Jean Gouranton

Switch from an Aquaporin to a Glycerol Channel by Two Amino Acids Substitution

The MIP (major intrinsic protein) proteins constitute a channel family of currently 150 members that have been identified in cell membranes of organisms ranging from bacteria to man. Among these proteins, two functionally distinct subgroups are characterized: aquaporins that allow specific water transfer and glycerol channels that are involved in glycerol and small neutral solutes transport. Since the flow of small molecules across cell membranes is vital for every living organism, the study of such proteins is of particular interest. For instance, aquaporins located in kidney cell membranes are responsible for reabsorption of 150 liters of water/day in adult human. To understand the molecular mechanisms of solute transport specificity, we analyzed mutant aquaporins in which highly conserved residues have been substituted by amino acids located at the same positions in glycerol channels. Here, we show that substitution of a tyrosine and a tryptophan by a proline and a leucine, respectively, in the sixth transmembrane helix of an aquaporin leads to a switch in the selectivity of the channel, from water to glycerol.

Electron microscopic observation of the respiratory tract of SPF piglets inoculated with Mycoplasma hyopneumoniae

Seven hysterectomy derived piglets were repeatedly challenged with Mycoplasma hyopneumoniae during the first week of life. Samples of trachea, bronchi and lung tissue collected 2-11 weeks post-inoculation (p.i.) were examined using light and electron microscopy. Autoradiography was used to study in more detail the site of M. hyopneumoniae multiplication. Gross lesions were observed in lung tissue and were characterized by hyperplasia of the epithelium and an increased mononuclear cell accumulation in perivascular and peribronchiolar areas. Mild lesions of the trachea and the bronchi, including epithelial hyperplasia and infiltration of the lamina propria by inflammatory cells, were noted. Electron microscopy showed that, 2-6 weeks p.i., changes in the mid-trachea and bronchi surface consisted of the loss of cilia. Mycoplasmas covered tufts of cilia remaining on the epithelial cell surface. Scanning and transmission electron micrographs showed that they were predominantly found closely associated with the top of cilia. No specialized terminal structure could be seen and no mycoplasma cells were identified lying free in the lumen nor in close contact with the plasma membrane of cells or microvilli. Some fine fibrils radiating from one mycoplasma to another or to cilia were seen at higher magnification by scanning electron microscopy. Six to eleven weeks p.i., a disrupted epithelial surface lacking cilia was observed. Cells were desquamated and shed into the lumen with cellular remains containing droplets of mucus. Autoradiography revealed that label corresponded to the observed mycoplasma distribution. At the top of cilia, a high density of labeling was visible in the zone of high mycoplasma concentration. Therefore, incorporation of the label in the mycoplasma is proof or their multiplication in the trachea. The intimate association between the mycoplasma and cilia may be an important factor in the pathogenesis of the disease caused by M. hyopneumoniae (swine enzootic pneumonia).

A Yeast Recombinant Aquaporin Mutant That Is Not Expressed or Mistargeted in Xenopus Oocyte Can Be Functionally Analyzed in Reconstituted Proteoliposomes

We have recently identified AQPcic (foraquaporin cicadella), an insect aquaporin found in the digestive tract of homopteran insects and involved in the elimination of water ingested in excess with the dietary sap (Le Cahérec, F., Deschamps, S., Delamarche, C., Pellerin, I., Bonnec, G., Guillam, M. T., Gouranton, J., Thomas, D., and Hubert, J. F. (1996) Eur. J. Biochem.241, 707–715). Like many other aquaporins, AQPcic is inhibited by mercury reagents. In this study, we have demonstrated that residue Cys82 is essential for mercury inhibition. Another mutant version of AQPcic (AQP-C134S), expression of which in Xenopus laevis failed to produce an active molecule, was successfully expressed in Saccharomyces cerevisiae. Using stopped-flow analysis of reconstituted proteoliposomes, we demonstrated that the biological activity and Hg sensitivity of yeast-expressed wild type and mutant type AQPcic was readily assessed. Therefore, we propose that the yeast system is a valid alternative to Xenopus oocytes for studying particular mutants of aquaporin.

Flow cytometric quantitative evaluation of phagocytosis by human mononuclear and polymorphonuclear cells using fluorescent nanoparticles

The use of fluorescent polymethacrylic nanoparticles (0.3 micron) as a flow cytometric reagent in the quantitative evaluation of phagocytosis by human mononuclear and polymorphonuclear cells is described. The preparation of the nanoparticles, by emulsion copolymerization of methacrylic monomers, and their physicochemical properties are briefly summarized. Nanoparticles coupled with a fluorescent agent (ethidium bromide) were used in a flow cytometric assay to study opsonin-independent phagocytosis by human polymorphonuclear cells and by human monocytes. The phagocytosis of nanospheres by monocytes was determined by flow cytometry from the fluorescence distribution and ingestion was visualized by scanning and transmission electron microscopy. One possible application of the fluorescent nanoparticles is the simultaneous analysis of cell surface antigens and cell phagocytic activity.

AQUAPORIN-RELATED PROTEINS IN THE FILTER CHAMBER OF HOMOPTERAN INSECTS

Abstract.In the Homopteran order of insects, the plant xylem feeders exhibit a highly differentiated part of their digestive tract known as the filter chamber. In this tissue, water crosses plasma membranes through a transepithelial osmotic gradient. In previous studies on the filter chamber of Cicadella viridis, we purified and characterized from the plasma membranes a 25 kDa protein that we demonstrated to be an aquaporin (or water channel, member of the major intrinsic protein family, a group of membrane channels for small solutes). We called this protein AQPcic for Cicadella aquaporin. In the present study, we used polyclonal antibody anti-AQPcic in Western blotting and immunocytochemical analysis of the intestinal tract of Cercopis sanguinolenta, Philaenus spumarius, Aphrophora alni (Cercopidae), Euscelidius variegatus, and Scaphoideus titanus (Jassidae). Western blotting experiments revealed that immunologically related AQPcic proteins are found in those species. The molecular weight of these proteins is 15–26 kDa. Immunocytochemical studies on ultrathin filter-chamber sections revealed that the anti-AQPcic antibody systematically labelled the membrane microvilli of epithelial cells. A good correlation thus exists between the physiology of these cells and the presence of aquaporin-related proteins in their membranes.

Structural and biochemical observations on specialized membranes of the “filter chamber”, a water‐shunting complex in sap‐sucking homopteran insects

Many homopteran insects feed on plant sap which contains solutes in very low concentration. Their digestive tract presents a complex called the “filter chamber” where the excess dietary water is believed to flow directly from the initial part of the midgut to the terminal part of the midgut and the proximal reions of the Malpighian tubules.

Fine structure and nature of the crystalloid intranuclear and intracytoplasmic inclusions in dog cauda epididymidis

Crystalloid inclusions are found both in nuclei and cytoplasm of epithelial cells in the dog cauda epididymidis. They are made up of layers of parallel filaments which form a 90° angle from a layer to the next one. The crystalloids are never surrounded with a membrane. Enzyme digestions on ultrathin sections show that they are proteinaceous in nature.

Expression of RNA isolated from the water-shunting complex of a sap-sucking insect increases the membrane permeability for water in Xenopus oocytes

The highly specialized membranes of the filter chamber found in the digestive tract of some homopteran insects could represent a favorable material for characterizing water channels. In order to demonstrate that membrane proteins of this epithelial complex serve as water channels, we have investigated the membrane permeability for water in Xenopus oocytes injected with RNA isolated from the filter chamber. Volumes of oocytes injected with filter chamber RNA were increased by 15% following a 16-min osmotic shock, while volumes of oocytes injected with RNA from midgut not of filter chamber or with water were increased only by 8.5 and 10%, respectively. This significant difference in oocyte swelling leads us to conclude that RNA isolated from the filter chamber contains mRNA coding for water channel proteins.

Globular intranuclear inclusions in the midgut cells of Carausius morosus: Ultrastructure, composition and kinetics of growth

Spherical electron-dense bodies, up to 2 μ m in diameter, are seen generally in clusters, in the nuclei of the midgut cells of Carausius morosus . The inclusions are never surrounded by a membrane and there seems to be no direct relationship between them and the nucleolus. Cytochemical methods on paraffin sections and on thin sections reveal that the inclusions are proteinaceous in nature and contain carbohydrates. In electron microscopical autoradiography the inclusions are densely labeled a few hours after an injection of tritiated lysine or arginine. The kinetics of inclusion growth, examined by quantitative ultrastructural autoradiography, suggest a migration of cytoplasmic proteins to the nucleus, where parts of them are then progressively incorporated into the intranuclear inclusions. The results of an immunocytochemical method utilizing cytosol antibodies are in agreement with these findings. Pellets of isolated intranuclear inclusions may be solubilized when treated by a combination of a hydrogen bond cleaving agent and a reducing agent. The amino acid composition reveals that the proteins of the inclusions are predominantly acidic. With SDS-polyacrylamide gel electrophoresis of the solubilized inclusions, up to 16 proteins can be detected; some of them are glycoproteins. A comparison between the electrophoretic profile of the inclusions and the profile of the cytosol proteins reveals that several proteins of the inclusions are major components of the cytosol.

Study of fast water movements in bacteria by cryoelectron microscopy

Movement of water into or out of cells is a fundamental process of life found throughout nature. However the molecular pathway of this transport remained elusive until discovery of the aquaporins, a large family of water channel proteins. In bacteria, the osmotic movement of water across the cytoplasmic membrane is one of the mechanisms triggered to maintain the cell turgor, a function essential for growth and survival. The first known prokaryotic aquaporin water channel gene, aqpZ, has been reported in Escherichia coli (Calamita et al., 1995) and functionally characterized (Borgnia et al., 1999; Delamarche et al., 1999). This indicates that, in spite of the high surface-to-volume ratio characterizing bacteria, the simple diffusion of water through the membrane lipids could not be always sufficient to preserve turgor. Presently, several microbial members of the MIP family have been identified by sequence homology, but only few microbial MIPs have been functionally studied (Maurel et al., 1994).