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  • 101
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 25-37 
    ISSN: 0886-1544
    Schlagwort(e): tubulin structure ; microtubule ; antibody ; microtubule poison ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Polyclonal antibodies have been raised against the peptide 28-38 of the β-subunit of the tubulin heterodimer in order to study the accessibility of this region in the tubulin heterodimer and in various tubulin assemblies. These antibodies were specific for all β'-tubulin subunits, except for β-tubulin isotypes, and did not recognize the α-tubulin subunit. The 28-38 region does not play a role in the interaction between the α-and β-subunits since it was accessible to the antibodies on the native heterodimer. The accessibility of the antibodies was not modified by several microtubular poisons. In contrast, in all tubulin assemblies obtained in the presence of microtubule associated proteins, the region 28-38 was not available to the antibodies. These antibodies did not react with microtubules or tubulin spirals assembled either from microtubule proteins or from pure tubulin when these tubulin assemblies were probed in the absence of free tubulin after centrifugation on glass coverslips. In addition, antibodies failed to interact with the microtubule cytoskeleton in cultured Ptk2 cells indicating that the 28-38 region of β-tubulin is also protected in cellular structures. These observations suggest that the 28-38 region of the β-tubulin subunit is either located in a zone of interaction between two successive tubulin dimers within a protofilament or hidden by an allosteric conformational change which occurs during tubulin assembly. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 102
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 160-169 
    ISSN: 0886-1544
    Schlagwort(e): actin polymerization ; myosin ; filament disassembly ; growth cones ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: A variety of mechanisms have been proposed to explain the forward extension of cytoplasm in advancing cells and axonal growth cones, including actin polymerization and osmotic swelling. Based on our observations of the filopodia of cultured neuronal growth cones, we propose a mechanism involving motor-induced extension and retraction. We observed that filopodia (actin-based protrusions 0.2-0.5 μ in diameter) extend and retract from growth cone lamellae at the same rate. Further, force is generated at the tips of filopodia which is sufficient to produce compressive buckling of the proximal portion of the filopodium. From our analysis of these movements we suggest that a motor protein powers both the extension and retraction of filopodia. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 103
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 104
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 147-158 
    ISSN: 0886-1544
    Schlagwort(e): depolymerization ; DNase I ; association rate constant ; dissociation rate constant ; polymor-phonuclear leukocyte ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Fluorescent derivatives of phallcidin are widely used to measure filamentous actin (F-actin) levels and to stabilize F-actin. We have characterized the kinetics and affinity of binding of tetramethylrhodaminy (TRITC)-phalloidin to rabbit skeletal muscle F-actin and to F-actin in lysates of rabbit polymorphonuclear leukocytes (PMNs). We have defined conditions where TRITC-phalloidin can be used to inhibit F-actin depolymerization and to quantify F-actin without prior fixation. By equi librium measurements, the affinity of TRITC-phalloidin binding to rabbit skeletal muscle F-actin (pyrene labeled) or to PMN lysate F-actin was 1-4 × 10-7 M. In both cases, the stoichiometry of binding was approximately 1:1. Kinetic measurements of TRITC-phalloidin binding to PMN lysate F-actin resulted in an association rate constant of 420 ± 120 M-1 sec-1 and a dissociation rate constant of 8.3 ± 0.9 ± 10-5 sec-1. The affinity calculated from the kinetic measurements. (2 ± 1 × 10-7 M) agreed well with that obtained by equilibrium measurements. The rate with which 0.6 μM TRITC-phalloidin inhibited 0.1 μM pyrenyl F-actin depolymerization (90% inhibition in 10 sec) was much faster than the rate of binding to pyrenyl F-actin (〈1% bound in 10 sec), suggesting that phalloidin binds to filament ends more rapidly than to the rest of the filament. We show that TRITC-phalloidin can be used to measure F-actin levels in cell lysates when G-actin is also present (i.e., in cell lysates at high concentrations) if DNase I is included to prevent phalloidin-induced polymerization.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 105
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 159-166 
    ISSN: 0886-1544
    Schlagwort(e): neoplastic cells ; mitotic cells ; metaphase ; protein kinase C ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: It has long been known that neoplastic cells are characterized by increases in cell motility. Earlier studies from this laboratory indicated that rnitotic events were also altered in many tumor and experimentally transformed cells and that this included increases in metaphase duration and a reduction in the duration of cytokinesis. The studies presented in this paper were done to determine whether or not transfection of normal rat embryo fibroblasts by the Ha-T24-ras oncogene could also produce such alterations in mitotic events. The results obtained with the use of time lapse video microscopy indicate that neither the duration of metaphase nor the rate of chromosome movement during anaphase was altered but that the rate of furrow progression during cytokinesis occurred at a significantly more rapid rate. Thus, the cellular alteratioons induced by transfection with Ha-T24-ras accelerate microfilament-dependent cytokinetic furrowing without significant effects on microtubule-dependent mitotic events. One of several possible mechanisms that could account for these observations involves a down regulation of protein kinase C which has been reported to occur in many neoplastic cells including those transformed by ras. Such a hypothesis could also have broader implications because it may be applicable to the increase in motility and metastatic activity generally observed in transformed cells.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 106
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 171-186 
    ISSN: 0886-1544
    Schlagwort(e): stable microtubules ; tubulin ; axonemes ; video-enhanced DIC microscopy ; microtubule poisons ; colchicine ; podophyllotoxin ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: We report a reliable method to prepare, in vitro, microtubules that are stabilized at both ends by axonemal structures, and report studies of their properties. Such “end-stabilized” microtubules neither grow nor shorten over times of several hours when tubulin subunits are present in the surrounding solution. When sub-units are removed, the microtubules eventually break. Breakage occurs within a sinuous and flexible region a few microns in length, that begins at a single point on the microtubule and grows. When breakage does occur, the resulting two free ends shorten very rapidly until the flexible part has depolymerized and the region of straight microtubule is reached. The remainder of the microtubule then shortens at rates comparable to those ordinarily observed in dynamic instability. Formation of the flexible region can be reversed if subunits are added to the buffer prior to breakage. End-stabilized microtubules are a useful tool for studying interactions of molecules with the microtubular wall. They may be a good model for interpreting stabilizing events that happen in the cell. A preliminary study of the effects of microtubule poisons on the wall is presented.
    Zusätzliches Material: 11 Ill.
    Materialart: Digitale Medien
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  • 107
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 187-198 
    ISSN: 0886-1544
    Schlagwort(e): amphibian ; respiratory ; axonemes ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Optimal conditions have been developed for the isolation and reactivation of highly coupled, demembranated ciliary axonemes from newt lungs [Hard, Cypher, and Schabtach, 1988, Cell Motil. Cytoskeleton 10:271-284]. In the present study, the motility of these cilia was further characterized by examining the effects of nucleotides, divalent cations, and temperature on beat frequency. When exposed to a reactivating solution containing Mg2+ and ATP, nearly 100% of the axonemes were motile and beat at frequencies of 0-50 Hz, depending on [MgATP] and temperature. Divalent cations were required for movement, with Mg2+ 2-3 times more effective than Ca2+. There was no absolute requirement for Ca2+ for motility. The beat frequencies obtained with fixed ATP and varying Mg2+ concentrations indicate that MgATP serves as the actual substrate. The effects of MgATP on beat frequency depended on the degree of mechanochemical coupling and temperature and MgATP-induced transition between two distinct states whose maximum beat frequencies differ by 200-300%.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 108
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 199-209 
    ISSN: 0886-1544
    Schlagwort(e): amphibian ; respiratory ; cilia ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Demembranated axonemes isolated from newt lung ciliated cells show a complex beat frequency response to varying [MgATP] and temperature [Hard and Cypher, 1992, Cell Motil. Cytoskeleton 21:187-198]. The present study was undertaken to ascertain whether the beat frequency of outer-arm-depleted newt lung axonemes is controlled in a manner similar to that of intact axonemes. Populations of demembranated ciliary axonemes were isolated by Triton X-100 extraction of lungs from the newt, Taricha granulosa. Aliquots of the demembranated axonemes were further treated with solutions containing high salt (0.375 M KCl) and 1.25 mM MgATP. This treatment resulted in the selective removal of outer dynein arms and a concomitant decrease in beat frequency to a stable level, 33-35% of control values. The effects of pH, salt concentration, nucleotides, and temperature on the beat frequency of reactivated outer-arm-depleted axonemes were ascertained and compared with those of intact axonemes. Some reactivation properties, such as nucleotide specificity, the effect of pH on beat frequency and the threshold [MgATP] required for reactivation (approximately 5 μM) were similar to those observed for intact axonemes. Other properties, such as the relationship between beat frequency and varying [MgATP] or salt concentration, differed both qualitatively and quantitatively from those of control axonemes, as did their response to temperature over the range, 5°-32°C. The nature of the results obtained with temperature and MgATP suggests that inner and outer dynein arms are not functionally equivalent in situ.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 109
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 110
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 223-234 
    ISSN: 0886-1544
    Schlagwort(e): actin ; acidic vesicles ; Ca2+ ; pH ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Unfertilized eggs of the sea urchin Arbacia punctulata contain pigment granules distributed throughout their cytoplasm. During the first 15 minutes after fertilization, these vesicles move out to the cortex where they become firmly anchored. We have used time-lapse video differential interference microscopy to analyze the motility of these organelles in unfertilized and fertilized Arbacia eggs. Pigment granules exhibit saltatory movement in both unfertilized and fertilized eggs. Quantitation of vesicle saltations before and after fertilization demonstrates that while there is no significant difference in the speed or pathlength of vesicle movement, there is a dramatic change in the orientation of these saltations. Saltations in the unfertilized egg are very non-radial and are as likely to be directed toward the cortex as away. In contrast, saltations in the fertilized egg are more radially oriented and more likely to be cortically directed. This transition must reflect underlying changes in the cellular structures necessary for pigment granule saltations. The change in the orientation of pigment granule saltations following fertilization requires both a transient increase in the cytoplasmic concentration of Ca2+ and an elevation of cytoplasmic pH. Similarly, the ability of pigment granules to adhere to the cortex requires both the transient elevation of cytoplasmic Ca2+ and the alkalinization of the cytoplasm. As the reorganization of cortical actin at fertilization is regulated by these ionic fluxes, and both movement and adhesion are sensitive to cytochalasins, we hypothesize that the alterations in directed motility and adhesion reflect underlying changes in the actin cytoskeleton.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 111
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 235-251 
    ISSN: 0886-1544
    Schlagwort(e): actin polymerization ; cell elongation ; photoreceptor ; cytoskeleton ; phalloidin ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: In the retinas of teleost fish, rod photoreceptors elongate in response to light. Light-activated elongation is mediated by the myoid of the rod inner segment and is actin-dependent. Inner segment F-actin filaments form bundles running parallel to the cell's long axis. We examined the mechanism of rod elongation using mechanically-detached rod fragments, consisting of the motile inner segment and sensory outer segment (RIS-ROS). When RIS-ROS are isolated from darkadapted green sunfish and cultured in the light, they elongate 15μm at 0.3-0.6μm/min. Elongation was inhibited 65% by 0.1μM Cytochalasin D, suggesting a requirement for actin assembly. To determine the extent of assembly during elongation, we used three approaches to measure the F-actin content in RIS-ROS: detection of pelletable actin by SDS-PAGE after detergent-extraction of RIS-ROS; quantification of fluorescein-phalloidin binding by fluorimetry, fluorescence-activated cell sorting and image analysis; estimation of total F-actin filament length by electron microscopy. All three assays indicated that no net assembly of RIS-ROS F-actin accompanied myoid elongation. An increase in F-actin content within the elongated myoid was counterbalanced by a decrease in F-actin content within the 13 microvillus-like calycal processes located at the end of the inner segment opposite to the growing myoid. O'Connor and Burnside (Journal of Cell Biology 89:517-524, 1981) showed that minus-ends of rod F-actin filaments are oriented towards the elongating myoid while plus-ends are oriented towards the shortening calycal processes. Our observations suggest that RIS-ROS elongation entails actin polymerization at the minus-ends of filaments coupled with depolymerization at the filament plus-ends.
    Zusätzliches Material: 11 Ill.
    Materialart: Digitale Medien
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  • 112
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 210-222 
    ISSN: 0886-1544
    Schlagwort(e): bioluminescence ; ATP depletion ; motility ; flagellum ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: The initiation of motility and modification of energy metabolism of rat caudal epididymal spermatozoa can be induced by dilution in a saline medium. We have investigated in these cells the relationships between the energy reserve (sperm ATP content measured by bioluminescence) and flagellar movement (high speed videomicrography, 200 frames/sec). A steady state was observed in sperm ATP content, progressive velocity (Vp) and flagellar beat frequence (F) with sperm dilution in a medium with glucose, lactate, pyruvate and acetate substrates after 30 minutes of incubation, without these substrates, changes in metabolic pathways occurred immediately and initially disturbed the relationship between ATP levels and F, suggesting differences in motility initiation when energy is from an endogenous origin via mitochondrial oxidative phosphorylation. This “energy crisis” was reversed by the addition of substrates to the medium.The three-dimensional flagellar movement observed in the presence of substrates quickly became two-dimensional in their absence. The flagellar beat envelope became more splayed, the mean amplitude of lateral head displacement increased and F decreased. The resulting high flagellar beat efficiency can be compared to that observed during hyperactivation which is a physiological event related to a fall in intracellular ATP level. In both media, the displacement of the flagellum in relation to the wave axis varied sinusoidally. The sine period increased with time when the spermatozoa were incubated in the medium without substrates. These results suggest a gradual slowing-down of the velocity of wave formation in the proximal part of the flagellum.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 113
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 38-50 
    ISSN: 0886-1544
    Schlagwort(e): cytomechanics ; invasion mechanisms ; kinematic analysis ; parasites ; protoplasm flow ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Spores of the microsporidium Nosema algerae were stimulated to germinate in vitro while observed with video-enhanced contrast microscopy. Field-by-field playback of tape-recorded sequences yielded the first serial illustrations and kinematic analysis of the explosive discharge of the polar filament and the sporoplasm. The filament emerges from the anterior pole of the spore in a regularly pitched helicoidal course along a nearly straight axis, with a mean maximum instant velocity of 105 μm/s. Just before elongation is completed the filament tip follows a tortuous path that often results in a curved or spiralling terminal configuration. Then elongation stops and, after a lag that may vary from less than 15 to over 500 ms, the sporoplasm pours out at the filament tip forming a globule that quickly grows up to a size larger than its original volume within the spore. Concomitantly, the helical filament becomes straightened and frequently the spore body is pulled forward. Thereafter a relaxed filament, usually 5-10% shorter than when maximally extended, remains connecting the empty spore case and the sporoplasmic droplet. Experiments with hyperosmolar media produced a considerable slowdown of filament extrusion and often precluded sporoplasm discharge. The present results are fully consistent with the hypothesis of a hydrostatic pressure-triggered mechanism of spore germination, and revealed that the process is composed of two discrete phases separated by a variable lag: (1) complete eversion of the polar filament, and (2) passage of the main sporoplasm mass along the tube. The data provide a preliminary basis toward the conception of a quantitative physical model of microsporidian spore germination. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 11 Ill.
    Materialart: Digitale Medien
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  • 114
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 115
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 155-159 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 116
    ISSN: 0886-1544
    Schlagwort(e): BHK-21 cells ; cytoskeleton ; microfilaments ; microtubules ; stress fibers ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: A monoclonal antibody was produced, using as antigen a BHK-21 cytoskeletal preparation enriched in intermediate filaments (IF) and their associated proteins. This antibody reacted exclusively with a reproducible set of 70-280kD polypeptides present in minor quantities in this preparation, as detected by immunoblot analysis. Based upon several criteria, this immunologically related group of polypeptides was designated as IFAP-70/280kD (IF-Associated Protein): (1) it coisolated with IF in vitro, (2) it co-localized (by both immunofluorescence and immunoelectron microscopy) with IF in situ in all stages of cell spreading, and (3) it segregated in vitro with the 54/55kD (desmin/vimentin) structural IF subunit proteins of BHK cells through two cycles of in vitro disassembly/assembly. Immunogold labeling further localized IFAP-70/280kD to regions of parallel or loosely bundled IF in situ, suggesting a role in regulating the supramolecular organization of IF. When this monoclonal antibody was used for double-label immunofluorescence observations of colchicine-treated BHK cells, it demonstrated the presence of colchicine-sensitive and colchicine-insensitive IF. Anti-IFAP-70/280kD localized entirely to the drug-induced juxtanuclear IF cap, while a polyclonal antibody directed against the desmin/vimentin structural IF subunits and the previously characterized monoclonal anti-IFAP-300kD [Yang et al., 1985; J. Cell Biol. 100:620] localized to both the juxtanuclear IF cap and a colchicine-insensitive IF network peripheral to the cap in the same cells. The colchicine-insensitive IF pattern often exhibited similarities to that observed for the actin-based stress fiber system, suggesting that stress fiber association may be an additional factor in IF organization. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 117
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 25-37 
    ISSN: 0886-1544
    Schlagwort(e): cytoskeleton ; human neutrophils ; actin binding proteins ; cytochalasins ; ultracentrifugation ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Filamentous (F) actin is a major cytoskeletal element in polymorphonuclear leukocytes (PMNs) and other non-muscle cells. Exposure of PMNs to agonists causes polymerization of monomeric (G) actin to F-actin and activates motile responses. In vitro, all purified F-actin is identical. However, in vivo, the presence of multiple, diverse actin regulatory and binding proteins suggests that all F-actin within cells may not be identical. Typically, F-actin in cells is measured by either NBDphallacidin binding or as cytoskeletal associated actin in Triton-extracted cells. To determine whether the two measures of F-actin in PMNs, NBDphallacidin binding and cytoskeletal associated actin, are equivalent, a qualitative and quantitative comparison of the F-actin in basal, non-adherent endo-toxin-free PMNs measured by both techniques was performed. F-actin as NBD-phallacidin binding and cytoskeletal associated actin was measured in cells fixed with formaldehyde prior to cell lysis and fluorescent staining (PreFix), or in cells lysed with Triton prior to fixation (PostFix). By both techniques, F-actin in PreFix cells is higher than in PostFix cells (54.25 ± 3.77 vs. 23.5 ± 3.7 measured as mean fluorescent channel by NBDphallacidin binding and 70.3 ± 3.5% vs. 47.2 ± 3.6% of total cellular actin measured as cytoskeletal associated actin). These results show that in PMNs, Triton exposure releases a labile F-actin pool from basal cells while a stable F-actin pool is resistant to Triton exposure. Further characterizations of the distinct labile and stable F-actin pools utilizing NBDphallacidin binding, ultracentrifugation, and electron microscopy demonstrate the actin released with the labile pool is lost as filament. The subcellular localization of F-actin in the two pools is documented by fluorescent microscopy, while the distribution of the actin regulatory protein gelsolin is characterized by immunoblots with antigelsolin. Our studies show that at least two distinct F-actin pools coexist in endotoxin-free, basal PMNs in suspension: (1) a stable F-actin pool which is a minority of total cellular F-actin, Triton insoluble, resistant to depolymerization at 4°C, gelsolin-poor, and localized to submembranous areas of the cell; and (2) a labile F-actin pool which is the majority of total cellular F-actin, Triton soluble, depolymerizes at 4°C, is gelsolin-rich, and distributed diffusely throughout the cell. The results suggest that the two pools may subserve unique cytoskeletal functions within PMNs, and should be carefully considered in efforts to elucidate the mechanisms which regulate actin polymerization and depolymerization in non-muscle cells.
    Zusätzliches Material: 12 Ill.
    Materialart: Digitale Medien
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  • 118
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 45-57 
    ISSN: 0886-1544
    Schlagwort(e): cell shape ; gene expression ; pleiotropic effects ; cell cycle ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: We have previously described stable mouse C127 cell lines in which a CaM mini-gene has been expressed in a bovine papilloma virus-based expression vector (Rasmussen and Means: EMBO J. 6:3961-3968. 1987). Elevation of CaM to levels five-fold higher than in control cells caused an acceleration in cell cycle progression by reducing the length of the G1 period. When these cell lines were originally isolated it was observed that cells in which CaM levels were increased had a flattened morphology. In this study we have examined the localization of actin, vimentin, and tubulin in these cells as compared to the BPV-transformed control cell line in order to determine if changes in shape were accompanied by differences in the cytoskeletal organization. Cell-cycle-dependent changes in the levels of mRNAs for histone H4, glyceraldehyde-3-phosphate dehydrogenase, β-actin, vimentin, and β-tubulin have also been examined. Our results indicate that increased CaM causes differences in the organization of microfilaments, intermediate filaments, and microtubules and that these changes are accompanied by selective differences in the cell-cycle-dependent expression of some mRNAs. Elevated CaM was also correlated with a reduced stability of β-tubulin mRNA. These studies indicate that CaM has pleiotropic effects on cell function and suggest that stable cell lines with altered CaM levels may provide a useful model system for understanding the moiecular basis of CaM-dependent regulation of cellular processes.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 119
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 120
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 65-73 
    ISSN: 0886-1544
    Schlagwort(e): N-cadherin ; L1 ; laminin ; neurite outgrowth ; neuronal guidance ; filopodia ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: The growth cone, a terminal structure on developing and regenerating axons, is specialized for motility and guidance functions. In vivo the growth cone responds to environmental cues to guide the axon to its appropriate target. These cues are thought to be responsible for position-specific morphological changes in the growth cone, but the molecules that control growth cone behavior are poorly characterized. We used scanning electron microscopy to analyze the morphology of retinal ganglion cell growth cones in vitro on different adhesion molecules that axons normally encounter in vivo. L1/8D9, N-cadherin, and laminin each induced distinctive morphological characteristics in growth cones. Growth cones elaborated lamellipodial structures in response to the cell adhesion molecules L1/8D9 and N-cadherin, whereas laminin supported filopodial growth cones with small veils. On L1/8D9, the growth cones were larger and produced more filopodia. Filopodial associations between adjacent growth cones and neurites were frequent on L1/8D9 but were uncommon on laminin or N-cadherin. These results demonstrate that different adhesion molecules have profoundly different effects on growth cone morphology. This is consistent with previous reports suggesting that changes in growth cone morphology in vivo occur in response to changes in substrate composition.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 121
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 122
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 175-184 
    ISSN: 0886-1544
    Schlagwort(e): protein phosphorylation ; signal transduction ; motility ; alpha-adrenoceptors ; microtubules ; pigment ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Melanophores of the cichlid Tilapia mossambica can be induced to aggregate pigment by addition of epinephrine to the medium, suggesting adrenergic control of this transport. The melanophore response to adrenergic stimulation was examined using agonists and antagonists that are highly specific for each alpha-adrenoceptor subclass. The signal transduction mechanism of each subclass is unique: stimulation of alpha1 receptors results in a rise in intracellular free Ca2+, while alpha2 stimulation results in decreased cAMP levels [Exton, 1985: Am. J. Physiol. 248:E633-E647 ]. Each alpha1 or alpha2 specific agonist tested showed a dose dependent ability to induce aggregation and each was able to effect complete aggregation of pigment, suggesting that aggregation can be mediated either by elevating Ca2+ or by lowering cAMP. However, in the presence of either an alpha1 or an alpha2 receptor antagonist, none of the agonists were able to induce significant aggregation, suggesting that changes in levels of both messengers are required for pigment aggregation in the melanophores. Moreover, experiments in which intracellular levels of Ca2+ or cAMP were perturbed, using BAPTA and forskolin, respectively, indicated that elevating Ca2+ in the presence of high cAMP is not sufficient to induce aggregation and, conversely, that lowering cAMP levels in the presence of reduced Ca2+ is not sufficient to induce pigment aggregation. These data indicate that the concentrations of both cAMP and Ca2+ are important in regulating pigment aggregation in teleost melanophores, and suggest that maximal aggregation of pigment requires altering the levels of both messengers. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 123
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 227-234 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 124
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 250-256 
    ISSN: 0886-1544
    Schlagwort(e): cytoskeletal localization ; signal transduction ; intermediate filaments ; rat basophilic leukemia cells ; translocation ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Protein kinase C (PKC) isoforms are key mediators in hormone, growth factor, and neurotransmitter triggered pathways of cell activation (Nishizuka: Science 233:305-312, 1986; Nature 334:661-665, 1988). Stimulation of kinase activity by diacylglycerol and calcium often leads to translocation of PKC from the cytosol to a particulate fraction (Kraft and Anderson: Nature 301:621-623, 1983). The β isoform of PKC is translocated and degraded much more rapidly than the β isoform in phorbolester-stimulated rat basophilic leukemia (RBL) cells (Huang et al.: J. Biol. Chem. 264:4238-4243, 1989). We report here immunofluorescence evidence that the distributions of PKC α and β are strikingly different in antigen-activated RBL cells. PKC β associates with perinuclear filaments and filaments that extend from the perinuclear area to the cell periphery whereas PKC β concentrates in regions of the cell periphery. This distribution of PKC β is distinctly different from that of actin filaments and microtubules as determined by phalloidin staining and by anti-tubulin antibody labeling. In contrast, the staining patterns obtained with antibodies to PKC β and to the intermediate filament protein vimentin are almost identical, indicating that PKC β associates with vimentin filaments. These bundles of 100 Å filaments may provide docking sites for interactions of PKC β with its substrates and thus confer specificity to the actions of this isoform. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 125
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 8-18 
    ISSN: 0886-1544
    Schlagwort(e): spermatozoa ; flagella ; motility ; epididymis ; maturation ; mammal ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Motility and flagellar movement of ram spermatozoa along the epididymis were analysed in vitro. From the caput to the cauda of the epididymis, the percentage of motile and progressive spermatozoa increases. No flagellar bending was observed in spermatozoa from the testis or the epididymal anterior caput. When spermatozoa reached the distal caput of the epididymis, a static curvature, associated with an initiation of the flagellar beating, appeared on the flagella. This curvature normally disappeared during epididymal transit. Its disappearance was associated with an increase in the flagellar beat efficiency. Our results suggest that the initiation of motility is related to two mechanisms involving: (1) the presence of a transient static curvature, and (2) the establishment of a symmetric regular beating of the flagellum. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 126
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 83-84 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 127
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 128
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 122-132 
    ISSN: 0886-1544
    Schlagwort(e): centrosome ; microtubule ; microfilament ; myogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Available data on the molecular composition of the centrosome, the typical microtubule-organizing center of animal cells, are still fragmentary. To address this important issue we have taken advantage of centrosome isolation from a human lymphoblastic cell line (KE37) to generate a monoclonal antibody (mAb) library. Here we present the characterization of one of these mAbs (CTR56). On the basis of both its immunofluorescence staining pattern and its reactivity with a major 200 kD antigen on immunoblots, CTR56 has been tentatively classified as an anticellular myosin heavy chain. In light of cytological and biochemical data obtained in parallel with two other well-characterized myosin antibodies, it appears that myosin cannot be considered as a genuine centrosomal protein. We have resolved the paradoxical results with CTR56 by showing that in addition to the cellular myosin heavy chain, this antibody also recognizes a high molecular weight protein specifically enriched in centrosomal fractions. The possible biological significance of this finding is discussed in structural and functional terms. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 129
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 169-187 
    ISSN: 0886-1544
    Schlagwort(e): nuclear actin ; nuclear myosin ; nuclear shell ; nuclear shape ; nuclear matrix ; silk gland ; nuclear structure ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: The branched nuclei from silk gland cells of larvae of Calpodes ethlius label with antibodies to actin and myosin and with rhodaminyl-phalloin, which is specific for f-actin. Optical sectioning localizes this actin and myosin to the nuclear periphery. Residual nuclear-associated fractions prepared from these cells contain sheets of nuclear lamina-like structures that bind heavy meromyosin and gold-tagged antibodies to actin and myosin. The results suggest that both actin and myosin, or a myosin-like protein, are components of a layer at the nucleocytoplasmic boundary that we call the nuclear shell. The nuclear shell appears to be associated with the nuclear envelope and may correspond to a zone on the cytoplasmic face of the envelope seen in electron micrographs of unextracted cells. The residual nuclear-associated fraction has a unique isoform of actin (43 kD, pl 6.45) that might allow the nuclei to associate with an actin network structurally and developmentally distinct from that of the cytoplasm. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 14 Ill.
    Materialart: Digitale Medien
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  • 130
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 12 (1992), S. 111-116 
    ISSN: 0887-3585
    Schlagwort(e): protein folding ; multiple minima problem ; peptide conformation ; energy calculation ; helices ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: We have investigated the conformational properties of a truncated analogue of mastoparan and of mastoparan X, both peptides from wasp venom. The electrostatically driven Monte Carlo method was used to explore the conformational space of these short peptides. The initial conformations used in this study, mainly random ones, led to α-helical conformations. The α-helical conformations thus found exhibit an amphipathic character. These results are in accord with experimental data from NMR and CD spectroscopy.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 131
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 12 (1992) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 132
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 12 (1992), S. 324-330 
    ISSN: 0887-3585
    Schlagwort(e): X-ray diffraction analysis ; hydrogen bonds ; peptide conformation ; 310/α-helix transition ; antiparallel helix packing ; leucyl-leucyl interaction ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The packing of peptide helices in crystals of the leucine-rich decapeptide Boc-Aib-Leu-Aib-Aib-Leu-Leu-Leu-Aib-Leu-Aib-OMe provides an example of ladder-like leucylleucyl interactions between neighboring molecules. The peptide molecule forms a helix with five 5→1 hydrogen bonds and two 4→1 hydrogen bonds near the C terminus. Three head-to-tail NH ċ O = C hydrogen bonds between helices form continuous columns of helices in the crystal. The helicial columns associate in an antiparallel fashion, except for the association of Leu ċ Leu side chains, which occurs along the diagonal of the cell where the peptide helices are parallel. The peptide, with formula C56H102N10O13, crystallizes in space group P212121 with Z = 4 and cell parameters a = 16.774(3) Å, b = 20.032(3) Å and c = 20.117(3) Å; overall agreement factor R = 10.7% for 2014 data with |Fobs| 〈 3σ(F); resolution 1.0 Å.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 133
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 12 (1992), S. 339-344 
    ISSN: 0887-3585
    Schlagwort(e): protein folding ; pro region ; protease inhibition ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: α-Lytic protease, an extracellular bacterial serine protease, is synthesized with a large pro region that is required in vivo for the proper folding of the protease domain. To allow detailed mechanistic study, we have reconstituted pro region-dependent folding in vitro. The pro region promotes folding of the protease domain in the absence of other protein factors or exogenous energy sources. Surprisingly, we find that the pro region is a high affinity inhibitor of the mature protease. The pro region also inhibits the closely related Streptomyces griseus protease B, but not the more distantly related, yet structurally similar protease, elastase. Based on these data, we suggest a mechanism in which pro region binding reduces the free energy of a late folding transition state having native-like structure.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 134
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 13 (1992) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 135
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 13 (1992), S. 1-25 
    ISSN: 0887-3585
    Schlagwort(e): aspartic proteinase zymogen ; molecular replacement ; structure-function ; activation peptide ; acid activation ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The structure of porcine pepsinogen at pH 6.1 has been refined to an R-factor of 0.173 for data extending to 1.65 Å. The final model contains 180 solvent molecules and lacks density for residues 157-161. The structure of this aspartic proteinase zymogen possesses many of the characteristics of pepsin, the mature enzyme. The secondary structure of the zymogen consists predominantly of β-sheet, with an approximate 2-fold axis of symmetry. The activation peptide packs into the active site cleft, and the N-terminus (IP-9P) occupies the position of the mature N-terminus (1-9). Thus changes upon activation include excision of the activation peptide and proper relocation of the mature N-terminus. The activation peptide or residues of the displaced mature N-terminus make specific interactions with the substrate binding subsites. The active site of pepsinogen is intact; thus the lack of activity of pepsinogen is not due to a deformation of the active site. Nine ion pairs in pepsinogen may be important in the advent of activation and involve the activation peptide or regions of the mature N-terminus which are relocated in the mature enzyme. The activation peptide-pepsin junction, 44P-1, is characterized by high thermal parameters and weak density, indicating a flexible structure which would be accessible to cleavage. Pepsinogen is an appropriate model for the structures of other zymogens in the aspartic proteinase family. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 17 Ill.
    Materialart: Digitale Medien
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  • 136
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 13 (1992) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 137
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 13 (1992), S. 70-85 
    ISSN: 0887-3585
    Schlagwort(e): major histocompatibility complex ; antigenic peptide ; molecular dynamics ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Computer simulation of the conformations of short antigenic peptides (5-10 residues) either free or bound to their receptor, the major histocompatibility complex (MHC)-encoded glycoprotein H-2 Ld, was employed to explain experimentally determined differences in the antigenic activities within a set of related peptides. Starting for each sequence from the most probable conformations disclosed by a pattern-recognition technique, several energy-minimized structures were subjected to molecular dynamics simulations (MD) either in vacuo or solvated by water molecules. Notably, antigenic potencies were found to correlate to the peptides propensity to form and maintain an overall α-helical conformation through regular i,i+4 hydrogen bonds. Accordingly, less active or inactive peptides showed a strong tendency to form i,i+3 hydrogen bonds at their N-terminal end. Experimental data documented that the C-terminal residue is critical for interaction of the peptide with H-2 Ld. This finding could be satisfactorily explained by a 3-D Q.S.A.R. analysis postulating interactions between ligand and receptor by hydrophobic forces. A 3-D model is proposed for the complex between a high-affinity nonapeptide and the H-2 Ld receptor. First, the H-2 Ld molecule was built from X-ray coordinates of two homologous proteins: HLA-A2 and HLA-Aw68, energy-minimized and studied by MD simulations. With HLA-A2 as template, the only realistic simulation was achieved for a solvated model with minor deviations of the MD mean structure from the X-ray conformation. Water simulation of the H-2 Ld protein in complex with the antigenic nonapeptide was then achieved with the template-derived optimal parameters. The bound peptide retains mainly its α-helical conformation and binds to hydrophobic residues of H-2 Ld that correspond to highly polymorphic positions of MHC proteins. The orientation of the nonapeptide in the binding cleft is in accordance with the experimentally determined distribution of its MHC receptor-binding residues (agretope residues). Thus, computer simulation was successfully employed to explain functional data and predicts α-helical conformation for the bound peptide. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 13 Ill.
    Materialart: Digitale Medien
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  • 138
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 13 (1992), S. 112-119 
    ISSN: 0887-3585
    Schlagwort(e): analytical affinty chromatography ; self-association ; HIV p24gag ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Analytical affinity chromatography (AAC) was used to detect and quantitate the self-association of p24gag, the major structural capsid protein of human immunodeficiency virus (HIV-1). p24gag was immobilized on a hydrophilic polymer (methacrylate) chromatographic support. The resulting affinity column was able to interact with soluble p24, as judged by the chromatographic retardation of the soluble protein upon isocratic elution undernonchaotropic binding conditions. The variation of elution volume with soluble protein concentration fit to a monomer-dimer model for self-association. The soluble p24-immobilized p24 association process was observed using both frontal and zonal elution AAC at varying pH values; the dissociation constant was 3-4 × 10-5 M at pH 7. That p24 monomer associates to dimers was determined in solution using analytical ultracentrifugation. The solution Kd was 1.3 × 10-5 M at pH 7. AAC in the zonal elution mode provides a simple and rapid means to screen for other HIV-1 macromolecules that may interact with p24 as well as for modulators, including antagonists, of HIV p24 protein assembly. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 139
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 13 (1992), S. 132-140 
    ISSN: 0887-3585
    Schlagwort(e): protein stability ; insertion mutations ; substitution mutations ; guanidine hydrochloride denaturation ; conformational changes ; circular dichroism spectroscopy ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: In a previous study, the small protein staphylococcal nuclease was shown to readily accommodate single alanine and glycine insertions, with average losses in stability comparable to substitutions at the same sites (PROT. 7:29-305, 1990). To more fully explore this unexpected adaptability to changes in residue spacing, 2 double amino acid insertions (alanyl-glycine, glycyl-glycine) and 3 additional single amino acid insertions with dissimilar side chains (proline, leucine, and glutamine) were constructed at 10 of the sites previously studied. At 8 of these sites, the type of amino acid side chain on the inserted residue significantly influenced the stability of the mutant protein. However, at 9 of the 10 sites, the double insertions were found to be no more destabilizing than the single alanine or glycine insertions. In contrast, double substitution mutations of staphylococcal nuclease, which replace two adjacent residues with alanine, do not show this striking degree of non-additivity. A comparison of the effects of single glutamine and single glycine insertions with alanyl-glycine insertions indicates that insertion of alanine into the peptide backbone is, on average, less destabilizing than appending the equivalent atoms onto the side chain of a glycine insertion. To explain their very different energetic effects, we propose that, unlike most substitutions, the inserted residue(s) must induce lateral displacements of the polypeptide chain, forcing the folded conformation away from that of wild type. The resulting obligatory shifts in the positioning of residues flanking the insertion generate a large number of degrees of freedom around which the mutant structure can relax. From the many alternative packing and bonding arrangements thus made available to the polypeptide chain, the energetically most favorable is selected. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 140
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 14 (1992), S. 213-223 
    ISSN: 0887-3585
    Schlagwort(e): protein folding ; protein structure ; rotamers ; simulated annealing ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: An unknown protein structure can be predicted with fair accuracy once an evolutionary connection at the sequence level has been made to a protein of known 3-D structure. In model building by homology, one typically starts with a backbone framework, rebuilds new loop regions, and replaces nonconserved side chains. Here, we use an extremely efficient Monte Carlo algorithm in rotamer space with simulated annealing and simple potential energy functions to optimize the packing of side chains on given backbone models. Optimized models are generated within minutes on a workstation, with reasonable accuracy (average of 81% side chain χ1 dihedral angles correct in the cores of proteins determined at better than 2.5 Å resolution). As expected, the quality of the models decreases with decreasing accuracy of backbone coordinates. If the backbone was taken from a homologous rather than the same protein, about 70% side chain X1 angles were modeled correctly in the core in a case of strong homology and about 60% in a case of medium homology. The algorithm can be used in automated, fast, and reproducible model building by homology. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 141
    ISSN: 0886-1544
    Schlagwort(e): cytoskeleton ; liposome ; focal contact ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: An F-actin binding protein was purified from bovine liver by means of DNase I affinity, hydroxylapatite and DEAE-cellulose column chromatographies. It consisted of a single polypeptide chain having an apparent molecular weight of 68,000 with a Stokes radius of 35Å. Electron microscopy of rotary shadowed specimens showed that the 68kD protein is a globular protein. This protein showed a higher affinity for F-actin in the presence of Ca2+ than in its absence, which is opposite to the actin-binding property shown by nonmuscle alpha-actinin or fimbrin. The 68kD protein had no F-actin severing and capping activity. Interestingly, the 68kD protein was found to aggregate liposomes at micromolar Ca2+ concentrations. Immunoblot analysis and partial protein sequence data identified the 68kD protein as an annexin VI (p68) homologue. Immunocytochemical studies showed that the 68kD protein was localized along stress fibers as well as membrane ruffles, microspikes and focal contacts, raising the possibility that annexin VI may contribute to control membrane-microfilament interaction in the cell. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 142
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 245-249 
    ISSN: 0886-1544
    Schlagwort(e): rhodamine phalloidin ; anti-chicken actin antibody ; plant actin ; DNase I ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Previous reports about phalloidin binding to plant actins have been indirect. We present here evidence showing that phalloidin does bind and stabilize filaments of actin extracted from pea roots. Criteria for the presence of actin included stabilization as a polymer in the presence of phalloidin, cross-reaction with antibody against chicken actin, affinity binding to DNase I, and ability to be decorated by the S1 fragment of rabbit muscle myosin.Phalloidin was able to stabilize polymers in pea root extracts against dissociation during SDS gel electrophoresis, and these polymers were shown to be composed exclusively of actin. Pea root actin isolated by affinity chromatography on a DNase I column was incubated with rhodamine phalloidin and electrophoresed on a native gel. The rhodamine fluorescence remained with the stabilized filaments, indicating clearly that phalloidin does bind to actin from a plant source. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
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  • 143
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 144
    ISSN: 0886-1544
    Schlagwort(e): epidermal keratinocytes ; cytoskeleton ; UV induced reorganization ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Indirect immunofluorescence microscopy has been used to investigate the ultraviolet (UV) radiation induced disruption of the organization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. Following irradiation, concurrent changes in the organization of the three major cytoskeletal components were observed in cells incubated under low Ca2+ (0.15 mM) conditions. UV irradiation induced a dose-dependent condensation of keratin filaments into the perinuclear region. This collapse of the keratin network was accompanied by the reorganization of microfilaments into rings and a restricted distribution of microtubules, responses normally elicited by exposure to high Ca2+ (1.05 mM) medium. The UV induced alteration of the keratin network appears to disrupt the interactions between keratin and actin, permitting the reorganization of actin filaments in the absence of Ca2+ stimulation.In addition to the perinuclear condensation of keratin filaments, UV irradiation inhibits the Ca2+ induced formation of keratin alignments at the membrane of apposed cells if UV treatment precedes exposure to high Ca2+ medium. Incubation of keratinocytes in high Ca2+ medium for 24 hours prior to irradiation results in the stabilization of membrane associated keratin alignments and a reduced susceptibility of cytoplasmic keratin filaments to UV induced disruption. Unlike results from investigations with isogenic skin fibroblasts, no UV induced disassembly of microtubules was discernible in irradiated human keratinocytes. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 145
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 45-60 
    ISSN: 0886-1544
    Schlagwort(e): cell motility ; cytoskeleton ; microtubule dynamics ; wound healing ; leading edge ; ruffling ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Fibroblasts migrating into an experimental wound contain an extensive array of detyrosinated microtubules (Glu MTs) oriented in the direction of migration, whereas nonmotile cells in the interior of a monolayer contain Glu MTs that are primarily coiled around the nucleus. To determine the role of cell-cell contact in the formation of these distinct arrays of Glu MTs, we studied the distribution of Glu MTs by immunofluorescence in NRK fibroblasts that had been fixed at different intervals after they had established contact with other cells. Time-lapse video recordings were made of the contacting cells to provide a record of cellular behavior. In motile cells that became completely surrounded by virtue of contact with other cells, Glu MTs were found mostly coiled around the nucleus. The proportion of cells whose Glu MTs extended to the original leading edge decreased dramatically after the cells had been surrounded for 10 min or more. At earlier times, when the contact was confined to a portion of the cell margin, Glu MTs were absent from the area behind the contact site, yet were still oriented toward the noncontacting and ruffling margins. The contact-induced alteration of Glu MTs was not due to the cessation of forward locomotion of cells per se, since immobilization of cells with cytochalasin D did not cause a dramatic change in Glu MTs. That cell-cell contact also specifies the type of Glu MTs formed in cells was shown by experiments in which MTs were regrown following complete depolymerization with nocodazole. The remodeling of Glu MTs during cell-cell contact may be involved in cellular repolarization during contact inhibition of locomotion and will be a useful marker for further dissecting the molecular events of contact inhibition of motility. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 146
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 61-70 
    ISSN: 0886-1544
    Schlagwort(e): activation ; fertilization ; microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Centrosomes are undetectable in unfertilized sea urchin eggs, and normally the sperm introduces the cell's microtubule-organizing center (MTOC) at fertilization. However, artificial activation or parthenogenesis triggers microtubule assembly in the unfertilized egg, and this study explores the reappearance and behavior of the maternal centrosome. During activation with A23187 or ammonia, microtubules appear first at the cortex; centrosomal antigen is detected diffusely throughout the entire cytoplasm. Later, the centrosome becomes more distinct and organizes a radial microtubule shell, and eventually a compact centrosome at the egg center organizes a monaster. In these activated eggs, centrosomes undergo cycles of compaction and decompaction in synchrony with the chromatin, which also undergoes cycles of condensation and decondensation. Parthenogenetic activation with heavy water (50% D2O) or the microtubule-stabilizing drug taxol (10 μM) induces numerous centrosomal foci in the unfertilized sea urchin egg. Within 15 min after incubation in D2O, numerous fine centrosomal foci are detected, and they organize a connected network of numerous asters which fill the entire egg. Taxol induces over 100 centrosomal foci by 15 min after treatment, which organize a corresponding number of asters. The centrosomal material in either D2O- or taxol-treated eggs aggregates with time to form fewer but denser foci, resulting in fewer and larger asters. Fertilization of eggs pretreated with either D2O or taxol shows that the paternal centrosome is dominant over the maternal centrosome. The centrosomal material gradually becomes associated with the enlarged sperm aster. These experiments demonstrate that maternal centrosomal material is present in the unfertilized egg, likely as dispersed undetectable material, which can be activated without paternal contributions. At fertilization, paternal centrosomes become dominant over the maternal centrosomal material. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 147
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 111-121 
    ISSN: 0886-1544
    Schlagwort(e): anti-α-spectrin immunocytochemistry ; phalloidin staining ; cortex ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Polypeptide of 240 kDa immunorelated to vertebrate α-spectrin was detected in Paramecium cells. The antigen was identified by monospecific antibody directed against α-subunit of spectrin, which was isolated from chicken erythrocytes by affinity and anion-exchange chromatography. Immunoblotting tests demonstrated that the anti-α-spectrin cross reacted with the 240 kDa polypeptide of Paramecium as well as that of various vertebrate cells. In Paramecium, the antigen was detected in cytoskeletal fraction and in contractile extract of the cells. Immuno-fluorescent and immunoelectron microscopy observations revealed cortical localization of α-spectrin immunoanalogue in Paramecium. The label was distinctly seen on the whole surface of trichocyst tips. The antigen was also distributed close to the inner alveolar membrane forming a regular, continuous, lattice-like structure. When stained with rhodamine-phalloidin, Paramecium cells displayed a similar fluorescent network, which underlay contours of cortical units. Basal bodies of cilia were labeled with phalloidin as well. Detection of α-spectrin immunoanalogue in Paramecium cortex may provide a new insight into arrangement of cortical elements in this organism. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 148
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 102-110 
    ISSN: 0886-1544
    Schlagwort(e): Chloroplast movement ; phytochrome ; near infrared laser ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: The cell of the green alga Mougeotia orients its chloroplast by rotation, according to the direction or polarization of incident red light. The mechanics of the rotation is described by the angle of rotation and the angular velocity of the rotator (i.e., the chloroplast). We developed a laser diffractometer to determine the angle of rotation of the chloroplast. The angle of rotation of the chloroplast shifted by a constant angular velocity, and hence, the net torque on the chloroplast was zero. This suggests that the driving torque acting on the chloroplast is always balanced by the viscous torque. The maximal driving force acting on the chloroplast was estimated to be nearly equal to the force generated by an actomyosin system. This is the first measurement of the driving force acting on the chloroplast in Mougeotia. The amplitude of the force supported the anchorage site hypothesis. However, it remains unclear whether or not the angular independence of the force also supports the hypothesis. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 149
    ISSN: 0886-1544
    Schlagwort(e): actin polymerization ; Dictyostelium ; F-actin capping proteins ; phospholipids ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: The fast and transient polymerization of actin in nonmuscle cells after stimulation with chemoattractants requires strong nucleation activities but also components that inhibit this process in resting cells. In this paper, we describe the purification and characterization of a new actin-binding protein from Dictyostelium discoideum that exhibited strong F-actin capping activity but did not nucleate actin assembly independently of the Ca2+ concentration. These properties led at physiological salt conditions to an inhibition of actin polymerization at a molar ratio of capping protein to actin below 1:1,000. The protein is a monomer, with a molecular mass of ∼ 100 kDa, and is present in growing and in developing amoebae. Based on its F-actin capping function and its apparent molecular weight, we designated this monomeric protein cap 100. As shown by dilution-induced depolymerization and by elongation assays, cap100 capped the barbed ends of actin filaments and did not sever F-actin. In agreement with its capping activity, cap100 increased the critical concentration for actin polymerization. In excitation or emission scans of pyrene-labeled G-actin, the fluorescence was increased in the presence of cap100. This suggests a G-actin binding activity for cap100. The capping activity could be completely inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and bound cap100 could be removed by PIP2. The inhibition by phosphatidylinositol and the Ca2+-independent down-regulation of spontaneous actin polymerization indicate that cap100 plays a role in balancing the G- and F-actin pools of a resting cell. In the cytoplasm, the equilibrium would be shifted towards G-actin, but, below the membrane where F-actin is required, this activity would be inhibited by PIP2. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 150
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 151
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 15-24 
    ISSN: 0886-1544
    Schlagwort(e): actin edge-bundle ; cortical tension ; cell shape ; microfilaments ; cell adhesion ; cell motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: We have previously described actin edge-bundles (AEBs) as cables of microfil-aments lining the webbed edges of 3T3 cells (Zand and Albrecht-Buehler: Cell Motil. Cytoskeleton 13:195-211, 1989). We have suggested that AEBs, along with their cell-substratum adhesions, resist cortical tension and prevent the collapse of cytoplasm towards the nucleus. In this paper, we report several stages of AEB disassembly and re-formation induced by the following micro-manipulations(1)Scoring of the webbed edge of a 3T3 cells with a microneedle. As a result the sides of the score retracted and the severed AEB appeared to disassemble down to its terminal adhesion points. The retraction stopped after 20-40 seconds and the cells formed a webbed edge with large curvature. Over a period of 20-80 minutes, the new web decreased in length and depth, until it regained its approximate original shape.(2)Bending of cell processes at acute angles. As a result the processes moved until they projected at right angles to the side of the cell and formed new webs gradually expanded their area. In both cases, the nascent webs were lined by actin edge-bundles.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 152
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 211-223 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: A 3-dimensional dynamic image analyzing system (3D-DIAS) has been developed in which a translocating cell is optically sectioned in the z-axis within a 2 sec period; the perimeter of the cell in each section is digitized into the 3D-DIAS data file, and the digitized perimeters are wrapped in order to reconstruct the cell image in three dimensions. Using 3D-DIAS, we have obtained the first dynamic 3-dimensional description of human polymorphonuclear leukocytes (PMN) translo-cating on a glass surface. A general behavior cycle has emerged which includes two phases. In the first, an ellipsoidal PMN with significant z-axis extends anteriorly and descends to the substratum. When the ventral surface of the anterior end contacts the substratum, there is rapid anterior expansion, which correlates with velocity peaks. In the second phase, the elongate PMN stops translocating along the substratum, the anterior end lifts off of the substratum, sometimes to heights greater than the length of the PMN at the substratum, and finally the PMN retracts into an ellipsoidal morphology still capable of random protrusions. During this second phase, which correlates with velocity troughs, turning usually occurs. The degree of turning is restricted by the continuous integrity of the posterior uropod. The period of the behavior cycle varies from roughly 0.5 to 2 min between PMNs, but is relatively constant within each individual PMN. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 12 Ill.
    Materialart: Digitale Medien
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  • 153
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 1-7 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 154
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 99-116 
    ISSN: 0886-1544
    Schlagwort(e): microinjection ; second messengers ; cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: We have directly evaluated the effects of various intracellular second messengers including cyclic nucleotides, calcium ion, and inositol polyphosphates on shape and motility of differentiating mouse neuroblastoma cells. The messengers were microinjected into cells and the responses of the soma, neurite, and growth cone were monitored using time-lapse video microscopy. Each messenger altered cell shape and motility in a characteristic manner. Cyclic AMP promoted lamellipodial expansion, neurite outgrowth, and motility. The other injected messengers opposed motility. Cyclic GMP caused motile structures to freeze and to retract permanently, while the inhibitory effects of calcium injection were concentrationdependent. Small calcium injections affected specifically actincontaining motile structures which froze and retracted temporarily. Intermediate calcium injections caused a strong contraction at the site of injection in all cells. With large injections, cells retracted long neurites, rounded up, and frequently began vigorous blebbing that continued to cell death. Injections of the inositol polyphosphates 1P3(1,4,5) and IP4(1,4,5,6) mimicked the effects of small calcium injections, as did electrical stimulation that elicited action potentials. The results suggest that in mouse neuroblastoma cells, intracellular CAMP elevation increases cytoskeletal organization and promotes neurite extension perhaps through an enhancement of cell-substratum adhesion. On the other hand, a rise of intracellular cGMP or intracellular calcium interferes directly with the function and organization of the actin-microfilament system. The integrated action of these second messenger systems may, therefore, operate in vivo to allow substances released from neighboring cells to regulate neuronal architecture. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 155
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 117-126 
    ISSN: 0886-1544
    Schlagwort(e): algae ; cell division ; cytokinesis ; mitosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: We have used two monoclonal antibodies to demonstrate the presence and localization of actin in interphase and mitotic vegetative cells of the green alga Chlamydomonas reinhardtii. Commercially available monoclonal antibodies raised against smooth muscle actin (Lessard: Cell Motil. Cytoskeleton 10:349-362, 1988; Lin: Proc. Natl. Acad. Sci. USA 78:2335-2339, 1981) identify Chlamydomonasactin as a ∼43,000-Mr protein by Western immunoblot procedures. In an earlier study, Detmers and coworkers (Cell Motil. 5:415-430, 1985) first identified Chlamydomonas actin using NBD-phallacidin and an antibody raised against Dictyostelium actin; they demonstrated that F-actin is localized in the fertilization tubule of mating gametes. Here, we show by immunofluorescence that vegetative Chlamydomonas cells have an array of actin that surrounds the nucleus in interphase cells and undergoes dramatic reorganization during mitosis and cytokinesis. This includes the following: reorganization of actin to the ante- rior of the cell during preprophase; the formation of a cruciate actin band in prophase; reorganization to a single anterior actin band in metaphase; rearrange- ment forming a focus of actin anterior to the metaphase plate; reextension of the actin band in anaphase; presence of actin in the forming cleavage furrow during telophase and cytokinesis; and finally reestablishment of the interphase actin array. The studies presented here do not allow us to discriminate between G and F-actin. None the less, our observations, demonstrating dynamic reorganization of actin during the cell cycle, suggest a role for actin that may include the movement of basal bodies toward the spindle poles in mitosis and the formation of the cleavage furrow during cytokinesis. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 156
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 101-110 
    ISSN: 0886-1544
    Schlagwort(e): F-actin ; silk gland ; phalloin ; periluminal circumferential actin bundles ; actin-coated vacuoles ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Labeling of silk glands with rhodaminyl-phalloin shows that most F-actin is restricted to parallel bundles that form rings around the gland lumen at the apical cell surface. The bundles are lost when larval feeding stops at moulting, and the F-actin is redistributed through the cytoplasm as coats to vacuoles and, occasionally, in variably oriented strands. After moulting there is a return to the distribution of filamentous actin in the apical periluminal rings of bundles. These events occur at the same time as F-actin in the nuclear shell [Henderson and Locke, submitted] undergoes its own set of changes. In silk gland cells two kinds of f-actin deployment take place concurrently.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 157
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 132-137 
    ISSN: 0886-1544
    Schlagwort(e): microtubules ; vesicles ; cytoplasmic movement ; monoclonal antibody ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: A cytoskeletal apparatus is involved in the movement of vesicles, organelles, and gametes in the pollen tube. The function of microfilaments has been defined quite precisely, but the role of microtubules needs to be further clarified. On the basis of immunological and biochemical investigations, we have identified a polypep-tide showing common properties with kinesin, a microtubule-based motor mainly described in nonplant tissues, in the pollen tube of Nicotiana tabacum. Like mammalian kinesin, the kinesin-immunoreactive homolog from Nicotiana tabacum pollen tubes binds to mammalian microtubules in an AMP-PNP dependent manner. The kinesin-like component is likely to be involved in the movement of vesicular material in the growing pollen tube.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 158
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 123-131 
    ISSN: 0886-1544
    Schlagwort(e): thrombin ; cytochalasin B ; phorbol-myristate-acetate ; aggregation ; secretion ; contractile gel ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Vinculin is an Mr 130 kDa protein that has been implicated in membrane-cytoskeleton interaction in various cell types. It has been demonstrated that vinculin is not a cytoskeletal component in resting platelets, but part of it becomes associated with the cytoskeleton during thrombin-induced activation. In this study, using a quantitative immunnoblotting technique, the relation of vinculin to the cytoskeleton in different phases of activation of bovine platelets was explored, and the process of incorporation of vinculin into the cytoskeleton was related to that of cytoskeletal assembly. The assembly of cytoskeleton proceeded at a significantly faster rate than the association of vinculin with it, which shows that the latter process is not due to passive trapping of vinculin into the Triton-insoluble residue, but certain biochemical changes had to occur before such an interaction became possible. When the formation of pseudopodia was prevented by cyto-chalasin B, but neither aggregation nor the release reaction induced by thrombin were inhibited, the recovery of vinculin in the Triton-insoluble residue even increased. In both time- and thrombin-concentration-dependent studies, poor correlation was found between vinculin-cytoskeleton association and the extent of aggregation. Activation with phorbol-myristate-acetate, which is a strong stimulus for aggregation but produces only a slight release in the granular content, resulted in the association of only a negligible amount of vinculin with the cytoskeletal fraction. The incorporation of vinculin into the cytoskeletal fraction of thrombin activated platelets started with the release reaction but still proceeded, and the greatest part of the reaction occurred after secretion had gone to completion. These findings suggest that platelet shape change and pseudopodium extrusion are not prerequisites for, and aggregation is not related to, vinculin-cytoskeleton interaction. The association of vinculin with the cytoskeleton correlates with the organization of contractile gel, which suggests a role for vinculin in secretion and clot retraction.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 159
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 281-292 
    ISSN: 0886-1544
    Schlagwort(e): ATPase ; CTPase ; minus-end-directed microtubule motility ; cytoplasmic dynein ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Extracts of unfertilized sea urchin eggs contain at least two isoforms of cytoplasmic dynein. One exhibits a weak affinity for microtubules and is primarily soluble. The other isoform, HMr-3, binds to microtubules in an ATP-sensitive manner, but is immunologically distinct from the soluble egg dynein (Porter et al.: Journal of Biological Chemistry 263:6759-6771, 1988). We have now further distinguished these egg dynein isoforms based on differences in NTPase activity. HMr-3 copurifies with NTPase activity, but it hydrolyzes CTP at 10 times the rate of ATP. The soluble egg dynein is similar to flagellar dynein in its nucleotide specificity; its MgCTPase activity is ca. 60% of its MgATPase activity. Non-ionic detergents and salt activate the MgATPase activities of both enzymes relative to their MgCTPase activities, but this effect is more pronounced for the soluble egg dynein than for HMr-3. Sucrose gradient-purified HMr-3 promotes an ATP-sensitive microtubule bundling, as seen with darkfield optics. We have also isolated a 20 S microtubule translocating activity by sucrose gradient fractionation of egg extracts, followed by microtubule affinity and ATP release. This 20 S fraction, which contains the HMr-3 isoform, induces a microtubule gliding activity that is distinct from kinesin. Our observations suggest that soluble dynein resembles axonemal dynein, but that HMr-3 is related to the dynein-like enzymes isolated from a variety of cell types and may represent the cytoplasmic dynein of sea urchin eggs.
    Zusätzliches Material: 6 Ill.
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  • 160
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 255-271 
    ISSN: 0886-1544
    Schlagwort(e): cytoskeleton ; globoside ; vimentin ; desmin ; keratin ; glial fibrillary acidic protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: We reported recently that two glycosphingolipids (GSLs), globoside (Gb4)and ganglioside GM3, colocalized with vimentin intermediate filaments of human umbilical vein endothelial cells. To determine whether this association is unique to endothelial cells or to vimentin, we analyzed a variety of cell types. Doublelabel immunofluorescent staining of fixed, permeabilized cells, with and without colcemid treatment, was performed with antibodies against glycolipids and intermediate filaments. Globoside colocalized with vimentin in human and mouse fibroblasts, with desmin in smooth muscle cells, with keratin in keratinocytes and hepatoma cells, and with glial fibrillary acidic protein (GFAP) in glial cells. Globoside colocalization was detected only with vimentin in MDCK and HeLa cells, which contain separate vimentin and keratin networks. GM3 ganglioside also colocalized with vimentin in human fibroblasts. Association of other GSLs with intermediate filaments was not detected by immunofluorescence, but all cell GSLs were detected in cytoskeletal fractions of metabolically labelled endothelial cells. These observations indicate that globoside colocalizes with vimentin, desmin, keratin and GFAP, with a preference for vimentin in cells that contain both vimentin and keratin networks. The nature of the association is not yet known. Globoside and GM3 may be present in vesicles associated with intermediate filaments (IF), or bound directly to IF or IF associated proteins. The prevalence of this association suggests that colocalization of globoside with the intermediate filament network has functional significance. We are investigating the possibility that intermediate filaments participate in the intracellular transport and sorting of glycosphingolipids.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 161
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 1-6 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 162
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 21 (1992), S. 313-325 
    ISSN: 0886-1544
    Schlagwort(e): pluripotent P19 EC cells ; immunoblotting ; indirect immunofluorescence microscopy ; microtubule-associated proteins ; MAP2 ; tau ; MAP IB ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Pluripotent P19 embryonal carcinoma (EC) cells were differentiated along the neuronal and muscle pathways. Comparisons of class I, II, III, and IV beta tubulin isotypes in total and colchicine-stable microtubule (MT) arrays from uncommitted EC, neuronal, and muscle cells were made by immunoblotting and by indirect immunofluorescence microscopy. In undifferentiated EC cells the relative amounts of these four isotypes are the same in both the total and stable MT populations. Subcellular sorting of beta tubulin isotypes was demonstrated in both neuronal and muscle differentiated cells. During neuronal differentiation, class II beta tubulin is preferentially incorporated into the colchicine-stable MTs while class III beta tubulin is preferentially found in the colchicine-labile MTs. The subcellular sorting of class II into stable MTs correlates with the increased staining of MAP IB. and with the expression of MAP 2C and tau. Although muscle differentiated cells express class II beta tubulin, stable MTs in these cells do not preferentially incorporate this isotype but instead show increased incorporation of class IV beta tubulin. Muscle cells do not show high levels of MAP IB and do not express MAP 2C or tau. These results are consistent with the hypothesis that a subcellular sorting of tubulin isotypes is the result of a complex interaction between tubulin isotypes and MT-associated proteins.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 163
    ISSN: 0886-1544
    Schlagwort(e): microtubule-organizing centers ; centrosomes ; microtubule cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: The molecular composition of two morphologically distinct microtubule-organizing centers (MTOCs) was compared by probing with monoclonal antibodies raised against (i) nucleus-associated bodies (NABs) isolated in a complex with nuclei from the cellular slime mold Dictyostelium discoideum and (ii) mammalian mitotic spindles isolated from Chinese hamster ovary (CHO) cells. The staining patterns observed by immunofluorescence microscopy in whole CHO cells and Dictyostelium amoebae showed that the distribution of thirteen MTOC antigens is heterogeneous. Not all antibodies recognized the MTOC in both interphase and mitosis. Most of the anti-MTOC antibodies cross-reacted with other cellular organelles such as nuclei, Golgi apparatus-like aggregates and cytoskeletal elements. Two antibodies, CHO3 and AX3, recognized phosphorylated epitopes present in both mammalian centrosomes and Dictyostelium NABs. On immunoblots, most of the antibodies showed multiple bands, often of high molecular weight, indicating that the antigenic determinants are shared among different molecules. One antibody inhibited the regrowth of microtubules onto centrosomes in vitro after addition of exogenous tubulin to detergent-lysed CHO cells on coverslips; this antibody binds to an antigen(s) that might be essential for the microtubule-nucleating activity of centrosomes. These observations demonstrate that molecular components in different MTOCs exhibit a variety of distinct subcellular localizations and functional properties, and that some antigenic molecules have been conserved among morphologically distinct MTOCs. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 164
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 170-174 
    ISSN: 0886-1544
    Schlagwort(e): nocodazole ; carbendazim ; antimicrotubule agents ; thiabendazole ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: We report the cloning and sequencing of 18 mutant alleles of the benA, β-tubulin gene of Aspergillus nidulans that confer resistance to the benzimidazole antifungal, antimicrotubule compounds benomyl, carbendazim, nocodazole, and thia-bendazole. In 12 cases, amino acid 6 was changed from histidine to tyrosine or leucine. In four cases, amino acid 198 was changed from glutamic acid to aspartic acid, glutamine, or lysine. In two cases, amino acid 200 was altered from phenylalanine to tyrosine. These data, along with previous data indicating that amino acid 165 is involved in the binding of the R2 group of these compounds [Jung and Oakley, 1990: Cell Motil. Cytoskeleton 17:87-94], suggest that regions of β-tubulin containing amino acids 6, 165, and 198-200 interact to form the binding site of benzimidazole antimicrotubule agents. These results also suggest that the presence of phenylalanine at amino acid 200 contributes to the great sensitivity of many fungi to benzimidazole antimicrotubule agents. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 1 Tab.
    Materialart: Digitale Medien
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  • 165
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 166
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 235-244 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 167
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 257-273 
    ISSN: 0886-1544
    Schlagwort(e): fungal cytoskeleton ; microtubules ; nocodazole ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: The microtubule system of the Sordaria macrospora ascus was examined by antitubulin immunofluorescence, without the removal of the cell wall. The complex cytoskeleton revealed three possible microtubule-organizing centers (MTOCs): the spindle pole body (SPB), the nuclear envelope, and an apical organizing center. MPM-2, a mitotic phosphoprotein antibody which reacts with MTOCs, stained the apical center in a developmentally specific manner, and the nuclear envelope and SPB in a cell cycle-dependent fashion. Nocodazole was used in both high (10-15 μg/ml) and low (0.5 μg/ml) concentrations to depolymerize the networks and reveal their points of origin and recovery. The apical center was active from prophase I to the end of first meiosis. The nuclear envelope was the site of microtubule nucleation in early prophase and at the telophase/interphase transition, while SPBs were active in both nuclear division and sporulation.Mutant strains deficient in sporulation and with aberrant morphology were analyzed by antitubulin and MPM-2 immunofluorescence. Shape mutants showed abnormal or absent apical organizing centers and abnormal cortical microtubule patterns, indicating a possible role for the cortical network in the establishment and maintenance of ascus shape. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 168
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 22 (1992), S. 274-280 
    ISSN: 0886-1544
    Schlagwort(e): motility assay ; gelation ; solation ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Regulation of actin/myosin II force generation by calcium [Kamm and Stull, Annu. Rev. Physiol. 51:299-313, 1989] and phosphorylation of myosin II light chains [Sellers and Adelstein, “The Enzymes,” Vol. 18, Orlando, FL: Academic Press, 1987, pp. 381-418] is well established. However, additional regulation of actin/myosin II force generation/contraction may result from actin-binding proteins [Stossel et al., Ann. Rev. Cell Biol. 1:353-402, 1985; Pollard and Cooper, Ann. Rev. Biochem. 55:987-1035, 1986] as they affect the gel state of the actin cytomatrix [reviewed in Taylor and Condeelis, Int. Rev. Cytol., 56:57-143, 1979]. Regulation of the gel state of actin may determine whether an isotonic or isometric contraction results from the interaction between myosin and actin. We have extended the single actin filament motility assay of Kron and Spudich [Proc. Natl. Acad. Sci. U.S.A. 83:6272-6276, 1986] by including filamin or α-actinin on the substrate with myosin II to examine how actin-crosslinking proteins regulate the movements of single actin filaments. Increasing amounts of actin-crosslinking proteins inhibit filament velocity and decrease the number of filaments moving. Reversal of crosslinking yields increased velocities and numbers of moving filaments. These results support the solation-contraction coupling hypothesis [see Taylor and Fechheimer, Phil. Trans. Soc. London B 299:185-197, 1982] which proposes that increased crosslinking of actin inhibits myosin-based contraction. This study also illustrates the potentially varied roles of different actin-crosslinking proteins and offers a novel method to examine actin-binding protein activity and their regulation of motility at the single molecule level. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 169
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 19-33 
    ISSN: 0886-1544
    Schlagwort(e): synaptic vesicle ; mitochondria ; coated vesicle ; immunogold electron microscopy ; motor protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: The family of molecular motors known as kinesin has been implicated in the translocation of membrane-bounded organelles along microtubules, but relatively little is known about the interaction of kinesin with organelles. In order to understand these interactions, we have examined the association of kinesin with a variety of organelles. Kinesin was detected in purified organelle fractions, including synaptic vesicles, mitochondria, and coated vesicles, using quantitative immunoblots and immunoelectron microscopy. In contrast, isolated Golgi membranes and nuclear fractions did not contain detectable levels of kinesin. These results demonstrate that the organelle binding capacity of kinesin is selective and specific. The ability to purify membrane-bounded organelles with associated kinesin indicates that at least a portion of the cellular kinesin has a relatively stable association with membrane-bounded organelles in the cell. In addition, immuno-electron microscopy of mitochondria revealed a patch-like pattern in the kinesin distribution, suggesting that the organization of the motor on the organelle membrane may play a role in regulating organelle motility. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 170
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 34-44 
    ISSN: 0886-1544
    Schlagwort(e): claret-nondisjunctional ; kinesin ; karyogamy ; genetic mosaics ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: We studied the timing of mitotic loss of maternally and paternally derived chromosomes among the progeny of Drosophila melanogaster females homozygous for an amorphic mutation in ncd, a gene encoding a kinesin-like protein. In order to determine the division at which chromosome loss occurs, we estimated the fraction of XO nuclei resulting from X chromosome loss by scoring the phenotype of 47 adult cuticular landmarks in 160 XX-XO mosaics (gynandromorphs) derived from maternal X chromosome loss, and 33 gynandromorphs derived from paternal X chromosome loss. The results show that while most of the mitotic loss of maternally derived chromosomes occurs at the first cleavage division, the mitotic loss of paternally derived chromosomes occurs only at the second and later divisions. This means that paternally derived chromosomes are immune from the effects of ncd prior to karyogamy, which occurs after the first cleavage division. We discuss the implications of these results for the function of the ncd gene product and for other kinesin-like proteins in Drosophila. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 171
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 71-82 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Zusätzliches Material: 14 Ill.
    Materialart: Digitale Medien
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  • 172
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 145-156 
    ISSN: 0886-1544
    Schlagwort(e): Dictyostelium ; cAMP wave ; temporal mechanism ; chemotaxis ; pseudopod formation ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: The instantaneous velocity plots of Dictyostelium discoideum amoebae responding to natural waves and simulated temporal waves of cAMP with periods of 7 min are highly similar. This similarity has been used to deduce the dynamics of a natural wave crossing an amoeba, and the behavior of amoebae has been characterized during the different phases of a natural wave with a computer-assisted dynamic image analyzing system. During the first ∼150 sec of the front of a natural wave, cells move persistently toward the aggregation center, with high instantaneous velocity and a decreased frequency of lateral pseudopod formation. During the last 30 sec of the front of the wave and the first 30 sec of the back of the wave, there is a “freeze” in cell shape and a dramatic depression in cell motility, pseudopod formation, and intracellular particle movement. During the last 180 sec of the back of the wave, there is a rebound in pseudopod formation, but it is random in direction and leads to no net cellular translocation. The data suggest that all of the behavior of a cell but orientation during the translocation phase is mediated by the temporal dynamics of the wave. The data also suggest that orientation toward the aggregation center occurs early in the front of the wave and that, once oriented, cells move in a blind fashion during the translocation phase. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 173
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992) 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 174
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 222-230 
    ISSN: 0886-1544
    Schlagwort(e): native electrophoresis ; tubulin isotypes ; dimerization ; complexes ; GTP binding ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: The functional subunit of microtubules is a heterodimer consisting of α- and β-tubulin. An understanding of tubulin dimerization has been hampered because it has not proved possible to purify native tubulin monomers. To study the process whereby tubulin dimers are formed, we made use of tubulins synthesized by in vitro transcription and translation. We present evidence that the in vitro synthesis of different mouse α-tubulin isotypes involves a multimolecular complex. The synthesis of mouse β-tubulin isotypes also involves the formation of multimolecular complexes, though different isotypes behave somewhat differently from one another. The properties of in vitro synthesized α- and β-tubulin multimolecular complexes strongly suggest that they are intermediates in the biosynthesis of tubulin monomers. Upon release, these monomers can exchange with pre-existing tubulin heterodimers. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 175
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 201-212 
    ISSN: 0886-1544
    Schlagwort(e): intercellular junctions ; desmosome ; assembly ; microtubules ; epithelia ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Desmosomes, complex multisubunit structures that assemble at sites of cell-cell contact, are important components of the epithelial junctional complex. Desmosome assembly requires the coordinated interaction at the plasma membrane of at least 8 cytoplasmic and integral membrane proteins organized into two structurally and functionally distinct domains, the cytoplasmic plaque and membrane core. Previous studies (Pasdar et al., J. Cell Biol., 113:645-655) provided evidence that cytokeratin filaments and microtubules may regulate transfer and assembly of cytoplasmic plaque and membrane core proteins, respectively. To determine directly the role of microtubules in these processes, Madin-Darby canine kidney (MDCK) cells were treated with nocodazole or colchicine to disrupt the microtubular network. Biochemical analysis of the different components of the cytoplasmic plaque and membrane core domains revealed little or no effect of nocodazole or colchicine on the kinetics of synthesis, post-translational modifications, transfer of proteins to the plasma membrane or their metabolic stability in the presence or absence of cell-cell contact. Likewise, immunofluorescence analysis of desmosome formation demonstratedan apparently normal desmosome assembly in the presence of nocodazole or colchicine upon induction of cell-cell contact. These results indicate that an intact microtubular network is not necessary for the processing or transport of the desmosomal membrane core glycoproteins to the plasma membrane in the absence or presence of cell-cell contact. Furthermore, the integration of the cytoplasmic plaque and membrane core domains induced by cell-cell contact at the plasma membranes of adjacent cells does not require the presence of functional microtubules. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 176
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 213-221 
    ISSN: 0886-1544
    Schlagwort(e): actin-activated ATPase ; LC20 cleavage ; phosphorylation ; HMM ; actin affinity ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Previous reports have shown that papain-digested gizzard subfragment-1 (PAP-S1) has a cleaved regulatory light chain (LC20), and Vmax similar to phosphorylated heavy meromyosin (HMM) Greene et al., Biochemistry 22:530-535, 1983; Sellers et al., J. Biol. Chem. 257:13880-13883, 1982; Umemoto et al., [J. Biol. Chem. 264:1431-1436, 1989], while S. aureus protease-digested S-1 (SAP-S1) has intact LC20, but Vmax closer to that of unphosphorylated HMM [Ikebe and Hartshorne, 1985]. To determine whether intact LC20 inhibits ATPase activity for subfragment- 1 (S1), we compared the kinetic properties and structures of unphosphorylated PAP-S1 and SAP-S1. SDS-PAGE showed that SAP-S1 had 68 and 24 KDa heavy chain and 20 and 17 KDa light chain components. PAP-S1 (15 minutes digestion at 20°C) also had 68 and 17 KDa bands, but the single 24 KDa band (24HC) was replaced by a group of 22-24 KDa fragments and LC20 was cleaved to a 16 KDa fragment. At 13 mM ionic strength, both PAP-S1 and SAP-S1 had Vmax similar to phosphorylated HMM (1.1-1.5 s-1). SAP-S1 had the same KATPase as phosphorylated HMM (38 μM actin). but KATPase for PAP-S1 was 3-fold stronger (11 μM actin). Subsequent digestion of SAP-S1 with papain did not significantly change Vmax, but as LC20 and 24HC were cleaved, both KATPase and Kbinding strengthened 3- to 5-fold. Thus, intact LC20 did not inhibit, and cleavage of LC20 did not increase Vmax for S1. Rather, papain cleavage of LC20 and 24HC was associated with strengthened actin binding. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 177
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 231-235 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 178
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 236-243 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 179
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 244-251 
    ISSN: 0886-1544
    Schlagwort(e): actin ; in vitro motility assay ; microtubule bundling ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: We have previously shown that nonmuscle caldesmon copurified with brain microtubules binds to microtubules in vitro [Ishikawa et al.: FEBS Lett. 299:54-56, 1992]. To explore the role of caldesmon in the functions of microtubules, further characterization was performed using smooth muscle caldesmon, whose molecular structure and function have been best-characterized in all caldesmon species.Smooth muscle caldesmon bound to microtubules with a stoichiometry of five tubulin dimers to one molecule of caldesmon with the binding constant of 1.1 × 106M-1. The binding of caldesmon to microtubules was inhibited in the presence of Ca2+ and calmodulin. Partial digestion of the caldesmon with α-chymotrypsin revealed that the binding site of the caldesmon for microtubules lay in the 34-kDa C-terminal domain. When the caldesmon was in the dimeric form in the absence of a reducing agent, the caldesmon cross-linked microtubules to form bundles. Further, the caldesmon potentiated the polymerization of tubulin, and inhibited the in vitro movement of microtubules on dynein. These results suggest that caldesmon may be involved in the regulation by Ca2+ of the functions of microtubules. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 5 Ill.
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  • 180
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 302-312 
    ISSN: 0886-1544
    Schlagwort(e): Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Materialart: Digitale Medien
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  • 181
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 252-264 
    ISSN: 0886-1544
    Schlagwort(e): Allium ; Tradescantia ; actin ; cell cortex ; division plane determination ; immunocytochemistry ; mitosis ; microtubules ; preprophase band ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: In order to assess the possible role of microfilaments (Mfs) in events preceding plant cell division, actin was localized in root cells of Allium cepa and Tradescantia virginiana by immunofluorescence microscopy. The distribution of Mfs was compared to that of microtubules (Mts) by means of dual localizations employing both antiactin and antitubulin. Cycling interphase cells contain Mfs that extend into all regions of the cytoplasm in random fashion. Prior to the rearrangement of the cortical Mt array into the initial broad preprophase band (PPB), the number of Mfs in the cytoplasm decreases, while a new population appears in the cortex. The cortical Mfs, which usually occupy the entire cell surface, are aligned parallel to the cortical Mts. When the initial PPB appears, these Mfs still cover the cortex or are arranged as a broad band encompassing the PPB. As the PPB narrows, the Mfs are also confined to an increasingly restricted zone usually wider than the PPB.than the PPB. When the PPB reaches its narrowest, densest configuration, aligned Mfs are excluded from the band proper, while others appear in flanking regions of the cortex. From prometaphase through anaphase, cortical Mfs are largely restricted to the ends of the cell overlying the spindle poles; they also tend to become more randomly oriented. Little or no actin is present in the spindle. During telophase, the two zones of aligned cortical Mfs over the ends of the cell gradually disappear and are replaced by new interphase networks. These changes provide additional data on the possible control of PPB organization by actin, and in addition indicate that the cortex may be the origin of the actin that aggregates at the spindle poles during cytochalasin treatment. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 182
    ISSN: 0886-1544
    Schlagwort(e): growth factors ; phorbol 12-myristate 13-acetate ; microtubule-tubulin equilibrium ; initiation of DNA synthesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Previous studies suggest that alterations in the microtubule (MT)-tubulin equilibrium during G0/G1 affect mitogenesis. To determine the effect of growth factors on the MT-tubulin equilibrium, we developed a radioactive monoclonal antibody binding assay (Ball et al.: J. Cell. Biol. 103:1033-1041, 1986). With this assay, 3H-Ab 1 - 1.1 binding to cytoskeletons in confluent populations of cultured cells is proportional to the number of tubulin subunits polymerized into MTs. We now show that purified α-thrombin increases 3H-Ab 1 - 1.1 binding to cytoskeletons of serum-arrested mouse embryo (ME) fibroblasts from 1.5- to 3-fold. This stimulation is dose-dependent and correlates with concentrations of thrombin required for initiation of DNA synthesis. Other mitogenic factors, epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA), also stimulate MT polymerization. Addition of colchicine (0.3 μM) eight hours after growth factor addition blocks stimulation of 3H-thymidine incorporation by thrombin, EGF, or PMA, suggesting that tubulin polymerization or subsequent events triggered by MT polymerization are required for cells to enter a proliferative cycle. Consistent with models for autoregulation of tubulin synthesis, thrombin, EGF, and PMA all increase tubulin synthesis 9 to 15 hr after growth factor addition, raising the possibility that the decrease in free tubulin and subsequent stimulation of tubulin synthesis is linked to progression of cells into a proliferative cycle. Colchicine addition to these cells also stimulates DNA synthesis, but colchicine-stimulated cells enter S phase 6 to 8 hr later than those stimulated by growth factors. This delayed stimulation may be related to the time required for degradation of tubulin- colchicine complexes below a critical level. These data suggest that regulation of cell proliferation may be linked to increased MT polymerization and the resulting decrease in free tubulin pools. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 183
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 23 (1992), S. 279-301 
    ISSN: 0886-1544
    Schlagwort(e): F-actin ; motile mass ; myosin II ; growth cone ; Life and Medical Sciences ; Cell & Developmental Biology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Medizin
    Notizen: Retraction similar to that occurring spontaneously in senescent axonal fields of goldfish regenerating ganglion cell axons is reliably induced by monoclonal antibody (mAb) 8A2. The retraction response is characterized by transformation of the growth cone into a nodular motile mass, which undergoes retrograde translocation in conjunction with the contiguous column of axoplasm, generating evacuated distal strands. The growth cone-to-motile mass transformation involves a reorganization of F-actin. In addition, the reorganization of F-actin is a necessary antecedent for retrograde bulk translocation of axoplasm. Contractile tension contributes to compaction within the motile mass, while that within the column of distal axoplasm is oriented longitudinally and appears to contribute to bulk movement. As a derivative of the growth cone, the motile mass exhibits protrusive activities and a capacity to translocate independently when microtubules are partially disrupted. Apparent compressive forces cause buckling of microtubules in the adjacent segment which appear as elbow-like protrusions. Cytochalasin D blocks mAb 8A2 induced retraction and immediately arrests retrograde translocation when it is in progress; however, neither nocodazole nor taxol blocks retraction. Phalloidin and immunofluorescence double labeling of retracted axons reveals that myosin 11, MLCK, and calmodulin co-localize with dense F-actin structures within the motile mass. These results suggest that microtubules play a subordinate, passive role, and that actomyosin interactions mediate the formation of the motile mass and the retraction response. Finally, axons grown on laminin exhibit a more robust retraction response than those grown on polylysine, implicating membrane-cytoskeletal interactions as modulating factors. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 17 Ill.
    Materialart: Digitale Medien
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  • 184
    ISSN: 0887-3585
    Schlagwort(e): protein structure ; protein sequences ; protein design de novo ; protein engineering ; computer algorithms ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: What is the current state of the art in protein design? This question was approached in a recent two-week protein design workshop sponsored by EMBO and held at the EMBL in Heidelberg. The goals were to test available design tools and to explore new design strategies. Five novel proteins were designed: Shpilka, a sandwich of two four-stranded β-sheets, a scaffold on which to explore variations in loop topology; Grendel, a four-helical membrane anchor, ready for fusion to water-soluble functional domains; Fingerclasp, a dimer of interdigitating β-β-α units, the simplest variant of the “handshake” structural class; Aida, an antibody binding surface intended to be specific for flavodoxin; Leather - a minimal NAD binding domain, extracted from a larger protein. Each design is available as a set of three-dimensional coordinates, the corresponding amino acid sequence and a set of analytical results. The designs are placed in the public domain for scrutiny, improvement, and possible experimental verification.
    Zusätzliches Material: 2 Ill.
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  • 185
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 12 (1992), S. 145-157 
    ISSN: 0887-3585
    Schlagwort(e): thermal diffuse X-ray scattering ; protein disorder ; molecular dynamics ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Diffuse scattering data have been collected on two crystal forms of lysozyme, tetragonal and triclinic, using synchrotron radiation. The observed diffraction patterns were simulated using an exact theory for simple model crystals which relates the diffuse scattering intensity distribution to the amplitudes and correlations of atomic movements. Although the mean square displacements in the tetragonal form are twice that in the triclinic crystal, the predominent component of atomic movement in both crystals is accounted for by short-range coupled motions where displacement correlations decay exponentially as a function of atomic separation, with a relaxation distance of ≈ 6 Å. Lattice coupled movements with a correlation distance ≈ 50 Å account for only about 5-10% of the total atomic mean square displacements in the protein crystals. The results contradict various presumptions that the disorder in protein crystals can be modeled predominantly by elastic vibrations or rigid body movements.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 186
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 12 (1992), S. 180-187 
    ISSN: 0887-3585
    Schlagwort(e): fibronectin ; domain ; collagen ; folding ; disulfide ; fluorescence ; GdmCl ; renaturation ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The gelatin-binding region of fibronectin is easily isolated as a stable and functional 42-kDa fragment (42-kDa GBF) containing four type I “finger” modules and two type II “kringle-like” modules arranged in the order I6-II1-II2-I7-I8-I9, where the numbers designate the order of these modules in each of the two polypeptide chains. Each module forms an independently folded domain stabilized by two disulfide bonds. Reduction of disulfides caused large changes in the intrinsic fluorescence and abolished the gelatin-binding activity of 42-kDa GBF and two nonoverlapping gelatin-binding subfragments, 30-kDa GBF (I6-II1-II2-I7) and 21-kDa GBF (I8-I9). However, high yields of active material could be regenerated, without diluting the protein, by dialysis into GdmCl followed by slow overnight removal of GdmCl while maintaining the redox potential with a mixture of oxidized and reduced glutathione. Fluorescence spectroscopic analysis indicated that the tertiary structure and thermodynamic stability of the refolded fragments were similar to those of the originals. The refolded fragments were quantitatively indistinguishable from the originals with respect to their dissociation constants for binding to a fluorescent-labeled collagen fragment. The results suggest that all or most of the cystines, a total of 24 in 42-kDa GBF, are correctly paired in the refolded products and that the tertiary structure was completely recovered. The fact that the 30- and 21-kDa fragments bind with a similar affinity proves the existence of at least two nonoverlapping sites in 42-kDa GBF that recognize gelatin.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 187
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 12 (1992), S. 278-298 
    ISSN: 0887-3585
    Schlagwort(e): folding nucleation ; hydrophobic cluster ; conserved loop length ; structure-sequence relationship ; sequence patterns ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The Greek key β-barrel topology is a folding motif observed in many proteins of widespread evolutionary origin. The arthropodan hemocyanins also have such a Greek key β-barrel, which forms the core of the third domain of this protein. The hemocyanin β-barrel was found to be structurally very similar to the β-barrels of the immunoglobulin domains, Cu,Zn-superoxide dismutase and the chromophore carrying antitumor proteins. The structural similarity within this group of protein families is not accompanied by an evolutionary or functional relationship. It is therefore possible to study structure-sequence relations without bias from nonstructural constraints. The present study reports a conserved pattern of features in these Greek key β-barrels that is strongly suggestive of a folding nucleation site. This proposed nucleation site, which we call a “β-zipper,” shows a pattern of well-conserved, large hydrophobic residues on two sequential β-strands joined by a short loop. Each β-zipper strand is near the center of one of the β-sheets, so that the two strands face each other from opposite sides of the barrel and interact through their hydrophobic side chains, rather than forming a hydrogen-bonded β-hairpin. Other protein families with Greek key β-barrels that do not as strongly resemble the immunoglobulin fold - such as the azurins, plastocyanins, crystallins, and prealbumins - also contain the β-zipper pattern, which might therefore be a universal feature of Greek key β-barrel proteins.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 188
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 12 (1992), S. 314-323 
    ISSN: 0887-3585
    Schlagwort(e): structure analysis ; graph theory ; protein structure ; β sheet ; retrieval ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: In order to find rules for the secondary structure prediction of proteins which describe the (sequentially) long-range interactions in sheet structures methods of applied graph theory were used. The so called β graph which describes the sheet topology was defined for every protein in the Brookhaven Data Bank containing β sheets. The resemblance of proteins at that topological level is discussed, and four notations and graphic representations of sheets which describe the sequential and topological neighborhoods of the strands were derived. This description level supports the usage of data structures which allow the implementation of efficient algorithms for the analysis and comparison of β structures in proteins. A computer program for the representation and retrieval of bibliographic data and β sheet structures was implemented. Some examples for substructure search illustrate the usefulness of the program. Two graphic catalogues were compiled: one contains all β graphs of PDB proteins and the other all occurring different greek key descriptions.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 189
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 12 (1992), S. 345-364 
    ISSN: 0887-3585
    Schlagwort(e): protein structure ; crystallography ; errors ; φ,ψ distribution ; χ1 angles ; stereochemical parameters ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Methods have been developed to assess the stereochemical quality of any protein structure both globally and locally using various criteria. Several parameters can be derived from the coordinates of a given structure. Global parameters include the distribution of φ,ψ and χ1 torsion angles, and hydrogen bond energies. There are clear correlations between these parameters and resolution; as the resolution improves, the distribution of the parameters becomes more clustered. These features show a broad distribution about ideal values derived from high-resolution structures. Some structures have tightly clustered distributions even at relatively low resolutions, while others show abnormal scatter though the data go to high resolution. Additional indicators of local irregularity include proline φ angles, peptide bond planarities, disulfide bond lengths, and their χ3 torsion angles. These stereochemical parameters have been used to generate measures of stereochemical quality which provide a simple guide as to the reliability of a structure, in addition to the most important measures, resolution and R-factor. The parameters used in this evaluation are not novel, and are easily calculated from structure coordinates. A program suite is currently being developed which will quickly check a given structure, highlighting unusual stereochemistry and possible errors.
    Zusätzliches Material: 17 Ill.
    Materialart: Digitale Medien
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  • 190
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 12 (1992), S. 382-399 
    ISSN: 0887-3585
    Schlagwort(e): protein secondary structure ; amino acid sequence ; distributions ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The conditional probability, P(σ|x), is a statement of the probability that the value of σ will be found given the prior information that a value of x has been observed. Here σ represents any one of the secondary structure types, α, β, τ, and ρ for helix, sheet, turn, and random, respectively, and x represents a sequence attribute, including, but not limited to: (1) hydropathy; (2) hydrophobic moments assuming helix and sheet; (3) Richardson and Richardson helical N-cap and C-cap values; (4) Chou-Fasman conformational parameters for helix, Pα, for sheet, Pβ, and for turn, Pτ; and (5) Garnier, Osguthorpe, and Robson (GOR) information values for helix, Iα, for sheet, Iβ, for turn, I,τ, and for random structure, Iρ.Plots of P (σ|x) vs. x are demonstrated to provide information about the correlation between structure and attribute, σ and x. The separations between different P (σ|x) vs. x curves indicate the capacity of a given attribute to discriminate between different secondary structural types and permit comparison of different attributes. P (α|x), P (β|x), P (τ|x) and P (ρ|x) vs. x plots show that the most useful attributes for discriminating helix are, in order: hydrophobic moment assuming helix 〉 Pα » N-cap 〉 C-cap ≈ Iα ≈ Iτ. The information value for turns, Iτ, was found to discriminate helix better than turns. Discrimination for sheet was found to be in the following order: Iβ » Pβ ≈ hydropathy 〉 Iρ ≈ hydrophobic moment assuming sheet.Three attributes, at their low values, were found to give significant discrimination for the absence of helix: Iα ≈ Pα ≈ hydrophobic moment assuming helix. Also, three other attributes were found to indicate the absence of sheet: Pβ » Iτ ≈ hydropathy. Indications of the absence of σ could be as useful for some applications as the indication of the presence of σ.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 191
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 13 (1992), S. 26-37 
    ISSN: 0887-3585
    Schlagwort(e): norcamphor ; P450CIA1 ; substrate specificity ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: While cytochrome P-450cam, catalyzes the hydroxylation of camphor to 5-exo-hydroxycamphor with 100% stereospecificity, norcamphor is hydroxylated by this enzyme yielding 45% 5-exo-, 47% 6-exo-, and 8% 3-exo-hydroxynorcamphor (Atkins, W.M., Sligar, S.G., J. Am. Chem. Soc. 109:3754-3760, 1987). The present study describes a 201-psec molecular dynamics (MD) simulation of norcamphorbound cytochrome P-450cam to elucidate the relationship between substrate conformational mobility and formation of alternative products. First, these data suggest that the product specificity is, at least partially, due to the mobility of the substrate within the active site. Second, the high mobility of norcamphor in the active site leads to an average increase in separation between the home iron and the substrate of about 1.0 Å; this increase in separation may be the cause of the uncoupling of electron transfer when norcamphor is the substrate. Third, the active site water located in the norcamphor-bound crystal structure possesses mobility that correlates well with the spin-state equilibrium of this enzyme-substrate complex. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 11 Ill.
    Materialart: Digitale Medien
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  • 192
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 13 (1992), S. 57-69 
    ISSN: 0887-3585
    Schlagwort(e): protein conformation ; aromatic contribution ; disulfide contribution ; CD spectra of the main secondary structural elements in proteins ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A recently developed algorithm, called Convex Constraint Analysis (CCA), was successfully applied to determine the circular dichroism (CD) spectra of the pure β-pleated sheet in globular proteins. On the basis of X-ray diffraction determined secondary structures, the original data set used (Perczel, A., Hollosi, M., Tusnady, G. Fasman, G.D. Convex constraint analysis: A natural deconvolution of circular dichroism curves of proteins, Prot. Eng., 4:-669-679, 1991), was improved by the addition of proteins with high β-pleated sheet content. The analysis yielded CD curves of the pure components of the main secondary structural elements (α-helix, antiparallel β-pleated sheet, β-turns, and unordered conformation), as well as a curve attributed to the “aromatic contribution” in the wavelength range of 195-240 nm. Upon deconvolution the curves obtained were assigned to various secondary structures. The calculated weights (percentages determining the contributions of each pure component curve in the measured CD spectra of a given protein) were correlated with the X-ray diffraction determined percentages in an assignment procedure and were evaluated. The Pearson product correlation coefficients (R) are significant for all five components. The new pure component curves, which were obtained through deconvolution of the protein CD spectra alone, are promising candidates for determining the percentages of the secondary structural components in globular proteins without the necessity of adopting an X-ray database. The CD spectrum of the CheY protein was interesting because it has the characteristic shape associated with the α-helical structure, but upon analysis yielded a considerable amount of β-sheet in agreement with the X-ray structure. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 193
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 13 (1992), S. 120-131 
    ISSN: 0887-3585
    Schlagwort(e): protein folding ; α-helix ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: A computational study of the role of all ionizable groups of the C-peptide in its helix-coil transition is performed within the framework of continuum electrostatics. The method employed in our computations involves a numeric solution of the Poisson equation with the Boundary Element Method. Our calculations correctly predict the experimentally observed trends in the helix-coil equilibrium of the C-peptide, and suggest that the mechanisms involved are more complex than usually presumed in the literature. Our results suggest that electrostatic interactions in the unfolded conformation are often more important than in the helix, total electrostatic contribution to the helix-coil transition due to the side chains of the C-peptide destabilizes the helix, changes in the helix stability produced by the changes in the ionization state of the side chains are dominated by side chain effects, the effect of the helix dipole on the energetics of the helix-coil transition of the C-peptide is either minor or similar to other contributions in magnitude; while the formation of a salt bridge is electrostatically favorable, formation of the hydrogen bond between a charged and a polar side chains is not. Factors limiting the accuracy of the computations are discussed. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 194
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 13 (1992), S. 152-157 
    ISSN: 0887-3585
    Schlagwort(e): Staphylococcus aureus ; bacterial toxins ; structure-function ; protein structure ; crystallography ; pyrogenic toxins ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The Type C staphylococcal enterotoxin produced by Staphylococcus aureus strain FRI-909 has been crystallized using a combination of two precipitants, ammonium sulfate and polyethylene glycol 400, with the addition of small amounts of detergent. Two related crystal forms have been obtained, one triclinic, and one tetragonal, both with one toxin molecule per asymmetric unit. These crystals are stable for at least 75 hr in the X-ray beam and diffract to at least 2.2 and 2.6 Å, respectively. The triclinic crystals have unit cell parameters a = 38.5 Å, b = 43.7 Å, c = 36.9 Å, and interaxial angles α = 99.9°, β = 95.8°, and γ = 98.5°. The tetragonal crystals are of space group P4122 with unit cell parameters a = 43.4 Å and c = 278.0 Å. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 195
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 13 (1992), S. 162-173 
    ISSN: 0887-3585
    Schlagwort(e): protein folding ; folding intermediates ; time resolved fluorescence ; nonradiative excitation energy transfer ; BPTI ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: The structure of BPTI and reduced BPTI in concentrated guanidinium HCI (GUHCl) in the presence of glycerol has been probed by measurements of dynamic nonradiative excitation energy transfer between probes attached to its amino groups. Inter probe distance distributions were obtained from analysis of donor fluorescence decay curves and used to characterize local structures in unordered states of the protein. Site specifically fluorescently labeled BPTI derivatives (1-n)BPTI (n = 15, 20, 41, 46) were used, each carrying a 2-methoxy-naphthyl-1-methylenyl group (MNA) at the N-terminal amino group of arg1 and 7-(dimethylamino)-coumarin-4-yl-acetyl residue (DA-coum) at one of its ε-NH2 groups of the lysine side chains. Analysis of donor fluorescence decay kinetics gave the interprobe distance distributions in the native and denatured states.The N-terminal-segment, residues 1-15, is in an extended conformation (with an average interprobe distance of 34 ± 2 Å) in the native state. Upon unfolding by reduction with DTT or β-mercapto ethanol in 6 M GUHCl/glycerol mixture, the conformation of this segment relaxed to a state characterized by a reduced averageinterprobe distance and a larger width of the distances distribution. The average distance between residues 1 and 26, i.e., between the N-terminus and the turn of the twisted β sheet element (residues 18-35), increased upon unfolding. At -30°C in the above solvent, the distribution between these two sites was probably composed of two conformational subpopulations. About 45 ±20% of the molecules were characterized by a short interprobe distance (like the native state) representing a compact conformation, and 55 ± 20% of the molecules showed large interprobe distances representing an expanded (unfolded) conformation.Thus local structures seem to exist in reduced denatured BPTI even underdenaturing conditions in 6 M GUHCl/glycerol mixtures. Some of those structures are unstable in guanidinium isothiocyanate (GUSCN). The method introduced here is suitable for probing local structures and very long range interactions in unfolded folded proteins and for search for folding initiation sites (FISs) and early folding intermediates. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 196
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 13 (1992), S. 206-222 
    ISSN: 0887-3585
    Schlagwort(e): molecular docking ; Monte Carlo ; simulated annealing ; rational drug-design ; dihydrofolate reductase ; proteinase inhibitors ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: We present a method to search for possible binding modes of molecular fragments at a specific site of a potential drug target of known structure. Our method is based on a Monte Carlo (MC) algorithm applied tothe translational and rotational degrees of freedom of the probe fragment. Starting from a randomly generated initial configuration, favorable bindingmodes are generated using a two-step process. An MC run is first prformed in which the energy in the Metropolis algorithm is substituted by a score function that measures the average distance of the probe to the targetsurface. This has the effect of making buried probes move toward the targetsurface and also allows enhanced sampling of deep pockets. In a second MC run, a pairwise atom potential function is used, and the temperature parameter is slowly lowered during the run (Simulated Annealing). We repeat this procedure starting from a large number of different randomly generated initial configurations in order to find all energetically favourable docking modes in a specified region around the target. We test this method using two inhibitor-receptor systems: Streptomyces griseus Proteinase B in complex with the third domain of the ovomucoid inhibitor from turkey, and dihydrofolate reductase from E. Coli in complex with methotrexate. The method could consistently reproduce the complex found in thecrystal structure searching from random initial positions in cubes ranging from 25 Å to 50 Å about the binding site. In the case of SGPB, we were also successful in docking to the native structure. In addition, we were successful in docking small probes in a search that included the entire protein surface. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 197
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 13 (1992), S. 272-272 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
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  • 198
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 13 (1992), S. 258-271 
    ISSN: 0887-3585
    Schlagwort(e): protein folding ; protein modeling ; knowledge-based prediction ; molecular force field ; statistical mechanics ; globins ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: We present an approach which can be used to identify native-like folds in a data base of protein conformations in the absence of any sequence homology to proteins in the data base. The method is based on a knowledge-based force field derived from a set of known protein conformations. A given sequence is mounted on all conformations in the data base andthe associated energies are calculated. Using several conformations and sequences from the globin family we show that the native conformation is identified correctly. In fact the resolution of the force field is high enough to discriminate between a native fold and several closely related conformations. We then apply the procedure to several globins of known sequence but unknown three dimensional structure. The homology of these sequences to globins of known structures in the data base ranges from 49 to 17%. Withone exception we find that for all globin sequences one of the known globinfolds is identified as the most favorable conformation. These results are obtained using a force field derived from a data base devoid of globins of known structure. We briefly discuss useful applications in protein structurlresearch and future development of our approach. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 199
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 14 (1992) 
    ISSN: 0887-3585
    Schlagwort(e): Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 200
    Digitale Medien
    Digitale Medien
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 13 (1992), S. 364-368 
    ISSN: 0887-3585
    Schlagwort(e): pancreatic spasmolytic polypeptide ; porcine pancreas ; hanging drop vapor diffusion method ; crystallization ; circular dichroism analysis ; porcine insulin ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Medizin
    Notizen: Pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapor diffusion method. Crystals suitable for X-ray diffraction analysis were grown at pH 4.7 from a solution of 6% saturated ammonium sulfate. The space group is orthorhombic I222 or I212121 with unit cell parameters a = 54.38 Å, b = 72.29 Å, and c = 180.85 Å. There are three molecules of PSP per asymmetric unit and a water content of 46.9%. The crystals diffracts to an estimated resolution of 2.7 Å.The far-UV CD spectrum of PSP shows some exceptional features which cannot be accounted for thoroughly in terms of standard secondary structures commonly seen in protein CD spectroscopy. With this limitation, the secondary structure analysis predicts 15% α-helix, between 10 and 20% antiparallel β-strand, 10% parallel β-strand, 15% turn, and 25 to 40% of other structures. © 1992 Wiley-Liss, Inc.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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