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  • 1
    ISSN: 1420-908X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The pharmacological actions of prostaglandins E1, F1α, E2 and A1 were studied on the longitudinal and circular muscles of the isolated small intestine of the toad. Prostaglandin E1 caused relaxation, whereas prostaglandin A1 was inactive and prostaglandins F1α and E2 caused contractions. Other substances which relaxed the toad intestine were adrenaline, noradrenaline and isoprenaline. On a molar basis, prostaglandin E1 was seven times less potent than isoprenaline, but it was three times more potent than adrenaline and seventeen times more potent than noradrenaline. Phentolamine and propranolol blocked the relaxant effects of catecholamines in concentrations which did not alter the relaxant effect of prostaglandin E1. The results suggested that prostaglandin E1 acted directly on the intestinal smooth muscle of the toad rather than by the local release of catecholamines.
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  • 2
    ISSN: 1420-9071
    Keywords: Quantitation of intracellular vanadium (free and bound) ; nuclear sequestration of vanadium ; nuclear microscopy ; cell cycle phase-specific evaluation of sub-2N DNA in flow cytometry ; programmed cell death ; autophagic autodigestion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Very little is known about the modulation of vanadium accumulation in cells, although this ultratrace element has long been seen as an essential nutrient in lower life forms, but not necessarily in humans where factors modulating cellular uptake of vanadium seem unclear. Using nuclear microscopy, which is capable of the direct evaluation of free and bound (total) elemental concentrations of single cells we show here that an NH4Cl acidification prepulse causes distinctive accumulation of vanadium (free and bound) in human Chang liver cells, concentrating particularly in the nucleus. Vanadium loaded with acidification but leaked away with realkalinization, suggests proton-dependent loading. Vanadyl(4), the oxidative state of intracellular vanadium ions, is known to be a potent source of hydroxyl free radicals (OH.). The high oxidative state of nuclei after induction of vanadyl(4) loading was shown by the redox indicator methylene blue, suggesting direct oxidative damage to nuclear DNA. Flow cytometric evaluation of cell cycle phase-specific DNA composition showed degradation of both 2N and 4N DNA phases in G1, S and G2/M cell cycle profiles to a solitary 1N DNA peak, in a dose-dependent manner, effective from micromolar vanadyl(4) levels. This trend was reproduced with microccocal nuclease digestion in a time response, supporting the notion of DNA fragmentation effects. Several other approaches confirmed fragmentation occurring in virtually all cells after 4 mM V(4) loading. Ultrastructural profiles showed various stages of autophagic autodigestion and well defined plasma membrane outlines, consistent with programmed cell death but not with necrotic cell death. Direct intranuclear oxidative damage seemed associated with the induction of mass suicide in these human Chang liver cells following vanadium loading and nuclear sequestration.
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  • 3
    ISSN: 0741-0581
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1573-0603
    Keywords: cell dissociation ; detachment ; rounding ; Na+/H+ antiport ; endocytosis ; sulphate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Activating the Na+/H+ antiporter causes a distinctive and rapid endocytosis which internalizes the plasma membrane leading to cell retraction and detachment as rounded cells with much reduced surface area. Antiport activation is by a combination of two individually effective motivations, viz. a) steep [Na+] and [H+] gradients across the plasma membrane and b) allosteric activation by second messengers initiated with simple inorganic sulphate. The dissociated cells are as viable as those released by trypsinization. Thus it provides an effective enzyme-free alternative to trypsin digestion in cell dissociation.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    The @Anatomical Record 235 (1993), S. 183-190 
    ISSN: 0003-276X
    Keywords: Mitosis ; Cell rounding ; Surface area ; Endocytosis ; Intracellular pH ; BCECF ; Neutral red ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The rounding up of mitotic human Chang liver cells in monolayer culture was studied quantitatively. It was surprising to find significant reduction in cell surface area considering that endocytosis has been demonstrated to be at a complete standstill in M phase. Uptake studies using impermeant BCECF (2′,7′-bis(2-carboxyethyl)-4(5)-carboxyfluorescein free acid) pH indicator and particulate neutral red dye in aqueous buffer showed preferential internalization into mitotic cells in direct contrast to expectation since interphase cells do not have arrested endocytosis. However, infolded plasma membrane ruffles and internalized extracellular material were demonstrated in prophase cells, much like those seen in interphase rounding via the induction of intracellular alkalinizations. Raised intracellular pH (pHi) is a universal and consistent finding in M phase cells. Despite cessation of small pit endocytosis, it remains possible for plasma membrane internalization to be a causal factor in the observed surface area reduction in mitotic rounding. © 1993 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Apoptosis 5 (2000), S. 29-36 
    ISSN: 1573-675X
    Keywords: Caspase-3 ; cell shrinkage ; DEVD-cho ; late mitosis cell cycle arrest ; megabase DNA fragmentations ; phosphatidylserine externalization ; zVAD-fmk.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Cell growth in human Chang liver cells was arrested in late mitosis with 600 μM zVAD-fmk. Associated cell death manifested cell shrinkage and phagocytic marking shown by phosphatidylserine (PS) externalization, in a dose-dependent manner. While low molecular weight internucleosomal ladder cleavages were suppressed, there were however high molecular weight DNA cleavages extending up to megabase level in association with chromatin condensation that appeared more marked than the staurosporine-induced positive-apoptosis control. Caspase-3 activity was suppressed. Specific caspase-3 inhibitor DEVD-cho also produced cell growth suppression with late mitosis arrest, and cell shrinkage which was expressed concomitantly with phagocytic PS marking in similar dose-dependent manners at 50 to 100 μM concentrations. Cell shrinkage, PS externalization, and high molecular weight DNA cleavages associated with chromatin condensation without 200 bp ladder fragmentations, were dissociated from caspase-3 activity. Anticaspases inducing late mitosis arrest provided fresh insights into late mitosis progression.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Apoptosis 5 (2000), S. 379-388 
    ISSN: 1573-675X
    Keywords: apoptosis ; ATP depletion ; cell acidification and shrinkage ; CpG-specific megabase fragmentations ; DCNP (2,6-dichloro-4-nitrophenol) ; housekeeping genes ; microarray (genechip) ; zVAD-fmk
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Previous suggestions of CpG-specific apoptotic commitment implied critical epigenetic modulation of house-keeping genes which have canonical CpG islands at 5′ promoter regions. Differential housekeeping gene activity however has not been shown. Using a focussed microarray (genechip) of 22 housekeeping genes we show this in apoptosis induced in human Chang liver cells by DCNP (2,6-dichloro-4-nitrophenol), a non-genotoxic inhibitor of sulfate detoxification. 3–7 folds downregulation of 9 genes in glycolysis, tricarboxylic acid cycle and the respiratory electron transport chain suggested gene-directed energy depletion which was correlated with observed ATP depletion. 4 folds downregulation of the pyruvate dehydrogenease gene suggested gene-directed metabolic acidosis which was correlated with observed cell acidification. Other differential housekeeping gene activity, including 4 folds upregulation of microtubular alpha-tubulin gene, and 2 folds upregulation of ubiquitin, also had a bearing on apoptosis. Broadspectrum zVAD-fmk caspase inhibition abolished 200 bp DNA ladder fragmentations but not the CpG-specific megabase fragmentations and other hallmarks of cell destruction, suggesting a caspase-independent cell death. Death appeared committed at gene-level.
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  • 8
    ISSN: 1573-0603
    Keywords: cultured cell microanalysis ; cold-stretched Pioloform substrate ; film-stripping in water ; Permount-coated target holder ; serum-prepulsed cell plating
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Accurate microanalysis of elemental concentrations and distributions especially with regard to trace elements have been difficult because the concentrations are usually below the threshold of the more commonly used electron microprobes. Cultured cell microanalysis with the aid of the nuclear microscope provides accurate quantitation of minor and trace elements, including variations when subjected to modulations and perturbations. We show here our highly reproducible technique of preparing monolayer cells for nuclear microscopy, using thin Pioloform supports stretched by cold treatment over dry ice. Cells are grown directly on these Permount-anchored Pioloform thin films using serum-prepulsed plating.
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  • 9
    ISSN: 0003-276X
    Keywords: Flow cytometric analysis of BCECF ratios ; Neutral red uptake and propidium iodide-DNA bindings ; Ao apoptotic peak ; two million mol.wt dextrans ; Macrophagic internalizations ; Large channel endocytosis ; Image analysis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Background: The early stages of apoptosis (programmed cell death) are said to be characterized by internucleosomal DNA fragmentation and “condensation of the cytoplasm” in which cells round up, detach, and increase in density. We studied the causation of apoptotic rounding.Methods: Human Chang liver cells in normal monolayer culture were compared with apoptotic counterparts derived from serum growth factor deprivation. Cell-by-cell analysis using the Coulter EPICS PROFILE II flow cytometer studied (1) the cell cycle from propidium iodide-DNA bindings, (2) uptake of neutral red (NR) dye, a viable cell marker, and (3) cytosolic pH (pHi) modulations from 2′,7′-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) fluorescence ratios with NH4Cl prepulsing and forward scatter bitmapping of cell surface area. Morphometric studies were done in the Quantimet 570 image analyser. Uptake of trypan blue, neutral red, and 2 million mol.wt fluoresceinated dextrans was studied by light microscopy. Cytological profiles were examined in light microscopy and transmission and scanning electron microscopy.Results: Three days of serum growth factor deprivation caused confluent flat substrate-attached cells to retract and round up, tethering tenuously to the substrate via thin microvillus attachments only. Ninety percent of cell surface area was lost with this flat-to-round change. There was high trypan blue staining with total loss of proliferative potential, and the entire genome was just fragmented DNA making up the solitary Ao (apoptotic) peak in cell cycle profiles. However, these rounded apoptotic cells also internalized huge 2 million mol.wt dextran particles and impermeant neutral red which is an established viable cell marker. The rounded apoptotic cells had an intensely acidic (pH 5.6) cytosol and therefore a steep [H+]i/[H+]o gradient promoting proton extrusion. The pHi upshifted dynamically upon acidification, recovering and even exceeding resting level by a whole pH unit. Surface area reduction occurred concomitantly in real time with pHi upshifts in these apoptotic cells. Acidification and recovery in apoptotic cells also produced enhanced uptake of neutral red. Cytological profiles showed abundant large endocytic channels and endosomes in the rounded apoptotic cells.Conclusion: Gross surface area reduction with evidence of distinctive endocytic activity including uptake of huge 2 million mol.wt dextran particles suggested large channel endocytic internalization as a causal factor in apoptotic rounding, in common with rounding in M-phase and interphase cells with pHi upshifting where concomitant surface area reduction and uptake of impermeant particles were similarly demonstrable. The reduction in size of the cell envelope, together with consequential concentration pressures, could account for the observed rise in cell density and shrinkage in cell size. As a symptom of continual pHi upshifting, apoptotic rounding appears to be a recovery-associated response rather than a direct consequence of the disruptive forces causing its death. © 1994 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
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