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  • 1
    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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  • 2
    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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  • 3
    ISSN: 1365-2559
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Aims : To evaluate the morphological features of 11 cases of breast ductal carcinoma in situ (DCIS) with spindle cells and to propose an approach to distinguish it from benign mimics. The association with neuroendocrine differentiation was also investigated.Methods : Cases of breast DCIS with a spindle cell component diagnosed in the Department of Pathology, Singapore General Hospital, between June 1996 and January 2003, were included in the study. The histological characteristics were documented, and immunohistochemistry for neuroendocrine markers, hormone receptors, cerbB2, smooth muscle actin (SMA) and high-molecular-weight (HMW) cytokeratins, was carried out. Electron microscopy was carried out on reprocessed paraffin-embedded material in three cases.Results : Of 11 women diagnosed with DCIS with spindle cells, four presented with nipple discharge, six with a breast lump, while one was discovered to have a screen detected density. The tumour size ranged from 3 to 41 mm. The proportion of spindle cells varied from 10% to 80% of the in-situ tumour cell population. Nuclear grade was low in seven cases and intermediate in four. Necrosis was observed in two cases. Architectural pattern was papillary in six cases, and mixed in the rest. Microinvasion was present in two cases, with possible microinvasion in another two. Immunohistochemistry for neuroendocrine markers synaptophysin and chromogranin showed positive reactivity for at least one marker in all but three cases; one of these latter cases demonstrated ultrastructural neurosecretory granules. Oestrogen and progesterone receptors were expressed in 10 and nine cases, respectively, while cerbB2 was positive in only one case. HMW cytokeratin immunoprofile revealed a general lack of immunostaining within the abnormal cell population; likewise, no positivity for SMA of the cellular proliferation was detected.Conclusions : Almost all DCIS lesions with spindle cells disclose neuroendocrine differentiation. Although the distinction from benign florid usual hyperplasia may pose a diagnostic histological problem, the presence of diffuse neuroendocrine expression, in conjunction with the pattern of HMW keratin profile on immunohistochemistry, supports an in-situ neoplastic process. The absence of SMA immunostaining, in conjunction with negative reactivity for cytokeratins 5/6 and 14, makes the possibility of a myoepithelial proliferation unlikely.
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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
    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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  • 7
    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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  • 8
    Publication Date: 2018-06-27
    Description: Thyroid hormone receptor β1 (THRB1) and estrogen-related receptor α (ESRRA; also known as ERRα) both play important roles in mitochondrial activity. To understand their potential interactions, we performed transcriptome and ChIP-seq analyses and found that many genes that were co-regulated by both THRB1 and ESRRA were involved in mitochondrial metabolic pathways. These included oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and β-oxidation of fatty acids. TH increased ESRRA expression and activity in a THRB1-dependent manner through the induction of the transcriptional coactivator PPARGC1A (also known as PGC1α). Moreover, TH induced mitochondrial biogenesis, fission, and mitophagy in an ESRRA-dependent manner. TH also induced the expression of the autophagy-regulating kinase ULK1 through ESRRA, which then promoted DRP1-mediated mitochondrial fission. In addition, ULK1 activated the docking receptor protein FUNDC1 and its interaction with the autophagosomal protein MAP1LC3B-II to induce mitophagy. siRNA knockdown of ESRRA , ULK1 , DRP1 , or FUNDC1 inhibited TH-induced autophagic clearance of mitochondria through mitophagy and decreased OXPHOS. These findings show that many of the mitochondrial actions of TH are mediated through stimulation of ESRRA expression and activity, and co-regulation of mitochondrial turnover through the PPARGC1A-ESRRA-ULK1 pathway is mediated by their regulation of mitochondrial fission and mitophagy. Hormonal or pharmacologic induction of ESRRA expression or activity could improve mitochondrial quality in metabolic disorders.
    Print ISSN: 1945-0877
    Topics: Medicine
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