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  • 1985-1989  (2,074)
  • 1988  (2,074)
  • Cell & Developmental Biology  (1,394)
  • Biochemistry and Biotechnology  (680)
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Years
  • 1985-1989  (2,074)
Year
Keywords
  • 101
    Electronic Resource
    Electronic Resource
    Dynamics of microtubules visualized by darkfield microscopy: Treadmilling and dynamic instability (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 229-236 
    Details
    ISSN: 0886-1544
    Keywords: microtubule ; treadmilling ; MAPs ; dynamic instability ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Individual microtubules undergoing treadmilling in vitro were visualized by darkfield light microscopy, and the relationship between treadmilling and dynamic instability was studied as a function of microtubule-associated proteins (MAPs). In order to demonstrate treadmilling directly by real-time observation, we constructed three-block microtubules, the center-block of which was decorated with Tetrahymena dynein. The decorated block can easily be distinguished from undecorated blocks in the darkfield microscope because the decorated one appears much thicker. At steady-state conditions, the length of an undecorated block at one end increased and that at another end decreased, while the decorated center-block did not change in its length. The results from these direct observations show that calf brain 3X-microtubules exhibit a treadmilling flux of 0.9 μm/h.Using a similar microscopy technique, we previously demonstrated that phosphocellulose PC-microtubules existed in either the growing or the shortening phase and alternated quite frequently at steady-state conditions (dynamic instability). How does treadmilling relate to dynamic instability? An image recording of individual 3X-microtubules containing MAPs revealed that the microtubules undergo treadmilling and do not exhibit any dynamic instability. This evidence shows that MAPs suppress the dynamic instability of microtubules. That is, treadmilling can take place in the steady state only after microtubules have been stabilized by MAPs.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100127
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  • 102
    Electronic Resource
    Electronic Resource
    Detection of single fluorescent microtubules and methods for determining their dynamics in living cells (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 237-245 
    Details
    ISSN: 0886-1544
    Keywords: video microscopy ; digital image processing ; fluorescence photobleaching ; microtubule dynamics ; living cell dynamics ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The ability to tag biological molecules fluorescently and to detect their distribution in living cells has promoted the study of cytoplasmic organization in general and microtubule dynamics in particular. The techniques that we have selected and developed allowed the determination of spatial and temporal changes of the microtubule network in living fibroblasts at the level of individual microtubules. We have employed two general approaches for determining pattern changes: direct video microscopy and photobleaching and subsequent observation. Direct observation of fluorescent microtubules by high-definition video microscopy provided good spatial resolution at several time points, but was limited to the less congested and thinner periphery of the cell. This approach was made possible by a relatively bright, photostable reporter, xrhodamine-tubulin, and showed that microtubules underwent rounds of assembly and disassembly from their ends. Bleaching and subsequent observation of lysed cells improved the signal to noise ratio by extracting soluble chromophore and permitted observations in congested areas, but was limited to a single time interval. This approach demonstrated that microtubule domains were replaced one by one and that turnover was most rapid at the cell periphery. Antibodies specific for nonbleached chromophore can be used to enhance the signal to noise ratio further or to extend spatial resolution by the use of immunoelectron microscopy. Direct video microscopy and photo-bleaching are two approaches to the study of dynamics that have complementary strengths and wide application to the biology of living cells.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100128
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  • 103
    Electronic Resource
    Electronic Resource
    Vesikin, a vesicle associated ATPase from squid axoplasm and optic lobe, has characteristics in common with vertebrate brain MAP 1 and MAP 2 (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 246-254 
    Details
    ISSN: 0886-1544
    Keywords: axoplasmic transport ; motility ; microtubules ; MAPs ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Vesikin, a protein that can associate with squid axoplasmic vesicles or optic lobe microtubuies, has been implicated as a force-generating molecule involved in microtubule-dependent vesicle transport [Gilbert and Sloboda, 1986, 1988]. Because vesikin crossreacts with an antibody to porcine brain microtubule associated protein 2 (MAP 2), studies were conducted to compare squid vesikin and brain MAPs. When taxol stabilized microtubules containing vesikin as a microtubule associated protein were incubated in the presence of ATP, vesikin dissociated from the microtubule subunit lattice. This behavior would be expected for an ATP-dependent, force generating molecule that serves as a crossbridge between vesicles and microtubules. When chick brain microtubules were treated under the same conditions, MAP 2 remained bound to the microtubules while MAP 1 dissociated in a manner similar to vesikin. One dimensional peptide mapping procedures revealed that, although digestion of vesikin and MAP 2 generated several peptides common to both proteins, vesikin and MAP 2 are clearly not identical. Furthermore, the addition of vesikin or MAPS 1 and 2 to purified tubulin stimulated microtubule assembly in a manner dependent on the concentration of added protein. These findings demonstrate that brain MAPs share characteristics common to squid vesikin and support the suggestion that brain MAPs 1 and 2 might act as a force generating complex for vesicle transport in higher organisms.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100129
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  • 104
    Electronic Resource
    Electronic Resource
    Microtubule-associated organelle and vesicle transport in fibroblasts (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 255-262 
    Details
    ISSN: 0886-1544
    Keywords: regulation of organelle transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Allen Video-enhanced constrast/differential interference constrast (AVEC-DIC) microscopy was used in conjunction with video intensification immunofluorescence microscopy to demonstrate that organelles and vesicle (particles) can move in either direction along microtubular linear elements in fibroblasts [Hayden et al., 1983]. Since it is not possible to determine the number of microtubules making up a linear element with light microscopy alone, AVEC-DIC microscopy was used in conjunction with whole-mount electron microscopy to show bidirectional transport along a single microtubule [Hayden and Allen, 1984]. These studies demonstrate that the structural polarity of the microtubule does not determine the direction of particle motion, and since dynein is an asymetric molecule, a simple microtubule-dynein-particle hypothesis cannot explain bidirectional transport along a single microtubule.Very little is known about regulation of particle transport in most cell types. Human embryonic lung fibroblasts grown on glass coverslips were serum-deprived for 24 hours and re-fed with serumless medium; the particle translocations/5 minutes were then determined The cells were then re-fed with either serumless medium, serum-containing medium, or serumless medium containing some bioactive factor, and the particle translocations/5 minutes were again determined for the same cells. Medium containing 10% fetal bovine serum inhibited particle translocation by 51.8%. Of the bioactive factors tested, only vasopressin produced a significant reduction in particle translocations (38%). This suggests that protein kinase C or calcium/calmodulin kinase could be involved in regulating particle transport.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100130
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  • 105
    Electronic Resource
    Electronic Resource
    Fast axonal transport alterations in amyotrophic lateral sclerosis (ALS) and in parathyroid hormone (PTH)-treated axons (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 321-330 
    Details
    ISSN: 0886-1544
    Keywords: organelle motion ; video microscopy ; computer motion analysis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Video-enhanced contrast techniques have been used to study fast axonal transport of organelles in diseased and normal human axons. A broad perspective on the importance of axonal transport in the pathogenesis of human neurological disorders is presented and problems in dealing with human nerve summarized. Results from analysis of organelle traffic in axons from motor nerve in patients with amyotrophic lateral sclerosis (ALS) show: (1) higher mean speed of anterograde organelles, (2) lower mean speed of retrograde organelles, and (3) lower retrograde organelle traffic density. Hyperparathyroidism, another human clinical syndrome, can mimic ALS. The effect of parathyroid hormone (PTH) on axons in vitro is to increase the mean speed of both anterograde and retrograde organelle traffic. The dose response curve and time course of the PTH effect are delineated. Dihydropyridine calcium channel antagonists block the PTH effect, implicating extracellular calcium in the alteration of organelle traffic speed. The results are discussed in relation to neuronal function and the regulation of fast axonal transport.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100136
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  • 106
    Electronic Resource
    Electronic Resource
    Localization of mitotic-apparatus-associated 51-kD protein in unfertilized and fertilized sea urchin eggs (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 496-505 
    Details
    ISSN: 0886-1544
    Keywords: centrosome ; spindle matrix ; postembedding immunofluorescent labeling ; mitotic apparatus ; sea urchin eggs ; 51-kD protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The 51-kD protein, a protein component of the mitotic apparatus in sea urchin eggs, is involved in the aster-forming activity previously shown in vitro [Toriyama et al., 1988]. Postembedding immunofluorescent labelings of eggs from fertilization through first cleavage showed that the 51-kD protein is localized in sperm asters, centrosomal regions, spindles, basal regions of astral microtubules, and regions surrounding daughter nuclei at telophase in situ. Immunofluorescence and immunoblot analyses detected the 51-kD protein uniformly in unfertilized eggs, but not in spermatozoa. When unfertilized eggs were treated with taxol, the 51-kD protein was shown to be associated with taxol-induced cytasters. Immunoblot analysis revealed that similar protein species are present in the mitotic apparatus of other species of sea urchin. It was suggested that the 51-kD protein may be involved in microtubule nucleation and microtubule matrix in sea urchin eggs in vivo.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100406
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  • 107
    Electronic Resource
    Electronic Resource
    The ciliary cycle during hyperpolarization-induced activity: An analysis of axonemal functional parameters (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 11 (1988), S. 275-290 
    Details
    ISSN: 0886-1544
    Keywords: ciliary motility ; inclination ; polarity of beating ; sliding velocity ; sliding translocation rate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Motor responses of the frontal cirri of the ciliate Stylonychia were recorded at the axial view of the ciliary base with high-speed cinematography. Voltage-clamp applying sustained hyperpolarizing voltage steps was used to explore the properties of the ciliary cycle modulated by the membrane potential. Upon hyperpolarization between - 1 and - 13 mV, a previously inactive frontal cirrus reoriented from a neutral posture and started beating so that the axis of the beating cone of a proximal cirral segment assumed an orientation near 100° (proceeding counterclockwise from posterior = 0°) and inclination near 60° (0° = perpendicular to the cell surface). The major beating amplitude was limited to about 150°. Increasing hyperpolarization increased the spatial polarity of the cycle (ratio of major over minor amplitude, from 2 to 2.4). Rates of the power stroke increased with hyperpolarizations up to - 4 mV but were consistently smaller than those of the return stroke during the ciliary cycle (ratio: 0.4 to 0.6; = temporal polarity). Comparison of different hypothetical beat forms (0-shape, D-shape, and egg-shape) showed that the orientation-time data are the major determinants of the angular velocity and rate of reorientation of the cilium during the cycle. Geometric transformation of these data led to descriptions of the cycle of a proximal ciliary segment in terms of active sliding velocities and rates of unidirectional sliding translocation between identified doublets. Three voltage-sensitive functional parameters of the cilium - the inclination (which is noncyclic) and the rates of active sliding and sliding translocation (both of which are cyclic in nature) - are discussed as generating the spatial and temporal properties of the ciliary beat.
    Additional Material: 13 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970110406
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  • 108
    Electronic Resource
    Electronic Resource
    Effect of metabolic inhibitors on sucrose-induced metaphase spindle elongation and spindle recovery (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 11 (1988), S. 291-302 
    Details
    ISSN: 0886-1544
    Keywords: mitosis ; mitotic apparatus ; microtubules ; kinetochores ; metabolic inhibitors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Hyperosmotic sucrose treatment of metaphase PtK-1 cells has been shown to produce a reversible concentration-dependent effect on spindle elongation linked to a functional alteration in the connection of the chromosome to the spindle (Pover et al.: European Journal of Cell Biology 39:366-372, 1985). Spindle elongation, similar to that which occurs at anaphase B, is thought to be driven by the compression stored in the form of microtubule curvature in the nonkinetochore (nkMT) population of microtubules at metaphase (Snyder et al.: European Journal of Cell Biology 35:62-69, 1984 and 39:373-379, 1985). Addition of metabolic inhibitors to Ham's F-12 salts with deoxyglucose (D/F-12 medium) containing 0.4 M sucrose and 1 mM DNP does not within statistical error affect the rate and extent of sucrose-induced spindle elongation; rates and extents are 60-75% of normal anaphase B motions. Electron microscopic analysis of metaphase cells treated with D/F-12 medium and 0.4 M sucrose with 1 mM DNP demonstrates that spindle microtubules lose curvature and become straight in appearance, typical of microtubule organization in untreated anaphase cells. Sucrose-treated cells released into D/F-12 medium show a rapid reduction in spindle length; however, cells treated with either 0.4 M sucrose or 0.4 M sucrose and 1 mM DNP-containing D/F-12 medium and released into DNP-containing D/F-12 medium do not exhibit a significant reduction in spindle length. Electron microscopic analysis links changes in spindle length with microtubule/kinetochore associations. These data suggest that energy required for the initial phases of spindle elongation during anaphase is preloaded into the mitotic spindle by metaphase and does not require additional energy to be expressed as examined by sucrose-induced spindle elongation in the presence of metabolic inhibitors. Second, energy is required to make or maintain (or both) functional chromosome associations with the spindle as measured by reduction in spindle length following sucrose removal.
    Additional Material: 14 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970110407
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  • 109
    Electronic Resource
    Electronic Resource
    Unique isoactins in the brush border of rat intestinal epithelial cells (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 11 (1988), S. 318-325 
    Details
    ISSN: 0886-1544
    Keywords: actin ; contractile proteins ; microvilli ; cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The mammalian genome contains 20-30 genes encoding a family of actins. To date, however, only six proteins (four muscle and two nonmuscle isoforms) encoded by this multigene complex have been identified. We have isolated two actins from the brush border of rat intestinal epithelial cells that have isoelectric points and N-terminal peptides characteristic of the cytoplasmic β- and γ-actins. However, using a panel of actin-specific monoclonal antibodies, we show that these actins contain a set of epitopes that distinguishes them from any of the known cytoplasmic or muscle isoforms. These unique actins share features of both the nonmuscle and muscle isoforms, suggesting that they represent an intermediate in the evolution of the specialized muscle actins.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970110409
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  • 110
    Electronic Resource
    Electronic Resource
    Isolation of an immunoreactive analogue of brain fodrin that is associated with the cell cortex of Dictyostelium amoebae (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 11 (1988), S. 303-317 
    Details
    ISSN: 0886-1544
    Keywords: spectrin ; actin ; membrane skeleton ; cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have used a polyclonal affinity-purified antibody made against chicken brain fodrin (both 240 and 235 Kd subunits) as a probe to determine if a fodrinlike protein exists in amoebae of Dictyostelium discoideum. In Western blots of whole cells and the isolated cell cortex, polypeptides measuring 220 and 70 Kd are recognized by the fodrin antibodies. In situ localization by indirect immunofluorescence with antifodrin indicates that the immunoreactive polypeptides are cortical. The immunoreactive analogues copatch and cocap with concanavalin A. At the level of resolution of the electron microscope, immunocytochemistry with antifodrin and colloidal gold confirms that the immunoreactive analogues are cortical proteins associated with microfilaments on the cytoplasmic side of the plasma membrane. We have isolated and characterized the 220 Kd protein to determine if it is similar to fodrin and to investigate its relationship to the 70 Kd polypeptide. The 220 Kd protein can be extracted from the cortex in the absence of detergent and isolated by gel filtration and sucrose density gradient sedimentation. The 220 Kd is a rod-shaped protein 118 ± 17.8 nm (N = 37) in length. It has a sedimentation coefficient of 9.3 S and Stokes' radius of 13 nm and exists as a dimer of approximately 500,000 daltons (Mr). Isolated 220 Kd binds to actin filaments in vitro when assayed by rotary shadowing. Morphological criteria distinguish 220 Kd from Dictyostelium myosin II heavy chain (215 Kd) and the filaminlike protein at 240 Kd. The 70 Kd polypeptide appears to be a cleavage fragment of the 220 Kd, since it is found after prolonged storage when formerly only the 220 Kd was present. Furthermore, the 220 and 70 Kd polypeptides exhibit similar one-dimensional peptide maps when treated with TPCK trypsin. On the basis of its physical and immunoreactive characteristics, and location in the cell, the 220 Kd may be a fodrinlike protein.
    Additional Material: 13 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970110408
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  • 111
    Electronic Resource
    Electronic Resource
    Announcement (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 11 (1988), S. 326-326 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970110410
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  • 112
    Electronic Resource
    Electronic Resource
    Maturation of hagfish gland thread cells: Composition and characterization of intermediate filament polypeptides (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 11 (1988), S. 31-45 
    Details
    ISSN: 0886-1544
    Keywords: cytoskeletal maturation ; keratinlike filaments ; holocrine secretion ; cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Previous studies with the hagfish, a primitive vertebrate, have shown that the gland thread cells (GTCs) each contain a single thread (∼60 cm long in average-sized cells) in the form of a concisely coiled cytoskeletal entity destined for export by holocrine secretion. The thread in relatively immature GTCs consists almost entirely of intermediate filaments (IFs) bundled in parallel alignment with far fewer microtubules (MTs). The three thread polypeptides described earlier (α, basic; β acidic; γ, most acidic; each with a Mr of 63-64 kD) are now further evaluated with respect to in vitro assembly, cross-reactivity with IF polypeptides from higher vertebrates, and peptide sequence homology with known IF polypeptides. The overall results mainly suggest that the hagfish polypeptides are keratinlike substances but lamins or a new type of IF is not ruled out. However, cross-reactivity is weak with mammalian keratins; the 8-11-nm filaments formed from mixtures of α and γ in vitro are generally linear rather than the curvilinear structures usually formed by keratin and nonkeratin IFs; and mixtures of α and β tend to yield 9-12-nm granules or granular strings. Polypeptide analyses on GTCs segregated on the basis of maturational stage show a progressive increase in β/γ values which correlates with cell maturation, but the α/(β+γ) ratios remain near 1. Inasmuch as β and γ have many similar properties, the documented increase in the amount of the β component in aging GTCs might in part be the result of a failure in a posttranslational modification system and may contribute to the ultrastructural changes that accompany thread maturation in preparation for holocrine secretion and subsequent modulation of the viscoelastic properties of mucus.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970110105
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  • 113
    Electronic Resource
    Electronic Resource
    Intermolecular localization of epitopes within an oligomeric protein by immunoelectron microscopy and image processing (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 161-183 
    Details
    ISSN: 0887-3585
    Keywords: hemocyanin ; correspondence analysis ; monoclonal antibodies ; electron microscopy ; images analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Three epitopes have been localized by immunoelectron microscopy on subunits Aa6 of the 4 × 6-meric hemocyanin of the scorpion Androctonus australis. Soluble immunocomplexes composed of monoclonal antibodies and of native hemocyanin were purified, negatively stained with uranyle acetate by the single-layer technique, and examined under the electron microscope (EM). The molecule images were digitized, aligned, and submitted to correspondence analysis according to the method of Van Heel and Frank (Ultramicroscopy 6: 187-194, 1981). A high-precision localization of the attachment point of the Fab arm to the antigen was achieved through a careful analysis of the average images. This method easily allowed the discrimination of epitopes located in different domains (Mr 20 kDa) of the same subunit. Nonoverlapping epitopes located in the same structural domain of subunit Aa6 could be distinguished by the stain exclusion patterns of their Fab arms. The method is general and may be used for epitope mapping in any antigen producing definite EM views.
    Additional Material: 12 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/prot.340030305
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  • 114
    Electronic Resource
    Electronic Resource
    Crystallization and preliminary X-ray diffraction study of a protein with a high potential rubredoxin center and a hemerythrin-type Fe center (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 184-186 
    Details
    ISSN: 0887-3585
    Keywords: new Fe-protein ; rubredoxin ; hemerythrin ; crystals ; X-ray diffraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A newly discovered iron-containing protein, isolated from the bacterium Desulfovibrio vulgaris (Hildenborough, NCIB 8303), has been crystallized. The molecule appears to be a dimer of mass 44kDa. This protein has iron centers with spectroscopic similarities to those in rubredoxins and in hemerythrins.The X-ray diffraction shows symmetry consistent with space group I222 or I212121. Cell parameters are a = 49.2 Å, b = 81.3 Å, c= 100.1 Å, and α, β, γ = 90°. X-ray diffraction data have been collected to 3.0 Å, and a search for useful heavy atom derivatives is in progress for the analysis of the crystal structure of this Fe-protein.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/prot.340030306
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  • 115
    Electronic Resource
    Electronic Resource
    Lecithin: Cholesterol acyltransferase activation by synthetic amphipathic peptides (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 187-198 
    Details
    ISSN: 0887-3585
    Keywords: amphipathic peptide ; liposomes ; peptide ; serum apolipoproteins ; synthetic ; LCAT ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The amphipathic helical theory of Segrest and colleagues (FEBS Lett.: 38: 247-253, 1974) proposes that the lipid-binding segments of serum apolipoproteins are in an alpha helical conformation. Furthermore the helices have a hydrophobic face and a hydrophilic face with a specific distribution of positively and negatively charged residues. The importance of the pattern of the charged residues in the lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation by the segments is still debated. We designed a 30-residue peptide, GALA, which in the alpha helical conformation hs a hydrophilic face composed of glutamic acid residues (Sabbarao et al.: Biochemistry 26: 2964-2972, 1987). GALA behaves like the serum apolipoproteins in its interaction with dimyristoylphospatidylcholine (DMPC) at neutral pH; the amino terminal tryptophan of GALA undergoes a blue shift in its fluorescence emission spectrum, and the circular dichroism (CD) spectrum indicates that GALA acquires alpha helical structure in the presence of DMPC. A DMPC-GALA:19/1 (molar ratio) complex can be isolated by gel-permeation chromatography. This complex has a discoidal structure with the approximate dimensions of 44-Å diameter. GALA edge thickness and a 170- to 350-Å diameter. GALA activates LCAT with DMPC but not with unsaturated phospholipids as the substrate. The apparent partition coefficient of GALA into DMPC vesicles is 100-fold larger than into egg phosphatidlylcholine vesicles. The interaction of GALA with unsaturated lipids at neutral pH is so weak that no detectable change in the spectroscopic properties of GALA or the structure of the liposomes can be detected under the conditions used here. The sequence of GALA differs from previously studied model Apo A1 peptides by the absence of positively charged residues on the hydrophilic face. This indicates that positive charges in Apo A1-like peptides are not required in order to form discoidal structures with saturated phospholipids or to activate LCAT with such lipid substrates.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/prot.340030307
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  • 116
    Electronic Resource
    Electronic Resource
    In memorium: Emil Thomas Kaiser, 1938-1988 (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. i 
    Details
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/prot.340040102
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  • 117
    Electronic Resource
    Electronic Resource
    Photochemical crosslinking of bacteriophage T4 single-stranded DNA-binding protein (gp32) to oligo-p(dT)8: Identification of phenylalanine-183 as the site of crosslinking (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 1-6 
    Details
    ISSN: 0887-3585
    Keywords: single-standard DNA-binding protein ; protein-nucleic acid interactions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Using ultraviolet light, both the 33,000-dalton single-standard DNA-binding protein from T4 bacteriophage (gp32)as well as a 25,000-dalton limited trypsin cleavage product of gp32 (core gp32*) that retains high affinity for single-stranded DNA can be crosslinked to an oligodeoxynucleotide, p(dT)8. After photolysis, a single tryptic peptide crosslinked to p(dT)8 was isolated by anion-exchange high-performance liquid chromatography. Gas-phase sequencing of this modified peptide gave the following sequence: Gln-Val-Ser-Gly-(X)-Ser-Asn-Tyr-Asp-Glu-Ser-Lys, which corresponds to residues 179-190 in gp32. Based on the absence of the expected phenylthiohydantoin derivative of phenylalanine 183 at cycle 5 (X) we infer that crosslinking has occurred at this position and that phenylalanine 183 is at the interface of the gp32:P(dT)8 complex in an orientation that allows covalent bond formation with the thymine radical produced by ultraviolet irradiation.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340040103
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    Structure-function relationships in 3-phosphoglycerate kinase: Role of the carboxy-terminal peptide (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 56-62 
    Details
    ISSN: 0887-3585
    Keywords: Protein engineering ; mutagenesis ; enzyme catalysis ; conformational changes ; domain movement ; hinge bending ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Yeast 3-phosphoglycerate kinase (PGK) is a monomeric enzyme (Mr ∼ 45,000) composed of two globular domains. Each domain corresponds approximately to the amino- and carboxyterminal halves of the polypeptide chain. The carboxy-terminal end extends over the interdomain “hinge” region and packs against the amino-terminal domain. It has been proposed that domain movement, resulting in closure of the active site left, is essential for the catalytic of PGK. Large-scale conformational changes have also been postulated to explain activation of the enzyme by sulfate ions. Using site-specific mutagenesis, we have removed a 15-amino-acid carboxy-terminal fragment, in order to probe its role in the substrate- and sulfate-induced conformational changes. The truncated enzyme exhibited approximately 1% of the activity of native PGK and lost the ability to undergo sulfateinduced activation. The Km for ATP was essentially unchanged (Km = 0.23mM), whereas the Km value for 3-phosphoglycerate was increased about eightfold (Km = 3.85 mM and 0.50 mM, respectively). These results suggest that the carboxy-terminal segment is important for the mechanism of substrate- and specific-induced conformational transitions. CD spectra and sedimentation velocity measurements indicate that the carboxy-terminal peptide is essential for structural integrity of PGK. The increased susceptibility of the truncated enzyme to thermal inactivation implies that the carboxy-terminal peptide also contributes to the stability of PGK.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340040108
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    Model of a complex between the tetrahemic cytochrome c3 and the ferredoxin I from Desulfovibrio desulfuricans (Norway strain) (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 63-70 
    Details
    ISSN: 0887-3585
    Keywords: molecular graphics ; protein complex ; electron transfer ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A three-dimensional model of an electron-transfer complex between the tetrahemic cytochrome c3 and the ferredoxin I from the sulfatereducing bacterium Desulfovibrio desulfuricans (Norway strain) has been generated through computer graphics methods. The model is based on the known X-ray structure of the cytochrome and on a model of the ferredoxin that has been derived through computer graphics modeling and energy minimization methods, from the X-ray structure of the homologous ferredoxin from Peptococcus aerogenes. Four possible models of interaction between the two molecules were examined by bringing in close proximity each of the four hemes and the redox center (4Fe-4S) of the ferredoxin and by optimizing the ion pairs interactions. One of these models shows by far the “best” structure in terms of charges, interactions, and complementary f the topology of the contact surfaces. In this complex, the distance between the iron atoms of the ferredoxin redox center and the hemic iron atom is 11.8 Å, which compares well with those found between redox centers in other complexes. The contact surface area between the two molecules is 170 Å2.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340040109
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    Masthead (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988) 
    Details
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/prot.340040201
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    Crystallization and preliminary investigation of single crystals of deoxyuidine triphosphate nucleotidohydrolase from Escherichia coli (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 71-75 
    Details
    ISSN: 0887-3585
    Keywords: dUTPase ; nucleotide binding enzyme ; X-ray crystallography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), an enzyme in the nucleotide metabolism that is a pyrophosphatase hydrolyzing dUTP, has been crystallized. The crystals belong to the trigonal space group R3 and diffract beyond 2 Å. The native dUTPase crystals and a mercury derivative are stable in the X-ray beam and are suitable for a high resolution X-ray structure analysis.
    Additional Material: 3 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340040110
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    Crystallographic studies of inhibitor binding sites in human carbonic anhydrase II: A pentacoordinated binding of the SCN- ion to the zinc at high pH (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 283-293 
    Details
    ISSN: 0887-3585
    Keywords: crystallography ; structure ; refinement ; sulfonamide ; thiocyanate ; mercury ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The binding of four inhibitors - mercuric ion, 3-acetoxymercuri-4-aminobenzenesulfonamide (AMS), acetazolamide (Diamox), and thiocyanate ion - to human carbonic anhydrase II (HCA II) has been studied with X-ray crystallography.The binding of mercury to HCA II at pH 7.0 has been investigated at 3.1 Å resolution. Mercuric ions are observed at both nitrogens in the His-64 ring. One of these sites is pointing toward the zinc ion. The only other binding site for mercury is at Cys-206.The binding of the two sulfonamide inhibitors AMS and Diamox, has been reinvestigated at 2.0 and 3.0 Å, respectively. Only the nitrogen of the sulfonamide group binds to the zinc ion replacing the hydroxyl ion. The sulfonamide oxygen closet to the zinc ion is 3.1 Å away. Thus the tetrahedral geometry of the zinc is retained, refuting earlier models of a pentacoordinated zinc.The structure of the thiocyanate complex has been investigated at pH 8.5 and the structure has been refined at 1.9 Å resolution using the least-squares refinement program PROLSQ. The crystallographic R factor is 17.6%. The zinc ion is pentacoordinated with the anion as well as a water molecule bound in addition to the three histidine residues. The nitrogen atom of the SCN- ion is 1.9 Å from the zinc ion but shifted 1.3 Å with respect to the hydroxyl ion in the native structure and at van der Waals' distance from the Oγl atom of Thr-199. This is due to the inability of the Oγl atom of Thr-199 to serve as a hydrogen bond donor, thus repelling the nonprotonated nitrogen. The SCN- molecule reaches into the deep end of the active site cavity where the sulfur atom has displaced the so-called “deep” water molecule of the native enzyme. The zinc-bound water molecule is 2.2 Å from the zinc ion and 2.4 Å from the SCN- nitrogen. In addition, this water is hydrogen bonded to the Oγl atom of Thr-199 and to another water molecule.We have observed that solvent and inhibitor molecules have three possible binding sites on the zinc ion and their significance for the catalysis and inhibition of HCA II will be discussed. All available crystallographic data are consistent with a proposed catalytic mechanism in which both the OH moiety and one oxygen of the substrate HCO3- ion are ligated to the zinc ion.
    Additional Material: 9 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340040407
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    Further experimental studies of the disulfide folding transition of ribonuclease A (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 251-261 
    Details
    ISSN: 0887-3585
    Keywords: protein folding kinetics ; disulfide bonds ; thiol-disulfide exchange ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Two very different mechanisms of folding have been proposed from experimental studies of disulfide formation in reduced ribonuclease A. (1) A pathway in which the rate-limiting step separates fully folded protein from all other disulfide intermediates and occurs solely in three-disulfide intermediates. (2) A multiple pathway mechanism with different rate-limiting steps for each pathway. The various rate-limiting steps involve disulfide breakage, formation, and rearrangement in intermediates with one, two, three, and four protein disulfides. To distinguish between these two mechanisms, we have carried out further studies of both unfolding and refolding.Refolding of reduced ribonuclease A requires three-disulfide intermediates to accumulate; negligible refolding occurs when only the nearly random one- and two-disulfide intermediate species are populated. Therefore, no rate-limiting steps of the type postulated in mechanism (2) occur in intermediates with one and two protein disulfides. Unfolding and disulfide reduction is an all-or-none process; no disulfide intermediates accumulate to detectable to detectable levels or precede the rate-limiting step. Mechanism (2) requires that such intermediates precede the rate-limiting step and accumulate to substantial levels.The different proposal were shown not to result from the use of different solution conditions or disulfide reagents; the two sets of data are not inconsistent. Instead, the inappropriate mechanism (2) resulted from an incorrect kinetic analysis and misinterpretation of the kinetics of disulfide formation are breakage.
    Additional Material: 6 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340040404
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    A monoclonal antibody to the Ca2+ -ATPase of cardiac sarcoplasmic reticulum cross-reacts with slow type I but not with fast type II canine skeletal muscle fibers: An immunocytochemical and immunochemical study (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 164-174 
    Details
    ISSN: 0886-1544
    Keywords: Ca2+-ATPase of sarcoplasmic reticulum ; immunofluorescence ; myofibers types I (slow) and II (fast) ; II D8 monoclonal antibody ; II H11 monoclonal antibody ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Ca2+ -ATPase of the sarcoplasmic reticulum was localized in cryostat sections from three different adult canine skeletal muscles (gracilis, extensor carpi radialis, and superficial digitalis flexor) by immunofluorescence labeling with monoclonal antibodies to the Ca2+ -ATPase Type I (slow) myofibers were strongly labeled for the Ca2+ -ATPase with a monoclonal antibody (II D8) to the CA2+ -ATPase of canine cardiac sarcoplasmic reticulum; the type II (fast) myofibers were labeled at the level of the background with monoclonal antibody II D8. By contrast, type II (fast) myofibers were strongly labeled for Ca2+ -ATPase of rabbit skeletal sarcoplasmic reticulum. The subcellular distribution of the immunolabeling in type I (slow) myofibers with monoclonal antibody II D8 corresponded to that of the sarcoplasmic reticulum as previously determined by electron microscopy. The structural similarity between the canine cardiac Ca2+ -ATPase present in the sarcoplasmic reticulum of the canine slow skeletal muscle fibers was demonstrated by immunoblotting. Monoclonal antibody (II D8) to the cardiac Ca2+ -ATPase binds to only one protein band present in the extract from either cardiac or type I (slow) skeletal muscle tissue. By contrast, monoclonal antibody (II H11) to the skeletal type II (fast) Ca2+ -ATPase binds only one protein band in the extract from type II (fast) skeletal muscle tissue. These immunopositive proteins coelectrophoresed with the Ca2+ -ATPase of the canine cardiac sarcoplasmic reticulum and showed an apparent Mr of 115,000. It is concluded that the Ca2+ -ATPase of cardiac and type I (slow) skeletal sarcoplasmic reticulum have at least one epitope in common, which is not present on the Ca2+ -ATPase of sarcoplasmic reticulum in type II (fast) skeletal myofibers. It is possible that this site is related to the assumed necessity of the Ca2+ -ATPase of the sarcoplasmic reticulum in cardiac and type I (slow) skeletal myofibers to interact with phosphorylated phospholamban and thereby enhance the accumulation of Ca2+ in the lumen of the sarcoplasmic reticulum following β-adrenergic stimulation.
    Additional Material: 7 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090208
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    Microtubules in ascidian eggs during meiosis, fertilization, and mitosis (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 219-230 
    Details
    ISSN: 0886-1544
    Keywords: fertilization ; ooplasmic segregation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The sequential changes in the distribution of microtubules during germinal vesicle breakdown (GVBD), fertilization, and mitosis were investigated with antitubulin indirect immunofluorescence microscopy in several species of ascidian eggs (Molgula occidentalis, Ciona savignyi, and Halocynthia roretzi). These alterations in microtubule patterns were also correlated with observed cytoplasmic movements. A cytoplasmic latticework of microtubules was observed throughout meiosis. The unfertilized egg of M. occidentalis had a small meiotic spindle with wide poles; the poles became focused after egg activation. The other two species had more typical meiotic spindles before fertilization. At fertilization, a sperm aster first appeared near the cortex close to the vegetal pole. It enlarged into an unusual asymmetric aster associated with the egg cortex. The sperm aster rapidly grew after the formation of the second polar body, and it was displaced as far as the equatorial region, corresponding to the site of the myoplasmic rescent, the posterior half of the egg. The female pronucleus migrated to the male pronucleus at the center of the sperm aster. The microtubule latticework and the sperm aster disappeared towards the end of first interphase with only a small bipolar structure remaining until first mitosis. At mitosis the asters enlarged tremendously, while the mitotic spindle remained remarkably small. The two daughter nuclei remained near the site of cleavage even after division was complete. These results document the changes in microtubule patterns during maturation in Ascidian oocytes, demonstrate that the sperm contributes the active centrosome at fertilization, and reveal the presence of a mitotic apparatus at first division which has an unusually small spindle and huge asters.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970090304
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    Dynamics of a fluorescent calmodulin analog in the mammalian mitotic spindle at metaphase (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 231-242 
    Details
    ISSN: 0886-1544
    Keywords: tubulin ; microtubules ; photobleaching ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have compared the exchange kinetics of fluorescein-labeled calmodulin and tubulin in the spindles of living mitotic cells at metaphase. Cultured mammalian cells in early stages of mitosis were microinjected with labeled calmodulin or tubulin and returned to an incubator to allow equilibration of the fluorescent protein with the endogenous protein pools. Calmodulin becomes concentrated in the mitotic spindle, and treatments with inhibitors of tubulin assembly show that this concentration is dependent on the presence of microtubules. The steady-state exchange rates of both tubulin and calmodulin were measured by an analysis of fluorescence redistribution after photobleaching (FRAP), using cells preequilibrated to either 26 ± 2°C or 36 ± 2°C. A pulse of laser light focused to a 5-μm diameter column was used to destroy the fluorescence at one pole of a metaphase mitotic spindle. Ratios of fluorescence intensity from the two half-spindles and from the two polar regions were calculated for each image in a post-bleach time series to determine the rates and extents of FRAP. For tubulin, we confirm earlier observations concerning the temperature dependence of the extent of FRAP, but our data do not show a significant temperature dependence for the rate of FRAP. We hypothesize that the reduced extent of tubulin FRAP at the lower temperatures is a result of microtubules that are stable to depolymerization at 26°C and are thus less likely to exchange subunits. Calmodulin's FRAP, however, does not exhibit any of the temperature dependence observed with fluorescent tubulin. At 26 ± 2°C calmodulin exchanges rapidly with the relatively stable population of microtubules, suggesting that calmodulin is bound, either directly or indirectly, to microtubule walls.
    Additional Material: 6 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090305
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    Distribution of acetylated α-tubulin in retina and in in vitro - assembled microtubules (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 243-253 
    Details
    ISSN: 0886-1544
    Keywords: neurons ; posttranslational modification ; tubulin isoforms ; rod and cone photoreceptors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have used the mouse monoclonal antibody 6-11B-1, specific for acetylated α-tubulin, to determine the distribution of acetylated α-tubulin in in vitro-assembled microtubules and retinal tissue. Analysis by immunoblots revealed that microtubules assembled from bovine brain extracts contain both acetylated and nonacetylated α-tubulin. Immunofluorescence, using 6-11B-1 and antitubulin B-5-1-2, a monoclonal antibody specific for α-tubulin, demonstrated the colocalization of both α-tubulin species in neurons of the retina and that acetylated microtubules are relatively abundant in neurons. However, analysis at higher resolution revealed that rod photoreceptors contain spatially distinct microtubule arrays which differ in content of acetylated α-tubulin and differ in stability. Acetylated microtubules which composed those of the rod outer segment and connecting cilium were resistant to depolymerization in nocodazole or colchicine. In contrast, the nonacetylated microtubules which composed those of the rod-inner segment were depolymerized in nocodazole or colchicine. Therefore, these acetylated microtubules are more resistant to depolymerization than non-acetylated microtubules.
    Additional Material: 6 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090306
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    Microtubule rearrangements during mitosis in multinucleate cells (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 254-263 
    Details
    ISSN: 0886-1544
    Keywords: microtubule interphase-mitosis transition ; mitotic asynchrony ; maturing centrosomes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The peroxidase-antiperoxidase (PAP) method for the detection of polymerized tubulin has been used to study the microtubule rearrangements during mitosis in PtK1 and HeLa multinucleate cells obtained by polyethyleneglycol (PEG)-mediated fusion. We demonstrate here that the transition of the microtubular cytoskeleton from interphase to mitosis is an inducible event and independent of the factor(s) responsible for chromatin condensation and nuclear envelope breakdown. However, for the induction of the microtubule rearrangements nuclear envelope breakdown is required. At midprophase, cytoskeletal microtubule rearrangements start for multinucleate PtK1 cells, whereas in HeLa cells such changes are delayed, and a more abrupt transition is observed here. After complete nuclear envelope breakdown (prometaphase) mitotic asters and spindles but no cytoplasmic (interphase) microtubuli can be observed in both systems. Metaphase is characterized by an interaction between the different mitotic poles which show the form of bipolar spindles, but individual separated mitotic poles far removed from the chromatin can also be seen.
    Additional Material: 7 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090307
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    Comparative study of the beat patterns of american and asian horseshoe crab sperm: Evidence for a role of the central pair complex in forming planar waveforms in flagella (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 264-270 
    Details
    ISSN: 0886-1544
    Keywords: axoneme ; flagellar movement ; helical wave ; planar wave ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: American horseshoe crab sperm flagella have the typical 9+2 structure whereas Asian horseshoe crab sperm flagella have a 9+0 axoneme lacking central pair and central sheaths. Beat patterns of the American and the Asian horseshoe crab sperm were recorded by means of a high-speed video system (200 fields/second) and were compared in order to study the role of the central pair of the axoneme in ciliary and flagellar movement.The American horseshoe crab sperm beat with relatively planar waves, whereas the Asian horseshoe crab sperm beat with right-handed helical waves. These results suggest that the central complex plays an important role in forming planar waves, whereas it is not essential for the conversion of microtubule sliding into axonemal bends.
    Additional Material: 3 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090308
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    Cytochalasin-Induced reorganization of actin in allium root cells (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 283-298 
    Details
    ISSN: 0886-1544
    Keywords: colchicine ; microtubule ; mitosis ; rhodamine-phalloidin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effect of cytochalasins on F-actin was investigated in Allium root cells stained with rhodamine-phalloidin. With cytochalasin D (CD), the normal interphase network of actin fibers is replaced by dispersed rods and specks similar to those seen in animal cells. However, during division, the specks accumulate at the poles in the form of one to a few large aggregates. The effects intensify with increasing concentration (0.5-5 m̈g/ml) and exposure time (0.5-3 hr). Further, similar behavior is observed with cytochalasin B, but dihydroCB has little effect. Double localizations show that during preprophase, aggregates cluster in association with microtubule foci at the new poles located near the nuclear envelope. From metaphase through anaphase, the aggregates are often found near the tips of kinetochore fibers, while in telophase they are often appressed to the pole side of the daughter nuclei. No association is seen between actin and the other microtubule arrays. The reorganization of F-actin into small specks is unaffected by sodium azide, but aggregation at the poles is very sensitive to this agent. Polar clustering is also blocked by oryzalin, colchicine, and isopropyl n-(3-chlorophenyl) carbamate, but taxol has no effect. Experiments with scleroderma serum 5051 show that CD-induced aggregates are embedded in centrosomal material at the poles. The results reveal that the reorganization of actin in response to cytochalasins differs during the cell cycle. Furthermore, the aggregation of actin during division is probably governed by an energy-dependent interaction with microtubules.
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    URL: http://dx.doi.org/10.1002/cm.970090402
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    Micromanipulation studies of the mitotic apparatus in sand dollar eggs (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 172-184 
    Details
    ISSN: 0886-1544
    Keywords: chromosome movement ; spindle elongation ; micromanipulation ; mechanical properties ; mitosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Mechanical properties of the mitotic spindle and the effects of various operations of the mitotic apparatus on the chromosome movement and spindle elongation were investigated in fertilized eggs and blastomeres of the sand dollar, Clypeaster japonicus. On the basis of results with mechanical stretching and compression of the spindle with a pair of microneedles and the behavior of an oil drop microinjected into the spindle, it was concluded that the equatorial region of the spindle is mechanically weaker than the half-spindle region. Anaphase chromosome movement occurred in the spindle from which an aster had been removed or separated with its polar end and in the spindle in which the interzonal region had been removed. This fact indicates that chromosomes move poleward in anaphase by forces generated near the kinetochores in the half-spindle. Because of the effects of separation or removal of an aster from the spindle on the spindle elongation in anaphase and the behavior of the aster, it was concluded that the spindle elongation in anaphase is caused by pulling forces generated by asters attached to the ends of the spindle.
    Additional Material: 11 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970100122
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    Dynamics of the endoplasmic reticulum in living onion epidermal cells in relation to microtubules, microfilaments, and intracellular particle movement (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 153-163 
    Details
    ISSN: 0886-1544
    Keywords: intracellular particle motions ; cytoplasmic streaming ; onion (Allium) epidermal cells ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The endoplasmic reticulum (ER) and associated organelle and particle movements in onion (Allium cepa) bulb scale epidermal cells were observed, recorded, and analyzed using computer-assisted video (AVEC-DIC, AVEC-POL and fluorescence) microscopy. The ER is composed of two interconnected sets of filamentous membrane tubules with diameters ranging from 0.1 to 0.5 μm. The first form a more stable, stationary network of intersecting polygonal membrane tubules lying closely appressed to the plasma membrane and continuous with a second very dynamic set of longer membrane tubules that often are located parallel to each other, shifting rapidly around the cytoplasm and forming dynamic knots or organization centers. The ER, mitochondria, and spherosomes fluoresced upon chlortetracycline treatment and are therefore presumed to sequester calcium. ER and mitochrondria also stain with the fluorescent dye, rhodamine 123. Mitochrondria and spherosomes are seen to move in the cytoplasm only along paths parallel to the axis of the ER tubules. Smaller particles (0.5 μm) tend to follow these same paths but may occasionally move independently. Particles and organelles move in close, but not in direct, association with the ER tubules. In optically favored cells, actin filaments were occasionally recorded located in parallel with the ER tubules and directly associated with moving particles. Streaming ceased promptly and reversibly upon treatment with cytochalasin B, which did not visibly disrupt the ER. Short-term treatment with colchicine did not inhibit streaming or disrupt the ER network, whereas long-term (hours) colchicine treatments caused the disappearance of the stationary, cortical polygonal networks and an aggregation of still slowly moving organelles and particles onto now visible actin filaments. This suggests that microtubule breakdown disrupts the three-dimensional distribution of the ER and rearranges actin filaments in the cell's cytoplasm. Actin filaments must be directly involved in generation of movement of the particles and organelles. A three-dimensional model, based on optical sectioning of the epidermal cells, is proposed to illustrate the distribution of the endoplasmic reticulum in onion epidermal cell cytoplasm.
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    URL: http://dx.doi.org/10.1002/cm.970100120
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    Microtubule dynamics in the chromosomal spindle fiber: Analysis by fluorescence and high-resolution polarization microscopy (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 185-196 
    Details
    ISSN: 0886-1544
    Keywords: mitosis ; kinetochore ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23-24°C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half-time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half-life of microtubule attachment to the kinetochore is less than several min at 23-24°C.In the second study, we used high-resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150-nm-thick optical sections that microtubules throughout the spindle laterally associate for several sec into “rods” composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970100123
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    Direct observation of mitotic spindle elongation in vitro (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 210-216 
    Details
    ISSN: 0886-1544
    Keywords: anaphase-B ; diatom spindle ; microtubule sliding ; mitosis in vitro ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Successful reactivation in vitro of anaphase B has recently been achieved with mitotic spindles isolated from the diatom Stephanopyxis turris. When a population of isolated spindles was studied indirectly by using immunofluorescence, nearly all of them were found to have elongated; however, when studied directly by using video microscopy, only a small proportion of spindles elongated. We report here conditions that allow nearly all of the spindles to elongate when observed directly with video microscopy. These direct observations validate previous ones made using indirect immunofluorescence. In addition, we find that the isolated spindles elongate with a linear rate, that the elongation is unchanged after the chromatin surrounding the spindles is digested with DNase I, and that during elongation a phase-dense matrix may accumulate in the spindle midzone.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970100125
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  • 135
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    Masthead (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988) 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970090101
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  • 136
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    Adaptation in the motility response to camp in dictyostelium discoideum (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 9-16 
    Details
    ISSN: 0886-1544
    Keywords: adaptation ; cAMP ; cell motility ; chemotaxis ; Dictyostelium discoideum ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When developing amebae of Dictyostelium discoideum are treated with constant concentrations of cAMP above 10-8 M, the average rate of motility is depressed, with maximum inhibition at roughly 10-6 M. It is demonstrated that shifting the concentration of cAMP from 0 M to concentrations ranging from 10-8 to 10-6 M in a perfusion chamber results in the immediate inhibition of motility. After shifting from 0 M to 10-8 or 10-7 M, the rate of cell motility remains low, then rebounds to a higher level, exhibiting a standard adaptation response. No adaptation is exhibited after a shift from 0 M to 10-6 M, a concentration resulting in maximum inhibition. It is demonstrated that the level of inhibition and the extent of the adaptation period are dependent upon the concentration of cAMP after the shift, and that submaximal inhibition is additive. The characteristics of adaptation in this motility response are very similar to the characteristics of adaptation for the relay system and phosphorylation of the putative cAMP receptor.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090103
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  • 137
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    Actin filament patterns in mouse lens epithelium: A study of the effects of aging, injury, and genetics (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 17-29 
    Details
    ISSN: 0886-1544
    Keywords: sequestered actin bundles ; polygonal arrays ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Using mainly fluorescence microscopy after rhodamine-phalloidin staining, the F-actin distribution in the mouse lens epithelium was studied with regard to the effects of age, genetic strain, and mechanical injury.These studies have revealed that aside from its association with the plasma membrane the structural organization of F-actin in the mouse lens epithelium in situ is characterized by two major configurations: (1) a filamentous arrangement in such patterns as stress fibers, polygonal arrays (PAs), and meshworks, and (2) a highly concentrated structure called a sequestered actin bundle (SAB).The aging study indicated that the SAB is a consistent character in C57BL/6 mice from the age of 5 wk on, but not in CF1 mice. The size and shape of the SAB change gradually with age as inferred from two-dimensional measurements. The genetic study on the SAB character using hybrids and congenic strains showed that it is inherited as a Mendelian dominant, probably multigenic mode. Finally, the injury study revealed a structural modification in cells around the wound, including flattening of cells at the edge and extension of processes into the wound space. In the rest of the epithelium, injury amplified membrane infolding and fluorescence of polygonal arrays but diminished the size and fluorescence intensity of SABs. These changes are thought to be correlated with wound repair involving cell division and migration.These studies illustrate the variability in F-actin expression in situ in lens epithelial cells that can be induced by intrinsic and extrinsic factors.
    Additional Material: 21 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090104
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    Dynamic behavior of the transferrin receptor followed in living epidermoid carcinoma (A431) cells with nanovid microscopy (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 30-47 
    Details
    ISSN: 0886-1544
    Keywords: video microscopy ; colloidal gold ; microtubules ; saltatory movement ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Transferrin receptors labeled with the B3/25 monoclonal antibody-gold complexes were followed in living A431 cells by using video-enhanced contrast microscopy. Initially, the antibody-gold complexes bind to receptors which are freely mobile on the upper cell surface; they then become trapped at the inner margins of the peripheral lamellae and internalize. During endocytosis discrete gold-loaded vesicular elements first appear, and then, as they fuse, a heterogenous peripheral endosomal compartment forms. The endosomes from this compartment then begin to migrate centripetally through the cytoplasm in a saltatory way so that within 15 min gold label accumulates in a juxtanuclear endosome compartment. This compartment, which consists mainly of multivesicular bodies, is thus formed by the influx and retention of peripheral endosomal elements and their continued fusion in the juxtanuclear area. Although their overall migration is inward, saltating endosomes frequently reverse their direction of movement. As label builds up in the juxtanuclear area, small vesicles containing gold label continuously pinch off from the larger elements and migrate toward the cell periphery.Experiments with nocodazole and sodium azide show that the saltatory movements, the accumulation and retention of endosomes in the juxtanuclear area, and the separation of vesicles from endosomes are driven by a microtubule-associated, ATP-dependent, motility-generating mechanism.Analysis of the movements shows that although each individual vesicle saltation can occur unpredictably toward the centre or the periphery of the cell, a net centripetal flux is observed. Moreover, it is evident that the probability of migration toward and maintenance in the juxtanuclear area is related to the diameter of the vesicles. We propose a mechanism by which bidirectional saltation along microtubules forming a radial network may be instrumental in the selective concentration of large endosomes in the juxtanuclear area while small vesicles are left free to return to the periphery. This process may be responsible for the sorting of receptors and ligands destined either for intracellular degradation in juxtanuclear lysosomes or, alternatively, for recycling to the plasma membrane.
    Additional Material: 13 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090105
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    Ultrastructure and motion analysis of permeabilized paramecium capable of motility and regulation of motility (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 73-84 
    Details
    ISSN: 0886-1544
    Keywords: cilia ; metachronal waves ; electron microscopy ; calcium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Structural and behavioral features of intact and permeabilized Paramecium tetraurelia have been defined as a basis for study of Ca2+ control of ciliary reversal. Motion analysis of living paramecia shows that all the cells in a population swim forward with gently curving spirals at speeds averaging 369 ± 19 μm/second. Ciliary reversal occurs in 10% of the cell population per second. Living paramecia, quick-fixed for scanning electron microscopy (SEM), show metachronal waves and an effective stroke obliquely toward the posterior end of the cell. Upon treatment with Triton X-100, swimming ceases and both scanning and transmission electron microscopy reveal cilia that uniformly project perpendicularly from the cell surface. Thin sections of these cells indicate that the ciliary, cell, and outer alveolar membranes are greatly disrupted or entirely missing and that the cytoplasm is also disrupted. These permeabilized paramecia can be reactivated and are capable of motility and regulation of motility. Motion analysis of cells reactivated with Mg2+ and ATP in low Ca2+ buffer (pCa7) shows that 71% swim forward in straight or curved paths at speeds averaging 221 ± 20 μm/second. When these cells are quick-fixed for SEM the metachronal wave patterns of living, forward swimming cells reappear. Motion analysis of permeabilized cells reactivated in high Ca2+ buffers (pCa 5.5) shows that 94% swim backward in tight spirals at a velocity averaging 156 ± 7 μm/second. SEM reveals a metachronal wave pattern with an effective stroke toward the anterior region. Although the permeabilized cells do not reverse spontaneously, the pCa response is preserved and the Ca2+ switch remains intact. The ciliary axonemes are largely exposed to the external environment. Therefore, the behavioral responses of these permeabilized cells depend on interaction of Ca2+ with molecules that remain bound to the axonemes throughout the extraction and reactivation procedures.
    Additional Material: 6 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090108
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    Motility of the spirochete leptospira (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 101-110 
    Details
    ISSN: 0886-1544
    Keywords: prokaryotic motility ; periplasmic flagella ; hydrodynamics ; model ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Spirochetes are a group of bacteria with a unique ultrastructure and a fascinating swimming behavior. This article reviews the hydrodynamics of spirochete motility, and examines the motility of the spirochete Leptospira in detail. Models of Leptospira motility are discussed, and future experiments are proposed.The outermost structure of Leptospira is a membrane sheath, and within this sheath are a helically shaped cell cylinder and two periplasmic flagella. One periplasmic flagellum is attached subterminally at either end of the cell cylinder and extends partway down the length of the cell. In swimming cells, each end of the cell may assume either a spiral or a hook shape. Translational cells have the anterior end spiral shaped, and the posterior end hook shaped. In the model of Berg et al., the periplasmic flagella are believed to rotate between the sheath and the cell cylinder. Rotation of the anterior periplasmic flagellum causes the generation of a gyrating spiral-shaped wave. This wave is believed sufficient to propel the cells forward in a low-viscosity medium. The cell cylinder concomitantly rolls around the periplasmic flagella in the opposite direction - which allows the cell to literally screw through a gel-like viscous medium without slippage. This model is presented, and it is contrasted to previous models of Leptospira motility.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090202
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    51-kd protein, a component of microtubule-organizing granules in the mitotic apparatus involved in aster formation in vitro (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 117-128 
    Details
    ISSN: 0886-1544
    Keywords: centrosome ; aster-forming activity ; tubulin polymerization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Mitotic apparatuses (MAs) isolated from sea urchin metaphase eggs were chilled on ice to depolymerize microtubules, homogenized, and incubated with tubulin. This caused formation of many small asters with microtubules focusing on granules which were probably fragments of the centrosome. The aster-forming protein components of the granules in the homogenized MAs were solubilized in 0.5 M KCl containing 50% glycerol. After dialysis against low-ionic-strength buffer solution, proteins congregated to form granular assembly capable of initiating aster formation. Phosphocellulose column chromatography enabled the separation of the aster-forming protein fraction which contained a 51,000 molecular weight protein (51-kd protein) as a major component. The protein fraction possessing the aster-forming activity was also prepared from methaphase whole egg homogenate, and the elution profile of the 51-kd protein on phosphocellulose column also coincided with that of the aster-forming activity. The granular assembly reconstituted from the phosphocellulose fraction formed asters whose microtubules show the same growth rate and length distribution as those of asters reconstructed from the granules in the homogenized MAs. Anti-51-kd protein antibody that was raised in rabbit and affinity-purified stained the center of asters which were reconstructed either from the granules in the homogenized MAs or from the granular assembly reconstituted from the phosphocellulose fraction. These results suggest that the 51-kd protein is a component in the aster-forming activity of the centrosomal component in vitro.
    Additional Material: 9 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090204
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    Subcellular sequestration of an antigenically unique β-tubulin (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 175-183 
    Details
    ISSN: 0886-1544
    Keywords: cytoskeleton ; microtubules ; monoclonal antibodies ; cell morphogenesis ; tubulin ; Trypanosoma brucei ; subflagellar microtubule quartet ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Tubulin from Trypanosoma brucei was characterized by Western blotting using well defined monoclonal antibodies reacting with α- or β-tubulin and a new monoclonal antibody, 1B41, raised against a microtubule-enriched fraction of T. brucei, which specifically reacts with the β-subunit of tubulin from either T. brucei or rat brain. This antibody has been used to examine the subcellular distribution of the corresponding antigen in T. brucei by indirect immunofluorescence. The epitope recognized by 1B41 is restricted to a thin line extending from the basal body region to the anterior end of the cell body. To determine the relationship between the immunoreactive zone and the flagellum, double-label immunofluorescence was performed in both interphase and mitotic cells with 1B41 and a flagellar marker, the monoclonal antibody 5E9, specific for the paraflagellar rod polypeptides of trypanosomes. These experiments revealed that the immunoreactive tubulin was contained in a part of the subpellicular cytoskeleton that remained in a constant spatial correspondence with the flagellum throughout the cell division cycle. The β-tubulin recognized by 1B41 may be segregated into the microtubular structures associated with a cisterna of the endoplasmic reticulum forming the subflagellar microtubule quartet (SFMQ). These results suggest that the presence of an antigenically unique β-tubulin defines a subpopulation of microtubules possessing specfic dynamic properties that may be involved in the morphogenesis of daughter cells during the division of T. brucei.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090209
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    Polymorphonuclear leukocyte locomotion is insensitive to lowered cytoplasmic calcium levels (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 184-189 
    Details
    ISSN: 0886-1544
    Keywords: cell locomotion ; cell motility ; calcium ; polymorphonuclear leukocyte ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Chemotactic factors stimulate the rate of locomotion of polymorphonuclear leukocytes (PMNs). To investigate the importance of cytoplasmic calcium we have examined the ability of the chemotactic peptide N-formylnorleucyl eucylphenalanine (FNLLP) to stimulate the locomotion of PMNs whose cytoplasmic calcium levels were reduced by incubation in EGTA or in EGTA plus the calcium ionophores, ionomycin or A23187. Locomotion was assayed by migration through micropore filters and by time-lapse videomicroscopy. Cells in EGTA exhibited similar or slightly reduced rates of locomotion compared to cells in Hanks' balanced salt solution (HBSS). The peptide dose dependence for the stimulation of locomotion was similar in medium containing calcium or EGTA. The presence of 1 μM ionophore plus EGTA had no effect on the stimulation of locomotion by peptide. The presence of ionophores (1 μM) plus external calcium inhibited locomotion.
    Additional Material: 3 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090210
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  • 144
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    Acrylamide treatment of PtKl cells causes dephosphorylation of keratin polypeptides (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 11 (1988), S. 24-30 
    Details
    ISSN: 0886-1544
    Keywords: intermediate filaments ; phosphorylation ; cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Treatment of PtKl cells with 5 mM acrylamide for 4 hr results in alterations in the distribution of keratin filaments within the cells. This effect is reversible within 18 hr. Labeling of PtKl cells with 32P demonstrates that there are four phosphorylated keratins, having Mr of 56 k, 53 k, 45 k, and 40 k. Phosphate associated with these polypeptides appears to turn over with a t1/2 of 12 hr. Incubation of labeled cells in 5 mM acrylamide results in approximately 50% dephosphorylation of keratins within 2 hr, which is 3 times faster than normal turnover. Recovery of cells from acrylamide is accompanied by rephosphorylation of keratins within 18 hr. Analysis by 31P NMR spectroscopy shows that acrylamide treatments are accompanied by a transient decrease in soluble inorganic phosphate. This is followed by a rapid increase in Pi which gradually returns to normal levels. These studies show a strong correlation between phosphorylation of PtKl cell keratins and morphological response of keratin filaments to acrylamide. These observations suggest that normal distribution of keratin filaments may be, in part, mediated by protein phosphorylation.
    Additional Material: 3 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970110104
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    Masthead (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 11 (1988) 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970110201
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  • 146
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    Spindle microtubule dynamics: Modulation by metabolic inhibitors (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 11 (1988), S. 97-105 
    Details
    ISSN: 0886-1544
    Keywords: spindle microtubules ; mitosis ; FRAP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Recent experiments have shown that spindle microtubules are exceedingly dynamic. Measurements of fluorescence recovery after photobleaching (FRAP), in cells previously microinjected with fluorescent tubulin, provide quantitative information concerning the rate of turnover, or exchange, of tubulin subunits with the population of microtubules in living cells at steady state. In an effort to elucidate the pathways and factors that regulate tubulin exchange with microtubules in living cells, we have investigated the energy requirements for tubulin turnover as measured by FRAP. Spindle morphology was not detectably altered in cells incubated with 5 mM sodium azide and 1 mM 2-deoxyglucose (Az/DOG) for 5 minutes, as assayed by polarized light microscopy and antitubulin immunofluorescence. In FRAP experiments on these ATP-depleted cells, the average rate of recovery and the average percent of bleached fluorescence recovered were reduced to 37% and 30% of controls, respectively. When the inhibitors were removed, cells continued through mitosis, and rapid FRAP was restored. In the presence of azide and glucose, the rate of recovery and percent of fluorescence recovered were only slightly reduced, demonstrating that energy production via glycolysis can support microtubule turnover. Longer incubations with Az/DOG altered the microtubule organization in mitotic cells: astral microtubules lengthened and spindle fibers shortened. Furthermore, both astral and spindle microtubules became resistant to nocodazole-induced disassembly under these conditions. Together these observations indicate that microtubule dynamics require ATP and suggest a relationship between microtubule organization and turnover.
    Additional Material: 7 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970110203
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    Iontophoretic localization of Ca-sensitive sites controlling activation of ciliary beating in macrocilia of Beroë: The ciliary rete (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 11 (1988), S. 126-138 
    Details
    ISSN: 0886-1544
    Keywords: Ca sensitivity ; macrocilia activation ; membrane rete ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Macrocilia are thick compound ciliary organelles found on the lips of the ctenophore Beroë. Each macrocilium contains several hundred axonemes enclosed by a single common membrane around the shaft of the organelle. Macrocilia are activated to beat rapidly and continuously in the normal direction by stimulus-triggered Ca influx through voltage-dependent Ca channels (Tamm, 1988). Heat-dissociated macrociliary cells are spontaneously active without depolarizing stimuli, providing Ca is present (Tamm, 1988). Here we investigate the spatial distribution of macrociliary Ca channels by iontophoretic application of extracellular Ca to different sites along quiescent, “potentially activated” macrocilia of dissociated cells in Ca-free medium. We find that Ca sensitivity for eliciting motility is highest or resides exclusively on the basal portion of the macrociliary surface. This is the first demonstration of local differences in Ca morphologically with a reticulum of unfused ciliary membranes at the base of the macrocilium. This ciliary rete is in direct communication with the surrounding sea water. It is likely that the ciliary rete provides the necessary Ca influx to trigger beating by virtue of its greater Ca conductance (i.e., density of Ca channels) and/or greater total membrane area.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970110206
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  • 148
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    Cytokinesis occurs at boundaries of domains delimited by nuclear-based microtubules in sporocytes of Conocephalum conicum (Bryophyta) (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 11 (1988), S. 139-146 
    Details
    ISSN: 0886-1544
    Keywords: phragmoplast ; sporogenesis ; indirect immunofluorescence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Studies of the microtubular cytoskeleton during sporogenesis in the hepatic Conocephalum conicum (Bryophyta) have revealed several unusual phenomena that contribute to understanding the cytokinetic apparatus in plant cell division. Although a typical phragmoplast forms in the interzonal microtubules of the first division spindle and expands to the cell periphery, no cell plate develops. There is no evidence of predetermined division sites and the orientation of both first and second meiotic spindles is imprecise. Simultaneous division of the cytoplasm follows second nuclear division. Equal apportionment of the cytoplasm appears to be a function of the establishment of cytoplasmic domains in the coenocyte, the boundaries of which are delimited by interaction of postmeiotic microtubule systems radiating from the four nuclei. Primary phragmoplasts are initiated in phragmoplasts that are initiated between nonsister nuclei. Depending upon the arrangement of nuclei in the nonpolar sporocyte, from one to three secondary phragmoplasts develop in the zones of contact between opposing sets of microtubules. Except for the site and time of initiation, the two types of phragmoplasts are identical. Eventually the phragmoplasts become confluent and cell plates form in all second division phragmoplasts. It is clear that typical functional phragmoplasts can form in sites determined by interaction of postmeiotic microtubule systems as well as in interzonal spindles as is common in plant cell division.
    Additional Material: 18 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970110207
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    Optical approaches to the study of foraminiferan motility (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 126-136 
    Details
    ISSN: 0886-1544
    Keywords: microtubules ; Allogromia ; intracellular transport ; surface motility ; actin ; morphogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubules are the major cytoskeletal component of foraminiferan reticulopodia. Video-enhanced differential interference contrast light microscopy has demonstrated that the microtubules serve as the intracellular tracks along which rapid bidirectional organelle transport and cell surface motility occurs. Microtubules appear to move, both axially and laterally within the pseudopodial cytoplasm, and these microtubule translocations appear to drive the various reticulopodial movements. F-actin is localized to discrete filament plaques form at sites of pseudopod-substrate adhesion. Correlative immunofluorescence and electron microscopy reveals a structural interaction between microtubules and the actin-containing filament plaques. Our recent data on reticulopodial motility are discussed in an historical context, and a model for foram motility, based on motile microtubules, is presented.
    Additional Material: 6 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970100117
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  • 150
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    Biophysics of the leading lamella (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 164-171 
    Details
    ISSN: 0886-1544
    Keywords: motility ; lamella ; cytoskeleton ; membrane ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Additional Material: 13 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100121
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  • 151
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    Regulation of the distribution of carotenoid droplets in goldfish xanthophores and possible implication to secretory processes (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 143-152 
    Details
    ISSN: 0886-1544
    Keywords: pigment organelle ; xanthophore ; microtubule ; F-actin ; intermediate filament ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In goldfish xanthophores, the formation of pigment aggregate requires: (1) that a pigment organelle (carotenoid droplet) protein p57 be in the unphosphorylated state; (2) that self-association of pigment organelles occur in a microtubule-independent manner; and (3) that pigment organelles via p57 associate with microtubules. In the fully aggregated state, the pigment organelles are completely stationary. Pigment dispersion is initiated by activation of a cAMP-dependent protein kinase, which phosphorylates p57 and allows pigment dispersion via an active process dependent on F-actin and a cytosolic factor. This factor is not an ATPase, and its function is unknown. However, its abundance in different tissues parallels secretory activity of the tissues, suggesting a similarity between secretion and pigment dispersion in xanthophores. The identity of the motor for pigment dispersion is unclear. Experimental results show that pigment organelles isolated from cells with dispersed pigment have associated actin and ATPase activity comparable to myosin ATPase. This ATPase is probably an organelle protein of relative molecular mass ∼72,000, and unlikely to be an ion pump. Isolated pigment organelles without associated actin have 5× lower ATPase activity. Whether this organelle ATPase is the motor for pigment dispersion is under investigation. The process of pigment aggregation is poorly understood, with conflicting results for and against the involvement of intermediate filaments.
    Additional Material: 10 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970100119
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    Abstracts (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 11 (1988), S. 187-217 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970110307
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    Masthead (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988) 
    Details
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/prot.340030201
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    Conformational characteristics of the complete cequence of group A streptococcal M6 protein (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 60-69 
    Details
    ISSN: 0887-3585
    Keywords: coiled-coli ; alpha-helix ; antiphagocytic ; heptad ; antigenic variation ; sequence repeats ; cell wall protein ; intermediate filaments ; myosin ; tropomyosin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: M protein is considered a virulence determinant on the streptococcal cell wall by virtue of its ability to allow the organism to resist attack by human neutrophils. The complete DNA sequence of the M6 gene from streptococcal strain D471 has allowed, for the first time, the study of the structural characteristics of the amino acid sequence of an entire M protein molecule. Predictive secondary structural analysis revealed that the majority of this fibrillar molecule exhibits strong alpha-helical potential and that, except for the ends, nonpolar residues in the central region of the molecule exhibit the 7-residue periodicity typical for coiled-coil proteins. Differences in this heptad pattern of nonpolar residues allow this central rod region to be divided into three subdomains which correlate essentially with the repeat regions A, B, and C/D in the M6 protein sequence. Alignment of the N-terminal half of the M6 sequence with PepM5, the N-terminal half of the M5 protein, revealed that 42% of the amino acids were identical. The majority of the identities were “core” nonpolar residues of the heptad periodicity which are necessary for the maintenance of the coiled coil. Thus, conservation of structure in a sequence-variable region of these molecules may be biologically significant. Results suggest that serologically different M proteins may be built according to a basic scheme: an extended central coiled-coil rod domain (which may vary in size among strains) flanked by functional end domains.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340030106
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    Identification of structural motifs from protein coordinate data: Secondary structure and first-level supersecondary structure*A FORTRAN version of the program DEFINE_STRUCTURE, which defines the secondary structure and characterizes the first level of supersecondary structure, is available from the authors (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 71-84 
    Details
    ISSN: 0887-3585
    Keywords: Cα coordinates ; distance matrix ; difference distance matrix ; helix axes, strand axes ; interaxial angles ; turns ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A computer program is described that produces a description of the secondary structure and supersecondary structure of a polypeptide chain using the list of alpha carbon coordinates as input. Restricting the term “secondary structure” to the conformation of contiguous segments of the chain, the program determines the initial and final residues in helices, extended strands, sharp turns, and omega loops. This is accomplished through the use of difference distance matrices. The distances in idealized models of the segments are compared with the actual structure, and the differences are evaluated for agreement within preset limits. The program assigns 90-95% of the residues in most proteins to at least one type of secondary elementIn a second step the now-defined helices and strands are idealized as straight line segments, and the axial directions and locations are compiled from the input Cα coordinate list. These data are used to check for moderate curvature in strands and helices, and the secondary structure list is corrected where necessary. The geometric relations between these line segments are then calculated and output as the first level of supersecondary structure. A maximum of six parameters are required for a complete description of the relations between each pair. Frequently a less complete description will suffice, for example just the interaxial separation and angle. Both the secondary structure and one aspect of the supersecondary structure can be displayed in a character matrix analogous to the distance matrix format. This allows a quite accurate two-dimensional display of the three-dimensional structure, and several examples are presentedA procedure for searching for arbitrary substructures in proteins using distance matrices is also described. A search for the DNA binding helix-turnhelix motif in the Protein Data Bank serves as an exampleA further abstraction of the above data can be made in the form of a metamatrix where each diagonal element represents an entire secondary segment rather than a single atom, and the off-diagonal elements contain all the parameters describing their interrelations. Such matrices can be used in a straightforward search for higher levels of supersecondary structure or used in toto as a representation of the entire tertiary structure of the polypeptide chain.
    Additional Material: 5 Ill.
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    Pattern descriptors and the unidentified reading frame 6 human mtDNA dinucleotide-binding site (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 97-101 
    Details
    ISSN: 0887-3585
    Keywords: URF ; nucleotide-binding sites ; pattern descriptor ; computer search ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In an effort to identify the structural elements essential to a given protein function a new pattern-directed inference system has been developed. It has been employed to identify a potential dinucleotide-binding domain within the human mitochondrial unidentified reading frame 6 product, thereby supporting an earlier study that this gene may encode a NADH dehydrogenase subunit.
    Additional Material: 2 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340030204
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    Lysine/fibrin binding sites of kringles modeled after the structure of kringle 1 of prothrombin (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 85-96 
    Details
    ISSN: 0887-3585
    Keywords: molecular modeling ; energy minimization ; lysine/fibrin binding ; kringle structures ; plasminogen ; tissue plasminogen activator ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The Lys binding site of kringle 1 and 4 (K1 and K4) of plasminogen (PG) has been modeled on the basis of the three-dimensional structure of kringle 1 of prothrombin and 300- and 600-MHz proton nuclear magnetic resonance observations. These structures were then compared to the corresponding regions of modeled kringle 1 and 2 of tissue plasminogen activator (PA). The coordinates of the modeled structures have been refined by energy minimization in the presence and absence of ∊-aminocaproic acid ligand in order basically to remove unacceptable van der Waals contacts. The binding site is characterized by an apparent dipolar surface, the polar parts of which are separated by a hydrophobic region of highly conserved aromatic residues. Zwitterionic ligands such as Lys and ∊-aminocaproic acid form ion pair interactions with Asp55 and Asp57 located on the dipolar surface; the latter are also conserved in all the Lys binding kringles. The cationic center of the dipolar surface is Arg71, in the case of PGK4, and is composed of Arg34 and Arg71 in PGK1. The doubly charged anionic/cationic interaction centers of the latter might account for the larger binding constants of PGK1 for like-ligands but the modeling suggests that PGK4 might be kinetically faster in binding bulkier ligands. The binding site region of PAK2, which also binds Lys, resembles those of PGK1 and PGK4. Since PAK2 lacks both cationic center Arg residues, ligand carboxylate binding appears to be accomplished though an imidazolium ion of His64, which is located just below the outer surface of the kringle.
    Additional Material: 6 Ill.
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  • 158
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    Site-directed mutations altering methyl-accepting residues of a sensory transducer protein (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 102-112 
    Details
    ISSN: 0887-3585
    Keywords: bacterial chemotaxis ; sensory adaptation ; protein modification ; membrane protein ; receptor protein ; transmembrane signalling ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The Trg protein is one of a family of transducer proteins that mediate chemotactic response in Escherichia coli. Transducers are methylaccepting proteins that gain or lose methyl esters on specific glutamyl residues during sensory adaptation. In this study, the significance of multiple sites of methylation on transducer proteins was addressed by using oligonucleotide-directed, site-specific mutagenesis to substitute an alanyl residue at each of the five methyl-accepting sites in Trg. The resulting collection of five mutations, each inactivating a single site, was analyzed for effects on covalent modification at the remaining sites on Trg and for the ability of the altered proteins to mediate sensory adaptation. Most of the alanyl substitutions had substantial biochemical effects, enhancing or reducing methyl-accepting activity of other sites, including one case of activation of a site not methylated in wild-type protein. Analysis of the altered proteins provided explanations for many features of the complex pattern of electrophoretic forms exhibited by Trg. The mutant proteins were less efficient than normal Trg in mediating adaptation. Correlation of biochemical and behavioral data indicated that reduction in the number of methyl-accepting sites on the transducer lengthened the time required to reach an adapted state.
    Additional Material: 10 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340030205
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    Human myeloperoxidase and thyroid peroxidase, two enzymes with separate and distinct physiological functions, are evolutionarily related members of the same gene family (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 113-120 
    Details
    ISSN: 0887-3585
    Keywords: evolution ; proximal histidine ; distal histidine ; heme enzyme ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Human myeloperoxidase and human thyroid peroxidase nucleotide and amino acid sequences were compared. The global similarities of the nucleotide and amino acid sequences are 46% and 44%, respectively. These similarities are most evident within the coding sequence, especially that encoding the myeloperoxidase functional subunits. These results clearly indicate that myeloperoxidase and thyroid peroxidase are members of the same gene family and diverged from a common ancestral gene. The residues at 416 in myeloperoxidase and 407 in thyroid peroxidase were estimated as possible candidates for the proximal histidine residues that link to the iron centers of the enzymes. The primary structures around these histidine residues were compared with those of other known peroxidases. The similarity in this region between the two animal peroxidases (amino acid 396-418 in thyroid peroxidase and 405-427 in myeloperoxidase) is 74%; however, those between the animal peroxidases and other yeast and plant peroxidases are not significantly high, although several conserved features have been observed. The possible location of the distal histidine residues in myeloperoxidase and thyroid peroxidase amino acid sequences are also discussed.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340030206
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    Peptide synthesis catalyzed by polyethylene glycol-modified chymotrypsin in organic solvents (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 130-137 
    Details
    ISSN: 0887-3585
    Keywords: peptide synthesis ; chymotrypsin specificity ; polyethylene glycol ; nonaqueous solvents ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Chymotrypsin modified with polyethylene glycol was successfully used for peptide synthesis in organic solvents. The benzene-soluble modified enzyme readily catalyzed both aminolysis of N-benzoyl-L-tyrosine p-nitroanilide and synthesis of N-benzoyl-L-tyrosine butylamide in the presence of trace amounts of water. A quantitative reaction was obtained when either hydrophobic or bulky amides of L- as well as D-amino acids were used as acceptor nucleophiles, while almost no reaction occurred with free amino acids or ester derivativesThe acceptor nucleophile specificity of modified chymotrypsin as a catalyst in the formation of both amide and peptide bonds in organic solvents was quite comparable to that in aqueous solution as well as to that of the leaving group in hydrolysis reactions. By contrast, the substrate specificity of modified chymotrypsin in organic solvents was different from that in water since arginine and lysine esters were found to be as effective as aromatic amino acids to form the acyl-enzyme with subsequent synthesis of a peptide bond.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340030208
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    Vibrational spectroscopy of bacteriorhodopsin mutants: I. Tyrosine-185 protonates and deprotonantes during the photocycle (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 219-229 
    Details
    ISSN: 0887-3585
    Keywords: proton transport ; energy transduction ; purple membrane ; proton wire ; Schiff base counter-ion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The techniques of FTIR difference spectroscopy and site-directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton-pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr→Phe substitution, difference spectra have been obtained for the bR→K and bR→M photoreactions. Only the Tyr-185→Phe mutation results in the disappearance of a set of bands that were previously shown to be due to protonation of a tryosinate during the br→K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United states of America 83:347, (1986)]. The Tyr-185→Phe mutation also eliminates a set of bands in the bR→M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272-cm-1 peak) are completely unaffected by the other ten Tyr→Phe mutations. Thus, tyrosinate-185 gains a proton during the bR→K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr-185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative C=NH+ stretch band shifts from 1640 cm-1 in the wild type to 1636 cm-1 in the Tyr-185→Phe mutant. A model that is consistent with these results is that Tyr-185 is normally ionized and serves as a counter-ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr-185, which raises the pKa of the latter group and results in its protonation.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340030403
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    Structural comparison of the prokaryotic ribosomal proteins L7/L12 and L30 (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 3 (1988), S. 243-251 
    Details
    ISSN: 0887-3585
    Keywords: protein evolution ; structural homology ; ribosome structure ; x-ray crystallography ; common motif ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structure of two prokaryotic ribosomal proteins, the carboxyterminal half of L7/L12 from Escherichia coli (L12CTF) and 1.30 from Bacilus Stearothermophilus display a remarkably similar fold in which alpaha-helices pack onto one side of an antiparallel, three-stranded, beta-pleated sheet. A detailed comparison of the structures by least-squares methods reveals that more than two-thirds of the alpha carbons can be superimposed with a root mean square distance of 2.33 Å. The principal difference is an extra alpha-helix in L12 CTF. The sequences of the proteins display a distinct conservation in regions which are crucial to the common fold, in particular the hydrophobic core. It is proposed that the similarity is a result of divergent evolution.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340030405
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    Crystallographic structure analysis of lamprey hemoglobin from anomalous dispersion of synchrotron radiation (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 77-88 
    Details
    ISSN: 0887-3585
    Keywords: protein structure ; diffraction ; anomalous scattering ; x-ray crystallography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The molecular structure of lamprey hemoglobin was previously determined and refined by conventional crystallographic analysis. In this study, the structural analysis has been repeated in the course of developing the method of multiwavelength anomalous diffraction (MAD) for phase determination. New experimental and analytical procedures that were devised to perform this determination should have general applicability. These include an experimental design to optimize signal strength and reduce systematic errors, experimental evaluation of anomalous scattering factors, and a least-squares procedure for analyzing the MAD data. MAD phases for the structure at 3Å resolution are as accurate overall as the multiple isomorphous replacement (MIR) phases determined previously.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340040202
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    Conformation of alamethicin in phospholipid vesicles: Implications for insertion models (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 89-98 
    Details
    ISSN: 0887-3585
    Keywords: membrane proteins ; channels ; circular dichroism spectroscopy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The secondary structure of alamethicin, a membrane channel-forming polypeptide, has been examined by circular dichroism spectroscopy to determine the relationship of its conformation in organic solution to its conformation in a membrane-bound state. The spectrum of alamethicin in small unilamellar dimyristoyl phosphatidylcholine vesicles is significantly different from its spectrum in 10% methanol/acetonitrile, the solvent from which it was crystallized (Fox and Richards: Nature 300:325-330, 1982), as well as its spectrum in methanol, the solvent in which NMR studies have been done (Banerjee and Chan: Biochemistry 22:3709-3713, 1983). This suggests that structural models based on studies of the molecule in organic solvents may not be entirely appropriate for the membrane-bound state. To distinguish between different models for channel formation and insertion, two different methods were used to associate the alamethicin with vesicles; in addition, the effect of oligomerization on the conformation of the membrane-bound state was investigated. These studies are consistent with a modified insertion model in which alamethicin monomers, dimers, or trimers associate with the bilayer and then spontaneously oligomerize to form a prechannel with a higher helix content. This aggregate could then “open” upon application of an appropriate gating transmembrane potential.
    Additional Material: 4 Ill.
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    Internal motion of a tryptophan residue in Streptomyces subtilisin inhibitor: Deuterium nuclear magnetic resonance in solution (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 131-136 
    Details
    ISSN: 0887-3585
    Keywords: 2H NMR ; selective deuteration ; tryptophan internal motion ; SSI-subtilisin complex ; protein conformational equilibrium ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Deuterium NMR spectroscopy was used to study internal motions of a deuterium-labeled single tryptophan (Trp) residue (per subunit) of Streptomyces subtilisin inhibitor (SSI) in solution. The free inhibitor with the five ring protons of the Trp replaced with deuterons showed a narrow resonance component (56 Hz) of about one-quarter of the total intensity, in addition to the broad resonance component (about 600 Hz) at 25°C, showing that it exits in an equilibrium mixture of two conformers, in one of which the typtophan side chain is highly mobile. In analogy to the two structures of SSI found in the crystal, these two conformers were attributed to the one in which the contact between the α-lobe and the beta;-lobe of the subunit is tight and the other in which the same contact is loose. When SSI forms a complex with subtilisin BPN′, the broad component becomes invisibly broad, but the narrow component increases with even further narrowing, suggesting that the binding to the enzyme favors the “loose” conformer over the “tight” conformer.
    Additional Material: 4 Ill.
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    Surface interactions of γ-crystallins in the crystal medium in relation to their association in the eye lens (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 137-147 
    Details
    ISSN: 0887-3585
    Keywords: eye lens proteins ; protein association ; crystal packing ; surface area ; homologous proteins ; point mutations ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A comparative study of intermolecular interactions in crystals of two homologous low molecular weight proteins, γ-II and γ-IIIb crystallins, from calf eye lens was carried out. Crystal packings for these proteins are very different: intermolecular contact areas compose about 33% of the total accessible surface area of γ-II as compared with 13% in γ-III. Two key residues seem to be mainly responsible for the differences in protein association in the crystal medium. These are Ser 103 and Leu 155 in γ-II, which are replaced by Met 103 and His 155 in γ-IIIb. A similar substitution of these residues is observed in different gene products of γ-crystallins from a number of vertebrates. This is consistent with the existence of a genetically controlled mechanism for determining intermolecular association of γ-crystallins in the native medium of the lens.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340040207
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    Polar hydrogen positions in proteins: Empirical energy placement and neutron diffraction comparison (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 148-156 
    Details
    ISSN: 0887-3585
    Keywords: Protein structure ; empirical energy ; energy minimization ; molecular dynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A method for the prediction of hydrogen positions in proteins is presented. The method is based on the knowledge of the heavy atom positions obtained, for instance, from X-ray crystallography. It employs an energy minimization limited to the environment of the hydrogen atoms bound to a common heavy atom or to a single water molecule. The method is not restricted to proteins and can be applied without modification to nonpolar hydrogens and to nucleic acids. The method has been applied to the neutron diffraction structures of trypsin ribonuclease A, and bovine pancreatic trypsin inhibitor. A comparison of the constructed and the observed hydrogen positions shows few deviations except in situations in which several energetically similar conformations are possible. Analysis of the potential energy of rotation of Lys amino and Ser, Thr, Tyr hydroxyl groups reveals that the conformations of lowest intrinsic torsion energies are statistically favored in both the crystal and the constructed structures.
    Additional Material: 3 Ill.
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    Masthead (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988) 
    Details
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/prot.340040301
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    X-ray and model-building studies on the specificity of the active site of proteinase K (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 157-164 
    Details
    ISSN: 0887-3585
    Keywords: synthetic inhibitors ; serine proteinase crystallography ; active site geometry ; computer graphics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Proteinase K, the extracellular serine endopeptidase (E.C. 3.4.21.14) from the fungus Tritirachium album limber, is homologous to the bacterial subtilisin proteases. The binding geometry of the synthetic inhibitor carbobenzoxy-Ala-Phechloromethyl Ketone to the active site of proteinase K was the first determined from a Fourier synthesis based on synchrotron X-ray diffraction data between 1.8 Å and 5.0 Å resolution. The protein inhibitor complexes was refined by restrained least-squares minimization with the data between 10.0 and 1.8 Å. The final R factor was 19.1% and the model contained 2,018 protein atoms, 28 inhibitors atoms, 125 water molecules, and two Ca2+ ions. The peptides portion of the inhibitor is bound to the active center of proteinase K by means of a three-stranded antiparallel pleated sheet, with the side chain of the phenylalanine located in the P1 site. Model building studies, with lysine replacing phenylalanine in the inhibitor, explain the relatively unspecific catalytic activity of the enzyme.
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    URL: http://dx.doi.org/10.1002/prot.340040302
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    Structural analysis of the 2.8 Å model of xylose isomerase from Actinoplanes missouriensis (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 165-172 
    Details
    ISSN: 0887-3585
    Keywords: α/β barrels ; crystal structure ; glucose isomerase ; xylose isomerase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structure of Xylose isomerase (X.I.) from Actinoplanes missouriensis has been solved to 2.8 Angstroms resolution. Phases were determined from a single Eu3+ derivative and from the noncrystallographic 22 symmetry of the tetrameric molecule. An atomic model was built and subjected to restrained crystallographic refinement. The resulting model is shown to be closely similar to the recently reported X.I.'s structures from three other bacterial sources. Each monomer is found to be composed of an eight-stranded α/β “T.I.M.” barrel forming an N-terminal domain of 328 residues followed by a large loop of 66 residues embracing an adjacent subunit. Analysis of intersubunit packing shows that the X.I. tetramer is an assembly of two tight dimers. The β barrel fits a simple hyperboloid model as other T.I.M. barrels do. The active site, identified as the binding site for the inhibitor xylitol, is located at the carboxyl end of the beta strands in the barrel next to a pair binding site for Eu3+ ions, which are assumed to the sites for the divalent ions involved in catalysis. Active sites in the tetramer are oriented towards the interface between dimmers. It is suggested that subunit interfaces might stabilize the active site region and this might explain the oligomeric nature of the other α/β barrel enzymes.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340040303
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    Formation of heterodimers between wild type and mutant trp aporepressor polypeptides of Escherichia coli (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 173-181 
    Details
    ISSN: 0887-3585
    Keywords: DNA binding protein ; ligand binding ; equilibrium dialysis ; dimer ; stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Availability of the three-dimensional structure of the trp repressor of Escherichia coli and a large group of repressor mutants has permitted the identification and analysis of mutants with substitutions of the amino acid residues that from the tryptophan binding pocket. Mutant aporepressors selected for study were overproduced using a multicopy expression plasmid. Equilibrium dialysis with 14C-tryptophan and purified mutant and wild type aporepressors was employed to determine tryptophan binding constants. The results obtained indicate that replacement of theronine 44 by methionine (TM44) or arginine 84 by histidine (RH84) lowers the affinity for tryptophan approximately two-and four-fold, respectively. Replacement of ariginine 54 by histidine (RH84) or glycine 85 by ariginine (GR85) results in complete loss of tryptophan binding activity. Purified mutant and wild type aporepressors were used in vitro heterodimer studies. The trp repressor of E. coli functions as a stable dimer. A large number of trp repressor mutants prduces defective repressors that are transdominant to the wild type repressor in vivo. The transdominance presumably results from the formation of inactive or slightly active heterodimers between the mutant and wild type polypeptide subunits. An in vitro assay was developed to detect and measure heterodimer formation. Heterodimer formation was thermally induced, and heterodimers were separated on nondenaturing polyacrylamide gels. Aporepressors readily formed heterodimer formation upon treatment at 65°C for 3 minutes. Heterodimer formation was significantly retarded by the presence of the corepressor, L-tryptophan. Indole-3-propionic acid, 5-methyl tryptophan, and other analogs of tryptophan, as well as indole, also inhibited heterodimer formation. These results indicate that the presence of the indole moiety in the corepressor binding pocket increases the stability of the dimer.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340040304
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  • 172
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    Interaction of peptide boronic acids with elastase: Circular dichroism studies (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 205-210 
    Details
    ISSN: 0887-3585
    Keywords: slow-binding inhibition ; transition-state analog ; conformational change ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Boronic acid derivatives of good peptide substrate of the serine proteases cause slow-binding inhibition, manifested as biphasic binding (Kettner and Shenvi: J. Biol. Chem. 259:15106-15114, 1984). These inhibitors are thought to act as reaction-intermediate analogs. Three peptides Boronic acids - Ac-Pro-boro-Val-OH, DNS-Ala-Pro-boro-Val-OH, and Ac-Ala-Ala-Pro-boro-Val-OH - were chosen for farultraviolet circular dichroism (CD) studies in order to determine whether the second phase involves a conformational change of pancreatic elastase. The dipeptide is a simple competitive inhibitors (Ki = 0.27 μM) and the latter are slow-binding inhibitors (Ki = 16.4 and 0.25 nM, respectively). Spectral deconvolution and correction for the formation of antiparallel β-sheet by the peptide inhibitors itself indicate that there is no significant change in the secondary structure of the enzyme in the either the initial or final inhibitors complex. A kinetic experiment confirmed that the slow-binding step was not associated with a CD spectral change, and that therefore a protein conformational change was not responsible for the sow binding.
    Additional Material: 2 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340040307
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  • 173
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    Masthead (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988) 
    Details
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/prot.340040401
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  • 174
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    Diffusion-collision model for the folding kinetics of myoglobin (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 211-227 
    Details
    ISSN: 0887-3585
    Keywords: protein-folding ; multiple pathways ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The diffusion-collision model has been used to analyze the folding kinetics of myoglobins. The microdomains, which are the basic units that coalesce during the folding, are identified with the helices and the stabilizing contacts between helices are determined form the native structure. Both association and disassociation reactions are included and a range of stabilization parameters is investigated to determine the variations in overall rate and the relative contributions made by the different intermediates during the folding process. In a comparison of folding to the native state and to the midpoint of the folding transitions. (i.e., 50% native protein at the completion of the reaction) significant differences in the contributing intermediates are found.
    Additional Material: 7 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340040308
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  • 175
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    Test of circular dichroism (CD) methods for crambin and CD-assisted secondary structure prediction of its homologous toxins (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 262-273 
    Details
    ISSN: 0887-3585
    Keywords: hierarchical assignment ; cereal grain ; mistletoe ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Methods that analyze protein circular dichroism (CD) spectra for fractions of secondary structure are evaluated for the plant protein crambin, which has a known high-resolution crystal structure. In addition, a two-step secondary structure prediction scheme is presented and used for the toxins homologous to crambin, shown by others to have secondary structures similar to crambin.The test of CD spectral analysis methods with the protein crambin employed two computer programs and several CD basis sets. Crambin's crystal structure, known to 0.945 Å resolution (Hendrickson, W.A., Teeter, M.M. Nature 290:107-113, 1981), allows accurate evaluation of results. Analysis with the protein spectra basis sets (Provencher, S. W., Glöckner, J. Biochemistry 20:33-37, 1981) as modified (Manavalan, P., Johnson, W. C., Jr. Anal. Biochem. 167:76-85, 1987) agreed most closely with crambin's crystal structure. This method was then applied to the CD spectra of the membrane-active toxins homologous to crambin (α1- and β-purothionin, phoratoxin A and B, an viscotoxin A3 and B).The new program SEQ (pronounced “seek”) was developed to assign the secondary structure along the protein chain in a hierarchical fashion and applied to the plant toxins. The method constrained the secondary structure fractions to those from CD analysis and combined standard statistical methods with amphipathic helix location.Both CD-arrived secondary structure percentages and sequence assignment indicate that the viscotoxins are structurally most similar to crambin. Purothionin's secondary structure was predicted to be fundamentally similar to crambin's with a difference at the start of the first helix. This assignment agreed with Raman and NMR analyses of Purothionin and lends validity to the method presented here. Differences from the NMR in the CD secondary structure fraction analysis for phoratoxin suggest interference in the CD from tryptophan residues.
    Additional Material: 2 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340040405
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    Refined structure of human carbonic anhydrase II at 2.0 Å resolution (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 274-282 
    Details
    ISSN: 0887-3585
    Keywords: crystallography ; refinement ; structure ; carbonic anhydrase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structure of human erythrocytic carbonic anhydrase II has been refined by constrained and restrained structure-factor least-squares refinement at 2.0 Å resolution. The conventional crystallographic R value is 17.3%. Of 167 solvent molecules associated with the protein, four are buried and stabilize secondary structure elements. The zinc ion is ligated to three histidyl residues and one water molecule in a nearly tetrahedral geometry. In addition to the zinc-bound water, seven more water molecules are identified in the active site. Assuming that Glu-106 is deprotonated at pH 8.5, some of the hydrogen bond donor-acceptor relations in the active site can be assigned and are described here in detail. The Oγ1 atom of Thr-199 donates its proton to the Oε1 atom of Glu-106 and can function as a hydrogen bond acceptor only in additional hydrogen bonds.
    Additional Material: 10 Ill.
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    URL: http://dx.doi.org/10.1002/prot.340040406
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    Stereochemistry of salt-bridge formation in α-helices and β-strands (1988)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 4 (1988), S. 294-295 
    Details
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/prot.340040408
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  • 178
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    Unfertilized sea urchin eggs contain a discrete cortical shell of actin that is subdivided into two organizational states (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 85-96 
    Details
    ISSN: 0886-1544
    Keywords: fertilization ; echinoderm eggs ; egg cortex ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Changes in the distribution and organizational state of actin in the cortex of echinoderm eggs are believed to be important events following fertilization. To examine the initial distribution and form of actin in unfertilized eggs, we have adapted immunogold-labeling procedures for use with eggs of Strongylocentrotus purpuratus. Using these procedures, as well as fluorescence microscopy, we have revealed a discrete 1-μm-thick concentrated shell of actin in the unfertilized egg cortex. This actin is located in the short surface projections of unfertilized eggs and around the cortical granules in a manner that suggests it is associated with the cortical granule surface. The actin in the short surface projections appears to be organized into filaments. However, most if not all of the actin surrounding the cortical granules is organized in a form that does not bind phalloidin, even though it is accessible to actin antibody. The lack of phalloidin binding is consistent with either the presence of nonfilamentous actin associated with the cortical granules or the masking of actin-filament phalloidin-binding sites by some cellular actin-binding component. In addition to the concentrated shell of actin found in the cortex, actin was also found to be concentrated in the nuclei of unfertilized eggs.
    Additional Material: 7 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090109
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  • 179
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    The position of the microtubule-organizing center relative to the nucleus is independent of the direction of cell migration in dictyostelium discoideum (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 111-116 
    Details
    ISSN: 0886-1544
    Keywords: aggregation ; cell-cell adhesion ; chemotaxis ; electron microscopy ; microcomputer analysis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Dictyostelium amoebae can migrate in several different modes. We tested for correlations of the direction of cell locomotion with the relative positions of the nucleus and microtubule-organizing center (MTOC). Five cases were analyzed on electron micrographs with a microcomputer. Each mode of movement showed characteristic locations of the MTOC relative to the nucleus; however, they differed in the various cases. In randomly migrating interphase amoebae, the number of cells with the MTOC located behind the nucleus was twice as great as those with the MTOC located ahead of the nucleus. During chemotactic migration toward folic acid, cells with the MTOC behind the nucleus were more numerous, with a concomitant reduction of anterior MTOCs.When amoebae aggregated on agar plates, a posterior location of the MTOC was most strikingly favored, whereas in cells aggregating under submerged conditions, the MTOC was indifferently anterior or posterior to the nucleus. (It may be significant that EDTA-resistant cell-cell adhesion was fully expressed in the former cells, but weaker in the latter.) Finally, in the case of chemotactically migrating cells from dissociated pseudoplasmodia, which adhere by means of other molecules, the MTOC was consistently ahead of the nucleus. Thus the MTOC shows no necessary preferential position anterior or posterior to the nucleus; its position, rather, correlates with the type of migration and perhaps with the nature of cell-cell adhesion.
    Additional Material: 4 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090203
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    Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 337-348 
    Details
    ISSN: 0886-1544
    Keywords: isoactins ; immunogold ; myofibrils ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In vertebrate skeletal muscle, the proliferating myoblasts synthesize nonmuscle isoforms of actin, and the cells begin to express muscle-specific actin isoforms during their myogenic differentiation. To study the distributions of the actin isoforms in myogenic cells and fully differentiated skeletal muscle, we prepared a peptide antibody specific for the skeletal α isoform of actin and used this antibody along with an antibody specifically reactive with nonmuscle γ actin to stain cultured myotubes and adult skeletal myofibrils by double-indirect immunofluorescence. At this level of resolution, no differences in isoform localization were seen: Both muscle and nonmuscle actins were detected in the myotubes and in the striations of mature myofibrils. Myotubes were also double-stained using immunogold electron microscopy, and the isoform distributions were determined quantitatively by counting the two sizes of gold particles that corresponded to labeling with each antibody. A quantitative analysis of immunoreactivity revealed that, although both forms were present in all actin-containing structures, nonmuscle actin was relatively more prevalent along the edges (cortical microfilaments) of the myotubes, whereas the muscle isoform predominated in the interior regions (containing forming myofibrils). Thus, we have found evidence of a heterogeneous distribution of muscle and nonmuscle actin isoforms in differentiating myogenic cells, and we have demonstrated that a nonmuscle actin isoform is a component of the muscle contractile apparatus.
    Additional Material: 6 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970090406
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  • 181
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    In vitro reactivation of anaphase B in isolated spindles of the sea urchin egg (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 197-209 
    Details
    ISSN: 0886-1544
    Keywords: GTP ; ATP ; tubulin ; spindle reactivation media ; birefringence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Spindles may be isolated from sea urchin eggs so that some mitotic processes can be reactivated in vitro. The isolation media allow spindles to remain stable for days. Transfer of the spindles to reactivation media results in loss of birefringence and breakdown of the matrix within which the microtubules function. If, however, tubulin and either guanosine triphosphate or adenosine triphosphate are present in these media so that tubulin can cycle, the spindles do not break down but grow in size and birefringence and show some of the movements of in vivo spindles. The most prominent is that of anaphase B if the mitotic apparatuses (MAs) have been isolated at a time when anaphase was initiated. When isolated during metaphase, MAs either do not show chromosome movement or, if they do, it is a random movement which causes redistribution of the chromosomes on the spindle surface. In either case, such metaphase spindles grow in size and birefringence. Thus under the proper conditions, cycling microtubules can interact with the spindle matrix to induce chromosome movements which resemble those seen in in vivo cells in the case of anaphase B and show some aspects of anaphase A in at least half the spindles isolated at metaphase, although such movements are not coordinated to show a true anaphase movement.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100124
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  • 182
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    Three-dimensional localization and redistribution of F-actin in higher plant mitosis and cell plate formation (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 217-228 
    Details
    ISSN: 0886-1544
    Keywords: immunogold ; microtubules ; optical sectioning ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The distribution of F-actin cables in dividing endosperm cells of a higher plant, Haemanthus, was visualized with the immunogold-silver-enhanced method and compared with the arrangement of immunogold-stained microtubules in the same cells. The three-dimensional distribution of F-actin cables and microtubules during mitosis and cell plate formation was analyzed using ultrathin optical sectioning of whole mounts in polarized light video microscopy. F-actin cables form a loose irregular network in the interphase cytoplasm. Much of this network remains outside of the spindle during mitosis. A few F-actin cables were detected within the spindle. Their pronounced rearrangement during mitosis appears to be related to the presence and growth of microtubule arrays. During prometaphase, actin cables located on the spindle surface and those present within the spindle tend to arrange parallel to the long axis of the spindle. Cables outside the spindle do not reorient, except those at the polar region, where they appear to be compressed by the elongating spindle. Beginning with mid-anaphase, shorter actin cables oriented in various directions accumulate at the equator. Some of them are incorporated into the phragmoplast and cell plate and are gradually fragmented as the cell plate is formed and ages. Actin cables adjacent to microtubule arrays often show a regular punctate staining pattern. Such a pattern is seldom observed in the peripheral cytoplasm, which contains few microtubules. The rearrangement of F-actin cables mimicks the behavior of spindle inclusions, such as starch grains, mitochondria, etc., implying that F-actin is redistributed passively by microtubule growth or microtubule-related transport. Thus F-actin or actomyosin-based motility does not appear to be directly involved in mitosis and cytokinesis in higher plants.
    Additional Material: 6 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970100126
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  • 183
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    Dynein as a microtubule translocator in ciliary motility: Current studies of arm structure and activity pattern (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 263-270 
    Details
    ISSN: 0886-1544
    Keywords: cilia ; axoneme ; ATP-induced microtubule sliding ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The dynein arms of ciliary doublet microtubules cause adjacent axonemal doublets to slide apart with fixed polarity. This suggests that there is a unique mechanochemistry to the dynein arm with unidirectional force generation in all active arms and also that not all arms are active at once during a ciliary beat. Negative stain and thin-section images of arms in axonemes treated with β, γ methylene adenosine triphosphate (AMP-PCP) show a consistent subunit construction where the globular head of the arm interacts with subfiber B of doublet N + 1. This interpretation differs from that provided by freeze etch and STEM interpretations of in situ arm construction and has implications for the mechanochemical cycle of the arm. A computer model of the arms in relation to other axonemal structures has been constructed to test these interpretations. Attachment of the head of the arm to subfiber B is directly demonstrable in splayed axonemes in AMP-PCP. About half of the doublets in an axoneme show such attachments, while half do not. This might imply that about half the doublets in an axoneme are active at any given instant and can be identified as such. This information may be useful in probing questions of how active arms differ biochemically from inactive arms and of how microtubule translocators in general become active.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100131
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  • 184
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    Isolation and reactivation of highly-coupled newt lung cilia (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 271-284 
    Details
    ISSN: 0886-1544
    Keywords: amphibian ; ciliary models ; isolation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The paired lungs of the newt, Taricha granulosa, are simple, unbranched sacs, 3.5-5.0 cm in length. The inner epithelium overlying the pulmonary vein is differentiated into a mucociliary tract that extends the entire length of the lung. Populations of single, demembranated ciliary axonemes, 12-13 μm in length, can be isolated by extracting whole lungs or primary cultures of the ciliated epithelium with Triton X-100. The motile capabilities of the isolated axonemes are the highest yet obtained for any ciliary model. When exposed to a suitable reactivating medium containing Mg2+ and ATP, nearly 100% of the axonemes become motile. Uniform reactivation of high quality requires short extraction times, minimization of mechanical damage, and strict adherence to optimal conditions throughout the extraction, storage, and reactivation procedures. Significant deviations from either pH 7.0 or 0.12 M salt can lead to a rapid, irreversible decrease in the beat frequency of reactivated axonemes. Both DTT and EDTA serve to stabilize their motility. The isolated axonemes beat at 29.5 Hz in the presence of 1.75 mM ATP at 21°C, matching the beat frequencies measured for cultured cells at the same temperature. With 5 mM ATP, beat frequencies over 40 Hz are measured. Our results show that neither the plasma membrane, accessory structures, nor hydrodynamic coupling of cilia are required for this activity and imply that the lack of these factors is not responsible for the low motile capabilities of ciliary models isolated previously.
    Additional Material: 9 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970100132
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  • 185
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    Studies on the mechanism of the reversal of rapid organelle transport in myelinated axons of Xenopus laevis (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 296-308 
    Details
    ISSN: 0886-1544
    Keywords: axonal transport ; organelles ; transport reversal ; myelinated axons ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Rapid organelle transport was studied by computer- and video-enhanced microscopy in the region of localized lesions in single myelinated axons of Xenopus laevis. Localized lesions were created that were either impermeable to small ions in the bathing medium or were permeable to agents with molecular weights up to 10,000. Providing the axons were bathed in a suitable “internal” medium, organelle transport continued to within a few micrometers of the lesion whether the lesion was permeable or not. Organelles undergoing anterograde and retrograde transport reversed their direction of transport on reaching the lesion. In preparations with lesions that were permeable, nonhydrolyzable analogs of ATP inhibited normally directed and reversed organelle transport. In permeable preparations, vanadate and EDTA inhibited retrograde and reversed retrograde transport at different intra-axonal concentrations; anterograde and reversed anterograde transport were also differentially inhibited. Anterograde and retrograde organelle transport were also shown to be inhibited at different intraaxonal concentrations of vanadate and EDTA.The results provide evidence for the existence of two different axonal transport mechanisms in myelinated axons. The two mechanisms can account for the normally directed and reversed transport of individual organelles.
    Additional Material: 9 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970100134
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  • 186
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    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.
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    URL: http://dx.doi.org/10.1002/cm.970100133
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  • 187
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    Rapid axonal transport as a chromatographic process: The use of immunocytochemistry of ligated nerves to investigate the biochemistry of anterogradely versus retrogradely transported organelles (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 309-320 
    Details
    ISSN: 0886-1544
    Keywords: immunofluorescence ; cytofluorimetric scanning ; composition of organelles ; rat motor axons ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The distribution and axonal transport of cholinergic organelles has been studied in the rat motor system, using immunofluorescence methods and a cytofluorimetric technique for quantification of immunoreactive material. Crush-operated spinal roots and sympathectomized sciatic nerves were sectioned longitudinally and incubated with antisera against p38, SV2, CGRP, chromogranin A (Chr A), synapsin I (SYN I), and with RASVA (rabbit anti-synaptic vesicle antiserum). Motor endplates were also studied. It was observed that proximally accumulating organelles - i.e., organelles which were in transport distally in the axons -  contained RASVA-like immunoreactivity (LI) p38, SV2, CGRP-LI, Chr A-LI, and SYN I-LI. Retrogradely transported organelles, however, contained only p38 and SV2 in addition to RASVA-LI, but virtually no CGRP-LI, ChrA-LI, or SYN I-LI. It is suggested that the rapid axonal transport mechanism operates in the nerves like a chromatographic process, which allows the concentration in the axons, proximal or distal to the crush, of organelles in anterograde or retrograde transport, respectively. The technique of nerve crushes in combination with immunocytochemistry can therefore be used to investigate the biochemical composition of organelles in transit along the axon, and give information on neurobiological events occurring in these long processes leading to the nerve endings. In this study, biochemical differences between anterogradely and retrogradely transported cholinergic crganelles in the motor neuron of the rat have been observed, and were related to suggested events in the endplate.
    Additional Material: 8 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970100135
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  • 188
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    Abstracts (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 341-348 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100138
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  • 189
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    Masthead (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 11 (1988) 
    Details
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970110101
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  • 190
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    Microtubule gelation-contraction in vitro and its relationship to component a of slow axonal transport (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 331-340 
    Details
    ISSN: 0886-1544
    Keywords: axonal transport ; SCAPs ; dynasomes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubule proteins, isolated by cycles of assembly, will undergo ATP-dependent gelation-contraction in vitro. A particulate component is present in these preparations, which is required for the gelation-contraction of microtubules assembled from purified tubulin. These particulates contain tubulin, neurofilament, spectrin, MAP2, and other as yet unidentified proteins. The particulates have a microtubule-stimulated ATPase that may be unique and is the likely motor for microtubule gelation-contraction. The basic structural unit of these particulates appears to be a crescent-shaped, or hemispherical, granule about 20 nm in diameter. The particles move along microtubule walls at a rate of about 1 μm. When compared to known physiological phenomena, microtubule gelation-contraction has striking similarities to component a of slow axonal transport (SCa), but displays no relationship to slow component b or to fast transport. On the basis of their similarities in composition, solubility, and rate of movement, we have proposed that the particulates responsible for microtubule gelation-contraction are the insoluble protein complexes, which have been suggested to be the transported component of SCa. We have termed these structures “slow component a particulates” or “SCAPs.” It is probable that similar motile protein complexes exist in cells other than neurons, and we propose the term “dynasome” to describe such structures in general.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100137
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  • 191
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    Cell model contraction in the ciliate spirostomum (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 278-282 
    Details
    ISSN: 0886-1544
    Keywords: myoneme ; microtubule ; Ca2+ ions ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: As the species name indicates, the large heterotrichous ciliate Spirostomum ambiguum is characterized by a twisting contraction of the cell body that is easily triggered by various kinds of external stimuli. On the basis of morphological studies, contraction and extension of this organism have been considered to result from antagonistic actions of myoneme and microtubular ribbons. After many trials, we have succeeded in preparing cell models to examine induced contractions and extensions of the cell body. The contraction of this model was induced by increasing the free Ca2+ concentrations even in the absence of Mg-ATP and was reversed by adding Mg-ATP without Ca2+. Using dynein ATPase inhibitors such as vanadate and ATP analogs, furthermore, the experiments revealed that the ATPase that generated the force between the two neighboring microtubular ribbons might be a dynein-like ATPase.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970090310
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  • 192
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    Wave of free calcium at fertilization in the sea urchin egg visualized with fura-2 (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 271-277 
    Details
    ISSN: 0886-1544
    Keywords: fertilization ; Ca2+ wave ; fura-2 ; sea urchin egg ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A wave front of increased free calcium traversing the egg at fertilization is demonstrated in the sea urchin Lytechinus pictus. The use of the fluorescent calcium chelator fura-2 in combination with low-light-level TV microscopy and image processing allows the visualization of the Ca2+ wave front with high spatial and temporal resolution. Such a wave is demonstrated as increased fluorescence after an excitation of 340-nm wavelength and as the reciprocal image in form of a reduced fluorescence when excited at 380 nm. The band-like appearance of the wave resembles the Ca2+ wave described for larger eggs of other species. In a dispermic egg the high resolution of the system used allows us to recognize two waves of Ca2+ originating from the respective points of sperm entry.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970090309
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  • 193
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    Determination of the average shape of flagellar bends: A gradient curvature model (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 312-324 
    Details
    ISSN: 0886-1544
    Keywords: flagella ; sea urchin spermatozoa ; waveform analysis ; Ciona spermatozoa ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Data obtained by manual digitization of photographs of flagellar bending waves have been analyzed by determining size parameters for the bends by least-squares fitting of a model waveform. These parameters were then used to normalize the data so that the average shape of the bends could be determined. Best fits were obtained with a model waveform derived from the constant curvature waveforms used previously but with provision for a linear change in curvature across the central region of the bend-the gradient curvature model (GCM). The central regions of the GCM bending waves are separated by transition regions with length determined by a parameter called the truncation factor (FT). Fitting the GCM to sine-generated bending waves give optimal fit when FT = 0.34. Fitting the GCM to four different samples of flagellar bending waves gave best fits with values of FT ranging from 0.17 for ATP-reactivated Lytechinus spermatozoa beating at approximately 10 Hz to 0.32 for live spermatozoa of Arbacia. The difference between the Arbacia waveforms and a sine-generated waveform is therefore very small, but a sine-generated waveform lacks the degree of freedom represented by FT that is required to fit other waveforms optimally.The residual differences between the waveform data and optimal GCM waveforms were averaged and found to be small. In most cases, the curvature in the central region of the optimal GCM decreased in magnitude towards the tip of the flagellum; however, this slope was highly variable and sometimes positive. Significant variations in both this slope and FT were found in individual bends as they propagated along a flagellum.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970090404
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  • 194
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    Video motion analysis of mitotic events in living cells of the fungus fusarium solani (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 325-336 
    Details
    ISSN: 0886-1544
    Keywords: anaphase ; aster ; mitosis ; motility ; spindle ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: An earlier, laser microbeam study produced evidence that, in Fusarium solani, extranuclear polar forces function at anaphase B of mitosis to pull apart the incipient daughter nuclei, whereas the central spindle functions primarily to limit the rate at which they separate. To elucidate further the various dynamics of mitotic anaphase, 8-14 mitoses in hyphae of F. solani were analyzed at 0.5-2.0-sec intervals using high-resolution, digitally processed, videotaped sequences. The spindle growth rate, although fluctuating frequently, averaged 0.6 μm/min during metaphase, increased to 3.6 μm/min during anaphase A and was maximal at 6.1 μm/min during anaphase B. Commonly, chromosomes migrated poleward during anaphase A at fluctuating rates, the average rate being an unprecedented 7.5 μm/min. During anaphase the mitotic apparatus migrated to and fro in the hyphae at rates of 3-15 μm/min, an apparent effect of opposing, fluctuating and typically unequal cytoplasmic forces applied to the two spindle poles. Thus, the molecular mechanisms underlying the various anaphase movements in F. solani do not operate entirely smoothly and uniformly. Accelerated growth of the central spindle is temporally associated with anaphase A and the development of asters. Thus, chromosome disjunction may allow the polar forces to increase the rate of spindle elongation. Microtubule dynamics and motor molecules appear to be adequate to account for the observed rates of mitotic movements.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970090405
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  • 195
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    The cytoskeletal microtubular system of some naked dinoflagellates (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 9 (1988), S. 361-374 
    Details
    ISSN: 0886-1544
    Keywords: microtubular cytoskeleton ; Dinoflagellates ; immunofluorescence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The cytoskeletal microtubule system has been studied in six species of unarmoured Dinoflagellates using immunofluorescence and electron microscopy. Several structures have been detected and described: (1) a subpellicular layer of microtubules, constituting the microtubular cytoskeleton, running singly or in bundles from the anterior part of the cell to the posterior; (2) a feeding apparatus, containing a ribbon of microtubules, which corresponds to a small peduncle in some species and is simply represented by a cytostome in some other species; and (3) the longitudinal flagellum that runs in a long intracytoplasmic pocket before becoming free at the extremity of the sulcus. A thorough study of the organization of the microtubular structures in a wide spectrum of Dinoflagellates is a prerequisite for understanding the evolutionary history of the group.
    Additional Material: 17 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970090408
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  • 196
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    High hydrostatic pressure effects in vivo: Changes in cell morphology, microtubule assembly, and actin organization (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 380-390 
    Details
    ISSN: 0886-1544
    Keywords: stress fiber ; cytoskeleton ; microvilli ; tubulin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We present the first study of the changes in the assembly and organization of actin filaments and microtubules that occur in epithelial cells subjected to the hydrostatic pressures of the deep sea. Interphase BSC-1 epithelial cells were pressurized at physiological temperature and fixed while under pressure. Changes in cell morphology and cytoskeletal organization were followed over a range of pressures from 1 to 610 atm. At atmospheric pressure, cells were flat and well attached. Exposure of cells to pressures of 290 atm or greater caused cell rounding and retraction from the substrate. This response became more pronounced with increased pressure, but the degree of response varied within the cell population in the pressure range of 290-400 atm, Microtubule assembly was not noticeably affected by pressures up to 290 atm, but by 320 atm, few microtubules remained. Most actin stress fibers completely disappeared by 290 atm. High pressure did not simply induce the overall depolymerization of actin filaments for, concurrent with cell rounding, the number of visible microvilli present on the cell surface increased dramatically. These effects of high pressure were reversible. Cells re-established their typical morphology, microtubule arrays appeared normal, and stress fibers reformed after approximately 1 hour at atmospheric pressure. High pressure may disrupt the normal assembly of microtubules and actin filaments by affecting the cellular regulatory mechanisms that control cytological changes during the transition from interphase into mitosis.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100305
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  • 197
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    Involvement of myxamoebal fragmin in the Ca2+-induced reorganization of the microfilamentous cytoskeleton in flagellates of Physarum polycephalum (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 410-419 
    Details
    ISSN: 0886-1544
    Keywords: calcium ; Ca2+ ; shape change ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When flagellates of Physarum polycephalum were treated with Triton X-100 and more than 10-5 M Ca2+, the microfilamentous cytoskeleton disintegrated, as seen by staining with rhodamine-phalloidin, and myxamoebal fragmin became associated with the Triton-insoluble cytoskeleton as demonstrated by immunofluorescence microscopy and immunoblotting. The association of myxamoebal fragmin with the cytoskeleton was reversed by the subsequent addition of excess EGTA. When flagellates were permeabilized in the absence of Ca2+, myxamoebal fragmin did not associate with the cytoskeleton and diffused out of the cells. Subsequent treatment of these cells with Ca2+ was ineffective in inducing either the association of myxamoebal fragmin with the cytoskeleton or the disintegration of the microfilamentous cytoskeleton. However, treatment of these permeabilized flagellates with 10 μg/ml purified myxamoebal fragmin and 1 mM Ca2+ caused the disintegration of the microfilaments. Therefore, we conclude that myxamoebal fragmin participates in the Ca2+-induced disintegration of the microfilamentous cytoskeleton in these permeabilized cells. Rapid cooling of flagellates caused the reversible association of myxamoebal fragmin with the Triton-insoluble cytoskeleton in vivo. Thus myxamoebal fragmin may also participate in the reorganization of the microfilamentous cytoskeleton induced in vivo by the cold treatment.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100308
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  • 198
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    Colocalisation of acetylated microtubules, glial filaments, and mitochondria in astrocytes in vitro (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 438-449 
    Details
    ISSN: 0886-1544
    Keywords: tyrosinated microtubules ; organelle distribution/transport ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have recently shown that acetylated α-tubulin containing microtubules (acety1-MTs; labeled by antibody 6-11B-1) constitute a cold-stable subset of the microtubule network of nonneuronal cells in rat primary forebrain cultures [Cambray-Deakin and Burgoyne: Cell Motil. 8(3):284-291, 1987b]. In contrast, tyrosinated α-tubulin containing MTs (tyr-MTs; labeled by antibody YL1/2) are cold-labile. Here we have examined the distribution of acety1-MTs and tyr-MTs in cultures of newborn rat forebrain astrocytes and simultaneously investigated the distribution of mitochondria and glial filaments. In double-label immunofluorescence experiments a marked colocalisation of acetyl-MTs and glial filament bundles was observed. Tyr-MTs did not show a similar colocalisation with glial filament bundles. Furthermore, the distribution of mitochondria closely followed that of the acetyl-MT and glial filament bundles. When cells were exposed to short-term (30-min) treatments with MT-disrupting agents such as colchicine and nocodazole, the tyr-MT network was removed but the distributions of acetyl-MTs, glial filaments, and mitochondria were unchanged. Increased exposure to colchicine (9-16 hr) caused a progressive disruption of the acetyl-MTs and the collapse of glial filaments and mitochondria to the perinuclear region. These results suggest that acetyl-MTs and glial filaments but not tyr-MTs may be involved in the intracellular transport of organelles and/or in the control of their cytoplasmic distribution.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970100311
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  • 199
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    Cultured cell extracts support organelle movement on microtubules in vitro (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 482-495 
    Details
    ISSN: 0886-1544
    Keywords: organelle motility ; kinesin ; cytoplasmic dynein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Directed movements of organelles have been observed in a variety of cultured cells. To study the regulation and molecular basis of intracellular organelle motility, we have prepared extracts from cultured chick embryo fibroblasts (CEF cells) which support the movement of membranous organelles along microtubules. The velocity, frequency and characteristics of organelle movements in vitro were similar to those within intact cells. Organelles and extract-coated anionic beads moved predominantly (80%) toward the minus ends of microtubules that had been regrown from centrosomes, corresponding to retrograde translocation. Similar microtubule-dependent organelle movements were observed in extracts prepared from other cultured cells (African green monkey kidney and 3T3 cells).Organelle motility was ATP and microtubule dependent. The frequency of organelle movement was inhibited by acidic (pH〈7) or alkaline (pH〉8) solutions, high ionic strength ([KCl] = 0.1 M), and the chelation of free magnesium ions. Treatment of the extracts with adenylyl imidodiphosphate (AMP-PNP, 7 mM), sodium orthovanadate (vanadate; Na3VO4, 20 μM), or N-ethylmaleimide (NEM, 2 mM) blocked all organelle motility. The decoration of microtubules with organelles was observed in the presence of AMP-PNP or vanadate. Motility was not affected by cytochalasin D (2 μM) or cAMP (1 mM). Kinesin (Mr= 116,000), an anterograde microtubule-based motor, was partially purified from the CEF extract by microtubule affinity purification in the presence of AMP-PNP, and was able to drive the movement of microtubule on glass coverslips. A similar preparation made in the presence of vanadate contained a different subset of proteins and did not support motility. These results demonstrate that intracellular organelle motility can be reproduced in vitro and provide the basis for investigating the roles of individual molecular components involved in the organelle motor complex.
    Additional Material: 6 Ill.
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    URL: http://dx.doi.org/10.1002/cm.970100405
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  • 200
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    Protease inhibitor and substrates block motility and microtubule sliding of sea urchin and carp spermatozoa (1988)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 10 (1988), S. 518-527 
    Details
    ISSN: 0886-1544
    Keywords: 9 + 2 flagellar beating ; aprotinin ; axonemes ; protease inhibitor ; sperm motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effects of protease substrates and inhibitor, which have been previously shown to inhibit mammalian sperm motility (de Lamirande, E., and Gagnon, C. [1986] J. Cell Biol. 102:1378-1383), were investigated using reactivated sea urchin and carp spermatozoa as models of “9 + 2” flagella. Aprotinin in the 2 to 20 μM range interfered with sperm motility by reducing both the beat frequency and the percentage of motile spermatozoa. These inhibitory effects of aprotinin were reversible either by dilution or by the addition of high concentrations of MgATP to the incubation medium. Protease substrates with a lys-ester bond, such as N-α-benzyloxycarbonyl-lys thiobenzyl ester (BLT), also affected motility, but in the 0.1 to 0.5 mM range. As with aprotinin, both the flagellar beat frequency and the percentage of motile spermatozoa were partially and completely decreased, respectively. Analysis of the beat frequencies as a function of MgATP concentration in the presence and absence of 6 μM aprotinin indicated that this protease inhibitor affects sperm motility by decreasing the maximal flagellar beat frequency rather than by altering the axoneme's apparent Km for MgATP. Furthermore, aprotinin concentrations that blocked flagellar reactivation completely inhibited the sliding of microtubules from trypsinized axonemes. Basic proteins or polypeptides of pI close to that of aprotinin (10.3) were also potent inhibitors of the reactivation of motility. However, the characteristics of their inhibition of flagellar beat frequencies and reversibility of their effects suggested that they might be acting on sites different from those sensitive to aprotinin. The inhibitory effects of protease inhibitor and substrates, as well as results of experiments showing the absolute requirement of an intact ester bond for the inhibitory action of protease substrates, suggest that the involvement of a protease in the reactivation of 9 + 2 flagellar beating might be considered as a possible mechanism to explain aprotinin and BLT actions.
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    URL: http://dx.doi.org/10.1002/cm.970100408
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