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Transport of water across the plasma membrane is a fundamental process occurring in all living organisms. In bacteria, osmotic movement of water across the cytoplasmic membrane is needed to maintain cellular turgor; however, the molecular mechanisms of this process are poorly defined. Involvement of aquaporin water channels in bacterial water permeability was suggested by the recent discovery of the aquaporin gene, aqpZ, in Escherichia coli. By employing cryoelectron microscopy to compare E. coli cells containing (AqpZ+) and lacking (AqpZ-) aquaporin, we show that the AqpZ water channel rapidly mediates large water fluxes in response to sudden changes in extracellular osmolarity. These findings (i) demonstrate for the first time functional expression of a prokaryotic water channel, (ii) evidence the bidirectional water channel feature of AqpZ, (iii) document a role for AqpZ in bacterial osmoregulation, and (iv) define a suitable model for studying the physiology of prokaryotic water transport.

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.

Objective: To determine whether the process of sperm nuclear destabilization would begin before sperm-oocyte fusion in humans., Design: Changes in the distribution of human protamine 1 were investigated in human spermatozoa from the ejaculate, in spermatozoa selected by swim-up or Percoll techniques, and in spermatozoa bound to zona pellucida (ZP) from oocytes that failed to fertilize in an IVF program., Setting: Center for Infertility and Assisted Reproductive Technology, and university departments., Patient(s): Fifteen couples undergoing an IVF program., Intervention(s): Women underwent a similar superovulation induction protocol that consisted of GnRH agonist associated with hMG., Main Outcome Measure(s): Comparative immunoelectron microscopic study of sperm nucleus labeling with an anti-human protamine 1 specific protamine monoclonal antibody., Result(s): After selection by swim-up or by Percoll, spermatozoa show a significantly lower nuclear labeling than in the ejaculate. After binding to the ZP, labeling increases, more in spermatozoa selected by swim-up than by Percoll, but, after Percoll selection, labeling in zona-bound spermatozoa is lower than in the ejaculate., Conclusion(s): In humans sperm binding to the ZP induces differences in the accessibility of the anti-human protamine 1 antibody, which are consistent with structural rearrangements of the DNA-nucleoproteins complex. These modifications must be a prelude to sperm decondensation, protamines replacement by histones, and subsequent reactivation of the sperm genome in the oocyte.

We previously described the structural organization of P25, a member of the major-intrinsic-protein family found in the digestive tract of homopteran sap-sucking insects [Beuron, F., Le Cahérec, F., Guillam, M. T., Cavalier, A., Garret, A., Tassan, J. P., Delamarche, C., Schultz, P., Mallouh, V., Rolland, J. P., Hubert, J.F., Gouranton, J. & Thomas, D. (1995) J. Biol. Chem. 270, 17414-17422]. We demonstrated, by means of introducing P25 tetramers into the membranes of Xenopus oocytes, that this protein exhibits functional properties similar to those of aquaporin 1, the archetypal water channel [Le Cahérec, F., Bron, P., Verbavatz, J. M., Garret, A., Morel, G., Cavalier, A., Bonnec, G., Thomas, D., Gouranton, J. & Hubert, J.F. (1996) J. Cell Sci. 109, 1285-1295]. In the present work, we cloned a full-length cDNA from a Cicadella viridis library with an open reading frame of 765 bp that encoded a 26-kDa protein whose sequence was 43, 40, 36 and 36% identical to aquaporins 1, 2, z and tonoplast intrinsic protein gamma, respectively. Translation of the corresponding RNA in Xenopus oocytes generated a polypeptide that was specifically recognized by polyclonal antibodies raised against native P25. Expression of the protein in Xenopus oocyte membranes was assessed by immunocytochemistry and led to a 15-fold increase of osmotic membrane water permeability. This increase was inhibited by HgCl2. The permeability had an Arrhenius activation energy of 11.7 kJ/mol. We called this protein Cicadella aquaporin (AQPcic). The oocytes expressing Cicadella aquaporin were less sensitive to HgCl2 than oocytes expressing aquaporin 1. In the Xenopus oocyte system, Cicadella aquaporin failed to transport glycerol, urea and ions. It exhibited permeabilities to ethylene glycol and formamide similar to those measured for aquaporin 1 under the same conditions.

Objective: To investigate the relationship between sperm preparation techniques and nuclear maturity, as evidenced by the electrophoretic profiles of sperm nuclear proteins., Design: Analysis of sperm nuclear quality in sperm populations used for IVF., Setting: Center for infertility and assisted reproductive technology and university departments., Patients: Twenty-seven men undergoing an infertility work-up., Main Outcome Measures: Comparative electrophoretic investigation of nucleoproteins extracted from spermatozoa selected by swim-up or Percoll techniques., Results: Nuclear maturity level is improved after the two methods of selection but is more improved after Percoll. In the two groups, selected spermatozoa contain less histones. Moreover, Percoll gradients appeared to enrich for spermatozoa with less intermediate proteins and more mature nucleoproteins of P2 family than swim-up spermatozoa., Conclusion: Percoll may offer advantages in terms of the quality of the selected spermatozoa that may influence the outcome of assisted conception techniques.

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.

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).

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 regions of the Malpighian tubules. Freeze-fracture experiments carried out on the filter chamber of Cicadella viridis revealed the presence of intramembrane particles on the whole surface of the microvilli and of basal membrane infoldings of the cells. Examination of negatively stained isolated membranes and of freeze-dried shadowed membranes revealed that the inner surface of the membrane is covered with particles protruding into the cytoplasm; they correspond to the numerous intramembrane particles observed on the P fracture face of the membrane. The outer surface of the membrane exhibits a regular network which corresponds to that observed on the E fracture face. SDS-PAGE analyses were performed on purified membranes of the filter chambers of C. viridis and Philaenus spumarius. In both cases 2 major components, 25 kDa and 75 kDa, were detected. These 2 components appear to be specific for the filter chambers since they were not found in membranes isolated from the other parts of the midgut. Thus, the membranes of these filter chambers, thought to be water-shunting complexes, possess structural and biochemical peculiarities which are probably related to water permeability.

The intranuclear filamentous inclusions of a human glioma were analysed with an electron microscope equipped with a goniometer stage. The inclusions consist of 6 to 8 filaments. Considering the organization of the constituent filaments we distinguish three basic types: 1. Filamentous bundles of more or less parallel filaments, forming a cigarshaped inclusion. 2. Crystalloid inclusions: a. Prisms. They consist of stacked layers of strictly parallel filaments. The angle formed by the filaments of adjacent layers if 60 degrees. b. Cylinders. The layers of filaments are bent up and may form either a circle or a spiral, when the inclusion is seen in cross-section. 3. Partially crystalloid or "intermediate" inclusions. We consider them to be transitional forms between types 1 and 2 inclusions. The crystalloid layers of such intermediate inclusions may form either prisms or cylinders. Finally, the similarity between the granulo-fibrillar capsules surrounding granular nuclear bodies and filamentous inclusions, as well as the existence of granular material dispersed between the filaments of some inclusions led us to investigate a relationship between these two structures.

The involvement of cyclic adenosine 3'-5' monophosphate (cAMP) in the regulation of human monocyte phagocytosis of staphylococcus aureus in vitro was demonstrated by assay of adenylate cyclase (AC) and cAMP phosphodiesterase (PDE). Phagocytosis was associated with diminished AC activity (p less than 0,05) and concurrently increased PDE activity (p less than 0,005). Transmission electron microscopy provided evidence that these changes coincide with peak phagocytic activity occuring 10-20 minutes after the start of phagocytosis.

Crystalline accumulations of ferritin-like particles are present within the cytoplasma and the nucleus in midgut epithelial cells of the homopteran Philaenus spumarius. A structural study at the electron microscope level reveals that these particles have the morphological characteristics of the ferritin molecule: crystals have a face-centered cubic structure with a lattice parameter of 14 +/- 1 nm; negatively stained isolated particles have the appearance of ferritin; on rotary-shadowed particles 3 axes of symmetry are clearly seen; image processing performed on selected molecules demonstrates a 4-fold symmetry. A semiquantitative electron microprobe analysis effected on aggregates of microcrystals in thin sections reveals a high atomic ratio Fe/P. Analyzed by SDS-PAGE, the protein subunit has a molecular weight of 18,600. The amino acid composition of the protein bears the general characteristics of the ferritin molecule in terms of polar and nonpolar residues. But in terms of sequences, this protein displays a strong dissimilarity to rat liver ferritin as demonstrated with a common amino acid index test and with immunoelectrophoresis experiments.

Involvement of cyclic adenosine 3',5'-mono-phosphate (cAMP) in the phagocytosis of staphylococci by human monocytes was demonstrated by assay of adenylate cyclase and cAMP phosphodiesterase (PDE). Monocyte adenylate cyclase and PDE activities were assayed on cell homogenates prepared from monocytes isolated by plate adherence. Phagocytosis of staphylococci was associated with diminished adenylate cyclase activity, which reached a minimum after 10 min of incubation (p less than 0.05), and an increase in PDE activity (p less than 0.005). Transmission electron microscopy provided evidence that these changes coincide with peak phagocytic activity occurring between 10 and 20 min after the start of phagocytosis but could not be assigned to a particular stage in the phagocytic process.

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.