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  • 1
    ISSN: 1432-2013
    Keywords: ADH ; Transepithelial ion net fluxes ; Na+, Cl−, K+, Ca2+ and Mg2+ transport ; Electron microprobe ; Mouse kidney ; Cortical and medullary thick ascending limb of Henle's loop ; In vitro microperfusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The effect of antidiuretic hormone (arginine vasopressin, AVP) on transepithelial Na+, Cl−, K+, Ca2+ and Mg2+ net transports was investigated in medullary (mTAL) and cortical (cTAL) segments of the thick ascending limb (TAL) of mouse nephron, perfused in vitro. Transepithelial net fluxes (J Na +,J Cl −,J K +,J Ca 2+,J Mg 2+) were determined by electron probe analysis of the collected tubular fluid. Transepithelial potential difference (PDte) and transepithelial resistance (Rte) were measured simultaneously. cTAL segments were bathed and perfused with isoosmolal, HCO 3 − containing Ringer solutions, mTAL segments were bathed and perfused with isoosmolal HCO 3 − free Ringer solutions. In cTAL segments, AVP (10−10 mol·l−1) significantly increasedJ Mg 2+ andJ Ca 2+ from 0.39±0.08 to 0.58±0.10 and from 0.86±0.13 to 1.19±0.15 pmol·min−1 mm−1 respectively. NeitherJ Na + norJ Cl −, (J Na +: 213±30 versus 221±28 pmol·min−1 mm−1,J Cl −: 206±30 versus 220±23 pmol·min−1 mm−1) nor PDte (13.4±1.3 mV versus 14.1±1.9 mV) or Rte (24.6±6.5Ω cm2 versus 22.6±6.4Ω cm2) were significantly changed by AVP. No significant effect of AVP on net K+ transport was observed. In mTAL segments, Mg2+ and Ca2+ net transports were close to zero and AVP (10−10 mol·l−1) elicited no effect. However NaCl net reabsorption was significantly stimulated by the hormone,J Na + increased from 107±33 to 148±30 andJ Cl − from 121±33 to 165±32 pmol·min−1 mm−1. The rise inJ NaCl was accompanied by an increase in PDte from 9.0±0.7 to 13.5±0.9 mV and a decrease in Rte from 14.4±2.0 to 11.2±1.7 Ω cm2. No K+ net transport was detected, either under control conditions or in the presence of AVP. To test for a possible effect of HCO 3 − on transepithelial ion fluxes, mTAL segments were bathed and perfused with HCO 3 − containing Ringer solutions. With the exception ofJ Ca 2+ which was significantly different from zero (J Ca 2+: 0.26±0.06 pmol·min−1 mm−1), net transepithelial fluxes of Na+, Cl−, K+ and Mg2+ were unaffected by HCO 3 − . In the presence of AVP,J Mg 2+ andJ Ca 2+ were unaltered whereasJ NaCl was stimulated to the same extent as observed in the absence of HCO 3 − . In conclusion our results indicate heterogeneity of response to AVP in cortical and medullary segments of the TAL segment, since AVP stimulates Ca2+ and Mg2+ reabsorption in the cortical part and Na+ and Cl− reabsorption in the medullary part of this nephron segment.
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  • 2
    ISSN: 1432-2013
    Keywords: Isolated perfused tubule ; cTAL ; Na+ 2Cl− K+ cotransporter ; piretanide ; macromolecular probe
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Piretanide blocks the Na+ 2Cl− K+ cotransporter protein in the thick ascending limb (TAL) of the loop of Henle reversibly. When tested from the luminal side in isolated perfused cTAL segments it leads to a half maximal inhibition (IC50) of the equivalent short circuit current (Isc) at a concentration of 10−6 mol/l. From the basolateral side it has no effect on Isc up to 10−4 mol/l. The present study was designed to search for high affinity blockers of the Na+ 2Cl− K+ cotransporter with large molecular weight in an attempt to use these macromolecules for antibody-labelling or affinity separation of this transport-protein. Amino-ethyl-dextran or amino-ethyl-polyethylene glycol (M.W. 5kd) were coupled to isothiocyanato-piretanide (ISO-PIR) at room temperature in DMSO. The resulting compounds dextran-sulfonylurea-piretanide (PIR-DEX) and polyethylene glycol-sulfonylurea-piretanide (PIR-PEG) (M.W. 5.38kd) were purified and tested in isolated perfused cTAL segments. IC50 values for ISO-PIR, PIR-DEX and PIR-PEG were estimated from dose response curves after their addition to the lumen or bath perfusate, respectively. ISO-PIR, PIR-DEX and PIR-PEG acted from the lumen side at 3·10−6, 6·10−6 and 2·10−6 mol/l. The inhibitory effect was easily reversible. From the basolateral side no effect for any compound was seen at up to 10−4 mol/l. In clearance experiments PIR-DEX was given to female Wistar rats as an i.v. bolus (25 μmol/kg) and the diuretic urine was collected. After dialysis (exclusion limit 2.5kd) the dialysed urine and the dialysate were tested in isolated perfused cTAL segments. The dialysates had no effect on Isc, but the dialysed urine inhibited Isc by 35% from the luminal side. The present data show: High molecular derivatives of piretanide with dextran or polyethylene glycol moieties block the Na+ 2Cl− K+ cotransporter in cTAL segments at roughly the same low concentration as piretanide itself. Our data exclude a metabolism of these piretanide compounds in the kidney. Since these macromolecular probes can probably not enter the cell their inhibitory effect indicates that the binding site for piretanide diuretics on the Na+ 2Cl− K+ cotransporter is exposed on the surface of the luminal cell membrane.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1432-2013
    Keywords: Glucagon ; Transepithelial ion net fluxes ; Na+, Cl−, K+, Ca2+, Mg2+ transport ; Electron microprobe ; Mouse kidney ; In vitro microperfusion ; Cortical and medullary thick ascending limb of Henle's loop ; In vivo micropuncture study
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The effects of glucagon on transepithelial Na+, Cl−, K+, Ca2+ and Mg2+ net fluxes were investigated in isolated perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. Transepithelial ion net fluxes (J Na +,J Cl −,J K +,J Ca 2+,J Mg 2+) were determined by electron probe analysis of the collected tubular fluid. Simultaneously the transepithelial voltage (PDte) and the transepithelial resistance (R te) were recorded. In cTAL-segments (n=8), glucagon (1.2×10−8 mol · l−1) stimulated significantly the reabsorption of Na+, Cl−, Ca2+ and Mg2+∶J Na + increased from 204±20 to 228±23 pmol · min−1 · mm−1,J Cl − from 203±18 to 234±21 pmol · min−1 · mm−1,J Ca 2+ from 0.52±0.13 to 1.34±0.30 pmol · min−1 · mm−1 andJ Mg 2+ from 0.51±0.08 to 0.84±0.08 pmol · min−1 · mm−1.J K+ remained unchanged: 3.2±1.3 versus 4.0±1.9 pmol · min−1 · mm−1. Neither PDte (16.3±1.5 versus 15.9±1.4 mV) norR te (22.5±3.0 versus 20.3±2.6 Ωcm2) were changed significantly by glucagon. However, in the post-experimental periods a significant decrease in PDte and increase inR te were noted. In mTAL-segments (n=9), Mg2+ and Ca2+ transports were close to zero and glucagon elicited no significant effect. The reabsorptions of Na+ and Cl−, however, were strongly stimulated:J Na + increased from 153±17 to 226±30 pmol · min−1 · mm−1 andJ Cl − from 151±23 to 243±30 pmol · min−1 · mm−1. The rise in NaCl transport was accompanied by an increase in PDte from 10.3±1.1 to 12.3±1.2 mV and a decrease inR te from 19.1±2.7 to 17.8±2.0 Ωcm2. No net K+ movement was detectable either in the absence or in the presence of glucagon. A micropuncture study carried out in hormone-deprived rats indicated that glucagon stimulates Na+, Cl−, K+, Mg2+ and Ca2+ reabsorptions in the loop of Henle. In conclusion our data demonstrate that glucagon stimulates NaCl reabsorption in the mTAL segment and to a lesser extent in the cTAL segment whereas it stimulates Ca2+ and Mg2+ reabsorptions only in the cortical part of the thick ascending limb of the mouse nephron. These data are in good agreement with, and extend, those obtained in vivo on the rat with the hormone-deprived model.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Medical microbiology and immunology 175 (1986), S. 1-13 
    ISSN: 1432-1831
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract A virus previously isolated from fledgling budgerigars (Melopsittacus undulatus) suffering from an acute disease, has been purified and the structural characteristics have been determined. The virions with a buoyant density of 1.34 g/ml are non-enveloped icosahedral particles with a diameter of about 46–48 nm. Their DNA genome has a molecular weight of about 3.3×106 d, and exists as supericoiled circular, relaxed circular, and linear molecules. There are eight structural proteins, the most abundant of which has a molecular weight of about 42,000 d. Empty capsid shells with buoyant densities of 1.31 g/ml are similar in size and shape, but lack DNA and histone-like polypeptides. Virus replication in chicken embryo cells results in cytopathic changes characterized by rounding and enlargement of the nucleus, and formation of intranuclear inclusion bodies. All these properties justify classification of the virus as polyoma-like.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    s.l. ; Stafa-Zurich, Switzerland
    Materials science forum Vol. 457-460 (June 2004), p. 143-146 
    ISSN: 1662-9752
    Source: Scientific.Net: Materials Science & Technology / Trans Tech Publications Archiv 1984-2008
    Topics: Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1662-9752
    Source: Scientific.Net: Materials Science & Technology / Trans Tech Publications Archiv 1984-2008
    Topics: Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics
    Notes: The aim of the present work is to grow 3C-SiC on (0001) 6H-SiC seeds using thePhysical Vapour Transport (PVT) method and to study the electrical and structural properties of the grown material. Photoluminescence (PL)-mappings reveal that the overgrown layer consists predominantly of the 3C-SiC polytype and capacitance-voltage (C-V) measurements result in a net nitrogen donor concentration of 1x1016cm-3. Transmission Electron Microscopy (TEM)observations also confirm that the overgrown layer is of the 3C-SiC polytype having the cubic [111] crystallographic direction parallel to the c-axis of the 6H-SiC substrate. In some cases, twin crystals of 3C-SiC are formed immediately after the interface and, in a few cases, small 6H-SiC inclusions are observed in the cubic film having the same orientation as the substrate. The film near the substrate/overgrown interface shows a high density of defects such as dislocations and stacking faults (SF’s), which propagate into the overgrown layer. Finally although there is a rapid decrease of the defect density within the first 60 µm from the interface, the SF density remains almost constant within the last 100 µm below the surface
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1432-2013
    Keywords: Ca2+ Cyclosporin A Fura-2 Kidney LLC-PK1-cells Nephrotoxicity Proximal tubule Signal transduction Tacrolimus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract. Here we have examined the effects of Cyclosporin A (CyA) on the free intracellular Ca2+ concentration ([Ca2+]i) of LLC-PK1/PKE20 cells to evaluate mechanisms of CyA nephrotoxicity using Fura-2 microspectrofluorometry or digital fluorescence video imaging. The CyA-associated changes were compared to the effects of tacrolimus (Tac), a structurally unrelated immunosuppressant with similar cellular pathways which also causes nephrotoxicity. CyA (EC50: 1 nmol/l, n=16) and Tac (EC50: 1 nmol/l, n=5) caused a concentration-dependent increase of [Ca2+]i which was substantially attenuated by reducing the external Ca2+ concentration (10–6 mol/l). Similarly Cyclosporin H, a non-immunosuppressive analogue of CyA, stimulated a Ca2+ influx. Nicardipine (10–6 mol/l) reduced the CyA- and the Tac-induced Ca2+ influx to 52±16% (n=10) and 13±10% (n=13) of control respectively. Diltiazem and verapamil (10–6 mol/l) were also effective, but flufenamate (10–4 mol/l), Gd3+ (10–5 mol/l) and La3+ (10–5 mol/l) were not. In the absence of extracellular Ca2+ CyA led to a small but significant [Ca2+]i increase, indicating additional release from internal stores. Depletion of inositol-1,4,5-trisphosphate- (InsP 3-) sensitive Ca2+ stores by extracellular ATP (10–4 mol/l) in low-Ca2+ solution completely suppressed the CyA-induced [Ca2+]i rise. CyA had no effect on the cellular InsP 3 concentration. Furthermore, inhibition of phospholipase-Cβ (PLCβ) by U73122 (2×10–5 mol/l) did not alter the CyA-stimulated [Ca2+]i rise. A direct effect of CyA on InsP 3-sensitive Ca2+ stores, the InsP 3 receptor, the Ca2+ content of the stores or involvement of additional stores is assumed. Incubation with CyA for 1, 12 and 24 h enhanced the rise in [Ca2+]i peak induced by ATP, arginine vasopressin (AVP) and angiotensin II. In summary, CyA stimulated a [Ca2+]i increase in LLC-PK1 cells through Ca2+ release from InsP 3-sensitive stores and Ca2+ influx via a nicardipine-sensitive pathway. The CyA-mediated [Ca2+]i increase is independent of PLCβ activity and InsP 3 metabolism. CyA caused long-term enhancement of the agonist-induced rise in [Ca2+]i. The effects of CyA on Ca2+ signaling appear to be independent of its immunosuppressive action.
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  • 8
    ISSN: 1432-2013
    Keywords: cAMP Carbachol Cl– secretion Exocrine secretion K+ channels Volume regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract. The Na+2Cl–K+ cotransporter accepts NH4 + at its K+-binding site. Therefore, the rate of cytosolic acidification after NH4 + addition to the bath (20 mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH4 + led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4 + uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (Δ fluorescence ratio units/1000 s). In pilot experiments it was shown that the pH signal caused by the Na+2Cl–K+ cotransporter could be amplified if the experiments were carried out in the presence of bath Ba2+ to inhibit NH4 + uptake via K+ channels. Therefore all subsequent experiments were performed in the presence of 1 mmol/l Ba2+. In the absence of any secretagogue, preincubation of RCC in a low-Cl– solution (4 mmol/l) for 10 min enhanced the uptake rate significantly from 1.70±0.11 to 2.54±0.27 U/1000 s (n=20). The addition of 100 mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93±0.17 to 2.84±0.43 U/1000 s (n=5). Stimulation of NaCl secretion by a solution containing 100 µmol/l carbachol (CCH) led to a small but significant increase in NH4 + uptake rate from 2.06±0.34 to 2.40±0.30 U/1000 s (n=11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100 µmol/l and 5 µmol/l, respectively) was from 2.39±0.24 to 3.06±0.36 U/1000 s (n=24). Whatever the mechanism used to increase the NH4 + uptake rate, azosemide (500 µmol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na+2Cl–K+ cotransporter: (1) lowering of cytosolic Cl– concentration; (2) cell shrinkage; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Medical microbiology and immunology 176 (1987), S. 113-121 
    ISSN: 1432-1831
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The molecular weights (mol. wts.) of the two double-stranded (ds) RNA segments of infectious bursal disease virus (IBDV) were determined using previously sequenced reovirus genes M3 and S2 as internal ds RNA reference molecules. Electrophoresis under fully denaturing conditions revealed mol. wts. of 2.26 × 106 daltons and 1.98 × 106 daltons. By direct length measurements under the electron microscope, using two different spreading conditions, the two segments were calculated to be composed of 3274 ± 79 base pairs (bp) and 2821 ± 59 bp or 3299 ± 68 bp and 2830 ± 73 bp, resulting in mol. wts. of 2.24– 2.26 × 106 daltons and 1.93–1.94 × 106 daltons, respectively. Base pair distances of 2.67 ± 0.08 Å and 2.71 ± 0.11 Å in ds RNA were close to those of the A-RNA form; in ds DNA included as a control, the rise per base pair was 3.18 Å, which is consistent with published results. Mol. wts. obtained for IBDV indicate that the RNAs of the other birnaviruses are also smaller than reported.
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