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  • 1
    Electronic Resource
    Electronic Resource
    Dynamic instability and motile events of native microtubules from squid axoplasm (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 285-295 
    Details
    ISSN: 0886-1544
    Keywords: organelle movement ; microtubule assembly/disassembly ; motion analysis ; MAPs ; force generation ; axonal transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Native microtubules from extruded axoplasm of squid giant axons were used as a paradigm to characterize the motion of organelles along free microtubules and to study the dynamics of microtubule length changes. The motion of large round organelles was visualized by AVEC-DIC microscopy and analyzed at a temporal resolution of 10 frames per second. The movements were smooth and showed no major changes in velocity or direction. During translocation, the organelles paused very rarely. Superimposed on the rather constant mean velocity was a velocity fluctuation, which indicated that the organelles are subject to considerable thermal motion during translocation. Evidence for a regular low-frequency oscillation was not found. The thermal motion was anisotropic such that axial motion was less restricted than lateral motion. We conclude that the crossbridge connecting the moving organelle to the microtubule has a flexible region that behaves like a hinge, which permits preferential movement in the direction parallel to the microtubule. The dynamic changes in length of native microtubules were studied at a temporal resolution of 1 Hz. About 98% of the native microtubules maintained their length (“stable” microtubules), while 2% showed phases of growing and/or shrinking typical for dynamic instability (“dynamic” microtubules). Gliding and organelle motion were not influenced by dynamic length changes. Transitions between growing and shrinking phases were low-frequency events (1-10 minutes per cycle). However, a new type of microtubule length fluctuation, which occurred at a high frequency (a few seconds per cycle), was detected. The length changes were in the 1-3 μm range. The latter events were very prominent at the (+) ends. It appears that the native axonal microtubules are much more stable than the purified microtubules and the microtubules of cultured cells that have been studied thus far. Potential mechanisms accounting for the three states of microtubule stability are discussed. These studies show that the native microtubules from squid giant axons are a very useful paradigm for studying microtubule-related motility events and microtubule dynamics.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100133
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  • 2
    Electronic Resource
    Electronic Resource
    Biologen in unserer Zeit (1993)
    Weinheim : Wiley-Blackwell
    Biologie in unserer Zeit 23 (1993), S. 81-95 
    Details
    ISSN: 0045-205X
    Keywords: Life and Medical Sciences
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/biuz.19930230606
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  • 3
    Electronic Resource
    Electronic Resource
    Towards a new classification of intracellular particle movements based on quantitative analyses (1986)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 6 (1986), S. 128-135 
    Details
    ISSN: 0886-1544
    Keywords: motion analysis ; axonal transport ; cytoplasmic transport ; Brownian motion ; AVEC-DIC microscopy ; saltatory particle motion ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A survey study of organelle movements in a variety of cell types of plant and animal origin was made with the aid of video-enhanced contrast, differential interference contrast (AVEC-DIC) microscopy followed by fine analysis of the motile behavior of the individual organelles. We found that there exists besides Brownian motion a wide spectrum of active motions in cells, i.e. motion that is directionally biased through the expenditure of metabolic energy. The types of active motion seen range from a continuous motion (sometimes appearing as streaming) in plant cells and neurons to various types of less ordered and less well directed motion. We did not see any clear-cut qualitative difference between plant and animal cells or between systems presumed to be actin- and microtubule-based. A preliminary classification of the types of active motion is presented. The ongoing research activities, which aim at a more precise definition of the different types of motion by a set of quantitative parameters, are described, and the progress made so far is reported.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970060210
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  • 4
    Electronic Resource
    Electronic Resource
    Movement of axoplasmic organelles on actin filaments from skeletal muscle (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 231-242 
    Details
    ISSN: 0886-1544
    Keywords: squid axoplasm ; organelle movement ; calmodulin ; actin filaments ; axonal transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: It was recently shown that, in addition to the well-established microtubule-dependent mechanism, fast transport of organelles in squid giant axons also occurs in the presence of actin filaments [Kuznetsov et al., 1992, Nature 356:722-725]. The objectives of this study were to obtain direct evidence of axoplasmic organelle movement on actin filaments and to demonstrate that these organelles are able to move on skeletal muscle actin filaments. Organelles and actin filaments were visualized by video-enhanced contrast differential interference contrast (AVEC-DIC) microscopy and by video intensified fluorescence microscopy. Actin filaments, prepared by polymerization of monomeric actin purified from rabbit skeletal muscle, were stabilized with rhodamine-phalloidin and adsorbed to cover slips. When axoplasm was extruded on these cover slips in the buffer containing cytochalasin B that prevents the formation of endogenous axonal actin filaments, organelles were observed to move at the fast transport rate. Also, axoplasmic organelles were observed to move on bundles of actin filaments that were of sufficient thickness to be detected directly by AVEC-DIC microscopy. The range of average velocities of movement on the muscle actin filaments was not statistically different from that on axonal filaments. The level of motile activity (number of organelles moving/min/field) on the exogenous filaments was less than on endogenous filaments probably due to the entanglement of filaments on the cover slip surface. We also found that calmodulin (CaM) increased the level of motile activity of organelles on actin filaments. In addition, CaM stimulated the movement of elongated membranous organelles that appeared to be tubular elements of smooth endoplasmic reticulum or extensions of prelysosomes. These studies provide the first direct evidence that organelles from higher animal cells such as neurons move on biochemically defined actin filaments. © 1994 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970280306
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  • 5
    Electronic Resource
    Electronic Resource
    Substructure of sidearms on squid axoplasmic vesicles and microtubules visualized by negative contrast electron microscopyPart of this work was performed at the Marine Biological Laboratory, Woods Hole, MA 02543. (1987)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 7 (1987), S. 20-30 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We present a high-resolution electron microscopic study of the sidearms on microtubules and vesicles that are suggested to form the crossbridges which produce the microtubule-based vesicle transport in squid axoplasm. The sidearms were found attached to the surfaces of the anterogradely transported vesicles in the presence of ATP. These sidearms were made of one to three filaments of uniform diameter. Each filament measured 5-6 nm in width and 30-35 nm in length. The filaments in some of the sidearms had splayed apart by pivoting at their base, thereby assuming a “V” shape. The spread configuration illustrated the independence of the individual filaments. The filaments in other sidearms were closely spaced and oriented parallel to each other, a pattern called the compact configuration. In axoplasmic buffer containing AMP-PNP, structures indistinguishable from the filaments of the sidearms on the vesicles were observed attached to microtubules. Pairs of filaments, thought to represent the basic functional unit, were observed attached to adjacent protofilaments of the microtubules by their distal tips. These data support a model of vesicle movement in which a pair of filaments within a sidearm forms two crossbridges and moves a vesicle by “walking” along the protofilaments of the microtubule.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970070104
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  • 6
    Electronic Resource
    Electronic Resource
    Intracellular guanosine-5′-0-(3-thiotriphosphate) blocks chemotactic motility of Dictyostelium discoideum amoebae (1993)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 25 (1993), S. 298-307 
    Details
    ISSN: 0886-1544
    Keywords: guanine nucleotides ; calcium ; chemotaxis ; pseudopods ; membrane traffic ; BAPTA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Starving amoebae of the cellular slime mold Dictyostelium discoideum react chemotactically towards the attractant cAMP. In this study, the effect of nonhydrolyzable analogs of GTP and GDP on the chemotactic behavior was analyzed with light microscopic techniques. Guanosine-5′-0-(2-thiotriphosphate) (GTPβS) or guanosine-5′-0-(2-thiodiphosphate) (GDPβS) was scrape-loaded into the cytoplasm of cells, together with a fluorescent marker. Stimulation with a cAMP-filled glass capillary revealed a reduced capacity of loaded cells to migrate to wards the capillary tip. Most cells still protruded filopods in the direction of the capillary tip, but full extension of pseudopods was inhibited in a dose-dependent and reversible manner. This indicates that in the presence of the analogs, chemotactic sensing still occurs, and that a more distal step of the cascade of events leading to the formation of the pseudopod is impaired.In cells loaded with the analogs together with the calcium indicator fura-2, stimulation with 10 μM cAMP led to a transient change in the intracellular free calcium concentration ([Ca2+]i), which was detectable in 28% of the cells. Furthermore, large vacuoles were found containing high amounts of calcium. On the other hand, clamping of [Ca2+]i at low levels with 1,2-bis(2-aminophenoxy) ethane N,N,N′,N′-tetraacetic acid (BAPTA) also inhibited motility, with neither filopods nor pseudopods formed.The data suggest that chemotactic migratory activity involves GTP-dependent processes that participate in the regulation of the Ca2+ homeostasis of the cell and in the regulation of membrane traffic that contributes to the directed locomotion. © 1993 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970250309
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  • 7
    Electronic Resource
    Electronic Resource
    Effects of vanadate on the assembly and disassembly of purified tubulin (1986)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 6 (1986), S. 314-323 
    Details
    ISSN: 0886-1544
    Keywords: vanadate ; microtubules ; tubulin polymerization ; taxol ; dynein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Sodium-orthovanadate (100-700 μM) added to purified pig brain microtubule protein (molar ratios 13-90 moles vanadate/mole tubulin) inhibits to a considerable extent the assembly (up to 65%) and the disassembly rates (up to 60%) of microtubules, as determined by turbidimetry. Vanadate added to preformed microtubules did not appreciably alter the turbidity level of the samples, however, the disassembly rates were decreased in the same manner as when vanadate was added prior to polymerization. Microtubule protein kept on ice for 3-6 hours became more susceptible to vanadate than freshly prepared protein. The effect of vanadate was independent of the GTP concentration at which the polymerization assays were performed (0.025 to 1 mM GTP). In the presence of taxol, which increases the rate and extent of microtubule formation, vanadate had no effect on assembly rates. Disassembly was inhibited, however, much less than in the presence of vanadate alone. Electron microscopy and polyacrylamide gel electrophoresis did not reveal differences between microtubules prepared in the presence or in the absence of vanadate. This is consistent with the notion that vanadate does not interfere with the interaction between tubulin and the high-molecular weight microtubule-associated proteins. Apparently vanadate brings about an allosteric change of the microtubule protein(s) resulting in the abnormal polymerization kinetics of tubulin found in our study. The above results may be relevant for studies where the effects of vanadate on intracellular motility are interpreted as being solely due to a specific inhibition of ATPases.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970060308
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  • 8
    Electronic Resource
    Electronic Resource
    Establishment and partial characterization of SV40 virus-immortalized hepatocyte lines of normal and lethal mutant mice carrying a deletion on chromosome 7 (1989)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 139 (1989), S. 599-609 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Deletions in chromosome 7 of the mouse have been shown to cause failure of expression of various hepatocyte-specific genes in newborn deletion homozygotes, including the gene encoding tyrosine amino transferase (TAT) (EC 2.6.1.5) (Gluecksohn-Waelsch, 1979). Primary liver cultures of newborn albino deletion mutant mice (C14CoS/C14CoS) and of phenotypically normal mice (C14CoS/Cch or Cch/Cch) were infected with SV40 virus and multiplying hepatocytes selected in arginine-deficient medium containing epidermal growth factor (EGF), insulin, and hydrocortisone (HC). Resulting normal (NMH-ch) and mutant (NMH-m14) hepatocyte lines expressing integrated viral transforming sequences did not senesce, they multiplied autonomously of EGF in medium with insulin plus HC, and they retained hepatocyte-specific functions. Both lines synthesized arginine and contained albumin and alpha-fetoprotein (AFP) mRNAs. TAT-specific mRNA was detected in normal but not in mutant hepatocyte lines. A fragment of the mouse tyrosinase gene, known to map at the albino locus (c) within the region deleted in the C14CoS mutant, hybridized with a 2.5 kb EcoRI fragment of normal NMH-ch DNA, whereas this fragment was undetectable in mutant NMH-m14 DNA. These immortalized hepatocyte lines reflect important properties of normal and mutant liver tissues from which they were derived. The deletion mutant mouse cell lines may be useful for complementation studies involving sequences corresponding to the deletions that encode regulatory gene(s) involved in the control of inducible expression of certain hepatocyte-specific genes such as TAT.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041390321
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  • 9
    Electronic Resource
    Electronic Resource
    Protein synthesis in embryonic tissues during mouse postimplantation development (1990)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 44 (1990), S. 19-37 
    Details
    ISSN: 0730-2312
    Keywords: cell lineage ; tissue separation ; gene expression ; 2-D gels ; mammalian embryology ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mouse embryos of the NMRI strain between the 7th and 9th day of gestation were isolated from the uterus and dissected into the various tissue derivatives in order to investigate newly synthesized proteins during morphogenesis. The day 7 embryo was fragmented into trophoblast and ectoplacental cone, distal and proximal endoderm, extraembryonic and embryonic ectoderm. The day 8 and day 9 embryos were divided into trophoblast and placental anlage, yolk sac, amnion, and allantois, as well as cranial, central, and caudal embryonic tissue. The intact embryos were incubated in Dulbecco's minimum essential medium in the presence of 35S-methionine for 4 h, then dissected into the various fragments, and further processed for two-dimensional gel electrophoresis. Protein synthesis of the isolated tissue derivatives was analyzed and compared for the three developmental stages. Concerning the proteins with isoelectric points in the range of 4.5 to 8.0 and molecular weight ratio (Mr) values between 20,000 and 200,000, we found several significant quantitative and qualitative differences in the various tissue fragments. In addition, we observed further quantitative and qualitative differences in protein synthesis during the postimplantation period investigated. We propose that the differences reflect some of the cell lineage- and developmental stage-specific changes in gene expression during early mammalian differentiation.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240440103
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  • 10
    Electronic Resource
    Electronic Resource
    Differential expression of the two VEGF receptors flt and KDR in placenta and vascular endothelial cells (1994)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 54 (1994), S. 56-66 
    Details
    ISSN: 0730-2312
    Keywords: VEGF ; PIGF ; KDR ; flt ; endothelial cells ; placenta ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Vascular endothelial growth factor (VEGF) is a newly identified growth and permeability factor with a unique specificity for endothelial cells. Recently the flt-encoded tyrosine kinase was characterized as a receptor for VEGF. A novel tyrosine kinase receptor encoded by the KDR gene was also found to bind VEGF with high affinity when expressed in CMT-3 cells. Screening for flt and KDR expression in a variety of species and tissue-derived endothelial cells demonstrates that flt is predominantly expressed in human placenta and human vascular endothelial cells. Placenta growth factor (PIGF), a growth factor significantly related to VEGF, is coexpressed with flt in placenta and human vascular endothelial cells. KDR is more widely distributed and expressed in all vessel-derived endothelial cells. These data demonstrate that cultured human endothelial cells isolated from different tissues express both VEGF receptors in relative high levels and, additionally, that all investigated nonhuman endothelial cells in culture are also positive for KDR gene expression.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240540107
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