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  • General Chemistry  (879)
  • Cell & Developmental Biology  (873)
  • 1985-1989  (1,752)
  • 1970-1974
  • 1985  (1,752)
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Publisher
Years
  • 1985-1989  (1,752)
  • 1970-1974
Year
  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 5 (1985), S. 1-15 
    ISSN: 0886-1544
    Keywords: contractile non-actin filaments ; dinoflagellates ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The motility and fine structure of the marine planktonic dinoflagellate Kofoidinium and members of other related genera have been investigated. Several types of shape change were found to occur: slow morphogenetic changes (which also occurred as restitution movements in response to injury), movements associated with the ingestion of food and the evacuation of wastes, and more rapid movements concerned with the capture of prey. All these movements seemed to be brought about by the contraction of refractile tracts within the cytoplasm, which were found to contain 2.3-nm filaments, some with a complex striated appearance. This and other evidence suggests that these filaments, which have counterparts in many other protists, are not actin filaments.
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  • 2
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    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 5 (1985) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 3
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    Cell Motility and the Cytoskeleton 5 (1985), S. 175-193 
    ISSN: 0886-1544
    Keywords: primary cilia ; connective tissues ; secretory organelles ; extracellular matrix ; cybernetic probe ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: More than 300 primary cilia have been identified electronmicroscopically in a variety of embryonic and mature connective tissue cells. To further define the enigmatic function of these cilia, we examined the interrelationships between the basal apparatus and cytoplasmic organelles and the ciliary shaft and the extracellular matrix. The basal diplosome was consistently associated with the secretory organelles including the maturing face of the Golgi complex, Golgi vacuoles and vesicles, the microtubular network, the plasma membrane, and coated pits and vesicles. Small vesicles and amorphous granules were also observed within the ciliary lumen and adjacent to the ciliary membrane. Microtubule-membrane bridges linked axonemal tubules to the ciliary membrane. The position, projection, and orientation of the axoneme were influenced by the structural organisation and mechanical properties of the matrix and frequently caused angulation of the ciliary shaft relative to the basal body. Located midway between the secretory apparatus and the extracellular matrix, primary cilia would appear ideally situated to mediate the necessry interaction between the cell and its surrounding environment prerequisite to the formation and maintenance of a functionally effective matrix. We propose that primary cilia in connective tissue cells could act as multifunctional, cellular cybernetic probes, receiving, transducing, and conducting a variety of extrinsic stimuli to the intracellular organelles responsible for effecting the appropriate homeostatic feedback response to changes in the extracellular microenvironment.
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  • 4
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    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 5 (1985), S. 239-249 
    ISSN: 0886-1544
    Keywords: tektins ; microtubules ; flagella ; cilia ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Affinity-purified antibodies against Strongylocentrotus purpuratus sperm flagellar tektin polypeptides have been tested for cross-reactivity with microtubules isolated from various sources, using indirect immunofluorescent staining and antibody binding to nitrocellulose replicas of SDS polyacrylamide gels. The antitektins reacted with sperm tail axonemes from four genera of sea urchins and with cilia from sea urchin embryos. Antibody binding was observed only if the specimens were prefixed by methods that would not preserve them well at an ultrastructural level. However, even after such fixation regimes, no antibody binding was detected to cytoplasmic microtubule arrays in the same embryos, to mitotic spindles isolated from sea urchin or to gill cilia from a mollusc. We conclude that, if tektins are present in sea urchin egg cytoplasmic microtubules, they are sufficiently different from the sperm tektins to have no common strongly antigenic determinants.
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  • 5
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    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 5 (1985), S. 333-350 
    ISSN: 0886-1544
    Keywords: eel sperm ; 9+0 flagellum ; motility ; helicoidal bending ; reactivation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The sperm flagella of the eel, Anguilla anguilla, are capable of vigorous motion in spite of having an axoneme with reduced structure that lacks the outer dynein arms, radial spokes and spoke heads, the two central tubules and the central tubule projections that are all part of the standard “9+2” axoneme. These sperm progress forward rapidly as a result of the propagation of helicoidal waves distally along the flagellum. Their flagellar beat frequencies are high, 93 Hz at 21°C, and they roll at a frequency of about 19 Hz. Eel sperm could be demembranated with Nonidet P-40 and reactivated with MgATP2- in 0.22 M K acetate at pH 8.1. The reactivated motility closely resembles that of the live sperm, with a beat frequency of 69 Hz, but the demembranated flagella are unusually fragile, and commonly disintegrate by a combination of splitting, coiling, and sliding within a few minutes. Little reactivation is obtained if acetate is replaced by Cl- in the reactivating medium. The Michaelis constant for beat frequency (0.2 mM) is similar to that obtained for several “9+2” flagella. These sperm, however, appear to lack the mechanism by which Ca2+ regulates waveform. Our results indicate that eel sperm flagella, which at rest are straight, are induced to bend helicoidally by ATP, as the result of sliding between tubules that is blocked at both the base and tip of the organelle. The flagellar waveform consists of a series of planar bends separated by short regions of right-handed twist, which give it an overall left-handed helicoidal form.
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  • 6
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    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 5 (1985), S. 377-392 
    ISSN: 0886-1544
    Keywords: newt ; lung ; cilia ; beat frequency ; waveform ; models ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Highly coupled newt lung ciliated cell models were used to study the effects of MgATP concentration on ciliary beat frequency and waveform. Models were prepared from ciliated lung cells of the newt Taricha granulosa by trypsin dissociation of the epithelium, demembranation with Triton X-100, and reactivation with MgATP, as described previously [Weaver and Hard, 1985]. Beat frequencies were measured stroboscopically. Ciliary waveforms of reactivated models and intact mucociliary epithelial sheets were determined by single frame analysis of high-speed movies. Waveform parameters calculated included the durations of the effective and recovery strokes, the angular velocities of the ciliary base and tip, the position of the bend along the ciliary shaft during the recovery stroke, the velocity of recovery stroke bend propagation, and the ratio of the duration of recovery stroke bend propagation to the duration of the recovery stroke itself. We found that beat frequency varied biphasically in response to MgATP at 21°C, as shown previously for isolated, individual, newt lung axonemes. Apparent Fmax (maximum beat frequency) and Km values of 25 Hz and 0.14 mM, and 35 Hz and 0.47 mM, respectively, were obtained for each linear segment of the biphasic double reciprocal plot. Demembranation did not alter either ciliary waveform or the pattern of coordination. In this system, metachrony is antilaeoplectic and ciliary waveform appears to be regulated independent of beat frequency.
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  • 7
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    Cell Motility and the Cytoskeleton 5 (1985), S. 17-29 
    ISSN: 0886-1544
    Keywords: cell motility ; membrane recycling ; immunofluorescence microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Mouse peritoneal macrophages subjected to gradients of activated mouse serum were found by immunofluorescence observations to have their Golgi apparatus and their microtubule-organizing center largely oriented in the direction of the gradient. By analogy with similar results obtained with motile fibroblasts, it is proposed that these two organelles are rapidly and coordinately reoriented inside the macrophages in order to direct the insertion of new membrane mass, via vesicles derived from the Golgi apparatus, into the leading edge of the cell. Consistent with the importance of such membrane insertion to cell migration, we found that the ionophore monensin, an inhibitor of Golgi functions, inhibited cell motility in the chemostactic gradient. It was further shown that several inhibitors of chemotaxis (monensin, cytochalasin D, cycloheximide) did not inhibit the reorientation of the Golgi apparatus/microtubule-organizing center in cells exposed to a chemotactic gradient, and that the reorientation required extracellular Ca+2.
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  • 8
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    Cell Motility and the Cytoskeleton 5 (1985), S. 491-506 
    ISSN: 0886-1544
    Keywords: Somitogenesis ; neurulation ; alpha-actinin ; morphogenesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A discrete stage in two different morphogenetic processes has been examined employing fluorescently labelled alpha-actinin as a probe to localize native alpha-actinin and antibodies to localize fibronectin and collagen type I. The stage of somitogenesis examined is the transition from the compact mesenchymal somitic mass to the epithelial somitic vesicle (ie, epithelialization of the somite). The stage of neurulation examined is the transition from the relatively flat neuroepithelium to the approximation of the neural folds. Before these morphogenetic movements begin, the neuroepithelium is sitting upon a basal lamina and interstitial collagen, and the somite is surrounded by a meshwork of interstitial collagen. During both of these processes, the cells become narrowed at their apices in the region of the tissue that is becoming concave, and alpha-actinin is localized in the apices. The localization of intracellular alpha-actinin and extracellular fibronectin, and the distribution of collagen, suggest that there is a coordinated appearance and distribution of these molecules that is temporally associated with these discrete morphogenetic events.
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  • 9
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    Cell Motility and the Cytoskeleton 5 (1985), S. 195-208 
    ISSN: 0886-1544
    Keywords: central pair ; radial spoke ; flagella ; mutant ; Chlamydomonas ; suppressor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Flash photomicrography at frequencies up to 300 Hz and computer-assisted image analysis have been used to obtain parameters describing the flagellar bending patterns of mutants of Chlamydomonas reinhardtii. All strains contained the uni1 mutation, to facilitate photography. The radial spoke head deficient mutant pf17, and the central pair deficient mutant, pf15, in combination with suppressor mutations that restore motility without restoring the ultrastructural or biochemical deficiencies, both generate forward mode bending patterns with increased shear amplitude and decreased asymmetry relative to the “wild-type” uni1 flagella described previously. In the reverse beating mode, the suppressed pf17 mutants generate reverse bending patterns with large shear amplitudes. Reverse beating of the suppressed pf15 mutants is rare. There is a reciprocal relationship between increased shear amplitude and decreased beat frequency, so that the velocity of sliding between flagellar microtubules is not increased by an increase in shear amplitude. The suppressor mutations alone cause decreased frequency and sliding velocity in both forward and reverse mode beating, with little change in shear amplitude or symmetry.
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  • 10
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    Cell Motility and the Cytoskeleton 5 (1985) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 11
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    Cell Motility and the Cytoskeleton 5 (1985), S. 265-265 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 12
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    Cell Motility and the Cytoskeleton 5 (1985), S. 463-473 
    ISSN: 0886-1544
    Keywords: sponge dissociates ; cell migration ; time-lapse analysis ; cell aggregation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The production of both lamellipodia and peculiar thin protuberances (scleropodia) characterizes the preaggregative motility of cells after dissociation of the sponge Clathrina.The locomotory paths taken by cells before aggregation were recorded by time-lapse microcinematography. Changes of direction in successive 50-s time intervals and 50-s mean velocities of each cell were both taken into account as statistical variables. Their distributions give probability density curves that seem to fit bilateral exponential functions. The analysis of the angles of turn indicates a tendency for the cells to persist in their direction of motion and to make counter-clockwise turns. Implications of such in vitro cellular behaviors in aggregative and in vivo processes are suggested.
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  • 13
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    Cell Motility and the Cytoskeleton 5 (1985), S. 81-101 
    ISSN: 0886-1544
    Keywords: fast axonal transport ; isolated axoplasm ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The development of AVEC-DIC microscopy and the application of this method to the study of fast axonal transport in isolated axoplasm extruded from the giant axon of the squid Loligo pealei provides a new paradigm for analyzing the intracellular transport of membranous organelles. The size of the axon, the number of transported particles, and the absence of permeability barriers like the plasma membrane in this preparation permit many experiments that are difficult or impossible to perform using other model systems. The use and features of this preparation are described in detail and a number of properties are evaluated for the first time. The process of extrusion is characterized. Particle movement is evaluated both in the interior of extruded axoplasm and along individual fibrils that extend from the periphery of perfused axoplasm. The role of divalent cations, particularly Ca2+, and the effects of elevated Ca2+ on axoplasmic organization and transport are analyzed. A series of pharmacological agents and polypeptides that alter cytoskeletal organization are used to examine the role of microfilaments and microtubules in fast transport. Finally, the effects of depleting ATP and of adding ATP analogues are discussed. The extruded axoplasm preparation is shown to be an invaluable model system for biochemical and pharmacological analyses of the molecular mechanisms of intracellular transport.
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  • 14
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    Cell Motility and the Cytoskeleton 5 (1985), S. 225-237 
    ISSN: 0886-1544
    Keywords: neural crest ; migratory behavior ; microfilaments ; stress fibers ; tractional force ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have investigated one aspect of the migratory behavior of quail neural crest (NC) cells by comparing the organization of microfilament bundles and the ability to distort migratory substrata by NC, somite, and notochord cells in vitro. In contrast to the numerous cytoplasmic stress fibers in somite-derived fibroblasts and notochord cells revealed by rhodamine-phalloidin staining and thin-section electron microscopy, microfilaments in NC cells are restricted to the cell cortex. To test the relative degrees of tension generated by these cell types on the underlying substratum, cells were cultured in collagen gels and on distortable silicone rubber sheets. Explanted somites and notochords produced dramatic radial alignment of 750 μg/ml collagen gels, whereas neural crest cells only aligned gels of lower concentrations. Fibroblasts did not migrate individually from explanted somites and notochords into 250 μg/ml collagen gels as readily as into higher concentration collagen lattices. In contrast, neural crest cells migrated into matrices of low concentration as well as into higher concentration collagen gels. Neural crest cells and their pigmented derivatives did not distort silicone rubber sheets, whereas somite and notochord-derived fibroblasts wrinkle this substratum after 4 days in culture. Thus, the differences in organization of the actin cytoskeleton reflect the tractional force exerted by these cells on their substratum. We hypothesize that the migratory behavior of NC cells in vivo may be related to their ability to translocate through embryonic extracellular matrices while generating relatively weak adhesions with the substratum, whereas the stronger forces generated by other embryonic cell types upon the delicate extracellular matrix may restrict their migration and may be associated with other morphogenetic events.
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  • 15
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    Cell Motility and the Cytoskeleton 5 (1985) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 16
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    Cell Motility and the Cytoskeleton 5 (1985), S. 31-51 
    ISSN: 0886-1544
    Keywords: microtubules ; birefringence ; flow birefringence ; tubulin ; polarization microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Understanding the molecular basis of mitotic movements in living cells will require correlative experiments on intact cells, cell models, purified tubulin, and perhaps other biopolymers. Birefringence is one assay that is useful in all of these experimental situations. Heretofore, studies of birefringence changes during mitosis have lacked a quantitiative basis for interpretation in terms of microtubule number and packing density. One of the aims of this work was to establish that relationship.Purified calf brain tubulin was polymerized to equilibrium and oriented in the hydrodynamic field of a microcapillary flow birefringence apparatus. The relationship between birefringence and microtubule packing density was determined by a combination of optical, electron microscopic, and biochemical methods. The data correlate surprisingly well with those obtained by others from in vitro measurements on isolated mitotic spindles. Using the flow birefringence data, the sensitivity of polarizing microscopes for detecting microtubules was examined and found to depend on microtubule packing density, object thickness, and instrumental factors that limit both the detection and measurement of weakly birefringent objects. Because of the dependence of measurement sensitivity on object thickness, a method of measuring the thickness of microtubule bundles using the dispersion of birefringence was developed. This method is capable of measuring thickness to within two or three Airy diffraction units and does not require any assumptions regarding object symmetry.
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  • 17
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    Cell Motility and the Cytoskeleton 5 (1985), S. i 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 18
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    Cell Motility and the Cytoskeleton 5 (1985), S. 209-224 
    ISSN: 0886-1544
    Keywords: flagellar dynein ; cyclic nucleotides ; sperm activation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Methods of demembranation and reactivation of Lytechinus pictus sperm were developed that result in non-motile sperm which take on a stable bend of about 3.5 radians at the proximal end of the cell. The middle and distal portion of the flagellum is relatively straight or slightly bent in the same direction forming a somewhat “C” shaped sperm cell. In these studies, we refer to this characteristic shape as the quiescent form, and as opposed to “rigor wave” sperm, the quiescent form is induced and maintained by a relatively high concentration of MgATP2- (〉 0.2 mM). Other conditions important to the production and maintenance of the quiescent form in demembranated sperm include: starting with concentrated, undiluted sperm, maintaining low Ca++ in the demembranation buffer, using a minimum of 0.2 mM MgATP2- and pH of 7.9-8.1 in the reactivation buffer. Deviation from some of these conditions results in a dramatic increase in motile, asymmetrically beating sperm. Addition of 0.4 mM CaCl2 to the reactivation buffer increased the proximal bend angle to 5 radians. The induction and maintenance of the stationary bend is mediated by dynein activity: “rigor wave” sperm were transformed to the quiescent form upon 0.2 mM ATP addition; micromolar vanadate abolished the quiescent form by “relaxation” of the proximal bend; and the vanadate relaxed sperm were restored to quiescent form by catechol. Importantly, 20 μM cAMP activated motility of the otherwise quiescent-form sperm. Quiescent-form, demembranated sperm were also activated by mild trypsin digestion. These and other data suggest that the quiescent-form sperm are trapped at the end of the principal bend, and these data are consistent with the proposal that the single stationary bend results from asymmetry of active microtubule sliding [Gibbons and Gibbons, (1980): J. Cell Biol. 84:13-27].
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  • 19
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    Cell Motility and the Cytoskeleton 5 (1985), S. 267-292 
    ISSN: 0886-1544
    Keywords: mammalian cilia ; respiratory tract ; mucociliary clearance ; laterofrontal cirri ; Mytilus edulis ; beat cycle ; computer analysis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The beat cycles of rabbit tracheal cilia in culture and Mytilus laterofrontal cirri were recorded using a phototransistor, transillumination, video, and phase-contrast microscopy. The photoelectronic signal and video image of the ciliary activity were simultaneously recorded as a composite image. The photoelectronic signal was converted into a digital signal by a data acquisition system for further computer processing.By the selection of a small detector area and accurate detector alignment, a simple, repetitive photoelectronic signal representing ciliary activity was obtained. This signal records the ciliary beat frequency and demonstrates the triphasic nature of the beat cycle. The photoelectronic signal can be precisely correlated with the ciliary activity by analysis of the composite video recordings to provide the duration of the effective, recovery, and rest phases of the beat cycle. The videophotoelectronic signal correlations were verified by high-speed cinematography. High-speed films of ciliary activity were digitized, and the image density of selected pixels was analyzed by computer with respect to time and ciliary motion.These studies indicate that duration of the phages of the beat cycle are differentially reduced with increased beat frequency; the effective phase duration was quickly reduced to a minimum. This was followed by the reduction of the duration of the recovery phase to a minimum. The rest phase continues to be reduced without reaching a minimum, over the range of beat frequencies observed. These results suggest that ciliary beat frequency may be regulated either by modifying the rates of dynein-microtubule interactions or the rate of transition from one beat phase to the next.
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  • 20
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    Cell Motility and the Cytoskeleton 5 (1985) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 21
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    Cell Motility and the Cytoskeleton 5 (1985), S. 351-354 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 22
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    Cell Motility and the Cytoskeleton 5 (1985) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 23
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    Cell Motility and the Cytoskeleton 5 (1985), S. 447-461 
    ISSN: 0886-1544
    Keywords: chemokinesis ; orthokinesis ; klinokinesis ; polymorphonuclear leucocytes ; locomotion ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Evidence is presented to show that klinokinesis, which was previously demonstrated in bacteria and amoeba only, may also occur in metazoan cells. The chemotactic peptide formyl-Met-Leu-Phe (fMLP) elicited orthokinetic and klinokinetic responses of human blood-borne polymorphonuclear leucocytes (PMNs) under the test conditions used. Increased speed (orthokinesis) was due to an increase in the proportion of migrating cells as well as in the speed of the locomoting subset. The klinokinetic effect was manifested by a decrease in the klinolocomotion index, the mean angle of changes in direction ≥ 90°, and the frequency of turns ≥ 90°. The klinolocomotion index was inversely related to speed. This explains the synergistic effect of klinokinesis and orthokinesis in this system. Colchicine alone had and orthokinetic effect which was exclusively due to alterations in the proportion of migrating cells and it altered the turning behaviour without exerting a klinokinetic effect. However, colchicine had marginal orthokinetic and klinokinetic effects on fMLP-stimulated cells resulting in reduced translocation. The relationship between klinokinesis and mean angle or frequency of turns has been analysed. Klinokinesis was a substantial though not the major element of the chemokinetic response to fMLP under the conditions used. No other metazoan cells have been shown to possess such a complete pattern of responses, including orthokinesis, klinokinesis, and chemotaxis, which regulate locomotion.
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  • 24
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    Cell Motility and the Cytoskeleton 5 (1985), S. 529-543 
    ISSN: 0886-1544
    Keywords: actin ; regulatory protein ; cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have isolated a 30,000-dalton protein from Dictyostelium which cosedimented with and affected the low shear viscosity of actin. At low concentrations, this protein increased the low shear viscosity to greater than that of the actin control, whereas higher concentrations decreased viscosity. The viscosity decrease correlated with the formation of actin filament bundles, as seen electron microscopically. This protein resembled a previously reported actin-binding protein from Dictyostelium [Fechheimer and Taylor, 84, J Biol Chem 259:4514] in electrophoretic mobility, Stokes radius, and ability to crosslink filaments, but was shown to be different by peptide mapping, lack of immunologic crossreactivity, and lack of sensitivity to calcium.
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  • 25
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    Cell Motility and the Cytoskeleton 5 (1985), S. 507-527 
    ISSN: 0886-1544
    Keywords: axonal transport ; microtubules ; organelles ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A model for fast axonal transport is developed in which the essential features are that organelles may interact with mechanochemical cross-bridges that in turn interact with microtubules, forming an organelle-engine-microtubule complex which is transported along the microtubules. Computer analysis of the equations derived to describe such a system show that most of the experimental observations on fast axonal transport can be simulated by the model, indicating that the model is useful for the interpretation and design of experiments aimed at clarifying the mechanism of fast axonal transport.
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  • 26
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    Cell Motility and the Cytoskeleton 5 (1985), S. 137-173 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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    Cell Motility and the Cytoskeleton 5 (1985), S. 475-489 
    ISSN: 0886-1544
    Keywords: digital image processing ; flagella ; cilia ; bends ; Hemicentrotus ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A novel method of digital image analysis of the bends of eukaryotic flagella and cilia was devised. In the analysis system, all image pixels were systematically extracted and processed to measure angular direction and curvature. Simulation experiments on theoretical model pictures of flagella with sine-generated or arcstraight line bending waves demonstrated that the method can be used with considerable high accuracy. This method then revealed abrupt changes in slope of the curvature in sperm flagella and embryo cilia of the sea urchin, Hemicentrotus pulcherrimus. This indicates that the digital image processing used may be helpful in the study of flagellar and ciliary movements.
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    Cell Motility and the Cytoskeleton 5 (1985), S. 61-75 
    ISSN: 0886-1544
    Keywords: dynein ; erythro-9-[3-2-(hydroxynonyl)]adenine (EHNA) ; ATPase ; inhibition ; axoneme ; cytoplasm ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In current purification strategies, affinity for microtubules or calmodulin is used to identify and purify cytoplasmic dynein-like ATPase from cell-free extracts of unfertilized sea urchin eggs. However, affinity purification procedures, though they define dynein-like ATPase activity, have not yet proven to be quantitative. An alternative purification strategy capable of producing a high yield of enzyme would require a specific assay in order to monitor cytoplasmic dynein purity at each step.In this study, we make a detailed comparison of the effects of EHNA on 22 different ATP-metabolizing enzyme activities, including 13 Mg++-ATPases. We isolate cytoplasmic dynein-like ATPase activity from three species of sea urchin eggs and sperm and show by means of dose-response curves that their sensitivities to inhibition by EHNA are very similar to one another. We demonstrate further that the EHNA dose-response characteristics of fourteen other ATP-metabolizing enzyme activities, including seven nondynein Mg++-ATPases, differ quantitatively from those of dynein-like ATPases.In studies of three other agents (vanadate, Ca++/calmodulin, and Triton X-100), we find that dynein-like ATPases vary by two orders of magnitude in their sensitivities to inhibition by vanadate, and little or no stimulation by either Ca++/calmodulin or Triton X-100 is seen. Our results suggest that inhibition by EHNA is a universal and specific property of dynein-like ATPases, which ultimately should prove useful in the quantitative purification and characterization of cytoplasmic dynein-like ATPase (s).
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  • 29
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    Cell Motility and the Cytoskeleton 5 (1985), S. 103-122 
    ISSN: 0886-1544
    Keywords: gliding ; cell motility ; cytoskeleton ; diatoms ; capping ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Gliding motility was investigated in the marine diatom, Amphora coffeaeformis. Ultrastructural, biochemical, and pharmacological protocols were employed to probe the possible involvement of cytoskeletal proteins and a secretory process in gliding motility. Motility rate was measured using a video recording apparatus, and the effects of various cytoskeleton-disrupting drugs on motility were tested. Cytochalasins D and E, podophyllotoxin, and vinblastine (all at 25 μUg/ml) reversibly inhibited motility, as did monensin (10 μUM) and pronase (25 μUg/ml). Biochemical protein analysis of whole-cell extracts by one- and two-dimensional polyacrylamide gel electrophoresis revealed polypeptides comigrating with rabbit skeletal muscle actin and bovine brain tubulin; however, specific assays used to separate actin from whole-cell preparations gave ambiguous results. Ultrastructural studies revealed the presence of extracellular material between the raphe canal and the substratum in motile cultures. An assay was devised for the detection of radioactively labeled material (MW 〉 1800 Daltons) released by motile cultures into the culture medium. When cultures were treated with either an anticytoskeletal drug or monensin, motility was inhibited while the amount of measured radioactivity increased over solvent-treated control groups. The results from this study indicate possible roles for both actin- and tubulin-based structures in gliding motility of Amphora. Though secretion may be necessary for gliding to occur, its exact relationship to motility was not deduced. The data obtained in this study are compatible with a theory for the mechanism of gliding which involves the surface translocation of externally exposed membrane proteins against an immobile matrix of substratum-attached secreted material to generate the force required for movement.
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    Cell Motility and the Cytoskeleton 5 (1985), S. 123-136 
    ISSN: 0886-1544
    Keywords: locomotion and shape control ; epithelial cells ; calcium ; reflection-interference contrast-microscopy ; cinematography ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The role of calcium in the induction of locomotion, control of direction of locomotion, and modulation of shape of epithelial cells derived from Xenopus laevis tadpole epidermis is investigated. Local influx of calcium is achieved by electrophoretic release of small amounts of calcium from a micropipette (tip diameter 0.1-0.5 μUm) closely apposed to the cell body or lamella. The cells are made permeable for calcium by calcium ionophore A23187, and they are kept in Ca++-free, Mg++-rich EGTA Ringer. Another method used to induce Ca++ influx is local application of A23187 while cells move in normal culture medium.Influx of Ca++ into the lamella induces a localised increase in thickness and enlargement of the lamella. Stationary cells become active and show movement in the direction of the Ca++ gradient. Fried-egg-shaped cells tend to acquire a semicircular shape and start moving. Moving cells change the direction of their locomotion, following the direction of Ca++ release. Influx of Ca++ in the cell body region induces its contraction concomitant with an increase in lamellar area.These observations suggest the presence of two different Ca++-sensitive components: an actomyosin meshwork in the cell body and an actin gel in the lamella. Influx of Ca++ induces contraction of actomyosin and solation of actin gel. Interaction of these two systems would explain modulation of shape and generation of locomotion in epithelial cells.
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    Cell Motility and the Cytoskeleton 5 (1985) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 32
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    Cell Motility and the Cytoskeleton 5 (1985), S. 293-309 
    ISSN: 0886-1544
    Keywords: Mytilus edulis ; 5-hydroxytryptamine ; lateral cilia ; laterofrontal cirri ; beat frequency ; methylxanthine ; filter-feeding ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The laterofrontal (LF) cirri on isolated gill filaments of Mytilus edulis, prepared in natural seawater, are active and initially beat with an average frequency of about 8 Hz (with a range of 6-14 Hz). However, the lateral (L) cilia on these filaments are arrested in a position at the end of their recovery stroke. Perfusion of the filament with artificial seawater (ASW), with or without 1% ethanol, has little or no biological effect on the activity of the LF cirri, although a transitory decrease in frequency often accompanies the perfusion process. The L cilia remain arrested during perfusion with ASW. The exposure of the gill to low levels of 5-hydroxytryptamine (5HT) (10-8 〈 5HT 〈 10-7 M) has no effect on the activity of the LF cirri but stimulates the L cilia to beat. Exposure to higher concentrations of 5HT (〉 10-7 M) elevates the beat frequency of the L cilia and simultaneously inhibits the activity of the LF cirri, leading to their arrest in a position at the end of the effective stroke. This arrest of the LF cirri occurs as the L cilia attain a 5HT-induced beat frequency between 12 to 14 Hz. The influence of 5HT on the L cilia and the LF cirri can be reversibly mimicked or enhanced by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). A concentration of 0.5 mM IBMX mimics low 5HT concentrations (about 10-7 M) by stimulating the L cilia to beat without affecting the beat frequency of the LF cirri. A combimation of 10-7 M 5HT and 0.5 mM IBMX in ASW mimics high (〉 10-6 M) 5HT concentrations by arresting the LF cirri and increasing the beat frequency of the L cilia. Under these conditions, the threshold of the LF cirri arrest response is again found to occur as the L cilia attain a beat frequency of 12 - 14Hz. These results suggest that the mechanisms of LF cirri arrest and L cilia activation are mediated by 5HT -induced changes in intracellular cyclic AMP levels.
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    Cell Motility and the Cytoskeleton 5 (1985), S. 393-413 
    ISSN: 0886-1544
    Keywords: chromosome movement ; Colcemid ; nocodazole ; meiotic prophase ; microtubules ; vinblastine ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effects of Colcemid, nocodazole, and vinblastine on microtubules and on the movement of chromosomes during late diakinesis were investigated in spermatocytes of the crane fly Nephrotoma suturalis. The kinds of movements observed in untreated cells - sex bivalent rotations, sex bivalent excursions, and rotations and positional changes of autosomal bivalents - also were observed in drug-treated cells. These results were obtained in living cells in which the disruption and inhibition of microtubule assembly had been confirmed with polarized light microscopy. Effects of Colcemid and nocodazole also were assessed in fixed cells using electron microscopy. The results are in agreement with a hypothesis that microtubules are not a force-generating component of the molecular machinery that brings about prophase movements.
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    Cell Motility and the Cytoskeleton 5 (1985), S. 53-60 
    ISSN: 0886-1544
    Keywords: calcium ; Chlamydomonas ; flagella ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Ca2+ has profound effects on the movement of cilia and eukaryotic flagella, including those of Chlamydomonas. Two clear changes seen in Chlamydomonas flagella with changes in Ca2+ are beat frequency and symmetry. Photographic and computer assisted analysis of flagellar bending patterns on a uniflagellate mutant of Chlamydomonas have been used to examine details of the effects of Ca2+ on the movement of ATP-reactivated, demembranated flagella. In addition to the forward mode bending pattern seen at low Ca2+ concentrations (10-9 M), which has a frequency of about 50 Hz and the reverse mode bending pattern seen at high Ca2+ concentrations (10-4 M) with a frequency around 70 Hz, we carefully examined bending patterns in the intermediate Ca2+ concentration range of 1-6.5 × 10-6 M. In this intermediate range, the bending patterns have significantly reduced asymmetry and slightly increased frequency, compared to the motility observed at low Ca2+ concentrations. These observations indicate that changes in these two parameters of motion do not occur in parallel and suggest that the effects of Ca2+ may be a multicomponent process. Physiologically, these changes in the beat pattern at intermediate Ca2+ may signal either (1) the beginning stages of transition to the symmetrical, high-frequency beating seen at high Ca2+, or (2) a more normal forward mode motility for the trans flagellum as suggested by Kamiya and Witman [1984]. No large amplitude bending patterns associated with transitions between forward and reverse mode beating in intact cells were seen at the intermediate Ca2+ concentrations.
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    Cell Motility and the Cytoskeleton 5 (1985), S. 415-430 
    ISSN: 0886-1544
    Keywords: Actin ; immunofluorescence ; NBD-phallacidin ; Chlamydomonas ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have localized actin in gametes of Chlamydomonas reinhardi by two approaches: (1) indirect immunofluorescence with an affinity-purified antibody and (2) staining with NBD-phallacidin, a fluorescent reagent that binds only to F-actin [Barak et al, 1980, Proc Natl Acad Sci, 77:980-984]. Staining of either mating type “plus” (mt+) or “minus” (mt-) gametes with antiactin antibody resulted in similar fluorescent images: most of the actin was located peripherally along the lateral and posterior aspects of the cells. There was diffuse staining centrally, but the flagella did not stain. No brightly stained spot was observed near the mt+ mating structure, the site where the fertilization tubule elongates with concomitant polymerization of actin [Detmers et al, 1983, J Cell Biol, 97:522-532]. Gametes stained prior to mating with NBD-phallacidin showed no fluorescence above background, indicating that there were no concentrations of F-actin in these cells. This suggested that the cytoplasmic staining observed with antiactin represented primarily a nonfilamentous form of the protein. In mating gametes staining with NBD-phallacidin was detected only in the fertilization tubule, indicating that this was the only dense accumulation of filamentous actin within the cells. Mating gametes stained with antiactin antibody exhibited cytoplasmic fluorescence that was slightly more punctate than prior to mating, and the fertilization tubule was brightly stained. Our observations suggest that the site-specific polymerization of actin within the fertilization tubule occurs in the absence of a concentrated pool of actin subjacent to the mating structure.
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    Cell Motility and the Cytoskeleton 5 (1985), S. 545-557 
    ISSN: 0886-1544
    Keywords: neutrophils ; cytoskeleton ; actin polymerization ; NBDphallacidin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The studies presented here characterize a simple, quantitative NBDphallacidin extraction assay for determining the F-actin content of fMLP-activated neutrophils. The NBDphallacidin extraction assay is based upon the specificity of NBDphallacidin binding to F-actin and the solubility of NBDphallacidin in methanol. Cells are fixed, permeabilized, and stained with NBDphallacidin; the cells are then pelleted, the bound NBDphallacidin is extracted into methanol, and the RFI (excite 465; emit 535) of the solution is determined. Binding of NBDphallacidin to neutrophils is saturable and 90% of bound NBDphallacidin is displaced by nonfluorescent phalloidin. The extraction of bound NBDphallacidin into methanol is complete and the excitation/emission characteristics of NBDphallacidin are not altered by extraction. The assay is relatively inexpensive, applicable to the study of cells in suspension or on substratum, allows kinetic studies with 5-10s time resolution, and is not affected by the shape of the cell or the distribution of the probe. We used the NBDphallacidin extraction assay to study the kinetics of fMLP-induced change in the F-actin content of neutrophils and the effect of tBOC peptide, an inhibitor of fMLP binding, on these changes. The extraction assay reveals a rapid, sequential fMLP-induced increase followed by a decrease in F-actin content. The tBOC peptide inhibits fMLP-induced actin polymerization. Addition of tBOC during fMLP-induced polymerization or at times when F-actin content is maximal enhances F-actin depolymerization. The rate of F-actin depolymerization is ≥ fourfold faster in the presence than in the absence of tBOC. The results show that (1) The NBDphallacidin extraction assay is useful for studying the kinetics of change in F-actin content of nonmuscle cells; (2) fMLP receptor occupancy is required for fMLP-dependent polymerization but not depolymerization; and (3) both the actin polymerizing and depolymerizing processes are active in the cell within 5 s after fMLP stimulation. Implications of these observations for understanding the observed increase and, then, decrease in F-actin content of fMLP-activated cells are discussed.
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    Cell Motility and the Cytoskeleton 5 (1985), S. 311-322 
    ISSN: 0886-1544
    Keywords: Spectrin ; TW 260/240 ; chicken intestinal brush border ; actin assembly ; actin filament cross-linking ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: TW 260/240 is a tissue-specific spectrin found in the terminal web region of the chicken intestinal bruish border. We have examined the effects of TW 260/240 on assembly rates and critical concentrations (Co's) for monomer addition at the barbed and pointed ends of the actin filament. For these studies, acrosomal processes (AP) from Limulus sperm were used as nuclei for actin assembly. Under conditions which favor the interaction of TW 260/240 for actin (20-75 mM KCl, 2 mM Mg++) no effect on either elongation rates or Co's at either end of the actin filament was observed in the presence of this spectrinlike protein. The Limulus AP nucleation assay also allowed visualization of the kinetics of filament binding and cross-linking by TW 260/240. Ultrastructural analysis of TW 260/240 binding to actin filaments at their growing ends indicates that TW 260/240 tetramers bind laterally to the filament. Finally, evidence is presented that indicates that filaments cross-linked by TW 260/240 are stabilized against shear-dependent breakage.
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    Cell Motility and the Cytoskeleton 5 (1985), S. 323-331 
    ISSN: 0886-1544
    Keywords: Tetrahymena ; cell model ; ATP concentration ; Ca sensitivity ; backward swimming ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Using Tetrahymena pyriformis, strain w, and Tetrahymena thermophila, B-1868, we prepared cell models that showed ciliary reversal with change in Ca-ion concentration, as was also noted for the Paramecium cell model. No differences could be found between these two strains in the reactivation state, and their response to environmental conditions was essentially the same. The reactivation rate was 90% or more. Swimming velocity of the cell model was found to be 200 ± 49 μm/sec at 25.0°C ± 0.5°C, while the velocity for the living cells was 527 ± 101 μm/sec. Swimming velocity with change in environmental conditions, such as pH, Mg-ATP, and Ca-ion concentrations, was studied. Compared to the cell model of Paramecium, the Tetrahymena cell model had higher sensitivity toward Ca-ion in the reactivation medium. The effects of chlorpromazine, and inhibitor of calmodulin, on the swimming behavior of the cell models were studied.
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    Cell Motility and the Cytoskeleton 5 (1985), S. 431-446 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubules assembled from sea urchin eggs with the use of taxol contain a 77,000-dalton protein as the major nontubulin component [Vallee and Bloom (1983): Proc Natl. Acad. Sci. U.S.A. 80:6259-6263]. We have raised five monoclonal antibodies to this protein to aid in its characterization. Immunoblot analysis of the sea urchin microtubule purification fractions indicated that the protein copurified quantitatively with microtubules. All five antibodies stained the mitotic spindle of dividing sea urchin eggs by immunofluorescence microscopy, indicating that the protein was a component of the mitotic spindle and suggesting that it was actually localized on microtubules in vivo. Immunofluorescent staining of higher resolution was observed in a subpopulation of the coelomic cells found in adult sea urchins, confirming that the 77,000-dalton protein is indeed present on microtubules in vivo. Because taxol was not used for the immunofluorescence experiments, we conclude that the microtubule-associated protein (MAP)-like behavior of the 77,000-dalton protein in vitro was not induced artifactually by taxol. To determine whether this protein is a component of sea urchin microtubules in general, cilia obtained from blastula stage embryos and sperm tail flagella were analyzed with the antibodies. The protein was undetectable by both immunoblot analysis and immunofluorescence microscopy in both preparations of axonemal microtubules. These results indicated that the 77,000-dalton MAP is restricted to cytoplasmic and mitotic microtubules in the sea urchin. Furthermore, in view of its particular abundance in embryos, whose microtubules are devoted substantially to mitosis, the 77,000-dalton MAP is likely to play an important role in regulating the activity of mitotic spindle microtubules in the sea urchin.
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    Cell Motility and the Cytoskeleton 5 (1985), S. 251-263 
    ISSN: 0886-1544
    Keywords: Chlamydomonas ; movement ; flagellar beat ; stigma ; high-speed microcinematography ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Using high-speed microcinematography (100-500 f/s) the movement of Chlamydomonas reinhardtii mutant 622 E has been studied with frame-by-frame analysis. The stigma lies in the cell equator, displaced out of the flagellar plane anticlockwise by an angle of about 45°. During forward movement the cells rotate anticlockwise about their long axis with a frequency of 1.4-2 Hz (maximum 2.5 Hz). The rotation is caused by a lateral component of 3-dimensional beating of the flagella during the effective strokes. The helical swimming path is a result of an unequal flagellar beating. This is normally synchronous, but synchrony is interrupted from time to time by an additional beat of the outward directed flagellum, in our study one after about every twenty beats on average. These results are discussed and compared with the results published by other groups.
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    Cell Motility and the Cytoskeleton 5 (1985), S. 355-375 
    ISSN: 0886-1544
    Keywords: Newt ; lung ; cilia ; cell models ; ciliary coordination ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Demembranated ciliated cell models are useful for studying mechanisms responsible for the regulation of ciliary coordination and waveform. This paper describes procedures for isolating ciliated cells from the newt, Taricha granulosa, by trypsin dissociation, their subsequent demembranation by Triton X-100, and their reactivation with MgATP to produce highly motile, coordinated, ciliated cell models. Reactivation of cell models with a high degree of mechanochemical coupling depended on avoiding mechanical damage and maintaining optimal conditions during all stages of isolation and reactivation. Highly motile models were prepared from cells incubated in trypsin, treated briefly with EDTA, separated by gentle agitation, and concentrated by centrifugation at low gravitational forces. Optimal demembranation and reactivation conditions were similar to those described previously for isolated newt lung axonemes. Under these conditions, nearly 100% of the models were reactivated when provided with MgATP and 90-95% beat with coordinated waves. The ciliary tufts beat at frequencies within the range measured in living cells and their reactivated motility was stable for at least 30 min at constant MgATP. These highly coupled models were used to show (1) that development of coordination in the ciliary tuft occurs at a higher substrate concentration range (10-25 μM) than that required to initiate motility per se (2-10 μM); (2) that outer dynein arms may not contribute to beat frequency at substrate concentrations below 35 μM; and (3) that vanadate has effects both on beat frequency and coordination of the tufts.
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  • 42
    ISSN: 0148-7280
    Keywords: spermatozoa ; acrosomal membrane ; membrane antigens ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Antiserum to purified boar spermatozoan outer acrosomal membrane (OAM) was raised in rabbits and adsorbed with boar liver and serum glutaraldehyde cross-linked immunoadsorbents. The IgG fraction of the antiserum was purified by (NH4)2SO4 precipitation followed by ion-exchange chromatography. Indirect immunofluorescence showed bright fluorescent staining of the acrosomal cap of boar spermatozoa and to a lesser extent of the acrosomes of bull and goat spermatozoa after incubation with anti-OAM-IgG. Immuno-electron microscopy further confirmed the specificity of the antibody for the OAM. Preincubation of the anti-OAM-IgG with isolated OAM, completely abolished its reactivity. When tested by ELISA, anti-OAM-IgG reacted with boar, bull, goat, and human spermatozoa; however, its binding activity to boar spermatozoa was significantly greater as compared to spermatozoa from the other species tested.In an effort to identify OAM antigens recognized by this antiserum, the isolated boar OAM was labeled either with 3H or with 125I and solubilized by mild detergent treatment. The extracted components were immunoprecipitated with anti-OAM-IgG and protein A-bearing S. aureus and the thus isolated antigens were analysed on SDS-PAGE. The results suggest that anti-OAM-IgG recognized one high molecular 3H-labeled glycoprotein (270 kd), and four 125I-labeled polypeptides of lower molecular weight of the boar OAM.
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    Gamete Research 11 (1985) 
    ISSN: 0148-7280
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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    Gamete Research 11 (1985), S. 169-178 
    ISSN: 0148-7280
    Keywords: spermatozoa ; motility ; fowl ; turkey ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An objective method of estimating the motility of fowl and turkey spermatozoa, depending on their rheotactic and light-scattering properties, has been developed. From a 1- to 2-minute recorder trace of the optical density of diluted semen before and after stopping its passage through a flow cuvette, three independent constants may be simply and graphically determined. These are: ODm, the maximum optical density of semen flowing through the cuvette, shown to be dependent on the concentration of spermatozoa; % (ΔOD)m, the maximal change of optical density following cessation of flow, which has been correlated with the percentage of motile spermatozoa in the sample; and t½, the time taken for the change of optical density to reach % (ΔOD)m. This latter parameter has been correlated with forward motility of both fowl and turkey spermatozoa.
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    Gamete Research 11 (1985), S. 223-236 
    ISSN: 0148-7280
    Keywords: oocytes ; maturation ; amphibian urodele ; glycogen ; exocytosis ; annulate lamellae ; vesicles ; electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The repartition and fate of glycogen β has been followed during progesterone-induced maturation of amphibian oocytes. The use of specific staining, both at the cytological and ultrastructural level, demonstrates that glycogen tends to be extruded from the oocyte during maturation of the urodeles Pleurodeles waltlii and Ambystoma mexicanum. No such effect of the hormone is observed in Xenopus laevis, where only a slight centrifuge migration of the glycogen could be recorded.Stacks of annulate lamellae increase during the early phase of in vitro progesterone-induced maturation (2 to 9 hours after progesterone application). After germinal vesicle breakdown (about 12 hours after beginning the progesterone treatment) annulate lamellae have disappeared and numerous masses of vesicles are present in the cytoplasm of Pleurodeles and Ambystoma matured oocytes. We never observed any close relation between the annulate lamellae and these vesicles.
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    Gamete Research 11 (1985), S. 271-281 
    ISSN: 0148-7280
    Keywords: GPI-1 ; oocyte growth ; in vitro culture ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The activity of the enzyme glucose-phosphate isomerase (GPI-1) in mouse oocytes is subject to regulation by the cis-acting gene Gpi-lta. Electrophoretic analysis of oocytes from 9- and 10-day-old mice showed that oocyte-specific regulation of GPI-1 is not observed in germ cells that have not started to grow (20 μm diameter) but appears as soon as oocyte growth begins (30 μm or larger). Three in vitro culture systems were used to examine the relation of GPI-1 expression to oocyte growth: culture of intact neonatal ovaries, and co-culture of dissociated oocytes and somatic cells from neonatal and from 13-day foetal ovaries. In all three systems modification of GPI-1 expression always occurred when oocyte growth began, showing that the presence of a normal follicle is not necessary for the expression of the gene Gpi-lta.
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  • 47
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    Gamete Research 11 (1985), S. 305-309 
    ISSN: 0148-7280
    Keywords: vitellus ; egg ; block to polyspermy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The block to polyspermy in golden hamster eggs is believed to operate only at the zona pellucida. However, changes in the egg vitellus also prevent further entry of capacitated sperm. When zona-free hamster eggs spontaneously activated in vitro, and in vivo fertilized eggs at pronuclear stage were inseminated with capacitated human sperm, penetration did not occur. In the case of a homologous system using hamster sperm and in vivo fertilized hamster eggs, slight attachment of sperm was observed but no penetration. The cortical granules were found to be released in spontaneously activated and in fertilized eggs as observed by phase contrast microscopy. These observations suggest that the egg vitellus plays a role in the block to poiyspermy in addition to that of the zona block.
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  • 48
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    Gamete Research 12 (1985), S. 301-304 
    ISSN: 0148-7280
    Keywords: sex ratio ; fast- and slow-developing embryos ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We cultured eight-cell mouse embryos to blastocyst stage, divided them into three groups according to the time of blastocoel formation, and transferred them separately into recipients. The proportion of live young from the fast-developing embryos was slightly high (49%) but not significantly different from those of other embryos (38% and 39%). However, the sex ratio of live young from the fast- and slow-developing embryos was significantly shifted toward the male (71%) and to the female (80%), respectively.
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  • 49
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    Gamete Research 12 (1985), S. 327-327 
    ISSN: 0148-7280
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 50
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    Gamete Research 12 (1985), S. 345-355 
    ISSN: 0148-7280
    Keywords: sperm ; acrosome reaction ; glycosaminoglycans ; seminal plasma ; heparin ; binding ; chondroitin sulfate ; heparan sulfate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Previous reports indicate that glycosaminoglycans (GAGs) would enhance the occurrence of acrosome reactions in sperm in vitro, but continuous exposure of those sperm to seminal plasma prevented a significant incidence of acrosome reactions. This study was designed to evaluate the interaction of GAGs and seminal plasma to promote acrosome reactions in bull sperm in vitro. Epididymal sperm required 22 hr to exhibit acrosome reactions in response to GAGs whereas only 9 hr were needed to achieve the same effect with washed ejaculated sperm. Exposure of epididymal sperm to seminal plasma for 20 min shortened the time for induction of the acrosome reaction to 9 hr. Scatchard analyses of displacement data suggested an alteration in the binding affinity of 3H-heparin to epididymal sperm membrane following the short-term exposure to seminal plasma. High doses (250 and 500 μg/ml) of heparin, heparan sulfate, and chondroitin-4-sulfate were without effect, but doses 〈100 μg/ml were stimulatory in terms of enhancing acrosome reactions. Compositional studies with seminal plasma revealed a total GAG content of 1.6 mg/ml, proportioned as 61.6% chondroitin sulfates, 17.6% heparin-like material, 0.3% hyaluronic acid, and 20.5% undetermined GAG. It is proposed that seminal plasma can alter the ability of sperm to respond to GAGs, and the high concentrations of GAGs endogenous to seminal plasma may prevent premature initiation of the membrane perturbations necessary for the acrosome reaction.
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  • 51
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    Gamete Research 11 (1985), S. 29-40 
    ISSN: 0148-7280
    Keywords: spermatozoa ; acrosome reaction ; lysolecithin ; guinea pig ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: When guinea pig spermatozoa are preincubated for 1 hr in Ca2+-free medium containing a low concentration of lysolecithin (LC, 85 μg/ml) and then exposed to 2 mM Ca2+ by diluting the preincubation medium with an equal volume of LC-free, 4 mM Ca2+-containing medium, the majority of the spermatozoa undergo acrosome reaction promptly. On the other hand, when the preincubated spermatozoa are exposed to 2 mM Ca2+ without reducing the original concentration of LC in the medium, none of them undergo acrosome reaction. These spermatoza can acrosome-react if they are transferred to an LC-free medium. These results and those of some other experiments suggest that in the presistent presence of a high concentration of LC in the medium, exogenous Ca2+ essential for the acrosome reaction either does not penetrate the sperm plasma membrane or, if it does, it cannot alter the membrane for the acrosome reaction, at least under the experimental conditions employed. Freeze-fracture examination of the sperm plasma membrane has revealed that small areas or patches free of intramembranous paarticles (IMPs) appear in the membrance during sperm preincubation, and these IMP-free areas expand drastically in response to Ca2+ when the LC conccentration in the medium is reduced at the time Ca2+ is added to the medium. In contrast, IMP-free areas remain unchanged even after exposure of spermatozoa to Ca2+ if the concentration of LC remains at its original level of 85 μg/ml.
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  • 52
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    Gamete Research 11 (1985), S. 59-68 
    ISSN: 0148-7280
    Keywords: zona solubility ; fertilization rate ; mouse ; embryonic development ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effects of in vitro aging of cumulus-intact versus cumulus-free metaphase II mouse oocytes were studied with respect to zona solubility and fertilization rates. Furthermore, zygotes from the in vitro fertilization studies were incubated and their developmental progress was recorded. The zona pellucida showed a gradual increase in resistance to dissolution by α-chymotrypsin with in vitro aging over a period of 6 hr. This effect was greater in cumulus-free as compared to cumulus-intact ova, but it was not nearly as profound as that seen in the control in vivo fertilized eggs. The fertilization rate of in vitro aging cumulus-intact ova compared favorably with the control in vivo aging group over a 6-hr time period. This was in sharp contrast to the decreased fertilization rate of in vitro aging cumulus-free ova over the same period of time. Lastly, development of zygotes to the blastocyst stage was also evaluated. The rate of first cleavage was similar in all experimental groups and compared favorably with the in vivo controls. However, further development to blastocysts of in vitro aged cumulus-free ova showed a marked decrease when compared to the cumulus-intact group and the in vivo fertilized controls. Thus we established a direct relationship between zona digestion time of in vitro aged cumulus-free oocytes and a decrease of fertilization rates in the mouse.
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  • 53
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    Gamete Research 11 (1985), S. 121-131 
    ISSN: 0148-7280
    Keywords: zona pellucida ; scanning electron microscope ; gamete interactions ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Hamster and mouse capacitated spermatozoa were interacted in vitro with hamster and mouse eggs in homologous and heterologous combinations. Also, fertilized and trypsin treated unfertilized hamster eggs, and unfertilized rat eggs were made to interact with capacitated hamster spermatozoa. The surface of the zona pellucida was then examined with the scanning electron microscope. It was found that sperm attachment, followed by sperm binding and penetration through the zona pellucida, was observed only when homologous gamete combinations were used. Binding of the spermatozoa to the zona was evidenced by the lytic effect of the acrosomal enzymes on the zona substance. When fertilized eggs and trypsin-treated unfertilized hamster eggs were mixed with capacitated hamster spermatozoa as well as in the heterologous gamete combinations, we found that the spermatozoa were able to establish attachment but not binding. Under these conditions the outer surface of the zona pellucida was never found to have penetration tracks made by the spermatozoa. Failure of heterologous spermatozoa to cross the foreign zona pellucida is believed to be associated with the inability of the foreign spermatozoa to establish binding and to the inability of their acrosomal enzymes to digest the zona. A similar mechanism is believed to work in zona-reacted and in trypsin-treated hamster eggs inseminated in vitro with homologous spermatozoa.
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  • 54
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    Gamete Research 11 (1985), S. 157-167 
    ISSN: 0148-7280
    Keywords: acrosomal reaction ; fertilization ; Asteroidea ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: When the spermatozoon of M glacialis contacts the mature oocyte jelly it adheres to it. Following this, there is a slight tumefaction of the acrosome, which is followed by the disruption of the apical acrosomal vesicle and cytoplasmic membranes. Acrosomal vesicle contents are liberated and spread along the outer surface of the oocyte jelly. Meanwhile, the acrosomal process begins to extend, penetrates all the jelly extension, then the vitelline layer, and finally contacts the cytoplasmic egg membrane. Nevertheless, the sperm cell continues lying at the outer border of the jelly. From the beginning of the acrosome reaction the dense and finely fibrillar subacrosomal material is connected, by some expansions, to the basal acrosomal vesicle membrane. Both nuclear and mitochondrial diameters have diminished.
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  • 55
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    Gamete Research 11 (1985), S. 253-259 
    ISSN: 0148-7280
    Keywords: TEST-yolk buffer ; capacitation ; human sperm chromosomes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Human sperm chromosomes were obtained after capacitation with TES-Tris (TEST) yolk buffer and fusion with Syrian hamster eggs. Semen samples could be stored at 4°C for 3 days and remain functional in the assay system. The efficiency of TEST yoik buffer for obtaining karyotypes was as good as, or greater than, the efficiency of standard BWW medium containing human serum albumin.
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  • 56
    ISSN: 0148-7280
    Keywords: sperm ; membrane ; plasma membrane ; polypeptides ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In this study a variety of properties of boar sperm plasma membrane proteins were examined. A qualitative and quantitative analysis of proteins washed from boar sperm revealed that large numbers and a variety of polypeptides (Ps) are easily removed from sperm upon washing. Initially (by the second wash), Ps are released from the plasma membrane (PM) of epididymal sperm and primarily correspond to those in epididymal fluid, but eventually (fifth wash) Ps are released that are not seen in epididymal fluid nor as components of the PM. These Ps appear to originate from the sperm cytosol and signal the damaging effects of extensive washing on sperm. Upon washing, ejaculated sperm release Ps characteristic of both epididymal fluid and accessory sex glands. Epididymal Ps are almost completely released by the fourth wash; accessory gland proteins appear to be more tenaciously bound and continue to be released with further washing. Most basic accessory gland Ps bind strongly enough to resist the series of washes necessary for the preparation of PM vesicles. About one-half of ejaculated sperm lose motility after five washes, but evidence of massive release of internal Ps, such as seen in epididymal sperm, is not noted. In the epididymis and after ejaculation, sperm are coated with numerous Ps which are released upon washing; many are released nonspecifically and rapidly, others are more firmly bound. These analyses extend the surface map of boar spermatozoa to include a description of loosely bound proteins and their origin. These results also indicate that the qualitative and quantitative changes in surface membrane protein composition, occurring after simple washing, are significant and may confound the interpretation of surface composition changes in studies which rely solely on immunological or radiolabelling procedures.In order to determine the nature of the binding of major polypeptides (Ps) to the lipid bilayer of boar sperm plasma membranes (PMs), the solubility of Ps in solutions of different ionic strength and in detergents was examined. Several major polypeptides (identified in previously published surface maps) were extracted by hypotonic and hypertonic salt solutions, suggesting that electrostatic interactions play a major role in their binding to the bilayer. Other major proteins were extracted only by detergents, suggesting that these proteins are embedded deeply into the bilayer. These extraction procedures also provided a new strategy for isolating specific Ps in large quantities. Radiolabelling procedures identified about 80 surface-exposed Ps, some of which are major constituents of the PM and others which are quantitatively minor components. Labelling of PM vesicles reveals about sixfold more Ps than does labelling of whole sperm. Increased labelling appears to be the result of surface accessibility of PM constituents after removal of loosely bound Ps from epididymal fluid and seminal plasma during the washings which accompany the preparation of PM vesicles from whole sperm. These results prescribe caution when interpreting changes in surface organization and membrane structure which are dependent solely on the use of radiolabels.
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  • 57
    ISSN: 0148-7280
    Keywords: in vitro fertilization ; human embryo ; embryo culture ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Between July 1982 and November 1983, two pregnancies were established using in vitro fertilization and embryo transfer (IVF and ET) procedures with three different schedules to induce follicular maturation. All women were cycling normally and had inoperable or absent fallopian tubes. Of 83 oocytes aspirated from 24 patients (31 cycles), 75% were considered mature and 25% immature by the morphological characteristics of the oocytes and cumulus cells. Oocytes were preincubated for 6-24 hours, and after insemination, 60% cleaved to the two-to-four-cell stage. The superovulation induction schedule employing hMG administered according to the individually adjusted treatment scheme established two pregnancies. This schedule was considered the superior regimen, as it gave the highest proportion of mature oocytes (89%) which cleaved (78%). The pregnancy-attaining follicle showed a high progesterone:estradiol-17β ratio (P4/E2) in its microenvironment of aspirated follicular fluid, culture media of granulosa cells, and oocyte-cumulus complex. Our observations indicate a high P4/E2 ratio in the pregnancy-attaining follicle, and thereby reflect a further parameter in influencing maturation of the oocytes most likely to implant.
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  • 58
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    Gamete Research 11 (1985), S. 311-319 
    ISSN: 0148-7280
    Keywords: oocytes ; cortical granule ; pig ; ultrastructure ; maturation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The structure, number, and distribution of cortical granules in porcine oocytes during maturation induced by human chorionic gonadotrophin (HCG) are reported.The number of granules remained constant for 30 hr following HCG. Thereafter, there was an approximate doubling by 50 hr. At all times examined, a dark and light form were present. Up to 40 hr the latter predominated while at 50 hr there was a marked increase in the number of the former. Movement of cortical granules to the plasma membrane took place between 20 and 30 hr post-HCG. The changes in cortical granules are correlated with the capacity of the oocytes to undergo a block to polyspermy.
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  • 59
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    Gamete Research 11 (1985), S. 330-330 
    ISSN: 0148-7280
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 60
    ISSN: 0148-7280
    Keywords: sperm maturation ; epididymis ; chromatin ; Feulgen-DNA ; uterine fluid ; cytophotometry ; ram ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Ram spermatozoa taken from the epididymal head, body, or tail or from the ejaculate were examined by microspectrometry after incubation in vitro with ewe uterine fluids at 37°C for 20 hours. Compared with incubation in Ringer's solution, uterine fluid incubation resulted in a decrease in nuclear Feulgen-DNA content. This decrease was greater for more immature spermatozoa (29.0 and 47.3% for spermatozoa from head and body, respectively) than for more mature spermatozoa (17.7 and 4.0% for spermatozoa from the tail and the ejaculate, respectively). In parallel with this decrease, there was a condensation of the chromatin which resulted in a decreased nuclear surface area, especially in spermatozoa taken from the epididymal body. Therefore, it would appear that, during epididymal maturation, changes in the ability of spermatozoa to maintain embryonic development as the spermatozoa mature are due to changes in chromatin structure.
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  • 61
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    Gamete Research 11 (1985), S. 349-365 
    ISSN: 0148-7280
    Keywords: spermatozoa ; mammalian ; fertilization ; mammalian ; nucleus ; spermatozoon ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 62
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    Gamete Research 11 (1985), S. 379-389 
    ISSN: 0148-7280
    Keywords: spermatozoon ; acrosome ; binding ; Ciona intestinalis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: This paper describes a study of the apical region of the spermatozoon of Ciona intestinalis before and during its binding to the vitelline coat of the egg. A combination of the techniques of thin sectioning, negative staining, and freeze fracture has revealed that in the apical-most region, where a small acrosomal vesicle lies on the flat tip of the nucleus, there is a cap-like region almost completely free of particles on the P face of the plasma membrane. The particle-free area is surrounded by two circlets of orderly arranged particles. Upon binding to the vitelline coat the particles of the distal circlet show a partial displacement, while the particles of the apical circlet remain unaltered. The relationship between the apical circlet and the binding process is discussed. The final step of the acrosome reaction, which occurs in only a few of the bound spermatozoa, consists in the fusion of the plasma membrane with the acrosomal membrane, in the dehiscence of the acrosomal contents and finally in the formation of membrane tubules.
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  • 63
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    Gamete Research 12 (1985), S. 75-84 
    ISSN: 0148-7280
    Keywords: rat ; enzyme ; esterase ; cumulus digestion ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Rat caproyl esterase (E.C.3.1.1.1), extracted from testis with Tween 80, was purified by cation exchange and lectin affinity chromatography. The 104-fold purified enzyme had an activity of 840 μmol/hr per mg protein.The purified esterase did not contain any hyaluronidase or N-acetyl-glucosaminidase activity. Electrophoresis on sodium dodecyl sulfate polyacrylamide gels revealed a single band of approximately 60,000 molecular weight. The esterase had an isoelectric point of 5.1. Inhibition experiments showed high sensitivity of the enzyme to sulfhydryl agents and complete inactivation by sodium aurothiomalate.The purified caproyl esterase was shown to digest the cumulus matrix from mouse ova.
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  • 64
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    Gamete Research 12 (1985) 
    ISSN: 0148-7280
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 65
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    Gamete Research 12 (1985), S. 101-116 
    ISSN: 0148-7280
    Keywords: fertillization ; spermatozoon ; gamete interaction ; zona pellucida ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In order to identify those proteins from the plasma membrane of hamster spermatozoa that exhibit an affinity for components of the zona pellucida we have used the Western blot technique. Zonae pellucidae from postovulatory hamster oocytes were solubilized by exposure to an acidic pH and then radiolabeled using the Bolton-Hunter reagent. These 125I-zona pellucida proteins retain their immunoreactivity and migrate in three heterogeneous bands when submitted to SDS-PAGE electrophoresis. Membrane proteins from epididymal spermatozoa of mature hamsters were extracted by treatment with Nonidet P-40 and then submitted to electrophoresis (SDS-PAGE). The proteins in the gel were electrophoretically transferred to a nitrocellulose membrane and then probed with the radiolabelled zone pellucida proteins. 125I-zona pellucida proteins bind preferentially to a sperm protein with a molecular weight of 26,400 ± 1,400 daltons (n = 9). Using a similar procedure it was shown that this protein also binds 125I-Concanavalin A. The interaction between the sperm protein and the 125I-zona pellucida proteins shows species specificity as demonstrated by the fact that the hamster 125I-zona pellucida proteins do not bind to proteins extracted from ram, bull, and stallion spermatozoa. Whether this sperm protein could be implicated be implicated in the process of sperm-egg interaction is under investigation.
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  • 66
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    Gamete Research 12 (1985), S. 151-163 
    ISSN: 0148-7280
    Keywords: aging sperm ; mouse zygotes ; chromosome anomalies ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The chromosome complement was studied in first-cleavage metaphases of mouse zygotes resulting from sperm aged in the male physiologically, after sexual rest. Females were inseminated by control males mating at 3-day intervals while experimentals mated to males that had had a sexual rest of 14 or more days. A total of 1954 eggs were collected 33-35 h post-HCG from 101 superovulated females mated to 42 controls and 43 experimental males. The fertilization rate was similar in both groups, being 84% and 85%, respectively. G-banded or Q-banded chromosomes were analyzed in 301 (68.3%) controls and 392 (49%) experimental first-cleavage metaphases. The overall rate of chromosome anomalies in controls was 4.45% as compared to 10.94% in experimentals, a highly significant difference. In the experimental group compared to controls, the frequency of trisomy, triploidy, structural rearrangements, and tetraploidy increased from 3.9% to 6.9%, 0% to 1.6%, 0.8% to 2.8%, and 0% to 1.3%, respectively. The genomic source of origin of the abnormalities was determined on the basis of differential condensation of the genomes. In the experimentals, grossly unbalanced sperm (diploids, disomics, double disomics, and those with large fragments) fertilized significantly more oocytes compared to controls. Our results implicate an advantage either in numbers or fertilizing capability for chromosomally abnormal sperm in a physiologically aged population.
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  • 67
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    Gamete Research 12 (1985), S. 225-225 
    ISSN: 0148-7280
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 68
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    Gamete Research 12 (1985), S. 385-398 
    ISSN: 0148-7280
    Keywords: in vitro fertilization ; pronuclear ova ; one-cell embryo ; monospermy ; polyspermy ; syngamy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The fine structure of pronuclear ova (monospermy and polyspermy) and one-cell embryos has been investigated in our IVF programme. Sixteen oocytes were collected at laparoscopy after appropriate hormonal stimulation and were matured and fertilized in vitro by methods that have given rise to normal pregnancies.Pronuclear ova showing monospermic fertilization had two vesicular pronuclei surrounded by aggregations of cellular organelles. The male pronucleus was closely associated with a sperm axoneme, while the female pronucleus was dismantling its envelope and condensing its chromatin ahead of its counterpart in late pronuclear ova. Each pronucleus had dispersed chromatin, dense compact nucleoli, and intranuclear annulate lamellae. Smooth endoplasmic reticulum, annulate lamellae, Golgi complexes, and mitochondria formed a conspicuous part of the perinuclear ooplasm. The one-cell embryos were either in syngamy or in the process of undergoing first cleavage. Positive evidence of cortical granule release and second polar bodies were detected in the perivitelline space. A block to polyspermy seemed to operate at the level of the inner zona.Dispermic and polyspermic ova had 3-16 pronuclei resembling those of monospermic ova and had sperm tails in the ooplasm. Sperm were also seen penetrating the inner zona and were occasionally found in the perivitelline space. Incomplete cortical granule release and early signs of cytoplasmic fragmentation were noted in polyspermic ova.Both normal and abnormal features of these ova are reported and compared with pronuclear structure in vivo and in vitro.
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  • 69
    ISSN: 0148-7280
    Keywords: spermatozoa ; acrosome reaction ; proteolytic enzyme ; hamster ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The involvement of a kallikrein-kinin system in the motility of mammalian spermatozoa has been suggested by several investigators. We found that incorporation of kallikrein (0.1-1.0) unit/ml) in the sperm incubation medium did not enhance the motility of hamster spermatozoa that were already active. However, this enzyme significantly increased the incidence of the acrosome reaction. Trypsin (1.8-18 units/ml) and chymotrypsin (0.34-3.4 units/ml) also increased the incidence of the acrosome reaction, and accelerated its onset. Kinins (bradykinin and kallidin) added to the medium in a wide concentration range (1 ng/ml to 1 mg/ml) had no marked effects on either the motility or the acrosome reaction. A kallikrein-kinin system is apparently not of primary importance at least for the acrosome reaction. The enhancement of the acrosome reaction by exogenous proteinases may be due in part to accelerated removal or alteration of the sperm surface coat (glycoprotein) by the enzyme peior to the acrosome reaction. Exogenous proteinases may also act synergistically with endogenous (acrosomal) proteinases (and other enzymes) in altering membrane proteins and dispersing the acrosome matrix during the course of teh acrosome reaction.
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  • 70
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    Gamete Research 11 (1985), S. 51-58 
    ISSN: 0148-7280
    Keywords: preimplantation embryo ; electron microscopy ; blastomeres ; cell adhesion ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In this report we describe a new method of immobilizing intact mouse preimplantation embryos and their dissociated blastomeres on a flat substrate so that they may be processed en masse for electron microscopy. The embryos can be arranged so that they will be coplanar and grouped together closely enough to be included within the same plane of section. The substrate consists of Thermonex plastic coverslips that have been coated with a layer of fibroblasts or with fibronectin. The plant lectin phytohemaggiutinin (PHA) is used as a ligand to adhere the embryos onto the coverslips. This method is compatible with other ligands and with a variety of immunocytochemical procedures and can be used with live or fixed embryos.
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  • 71
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    Gamete Research 11 (1985) 
    ISSN: 0148-7280
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 72
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    Gamete Research 11 (1985), S. 107-119 
    ISSN: 0148-7280
    Keywords: oocyte maturation ; follicular maturation ; cumulus maturation ; surrogate follicles ; xenogenous ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Pig oocytes obtained from slaughterhouse material and rat oocytes obtained from PMSG-treated immature females were incubated as isolated oocytes or injected into explanted pig follicles (5-8 mm). Free oocytes of both species, with or without their cumulus investment or gonadotropins during culture, matured at high rates after 30 hr or 9-10 hr of culture, respectively. Gonadotropic stimulation was necessary for maturation of both the native and injected cumulus-intact pig oocytes in follicle culture. Cumulus-free pig oocytes injected into follicle failed to mature in response to gonadotropic stimulation, suggesting an inability to perceive or respond to stimulation. Injected rat oocytes, however, matured irrespective of cumulus investment or gonadotropic stimulation. Their maturation was delayed and reduced at 9 hr. These results in the rat suggest that the pig follicular environment is incapable of regulating rat oocyte maturation but rather presents a permissive or supportive environment for their maturation. The explanted surrogate follicles from the pig or other species may provide a useful model for the study of oocyte-follicle interactions in oocyte maturation within or between species.
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  • 73
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    Gamete Research 11 (1985), S. 133-142 
    ISSN: 0148-7280
    Keywords: oocyte ; estradiol ; insulin ; meiosis ; Rana pipiens ; follicle wall ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Estradiol 17-β (E2) was found to either inhibit or synergize Na-insulin (Ins)-induced meiotic maturation of Rana oocytes. Inhibition of Ins activity occurred in the presence of the follicular investments of the oocyte; synergism with Ins occurred in oocytes denuded of the follicle wall. Similarly, co-incubation of E2 with frog pituitary homogenate (FPH) or pregnenolone (Pe) significantly decreased meiotic reinitiation as determined by germinal vesicle dissolution (GVD) in follicle-enclosed oocytes. By contrast, E2 had no consistently significant effect on progesterone (P)-induced meiosis in follicle-enclosed oocytes. Furthermore, E2 had no significant effect, either inhibitory or synergistic, on Pe- or P-induced GVD of denuded oocytes. Thus, of the meiotogens tested (Ins, P, Pe, FPH), all but P were consistently inhibited by E2 in the presence of the follicle wall. Na-insulin was the only meiotogen tested (Ins, P, Pe) which was potentiated by E2 in denuded oocytes, However, when E2 and Ins were spatially separated on the surface of individual intact follicles, the result was synergism of Ins-induced GVD rather than inhibition. These results suggest that Ins acts to induce GVD in the denuded oocyte through a mechanism distinct from that used by P (ie, Ins mechanism allows E2 synergism while the P mechanism does not). The E2 inhibitory effect on Ins-induced GVD appears to be dependent upon simultaneous exposure of follicle wall tissue to mixtures of E2 and Ins. The synergistic effect of E2 on Ins-induced GVD is dependent upon the simultaneous exposure of the oocyte surface to Ins and E2, either as a homogenous mixture in the case of denuded oocytes or as single substances at independent sites, for follicle-enclosed oocytes.
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  • 74
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    Gamete Research 11 (1985), S. 191-221 
    ISSN: 0148-7280
    Keywords: ovulation ; follicle cell ; granulose cell ; viviparity ; mammal arthropod ; lower vertebrate ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 75
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    Gamete Research 11 (1985), S. 179-189 
    ISSN: 0148-7280
    Keywords: spermatozoa ; capacitation ; sperm-egg adhesion ; sperm-oocyte fusion ; surface glycoproteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The nature and control of changes in surface carbohydrates in capacitating hamster spermatozoa were analysed by using five inhibitors of glycoprotein biosynthesis in an in vitro fertilization system. Epididymal spermatozoa were treated with amphomycin, bacitracin, tunicamycin, 2-deoxyglucose, and 2-deoxy-2-fluoro-D-glucose either during the entire period of capacitation or briefly at the end of capacitation before exposing to Con A-coated agarose beads or hamster eggs with or without their zonae pellucidae. Untreated 4½-5-hr spermatozoa exhibited nearly 100% fertilization and became bound to Con A-agarose beads mainly along the length of their flagellae, resulting in the formation of clumps on the beads. In the presence of inhibitors of glycosylation, spermatozoa did not bind to Con A-agarose beads or zona-intact oocytes and they did not fuse with the zona-free oocytes. Sperm-zona binding was also inhibited by UDP-galactose and UDP-N-acetylglucosamine, but not by UDP-glucose. Sperm motility was not damaged by these inhibitors, and zona-intact and zona-free oocytes pretreated with these inhibitors underwent normal fertilization with untreated spermatozoa. These results further strengthen the view that glycoproteins on the sperm surface may be required during different stages of fertilization, including sperm-egg fusion.
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  • 76
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    Gamete Research 11 (1985), S. 261-270 
    ISSN: 0148-7280
    Keywords: Arbacia ; sperm motility ; transglutaminase ; calmodulin ; calcium ; chelators ; channels ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Sea urchin spermatozoa react to transglutaminase inhibitors, which may antagonize the function of calmodulin, in dose-dependent fashion. The optimum concentration of diallyl-amino propionyl benzothiophenc (DAPBT), a potent, noncompetitive inhibitor of transglutaminase, 0.01 mM, causes a 3 1/2-fold increase in the forward swimming speed of Arbacia sperm. This effect apparently involves calcium-dependent enzymes since manipulation of both extracellular and intracellular calcium by means of chelating agents, calcium-channel blockers, and calmodulin antagonists depresses the stimulatory effect of DAPBT. These results suggest that in the spermatozoa the interaction of flagellar sliding filaments may be mediated by reversible cross-linking of the contractile proteins catalyzed by a calcium- dependent transglutaminase.
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  • 77
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    Gamete Research 12 (1985), S. 183-224 
    ISSN: 0148-7280
    Keywords: spermatozoa ; capacitation ; acrosome reaction ; membrane lipids ; sterols ; phospholipids ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The abundance of data pertaining to the metabolism of lipids in relation to mammalian fertilization has warranted an effort to assemble a molecular membrane model for the comprehensive visualization of the biochemical events involved in sperm capacitation and the acrosome reaction. Derived both from earlier models as well as from current concepts, our membrane model depicts a lipid bilayer assembly of space-filling molecular models of sterols and phospholipids in dynamic equilibrium with peripheral and integral membrane proteins. A novel feature is the possibility of visualizing individual lipid molecules such as phosphatidylcholine, phosphatidylethanolamine, lysophospholipids, fatty acids, and free or esterified cholesterol. The model illustrates enzymatic reactions which are believed to regulate the permeability and integrity of the plasma membrane overlying the acrosome during interactions between the male gamete and capacitation factors present in fluids of the female genital tract. The use of radioactive lipids as molecular probes for monitoring the metabolism of cholesterol and phosphatidylcholine revealed the presence of (1) steroid sulfatase in hamster cumulus cells, (2) lecithin: cholesterol acyltransferase in human follicular fluid, (3) phospholipase A2, and (4) lysophospholipase in human spermatozoa. These enzymatic reactions can be integrated into a pathway that provides a link between the concepts of lysophospholipid accumulation in the sperm membranes and alteration of the cholesterol/phospholipid ratio as factors involved in the preparation of the membranes for the acrosome reaction. Capacitation is viewed as a reversible phenomenon which, upon completion, results in a decrease in negative surface charge, an efflux of membrane cholesterol, and an influx of calcium between the plasma and outer acrosomal membranes. Triggered by the entry of calcium, the acrosome reaction involves phospholipase A2 activation followed by a transient accumulation of unsaturated fatty acids and lysophospholipids implicated in membrane fusion which occurs during the formation of membrane vesicles in spermatozoa undergoing the acrosome reaction.
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  • 78
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    Gamete Research 12 (1985), S. 357-371 
    ISSN: 0148-7280
    Keywords: hybrid dysgenesis ; spermatogenesis ; transmission electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The mechanisms involved in the impaired spermatogenesis of male goldfinch x canary hybrids were investigated by transmission electron microscopy and compared with spermatogenesis in the testes of the parent species.In the parent species the testes were of normal structure, with the only unusual observation being that the Sertoli cells were variable in cytoplasmic electron density. In hybrid birds the Sertoli cells were either electron dense or electron lucent with respect to both nucleus and cytoplasm.In the hybrids examined in this study, no spermatozoa were produced. Spermatogenic stages were arrested without formation of synaptonemal complexes. Centrioles were abnormally arranged in both somatic and germ cells. When they moved away from the basement lamina the germ cells degenerated and were phagocytosed. No focal tight junctions were present between Sertoli cells overlying what would normally have been the basal compartment of the tubule. The basement lamina was unusually thickened, peritubular cells were abnormal in structure, and numerous plasma cells were present in the interstitial tissue.The observations reported here suggest that there was an immunological basis for degeneration of germ cells in the hybrid testis.
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  • 79
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    Gamete Research 11 (1985), S. 69-82 
    ISSN: 0148-7280
    Keywords: Acrosin ; inhibitors ; alkaline ; proteinase ; acrosomal ; azocoll ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A proteolytic enzyme capable of cleaving intact proteins and synthetic substrates α-N-benzoyl-DL-arginine β-naphthylamide (Bz-Arg-NNap), α-N-benzoyl-L-arginine p-nitroanilde (Bz-Arg-NPhNO2), and α-N-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt) was purified 92- fold from the rabbit testes. The enzyme exhibited optimal activity at pH 9.0 and 50°C. The polyacrylamide gel electrophoresis and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified enzyme demonstrated multiple forms; the major band in the SDS-polyacrylamide gel electrophoresis corresponded to a Mt 48,000. The same value was established by the gel filtration over Sephadex G-75. The rabbit testicular alkaline proteinase (TAP) resembled acrosin in the hydrolysis of Bz-Arg-OEt. However, CaCl2, a potential stimulator of acrosin activity, inhibited the alkaline proteinase. The strong inhibitors of acrosin, eg pheny methyl sulphonyl fluoride (PMSF), tosyl lysine chloromethyl ketone (TLCK), and benzamidine did not inhibit the alkaline proteinase. TAP was activated by an acrosin inhibitor isolated from the rabbit testes. Since 0.5 M KCl was necessary for complete extraction of the enzyme and the bulk of the activity was present in 9,000g pellet of the testicular homogenate. The alkaline proteinase appeared to be associated with the membranous structures.
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  • 80
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    Gamete Research 11 (1985) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 81
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    BioEssays 2 (1985), S. 32-34 
    ISSN: 0265-9247
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: During development some cells are migratory whilst others are stationary. However, the same cell may change its behaviour depending upon its environment. Recent evidence has implicated the extracellular matrix protein fibronectin in the regulation of migratory behaviour. As the structure of this molecule becomes elucidated, it is also becoming possible to interpret this regulation in precise molecular terms.
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  • 82
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    BioEssays 2 (1985), S. 34-36 
    ISSN: 0265-9247
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The bulk of the DNA in eukaryotic chromatin behaves as if it is topologically relaxed; however, a subfraction can be shown to be under suercoil tension. Endonuclease S1 cuts at specific hypersentive sites in chromatin (in the promoter regions of active genes) and this enzyme cuts in the same region in supercoiled plasmids, but not in relaxed or linearized molecules. A subfraction of the minichromosomes formed after SV40 infection or microinjection of plasmid DNA into oocytes contains supercoil tension and this subfraction is thought to play an important role in transcription.
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  • 83
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    BioEssays 2 (1985), S. 68-71 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The two theories of pancreatic enzyme secretion, those of exocytosis and transmembrane flow, are described. Data thought to support the theory of transmembrane flow of single molecules from pancreatic acinar cells are first reviewed, and the conditions which could allow these data to be explained by the theory of exocytosis of enzyme quanta, i.e. secretory granules, are then discussed.The evidence suggesting short-term modulation of the composition of pancreatic juice is also considered, and its possible explanations at the organ and cellular level are discussed, in the light of suggested theories on enzyme secretion pathways.
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    BioEssays 2 (1985), S. 244-249 
    ISSN: 0265-9247
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Chi sites are examples of special sites enhancing homologous recombination in their region of the chromosome. Chi, 5′ G-C-T-G-G-T-G-G3′, is a recognition site for the RecBC enzyme, which nicks DNA near Chi as it unwinds DNA. A molecular model of genetic recombination incorporating these features is reviewed.
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  • 85
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    BioEssays 2 (1985), S. 254-259 
    ISSN: 0265-9247
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: DNA transfer directly from cell to cell (conjugation) is common among prokaryotes, particularly Gram-negative bacteria like Escherichia coli. The phenomenon invariably requires a set of plasmid genes in the DNA donor cell. In addition, E. coli itself makes limited and specific contributions to the donor activity of strains carrying the conjugative plasmid F. These contributions have yet to be defined biochemically, but it is already clear that the cell envelope is an importan nexus between plasmid- and chromosome-encoded proteins required for the establishment and maintenance of DNA donor activity.
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    BioEssays 2 (1985), S. 273-276 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    BioEssays 2 (1985), S. 84-86 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    BioEssays 2 (1985), S. 78-80 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 89
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  • 90
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 91
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 92
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    BioEssays 2 (1985), S. 158-161 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Nuclear proteins are synthesized in the cytoplasm and must subsequently enter the nucleus. Recent experiments indicate some similarities and some differences between protein localization to the nucleus and localization to other organelles.
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  • 93
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 94
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    BioEssays 2 (1985), S. 250-254 
    ISSN: 0265-9247
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: To further comprehend how synthesis and assembly of myofibrillar components is regulated, several laboratories have undertaken genetic studies of muscle development in Drosophila melanogaster. This small fly lends itself well to classical and molecular genetic approaches, and possesses a set of muscle fibers, termed indirect flight muscles (IFM), which is particularly advantageous for such investigations. Structural and functional analyses of cloned Drosophila contractile protein genes have revealed that protein isoforms can be specified either by multigene families or by differentially splicing primary transcripts of a single gene. Characterization of mutations which disrupt flight muscle development has so far revealed that profound abnormalities are caused by defective alleles of actin, tropomyosin, and myosin heavy-chain genes. Muscle defects associated with particular alleles can be alleviated by integration of corresponding wild-type gene copies into germ-line chromosomes. Strategies for further applying genetic techniques to investigations of Drosophila muscle development are discussed.
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  • 95
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    BioEssays 2 (1985), S. 263-267 
    ISSN: 0265-9247
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The small, relatively constant size and conservative evolution of chloroplast DNA (cpDNA) make it an ideal molecule for tracing the evolutionary history of plant species. At lower taxonomic levels, cpDNA variation is easily and conveniently assayed by comparing restriction patterns and maps, while at higher taxonomic levels, DNA sequencing and inversion analysis are the methods of choice for comparing chloroplast genomes. The study of cpDNA variation has already yielded important new insights into the origin and evolution of many agriculturally important crop plants, and promises to significantly enhance our phylogenetic understanding of the major lines of descent among land plants and algae.
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  • 96
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    Gamete Research 11 (1985), S. 1-17 
    ISSN: 0148-7280
    Keywords: oocyte ; fertilization potential ; meiotic maturation ; crabs ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The electrophysiological properties of immature and mature oocytes of two crabs were analyzed. Growing immature oocytes of Carcinus maenas and fully grown immature oocytes of Maia squinado had essentially K+ dependent resting potentials, Em, of -61 ∓ 1 mV, n=19, and -67.3 ± 0.5 mV, n=68, respectively. Fully grown immature oocytes of Carcinus maenas showed an Em of -40 ± 1.5 mV, n=19, that was k+ and Cl- dependent. In mature oocytes of both species, the plasma membrane became exclusively permeable to cl- and the Em attained-41 ± 1 mV, n=49 and -34 ± 1.5 mV, n=27 for Carcinus maenas and Maia squinado, respectively. After in vitro insemination, a dramatic increase in egg membrane permeability to K+ was observed. This instantaneously caused a sustained hyperpolarization constituting, for these crabs, the fertilization potential. We observed that concurrently with this electrical response to fertilization, sperm reinitiated the oocyte meiotic maturation previously arrested at the first metaphase. The triggering mechanism of the fertilization potential as well as the possible occurrence of a physiological polyspermy are discussed.
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  • 97
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    Keywords: fertilization ; polyspermy ; sea urchin eggs ; leukotrienes ; arachidonic acid ; FPL-55712 ; BW-755C ; 5-lipoxygenase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Arachidonic acid can be oxidized via the cyclooxygenase pathway to produce prostaglandins and via the 5-lipoxygenase pathway to produce leukotrienes. These substances are known to be extremely potent regulators of cellular function in somatic tissues, particularly during inflammatory reactions. Recent studies have implicated cyclooxygenase-derived products in preventing polyspermy in sea urchins [Schuel et al, Gamete Res 10:9-19, 1984]. FPL-55712, a receptor antagonist for leukotrienes in somatic tissues, causes a dose-(1-10 μM) and sperm-density-dependent induction of polyspermic fertilization in sea urchins if added before the egg completes the cortical reaction (elevation of the fertilization envelope). Eggs pretreated with FPL-55712 become polyspermic upon subsequent insemination with untreated sperm in sea water. Sperm pretreated with the drug do not cause polyspermy when used to inseminate untreated eggs. The 5-lipoxygenase inhibitor BW775C also promotes polyspermy. FPL-55712 and BW755C do not retard elevation of the fertilization envelope. These findings imply that (1) leukotrienes may be produced via the 5-lipoxygenase pathway during fertilization in sea urchins, and (2) the reaction of leukotrienes with putative receptors on the egg's surface may modulate its receptivity to sperm during the cortical reaction, and thereby help prevent polyspermy.
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  • 98
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    Gamete Research 11 (1985), S. 83-97 
    ISSN: 0148-7280
    Keywords: oocyte maturation ; follicular fluid ; mouse ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effects of the putative maturation inhibitor in porcine follicular fluid on gonadotropinstimulated reversal of cyclic adenosine monophosphate (cAMP)-maintained meiotic arrest in mouse oocytes in vitro were assessed in this study. When cumulus cell-enclosed oocytes were cultured in a suboptimal inhibitory concentration of dibutyryl cAMP (dbcAMP), the effect of follicle-stimulating hormone (FSH) on oocyte maturation was initially inhibitory at 3 hr, but stimulatory at 6 hr. Supplementation of the medium with an ultrafiltrate of porcine follicuiar fluid (PM10-filtrate) completely suppressed FSH-promoted reversal of inhibition at 6 hr. Charcoal extraction eliminated this effect of the PM10-filtrate. FSH reversed the inhibition of maturation of cumulus cell-enclosed oocytes maintained by a high concentration of dbcAMP and suboptimal concentrations of the phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine (IBMX), during a 21-22-hr culture period. However, the effect of a completely inhibitory concentration of IBMX was not reversed by gonadotropin. A component of serum was also found to inhibit FSH reversal of dbcAMP-maintained meiotic arrest, and this activity was removed by charcoal extraction. In addition, when oocytes were cultured in medium containing a suboptimal concentration of dbcAMP plus a low molecular weight fraction (〈 1,000) of porcine follicular fluid, porcine serum, or fetal bovine serum, a synergistic inhibition of maturation was observed. Experiments with highly purified gonadotropins revealed that reversal of dbcAMP-maintained meiotic arrest occurred only in response to FSH; neither highly purified luteinizing hormone nor human chorionic gonadotropin could mimic this action of FSH. Also, this effect was mediated by the cumulus cells, since FSH could not reverse dbcAMP-maintained meiotic arrest in denuded oocytes. Furthermore, elevating cAMP levels in denuded oocytes augmented, rather than reversed, the inhibitory action of dbcAMP on oocyte maturation. These data therefore suggest that dbcAMP- or IBMX-maintained meiotic arrest in vitro is reversed by an FSH-stimulated, cAMP-dependent process mediated by the cumulus cells and demonstrate that a factor present both in follicular fluid and serum prevents this action of the gonadotropin.
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  • 99
    ISSN: 0148-7280
    Keywords: calcium-modulated proteins ; calmodulin ; calmodulin-binding proteins ; spermatozoa ; membranes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Reproducible concentrations of calmodulin representing approximately 0.1% of the membrane protein were detected in purified plasma membranes from bovine epididymal spermatozoa. When membranes were isolated in the presence of 1 mM EGTA, the amount of calmodulin associated with the plasma membranes was not reduced. Calmodulin-binding proteins were detected in both purified plasma membranes and in a mixed membrane fraction containing both plasma membranes and cytoplasmic droplet membranes. A calcium-dependent, calmodulin-binding protein of apparent molecular weight 123,000 was detected in both fractions. In the presence of 1 mM EDTA, putative calcium-independent calmodulin-binding proteins of apparent molecular weights 93,000, 32,000, 18,000, and 15,000 were detected in the plasma membrane fraction. The 15,000 Mr polypeptide was also present in the mixed membrane fraction but the three proteins of higher molecular weight were reduced or absent in this fraction.
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    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Gamete Research 11 (1985), S. 329-329 
    ISSN: 0148-7280
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Type of Medium: Electronic Resource
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