expression patterns in the adult head. During regeneration, cnox-1 was expressed as an early gene whatever the polarity, whereas cnox-2 was up-regulated later during head but not foot regeneration. Finally, cnox-3 expression was reestablished in the adult head once it was fully formed. These results suggest that the Hydra genes related to anterior Hox/paraHox genes are involved at different stages of apical differentiation. However, the positional information defining the oral/aboral axis in Hydra cannot be correlated strictly to that characterizing the anterior-posterior axis in vertebrates or arthropods.

Regulation of gap junctional conductance.

Gap junctional conductance is regulated by the number of channels between coupled cells (the balance between formation and loss of these channels) and by the fraction of these channels that are open (gating mechanisms). A variety of treatments are known to affect junction formation. Adenosine 3',5'-cyclic monophosphate (cAMP) is involved in some cases, and protein synthesis may be required but precursor molecules can also exist. Junction removal occurs both by dispersion of particles and by internalization of junctional membrane. Factors promoting removal are not well understood. A variety of gating mechanisms exist. Coupling may be controlled by changes in conductance of nonjunctional membranes. Several kinds of voltage dependence of junctional conductance are known, but rat ventricular junctions at least are electrically linear. Cytoplasmic acidification decreases conductance of most gap junctions. Sensitivity in rat ventricular myocytes allows modulation of coupling by moderate changes near normal internal pH. Increasing intracellular Ca also decreases junctional conductance, but in the better studied cases sensitivity is much lower to Ca than H. A few data support low sensitivity to Ca in cardiac cells, but quantitative studies are lacking. Higher alcohols such as octanol block junctional conductance in a wide range of tissues including rat ventricular myocytes. An antibody to liver gap junctions blocks junctions between rat ventricular myocytes. Cross reactivity indicates at least partial homology between many gap junctions. Although differences among gap junctions are known, a general physiology is being developed, which may have considerable relevance to normal cardiac function and also to conduction disorders of that tissue.

Retinoic acid enhances connexin43 expression at the post-transcriptional level in rat liver epithelial cells.

The mechanism by which all-trans retinoic acid (RA) stimulates gap junctional intercellular communication (GJIC) in the rat liver epithelial cell line. IAR203, was investigated. When RA, at 0.1 microM for 24-48 h, enhanced the dye transfer in IAR203 cells (x 1.4), it increased the amount of connexin43 (Cx43) in the cell-cell contact regions of the plasma membrane, as evidenced by analysis by Western blot and by immunofluorescence. It had no effect on the level of Cx43 mRNA. Freeze-fracture analysis of the size of gap junctions revealed an increase of the proportion of small gap junctions in RA-treated cells. We conclude that, in IAR203 cells, RA stimulates GJIC by acting at the post-transcriptional level of Cx43 regulation. The possibility that RA acts indirectly on the regulation of Cx43 expression, and increases the half-life of Cx43 by inducing adhesion molecules is discussed.