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  • 1
    Electronic Resource
    Electronic Resource
    Two distinct isoforms of sea urchin egg dynein (1992)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 281-292 
    Details
    ISSN: 0886-1544
    Keywords: ATPase ; CTPase ; minus-end-directed microtubule motility ; cytoplasmic dynein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Extracts of unfertilized sea urchin eggs contain at least two isoforms of cytoplasmic dynein. One exhibits a weak affinity for microtubules and is primarily soluble. The other isoform, HMr-3, binds to microtubules in an ATP-sensitive manner, but is immunologically distinct from the soluble egg dynein (Porter et al.: Journal of Biological Chemistry 263:6759-6771, 1988). We have now further distinguished these egg dynein isoforms based on differences in NTPase activity. HMr-3 copurifies with NTPase activity, but it hydrolyzes CTP at 10 times the rate of ATP. The soluble egg dynein is similar to flagellar dynein in its nucleotide specificity; its MgCTPase activity is ca. 60% of its MgATPase activity. Non-ionic detergents and salt activate the MgATPase activities of both enzymes relative to their MgCTPase activities, but this effect is more pronounced for the soluble egg dynein than for HMr-3. Sucrose gradient-purified HMr-3 promotes an ATP-sensitive microtubule bundling, as seen with darkfield optics. We have also isolated a 20 S microtubule translocating activity by sucrose gradient fractionation of egg extracts, followed by microtubule affinity and ATP release. This 20 S fraction, which contains the HMr-3 isoform, induces a microtubule gliding activity that is distinct from kinesin. Our observations suggest that soluble dynein resembles axonemal dynein, but that HMr-3 is related to the dynein-like enzymes isolated from a variety of cell types and may represent the cytoplasmic dynein of sea urchin eggs.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970210404
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  • 2
    Electronic Resource
    Electronic Resource
    Vanadate reduces sodium-dependent glucose transport and increases glycolytic activity in LLC-PK1 epithelia (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 158 (1994), S. 459-466 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effect of vanadate pentoxide on apical sodium-dependent glucose transport in LLC-PK1 epithelia was examined. Epithelia grown in the presence or absence of 1 μM vanadate formed confluent monolayers and exhibited no differences in DNA, protein, or ultrastructure. Vanadate-supplemented epithelia demonstrated a lower steady-state α-methyl-D-glucopyranoside (AMG) concentrating capacity and a twofold reduction in apical AMG uptake Jmax. This decreased AMG transport occurred as a consequence of a reduction in the number of transport carriers and was not associated with a change in the sodium electrochemical gradient. The vanadate-induced reduction in apical glucose carrier functional activity and expression was accompanied by a stimulation of intracellular glycolytic flux activity, as evidenced by increased glucose consumption, lactate production, PFK-1 activity, and intracellular ATP. There was no difference in intracellular cAMP levels between vanadate-supplemented and non-supplemented epithelia. These results demonstrate an association between stimulation of glycolytic pathway activity and an adaptive response in the form of a reduction in the function and expression of the sodium-dependent apical glucose transporter in LLC-PK1 epithelia. © 1994 Wiley-Liss, Inc.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041580310
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  • 3
    Electronic Resource
    Electronic Resource
    Regulation of polyamine transport by polyamines and polyamine analogs (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 155 (1993), S. 399-407 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Regulation of polyamine transport in murine L1210 leukemia cells was characterized in order to better understand its relationship to specific intracellular polyamines and their analogs and to quantitate the sensitivity by which it is controlled. Up-regulation of polyamine uptake was evaluated following a 48-hr treatment with a combination of biosynthetic enzyme inhibitors to deplete intracellular polyamine pools. The latter declined gradually over 48 hr and was accompanied by a steady increase in spermidine (SPD) and spermine (SPM) transport as indicated by rises in Vmax to levels ∼4.5 times higher than control values. Restoration of individual polyamine pools during a 6-hr period following inhibitor treatment revealed that SPD and SPM uptake could not be selectively affected by specific pool changes. The effectiveness of individual polyamines in reversing inhibitor-induced stimulation of uptake was as follows: putrescine 〈 SPD 〈 SPM = the SPM analog, N1, N12-bis(ethyl)spermine (BESPM). In contrast to stimulation of transport, down-regulation by exogenous polyamines or analogs occurred rapidly and in response to subtle increases in intracellular pools. Following a 1-hr exposure to 10 μM BESPM, Vmax values for SPD and SPM fell by 70%, whereas the analog pool increased to only 400-500 pmol/106 cells - about 15-20% of the total polyamine pool (∼2.8 nmol/106 cells). SPM produced nearly identical regulatory effects on transport kinetics. Both BESPM and SPM were even more effective at down-regulating transport that had been previously stimulated four to fivefold by polyamine depletion achieved with enzyme inhibitors. A dose response with BESPM at 48 hr revealed a biphasic effect on uptake whereby concentrations of analog 〈 3 μM produced an increase in SPD and SPM Vmax values, whereas concentrations 3 μM and higher produced a marked suppression of these values. Cells treated with 3 μM BESPM for 2 hr and placed in analog-free medium recovered transport capability in only 3 hr. Thus, whereas stimulation of polyamine transport is a relatively insensitive and slowly responsive process that tends to parallel polyamine depletion, down-regulation of polyamine transport by exogenous polyamines and analogs and its reversal are rapidly responsive events that correlate with relatively small (i.e., 15-20%) changes in intracellular polyamine pools.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041550222
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  • 4
    Electronic Resource
    Electronic Resource
    Novel monoclonal antibodies identify antigenic determinants unique to cellular senescence (1990)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 142 (1990), S. 425-433 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Normal human diploid fibroblasts exhibit a limited lifespan in vitro and are used as a model to study in vivo aging. Monoclonal antibodies were generated against partially purified surface membranes from human diploid fibroblasts at the end of their lifespan (senescent). Three hybridomas were isolated that secreted antibodies reacting to cellular determinants expressed specifically on senescent human fibroblasts of different origin, including neonatal foreskin, embryonic lung, and adult skin punch biopsy, but not expressed on matched young cells. The antibodies did not bind to immortal human cells and normal young cells made reversibly nondividing, indicating the antigens are not expressed in cells that are not senescent. The antibodies identified senescent cells in a mixed cell population and expression of the senescent cell antigens correlated strongly with the cells inability to synthesize DNA at the onset of senescence. The antigens appeared to be cell surface or extracellular matrix associated, and the epitopes were destroyed by mild trypsin treatment. Western analysis indicated all three antibodies reacted with fibronectin. Though the antigenic determinants on the fibronectir molecule were not accessible in the intact young cell, the epitopes were present in fibronectin extracted from both senescent and young cells, as well as purified human plasma fibronectin. These antibodies and the senescent specific expression of the antigens provide powerful tools to investigate the mechanisms leading to in vitro senescence. This may enable us to investigate directly the relationship between cellular aging and aging of the individual.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041420228
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  • 5
    Electronic Resource
    Electronic Resource
    Common senescent cell-specific antibody epitopes on fibronectin in species and cells of varied origin (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 150 (1992), S. 545-551 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The phenomenon of in vitro cellular senescence has been demonstrated in cultured cells derived from humans and various other species. We have previously shown that monoclonal antibodies SEN-1, SEN-2, and SEN-3 react to epitopes on fibronectin that are exposed when human diploid fibroblasts become senescent. We here present results demonstrating that exposure of these epitopes is specific to senescence for a variety of human cells: epidermal keratinocytes, mammary epithelial cells, as well as fibroblasts. Fibronectin from 11 additional species was also analyzed by Western immunoblot for ability to bind the SEN antibodies. SEN-1 bound only human and gorilla fibronectin, whereas SEN-2 and SEN-3 bound fibronectin from those two species as well as the horse, cow, sheep, goat, dog, and chick. None of the antibodies reacted with fibronectin from the rabbit, rat, or mouse. These data indicated a correlation between the ability of the SEN antibodies to bind fibronectin from a particular species and the ability of cells from that species to exhibit a stable senescent phenotype in vitro. Therefore, exposure of this region of fibronectin may be important in the establishment and maintenance of cellular senescence. In addition, the ability of the SEN antibodies to react with fibronectin from a variety of senescent cells emphasizes their usefulness as markers for cellular senescence.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041500315
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  • 6
    Electronic Resource
    Electronic Resource
    Lectin and immunolabeling of microvascular endothelia (1991)
    New York, NY : Wiley-Blackwell
    Journal of Electron Microscopy Technique 19 (1991), S. 305-315 
    Details
    ISSN: 0741-0581
    Keywords: Endothelium ; Immunocytochemistry ; Electron microscopy ; Lectins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: A number of recently developed localization techniques are beginning to be applied in the study of endothelial cells and their structural components. In this article we will review a number of these cytochemical approaches as well as their advantages and disadvantages and their applications. The methods will be presented for processing tissues for either L.R. White embedding or semi-thin and thin frozen sections followed by subsequent lectin and immunolabeling for fluorescence and electron microscopic examination. These techniques are easily applied in the localization of perfused exogenous proteins and of endogenous endothelial-associated proteins. The results that can be obtained from such studies are presented and discussed.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jemt.1060190306
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