Lower urinary tract (LUT) symptoms become prevalent with aging and affect millions; however, therapy is often ineffective because the etiology is unknown. Existing assays of LUT function in animal models are often invasive; however, a noninvasive assay is required to study symptom progression and determine genetic correlates. Here, we present a spontaneous voiding assay that is simple, reproducible, quantitative, and noninvasive. Young female mice from eight inbred mouse strains (129S1/SvImJ, A/J, C57BL/6J, NOD/ShiLtJ, NZO/H1LtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) were tested for urination patterns on filter paper. Repeat testing at different times of the day showed minimal within-individual and within-strain variations, but all parameters (spot number, total volume, percent area in primary void, corner voiding, and center voiding) exhibited significant variations between strains. Calculation of the intraclass correlation coefficient, an estimate of broad-sense heritability, for each time of day and for each voiding parameter revealed highly significant heritability [spot number: 61%, percent urine in primary void: 90%, and total volume: 94% (afternoon data)]. Cystometrograms confirmed strong strain-specific urodynamic characteristics. Behavior-voiding correlation analysis showed no correlation with anxiety phenotypes. Diagnostically, the assay revealed LUT symptoms in several systems, including a demonstration of voiding abnormalities in older C57BL/6J mice (18-24 mo), in a model of protamine sulfate-induced urothelial damage and in a model of sucrose-induced diuresis. This assay may be used to derive pathophysiological LUT readouts from mouse models. Voiding characteristics are heritable traits, opening the way for genetic studies of LUT symptoms using outbred mouse populations.
We located fully sequenced putative genes of the plasma membrane intrinsic proteins (PIPs) family in the Populus trichocarpa (Torr. Gray), genome. Of 23 gene candidates, we assigned eight genes to the PIP2 subfamily. All eight putative genes were expressed in vegetative tissues (roots, leaves, bark and wood), and all of them showed water channel activity after being expressed in Xenopus oocytes. Six of eight proteins were affected by mercury ions. No proteins were affected by the presence of nickel or tungsten ions, or by lowering the pH of bathing external solution from 7.4 to 6.5. The presence of copper ions caused seven of eight PIP2 proteins to increase their water transport capacity by as much as 50%. This systematic study of the PIP2 subfamily of proteins in P. trichocarpa provides a basic overview of their activity as water channels and will be a useful reference for future physiological studies of plant water relations that use P. trichocarpa as a model system.
is a useful model system for studying expressed water and solute transporters but suffers from a number of limitations, most notably large unstirred layers and other intracellular diffusion barriers. To overcome these, we have developed a method for isolating plasma membrane vesicles from oocytes. This approach facilitates more precise control of the intravesicular environment and virtually eliminates the problem of unstirred layers in kinetic experiments. The isolation procedure results in 50.6-fold enrichment of the plasma membrane marker alkaline phosphodiesterase compared with the homogenate. Markers of late endosomes/lysosomes and mitochondria were not enriched, and the endoplasmic reticulum was enriched only modestly. Permeabilities of native plasma membrane to water and urea were 8.1 ϫ 10 Ϫ4 and 5.6 ϫ 10 Ϫ7 cm/s, respectively, values that are sufficiently low to classify them as barrier membranes. Phospholipid analysis by mass spectrometry showed the membrane, not including cholesterol, to be rich in phosphatidylcholine (35.8 mole percent), sphingomyelin (25.8 mole percent), and phosphatidylinositol (6.8 mole percent). Cholesterol concentration was 20.7 mole percent. Membrane vesicles isolated from oocytes expressing aquaporin-1 exhibited fourfold higher water permeability in stopped-flow experiments. Oocytes expressing mouse urea transporter A3 (UT-A3) exhibited 7.5-fold faster phloretin-inhibitable urea transport compared with water-injected controls. There was no difference in water permeability between these membrane vesicles, suggesting that UT-A3 is not a water carrier. In conclusion, we describe an improved method for the isolation of the oocyte plasma membrane that will allow the study of water and solute transport kinetics as well as substrate selectivity in heterologously expressed proteins.
Current models of cell polarity invoke asymmetric cues that reorganize the secretory apparatus to induce polarized protein delivery. An important step in this process is the stabilization of the protein composition in each polarized membrane domain. The spectrin-based membrane skeleton is thought to contribute to such stabilization by increasing the half-life of many proteins at the cell surface. Genetic evidence is consistent with a negative role for Drosophila βHeavy-spectrin in endocytosis, but the inhibitory mechanism has not been elucidated. Here, we investigated the membrane binding properties of the C-terminal nonrepetitive domain of βHeavy-spectrin through its in vivo expression in transgenic flies. We found that this region is a membrane-association domain that requires a pleckstrin homology domain for full activity, and we showed for the first time that robust membrane binding by such a C-terminal domain requires additional contributions outside the pleckstrin homology. In addition, we showed that expression of the βHeavy-spectrin C-terminal domain has a potent effect on epithelial morphogenesis. This effect is associated with its ability to induce an expansion in plasma membrane surface area. The membrane expansions adopt a very specific bi-membrane structure that sequesters both the C-terminal domain and the endocytic protein dynamin. Our data provide supporting evidence for the inhibition of endocytosis by βHeavy-spectrin, and suggest that the C-terminal domain mediates this effect through interaction with the endocytic machinery. Spectrin may be an active partner in the stabilization of polarized membrane domains.
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