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  • 1
    ISSN: 1432-1424
    Keywords: Cell culture ; Water permeability ; Epithelial barriers ; Medium hypertonicity ; Freeze-fracture ; Rabbit rectum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Caco-2 cells, originated in a human colonic cancer, are currently used as model systems to study transepithelial transports. To further characterize their water permeability properties, clone P1 Caco-2 cells were cultured on permeable supports. At confluence, the transepithelial net water movement (J W), mannitol permeability (P s), and electrical resistance (R) were simultaneously measured. The observed results were correlated with transmission and freeze-fracture electron microscopy studies and compared with those obtained, in similar experimental conditions, in a typical mammalian epithelial barrier: the rabbit rectum. When the serosal solution was made hypertonic (50 mm polyethylene glycol-PEG), the spontaneously observed secretory J w rapidly reversed, became absorptive and then stabilized. Simultaneously, the R values dropped and P s went up. In the case of the rabbit rectal epithelium, a similar treatment did not elicit significant changes in the water permeability during the first 20 min following the osmotic challenge while there was a significant increase in the transepithelial resistance. After exposure to serosal hypertonicity, several morphological modifications developed in the Caco-2 cells: Localized dilations in the intercellular spaces and vacuoles in the cytoplasm appeared. Nevertheless, most cells remained in contact and no evidence of cell shrinking was observed. Simultaneously, the tight-junction structure was more or less disorganized. The filament network lost its sharpness and “omega” figures appeared, bordering the intercellular spaces. In some cases the tight-junction network was completely disrupted. In the case of the rabbit rectum the structural modifications were completely different: Serosal hypertonicity rapidly induced cell shrinking and the opening of the intercellular spaces, with no noticeable change in the tight-junction structure. These results suggest that Caco-2-P1 cell membranes, contrary to the case of the basolateral membrane of rabbit rectal cells, have no water channels and that a paracellular route could play a central role in the water movements across this epithelial barrier.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 6 (1971), S. 353-367 
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary A special technique for the electron-microscope study of the fine structure of ultrathin artificial membranes is described. Membranes made of total phospholipids of the cerebral cortex and cholesterol showed globular elements of 40 Å embedded in a denser and diffuse matrix. These same elements were also seen organized in a periodic banded pattern. Identical patterns were observed with and without supporting films. Lipidic membranes containing small amounts of proteolipid fromElectrophorus showed a lower electron density, a finer and smoother texture and a decrease in electrical resistance. Lipidic membranes containing the cholinergic receptor proteolipid fromElectrophorus, upon addition of acetylcholine, showed a rapid and transient rise in conductance which was accompanied by changes in fine structure, consisting in a more uneven corrugated appearance of the membrane and the presence of dense spots of 20 Å. These results are discussed in relation to the channel hypothesis of ion permeability. It is postulated that the binding of acetylcholine by the receptor proteolipid results in conformational changes in this protein that facilitate the translocation of ions through the membrane.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 86 (1985), S. 239-245 
    ISSN: 1432-1424
    Keywords: water channels ; glutaraldehyde fixation ; frog urinary bladder ; unstirred layers ; osmotic and diffusional permeabilities ; Rana esculenta
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Unidirectional and net water movements were determined, in frog urinary bladders, before and after glutraldehyde fixation. Experiments were performed in three experimental conditions: 1) in nonstimulated preparations, 2) after the action of antidiuretic hormone (ADH) and 3) in nonstimulated preparations to which amphotericin B was incorporated from the luminal bath. As previously observed for net water fluxes, the increase in the unidirectional water movement induced by ADH was well preserved by glutaraldehyde fixation. After correction for the effects of unstirred layers and nonosmotic pathways, the observed correlation between the ADH-induced increases in the osmotic (Pf) and diffusional (Pd) permeability coefficients was not modified by the fixative action (before glutaraldehyde: slope 11.19,r:0.87±0.07;n=12; after glutaraldehyde: slope 10.67,r:0.86±0.04,n=39). In the case of amphotericin B, ΔPf/ΔPd=3.08 (r: 0.83±0.08), a value similar to that observed in lipid bilayers or in nonfixed toad urinary bladders. It is concluded that 1) The experimental approach previously employed to study water channels in artificial lipid membranes and in amphibian urinary bladders, can be applied to the glutaraldehyde-fixed frog urinary bladder. 2) Glutaraldehyde fixation does not modify the permeability properties of the ADH-induced water channels. 3) Any contribution of exo-endocytic processes or cell regulatory mechanisms to the observed permeability parameters can probably be excluded. 4) Glutaraldehyde-fixed preparations are a good model to characterize these water pathways.
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  • 4
    ISSN: 1432-1424
    Keywords: Diffusional permeability ; Image analysis ; Mercury chloride ; Osmotic permeability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract The water permeability properties of ovarian oocytes from Xenopus laevis and Bufo arenarum, a toad species found in the Buenos Aires region, were studied. We report that: (i) the water osmotic permeability (P f, cm/sec × 10−4) was significantly higher in Bufo (6°C=12.3±2.4; 18°C = 20.8±4.8) than in Xenopus oocytes (6°C=5.3±0.3; 18°C=6.2±1.6). The corresponding water diffusion permeability values (P d, cm/sec × 10−4) were: Xenopus = 2.3±0.3 (6°C) and 4.8±0.7 (18°C); Bufo=2.7±0.4 (6°C) and 6.0 ±0.5 (18°C). (ii) Amphotericin B increased the P f and P d values. The observed ΔP fΔP d ratio was not significantly different from the expected results (n=3), after amphotericin B incorporation in both species. This means that the influence of unstirred layers and other potential artifactual compounds did not significantly affect our experimental results, (iii) Preincubation with gramicidin during 12 hr induced a clear increase in the oocyte volume. After that, a hypotonic shock only slightly increased the oocyte volume. Conversely, a hypertonic challenge induced a volume change significantly higher than the one observed in control conditions, (iv) Mercury ions did not affect the osmotic permeability in Xenopus oocytes but clearly inhibited, in a reversible way, the osmotic permeability in oocytes from B. arenarum. (v) Mercury ions did not reduce P d values in either species, (vi) The ΔP fΔP d values calculated from the differences observed in these parameters between both species were 11.9±5.1 at 18°C and 15.5±2.4 at 6°C. These numbers are similar to those previously reported in the case of membranes having water channels. From these results, we propose that water channels are present in the ovarian oocyte from B. arenarum but not in the ovarian oocyte from X. laevis.
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  • 5
    ISSN: 1432-1424
    Keywords: Key words: Water flux — Forskolin — Vasopressin — LLC-PK1 cells — Mercury chloride — Toad urinary bladder
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Transepithelial water permeability was measured in LLC-PK1 cells stably transfected with aquaporins (AQPs): AQP1, AQP2, and a chimera of AQP1 and AQP2 containing 41 amino acids of the C-terminus of AQP2. Transepithelial water fluxes (Jw) were not previously reported in cells transfected with aquaporins. Jw were now recorded each minute using a specially developed experimental device. A significant increase in Posm after forskolin (FK) plus vasopressin (VP) was found in AQP2 transfected cells (39.9 ± 8.2 vs. 12.5 ± 3.3 cm · sec−1· 10−3), but not in cells transfected with AQP1 (15.3 ± 3.6 vs. 13.4 ± 3.6 cm · sec−1· 10−3). In the case of the AQP1/2 cells (chimera) the FK plus VP induced Posm was smaller than in AQP2 cells but significantly higher than in mock cells at rest (18.1 ± 4.8 vs. 6.7 ± 1.0 cm · sec−1· 10−3). The increases in Posm values were not paralleled by increases in 14C-Mannitol permeability. HgCl2 inhibited the hydrosmotic response to FK plus VP in AQP2 transfected epithelia. Results were comparable to those observed, in parallel experiments, in a native ADH-sensitive water channel containing epithelial barrier (the toad urinary bladder). Electron microscopy showed confluent LLC-PK1 cells with microvilli at the mucosal border. The presence of spherical or elongated intracellular vacuoles was observed in AQP2 transfected cells, specially after FK plus VP stimulus and under an osmotic gradient. These results demonstrate regulated transepithelial water permeability in epithelial cells transfected with AQP2.
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  • 6
    ISSN: 1432-1424
    Keywords: Frog and toad urinary bladder and skin ; Osmotic water permeability ; Channel-forming integral protein (CHIP28) ; Salt acclimation ; RT-PCR
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract A water channel, the frog aquaporin-CHIP (FA-CHIP) was recently cloned from Rana esculenta urinary bladder. The 28.9 kDa encoded protein shows 78.8%, 77.4%, 42.4% and 35.6% identity with rat CHIP28, human CHIP28, rat WCH-CD and γ-TIP, other members of the new transmembrane water channel family (Aquaporin-CHIP). We have now studied membranes from different frog (R. esculenta) organs employing semiquantitative PCR using FA-CHIP specific primers and an internal standard to quantify the PCR products. The FA-CHIP mRNA was abundantly expressed in the frog urinary bladder, skin, lung and gall bladder, while a lower expression was detected in the colon, liver and oviduct. FA-CHIP mRNA was not detected in the frog kidney, erythrocytes and brain but its expression was observed in the toad (Bufo arenarum) urinary bladder and skin, showing that FA-CHIP is probably a general amphibian water channel. Salt acclimation is known to increase the water permeability of frog and toad epithelia. We have now observed that salt acclimation for 1, 3, 4 or 5 days markedly increased skin and urinary bladder FA-CHIP mRNA expression. It is generally accepted that water permeability is controlled in these tissues by the rate of water channel transfer from subapical vesicles (aggrephores) to the apical membrane. Our results indicate that water permeability is also regulated at the level of the FA-CHIP transcription.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 176 (2000), S. 151-158 
    ISSN: 1432-1424
    Keywords: Key words: Proestrus — Estrus — Osmotic Permeability — Aquapoins — HgCl2, Diabetes mellitus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Important functional and structural modifications occur in mammalian oocytes during their arrival to maturity. In this process, oocytes switch from a high activity level, implying an important metabolic rate and a coordinated movement of water and solutes, to a lower functional state. The aim of this work was to study the mechanisms involved in water movements during oocyte arrival to maturity. Volume changes, induced by an osmotic gradient, were followed by video microscopy in rat oocytes. The water osmotic permeability (P osm ) of immature oocytes (proestrus) was sensitive to HgCl2 and phloretin. In contrast, mature oocytes (estrus) had a reduced P osm that was not sensitive to these compounds. When proestrus oocytes were incubated in vitro at 37°C they spontaneously arrived at maturity and its P osm decreased between four and six hours of incubation. RT-PCR experiments were performed using specific primers for all rat aquaporins that had been cloned. We found that aquaporin-9 transcript (AQP9) is present in proestrus oocytes but not in estrus oocytes. AQP9 has been recently described as a ``broad selective channel'' responsible for solute and water transfers in highly active cells. Our experiments showed that proestrus oocytes, but not estrus, are permeable to mannitol. It is concluded that during the process of maturation, P osm decreases and AQP9 transcripts disappear. We report here the first study correlating water permeability and aquaporin mRNA expression in mammalian oocytes.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 177 (2000), S. 149-157 
    ISSN: 1432-1424
    Keywords: Key words: T84 colon cells — Cell pH — Na+/H+ exchange — Cl−/HCO−3 exchange — Na+/HCO−3 cotransport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 ± 0.023 (n= 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 ± 0.011 pH units/min in the presence of Na+, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na+ independent H+ transport mechanisms in HEPES Ringer. In CO2/HCO− 3 Ringer, basal cell pH was 7.21 ± 0.020 (n= 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO2, followed by return to the initial pH at a rate of −0.14 ± 0.012 (n= 8) pH/min; this return was retarded or abolished in the absence of Cl− or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl−/HCO− 3 exchange. This exchange was not Na+ dependent. When Na+ was added to cells incubated in 0 Na+ Ringer while blocking Na+/H+ exchange by HMA, cell alkalinization by 0.19 ± 0.04 (n= 11) pH units was observed, suggesting the presence of Na+/HCO− 3 cotransport carrying HCO− 3 into these cells, which was abolished by DIDS. These experiments, thus, show that Na+/H+ and Cl−/HCO− 3 exchange and Na+/HCO− 3 cotransport participate in cell pH regulation in T84 cells.
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  • 9
    Electronic Resource
    Electronic Resource
    College Park, Md. : American Institute of Physics (AIP)
    Journal of Mathematical Physics 43 (2002), S. 1584-1597 
    ISSN: 1089-7658
    Source: AIP Digital Archive
    Topics: Mathematics , Physics
    Notes: In this work we establish a relationship between Cartan's geometric approach to third-order ordinary differential equations and the three-dimensional null surface formulation. We then generalize both constructions to allow for caustics and singularities that necessarily arise in these formalisms. © 2002 American Institute of Physics.
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  • 10
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Biochimica et Biophysica Acta (BBA)/Biomembranes 241 (1971), S. 716-718 
    ISSN: 0005-2736
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology , Chemistry and Pharmacology , Medicine , Physics
    Type of Medium: Electronic Resource
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