Measurement of absolute cell volume, osmotic membrane water permeability, and refractive index of transmembrane water and solute flux by digital holographic microscopy.

ABSTRACT. A dual-wavelength digital holographic microscope to measure absolute volume of living cells is proposed. The optical setup allows us to reconstruct two quantitative phase contrast images at two different wavelengths from a single hologram acquisition. When adding the absorbing dye fast green FCF as a dispersive agent to the extracellular medium, cellular thickness can be univocally determined in the full field of view. In addition to the absolute cell volume, the method can be applied to derive important biophysical parameters of living cells including osmotic membrane water permeability coefficient and the integral intracellular refractive index (RI). Further, the RI of transmembrane flux can be determined giving an indication about the nature of transported solutes. The proposed method is applied to cultured human embryonic kidney cells, Chinese hamster ovary cells, human red blood cells, mouse cortical astrocytes, and neurons.

A novel human aquaporin-4 splice variant exhibits a dominant-negative activity: a new mechanism to regulate water permeability.

Two major isoforms of aquaporin-4 (AQP4) have been described in human tissue. Here we report the identification and functional analysis of an alternatively spliced transcript of human AQP4, AQP4-Δ4, that lacks exon 4. In transfected cells AQP4-Δ4 is mainly retained in the endoplasmic reticulum and shows no water transport properties. When AQP4-Δ4 is transfected into cells stably expressing functional AQP4, the surface expression of the full-length protein is reduced. Furthermore, the water transport activity of the cotransfectants is diminished in comparison to transfectants expressing only AQP4. The observed down-regulation of both the expression and water channel activity of AQP4 is likely to originate from a dominant-negative effect caused by heterodimerization between AQP4 and AQP4-Δ4, which was detected in coimmunoprecipitation studies. In skeletal muscles, AQP4-Δ4 mRNA expression inversely correlates with the level of AQP4 protein and is physiologically associated with different types of skeletal muscles. The expression of AQP4-Δ4 may represent a new regulatory mechanism through which the cell-surface expression and therefore the activity of AQP4 can be physiologically modulated.

Inappropriate antidiuretic hormone secretion: long-term successful urea treatment.

Hyponatremia is the main complication of inappropriate antidiuretic hormone secretion (SIADH), sometimes fatal. Treatment strategy depends on the cause and the severity of the hyponatremia. Recent studies have shown the efficacy of urea in treating acute hyponatremia secondary to SIADH, by inducing an osmotic water drive. We describe an infant with chronic hyponatremia secondary to SIADH in which the long-term oral treatment with urea was successful and well tolerated. The aim of this paper is to highlight the potential benefits of urea treatment in case of chronic hyponatremia secondary to SIADH. CONCLUSION: Chronic oral urea treatment in children with SIADH allows an easy and safe water and sodium control and may permit a decrease in fluid restriction in this situation.

Subcellular Redistribution of Root Aquaporins Induced by Hydrogen Peroxide.

Aquaporins are water channel proteins that mediate the fine-tuning of cell membrane water permeability during development or in response to environmental stresses. The present work focuses on the oxidative stress-induced redistribution of plasma membrane intrinsic protein (PIP) aquaporins from the plasma membrane (PM) to intracellular membranes. This process was investigated in the Arabidopsis root. Sucrose density gradient centrifugation showed that exposure of roots to 0.5 mM H2O2 induces significant depletion in PM fractions of several abundant PIP homologs after 15 min. Analyses by single-particle tracking and fluorescence correlative spectroscopy showed that, in the PM of epidermal cells, H2O2 treatment induces an increase in lateral motion and a reduction in the density of a fluorescently tagged form of the prototypal AtPIP2;1 isoform, respectively. Co-expression analyses of AtPIP2;1 with endomembrane markers revealed that H2O2 triggers AtPIP2;1 accumulation in the late endosomal compartments. Life-time analyses established that the high stability of PIPs was maintained under oxidative stress conditions, suggesting that H2O2 triggers a mechanism for intracellular sequestration of PM aquaporins without further degradation. In addition to information on cellular regulation of aquaporins, this study provides novel and complementary insights into the dynamic remodeling of plant internal membranes during oxidative stress responses.

Comment on "Streaming potential dependence on water-content in Fontainebleau sand" by V. Allegre, L. Jouniaux, F. Lehmann and P. Sailhac

Allegre et al. recently presented new experimental data regarding the dependence of the streaming potential coupling coefficient with the saturation of the water phase. Such experiments are important to model the self-potential response associated with the flow of water in the vadose zone and the electroseismic/seismoelectric conversions in unsaturated porous media. However, the approach used to interpret the data is questionable and the conclusions reached by Allegre et al. likely incorrect

Vasopressin-dependent coupling between sodium transport and water flow in a mouse cortical collecting duct cell line.

Water balance is achieved through the ability of the kidney to control water reabsorption in the connecting tubule and the collecting duct. In a mouse cortical collecting duct cell line (mCCD(c11)), physiological concentrations of arginine vasopressin increased both electrogenic, amiloride-sensitive, epithelial sodium channel (ENaC)-mediated sodium transport measured by the short-circuit current (Isc) method and water flow (Jv apical to basal) measured by gravimetry with similar activation coefficient K(1/2) (6 and 12 pM, respectively). Jv increased linearly according to the osmotic gradient across the monolayer. A small but highly significant Jv was also measured under isoosmotic conditions. To test the coupling between sodium reabsorption and water flow, mCCD(c11) cells were treated for 24 h under isoosmotic condition with either diluent, amiloride, vasopressin or vasopressin and amiloride. Isc, Jv, and net chemical sodium fluxes were measured across the same monolayers. Around 30% of baseline and 50% of vasopressin-induced water flow is coupled to an amiloride-sensitive, ENaC-mediated, electrogenic sodium transport, whereas the remaining flow is coupled to an amiloride-insensitive, nonelectrogenic sodium transport mediated by an unknown electroneutral transporter. The mCCD(c11) cell line is a first example of a mammalian tight epithelium allowing quantitative study of the coupling between sodium and water transport. Our data are consistent with the 'near isoosmotic' fluid transport model.

The sigma factor AlgU (AlgT) controls exopolysaccharide production and tolerance towards desiccation and osmotic stress in the biocontrol agent Pseudomonas fluorescens CHA0.

A variety of stress situations may affect the activity and survival of plant-beneficial pseudomonads added to soil to control root diseases. This study focused on the roles of the sigma factor AlgU (synonyms, AlgT, RpoE, and sigma(22)) and the anti-sigma factor MucA in stress adaptation of the biocontrol agent Pseudomonas fluorescens CHA0. The algU-mucA-mucB gene cluster of strain CHA0 was similar to that of the pathogens Pseudomonas aeruginosa and Pseudomonas syringae. Strain CHA0 is naturally nonmucoid, whereas a mucA deletion mutant or algU-overexpressing strains were highly mucoid due to exopolysaccharide overproduction. Mucoidy strictly depended on the global regulator GacA. An algU deletion mutant was significantly more sensitive to osmotic stress than the wild-type CHA0 strain and the mucA mutant were. Expression of an algU'-'lacZ reporter fusion was induced severalfold in the wild type and in the mucA mutant upon exposure to osmotic stress, whereas a lower, noninducible level of expression was observed in the algU mutant. Overexpression of algU did not enhance tolerance towards osmotic stress. AlgU was found to be essential for tolerance of P. fluorescens towards desiccation stress in a sterile vermiculite-sand mixture and in a natural sandy loam soil. The size of the population of the algU mutant declined much more rapidly than the size of the wild-type population at soil water contents below 5%. In contrast to its role in pathogenic pseudomonads, AlgU did not contribute to tolerance of P. fluorescens towards oxidative and heat stress. In conclusion, AlgU is a crucial determinant in the adaptation of P. fluorescens to dry conditions and hyperosmolarity, two major stress factors that limit bacterial survival in the environment.

An eceriferum locus, cer-zv, is associated with a defect in cutin responsible for water retention in barley (Hordeum vulgare) leaves.

Drought limits plant growth and threatens crop productivity. A barley (Hordeum vulgare) ethylene imine-induced monogenic recessive mutant cer-zv, which is sensitive to drought, was characterized and genetically mapped in the present study. Detached leaves of cer-zv lost 34.2 % of their initial weight after 1 h of dehydration. The transpiration was much higher in cer-zv leaves than in wild-type leaves under both light and dark conditions. The stomata of cer-zv leaves functioned normally, but the cuticle of cer-zv leaves showed increased permeability to ethanol and toluidine blue dye. There was a 50-90 % reduction in four major cutin monomers, but no reduction in wax loads was found in the cer-zv mutant as compared with the wild type. Two F(2) mapping populations were established by the crosses of 23-19 × cer-zv and cer-zv × OUH602. More polymorphisms were found in EST sequences between cer-zv and OUH602 than between cer-zv and 23-19. cer-zv was located in a pericentromeric region on chromosome 4H in a 10.8 cM interval in the 23-19 × cer-zv map based on 186 gametes tested and a 1.7 cM interval in the cer-zv × OUH602 map based on 176 gametes tested. It co-segregated with EST marker AK251484 in both maps. The results indicated that the cer-zv mutant is defective in cutin, which might be responsible for the increased transpiration rate and drought sensitivity, and that the F(2) of cer-zv × OUH602 might better facilitate high resolution mapping of cer-zv.

Characterization and genetic mapping of eceriferum-ym (cer-ym), a cutin deficient barley mutant with impaired leaf water retention capacity.

The cuticle covers the aerial parts of land plants, where it serves many important functions, including water retention. Here, a recessive cuticle mutant, eceriferum-ym (cer-ym), of Hordeum vulgare L. (barley) showed abnormally glossy spikes, sheaths, and leaves. The cer-ym mutant plant detached from its root system was hypersensitive to desiccation treatment compared with wild type plants, and detached leaves of mutant lost 41.8% of their initial weight after 1 h of dehydration under laboratory conditions, while that of the wild type plants lost only 7.1%. Stomata function was not affected by the mutation, but the mutant leaves showed increased cuticular permeability to water, suggesting a defective leaf cuticle, which was confirmed by toluidine blue staining. The mutant leaves showed a substantial reduction in the amounts of the major cutin monomers and a slight increase in the main wax component, suggesting that the enhanced cuticle permeability was a consequence of cutin deficiency. cer-ym was mapped within a 0.8 cM interval between EST marker AK370363 and AK251484, a pericentromeric region on chromosome 4H. The results indicate that the desiccation sensitivity of cer-ym is caused by a defect in leaf cutin, and that cer-ym is located in a chromosome 4H pericentromeric region.

Biodiversity of amoebae and amoebae-resisting bacteria in a drinking water treatment plant

The complex ecology of free-living amoebae (FLA) and their role in spreading pathogenic microorganisms through water systems have recently raised considerable interest. In this study, we investigated the presence of FLA and amoebae-resisting bacteria (ARB) at various stages of a drinking water plant fed with river water. We isolated various amoebal species from the river and from several points within the plant, mostly at early steps of water treatment. Echinamoeba- and Hartmannella-related amoebae were mainly recovered in the drinking water plant whereas Acanthamoeba- and Naegleria-related amoebae were recovered from the river water and the sand filtration units. Some FLA isolates were recovered immediately after the ozonation step, thus suggesting resistance of these microorganisms to this disinfection procedure. A bacterial isolate related to Mycobacterium mucogenicum was recovered from an Echinamoeba-related amoeba isolated from ozone-treated water. Various other ARB were recovered using co-culture with axenic Acanthamoeba castellanii, including mycobacteria, legionella, Chlamydia-like organisms and various proteobacteria. Noteworthy, a new Parachlamydia acanthamoebae strain was recovered from river water and from granular activated carbon (GAC) biofilm. As amoebae mainly multiply in sand and GAC filters, optimization of filter backwash procedures probably offers a possibility to better control these protists and the risk associated with their intracellular hosts

In vitro engineering of human stratified urothelium: analysis of its morphology and function.

PURPOSE: Gastric or intestinal patches, commonly used for reconstructive cystoplasty, may induce severe metabolic complications. The use of bladder tissues reconstructed in vitro could avoid these complications. We compared cellular differentiation and permeability characteristics of human native with in vitro cultured stratified urothelium. MATERIALS AND METHODS: Human stratified urothelium was induced in vitro. Morphology was studied with light and electron microscopy and expression of key cellular proteins was assessed using immunohistochemistry. Permeability coefficients were determined by measuring water, urea, ammonia and proton fluxes across the urothelium. RESULTS: As in native urothelium the stratified urothelial construct consisted of basal membrane and basal, intermediate and superficial cell layers. The apical membrane of superficial cells formed villi and glycocalices, and tight junctions and desmosomes were developed. Immunohistochemistry showed similarities and differences in the expression of cytokeratins, integrin and cellular adhesion proteins. In the cultured urothelium cytokeratin 20 and integrin subunits alpha6 and beta4 were absent, and symplekin was expressed diffusely in all layers. Uroplakins were clearly expressed in the superficial umbrella cells of the urothelial constructs, however, they were also present in intermediate and basal cells. Symplekin and uroplakins were expressed only in the superficial cells of native bladder tissue. The urothelial constructs showed excellent viability, and functionally their permeabilities for water, urea and ammonia were no different from those measured in native human urothelium. Proton permeability was even lower in the constructs compared to that of native urothelium. CONCLUSIONS: Although the in vitro cultured human stratified urothelium did not show complete terminal differentiation of its superficial cells, it retained the same barrier characteristics against the principal urine components. These results indicate that such in vitro cultured urothelium, after being grown on a compliant degradable support or in coculture with smooth muscle cells, is suitable for reconstructive cystoplasty.

An ATP-binding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice.

Land plants have developed a cuticle preventing uncontrolled water loss. Here we report that an ATP-binding cassette (ABC) subfamily G (ABCG) full transporter is required for leaf water conservation in both wild barley and rice. A spontaneous mutation, eibi1.b, in wild barley has a low capacity to retain leaf water, a phenotype associated with reduced cutin deposition and a thin cuticle. Map-based cloning revealed that Eibi1 encodes an HvABCG31 full transporter. The gene was highly expressed in the elongation zone of a growing leaf (the site of cutin synthesis), and its gene product also was localized in developing, but not in mature tissue. A de novo wild barley mutant named "eibi1.c," along with two transposon insertion lines of rice mutated in the ortholog of HvABCG31 also were unable to restrict water loss from detached leaves. HvABCG31 is hypothesized to function as a transporter involved in cutin formation. Homologs of HvABCG31 were found in green algae, moss, and lycopods, indicating that this full transporter is highly conserved in the evolution of land plants.

Accurate performance of a rat model of schizophrenia in the water maze depends on visual cue availability and stability: a distortion in cognitive mapping abilities?

Rats were treated postnatally (PND 5-16) with BSO (l-buthionine-(S,R)-sulfoximine) in an animal model of schizophrenia based on transient glutathione deficit. The BSO treated rats were impaired in patrolling a maze or a homing table when adult, yet demonstrated preserved escape learning, place discrimination and reversal in a water maze task [37]. In the present work, BSO rats' performance in the water maze was assessed in conditions controlling for the available visual cues. First, in a completely curtained environment with two salient controlled cues, BSO rats showed little accuracy compared to control rats. Secondly, pre-trained BSO rats were impaired in reaching the familiar spatial position when curtains partially occluded different portions of the room environment in successive sessions. The apparently preserved place learning in a classical water maze task thus appears to require the stability and the richness of visual landmarks from the surrounding environment. In other words, the accuracy of BSO rats in place and reversal learning is impaired in a minimal cue condition or when the visual panorama changes between trials. However, if the panorama remains rich and stable between trials, BSO rats are equally efficient in reaching a familiar position or in learning a new one. This suggests that the BSO accurate performance in the water maze does not satisfy all the criteria for a cognitive map based navigation on the integration of polymodal cues. It supports the general hypothesis of a binding deficit in BSO rats.