bluuebunny/crossref_metadata_2025_split huggingface dataset:
The effects of serum immunoglobulins on the metachronal coordination of the lateral cilia of Mytilus edulis
Authors: Sanderson Michael J. , Sleigh Michael A. 
Published: February 2005

AbstractHuman IgM and a bovine, IgM‐enriched serum fraction isolated from normal adult serum at concentrations of 0.25–1 mg/ml protein induced a pronounced increase in the metachronal wavelength of the lateral (L) cilia of the sea mussel Mytilus edulis without altering their beat frequency. This change in activity was indistinguishable from that induced by 50% adult human or bovine serum. At protein concentrations ranging from 1–9 mg/ml, human IgG or a bovine, IgG‐enriched serum fraction had no or little effect on the activity of the L cilia. Similarly, neither monomeric (8S) human IgM (0.25 mg/ml) nor monospecific pentameric IgM (1 mg/ml) isolated from Waldenström's macroglobulinemia patients altered the metachrony of the L cilia. Indirect immunofluorescence demonstrated that both bovine and human IgM became attached almost exclusively to the L cilia, while very little bovine or human IgG was found to associate with these cilia.The results of this study suggest that serum IgM specifically binds to the L cilia of Mytilus in an antigen–antibody manner and agglutinates adjacent cilia into blocks or bundles, thereby increasing the coupling between cilia. As a result, the wavelength of the metachronal coordination is increased. The origin of these ciliary antibodies and their significance to ciliary bioassays used to monitor serum for the detection of cystic fibrosis are discussed.
Taxol stabilization of mitotic spindle microtubules: Analysis using calcium induced depolymerization
Authors: Salmon E. D. , Wolniak Stephen M. 
Published: February 2005

AbstractTaxol stabilizes or promotes the assembly of microtubules. In this report we characterize the rate, extent, and reversibility of taxol stabilization of calciumlabile microtubules in isolated mitotic spindles, principally from embryos of the sand dollar Echinarachnius parma. The intense depolymerizing action of 100 μM Ca2+ was used to assess the extent of stabilization by taxol. Changes in spindle microtubule assembly were evaluated and recorded by measuring changes in spindle birefringent retardation (BR). Membrane‐free mitotic spindles, isolated with a calcium‐chelating, nonionic detergent buffer, were stored in an EGTA‐gylcerol storage buffer to prevent microtubule depolymerization. When perfused with an EGTA‐buffer without glycerol, microtubules in these isolated spindles depolymerized gradually over 60–120 min; but in isolated spindles perfused with buffer that contained 100 μM Ca2+, BR decreased by 90% within 2–5 sec. In contrast, spindles that were pretreated for 3 min with 1 μM taxol, or for about 30 sec with 10 μM taxol, lost less than 10% of their initial BR when perfused with buffer containing 100 μM Ca2+. The rate and extent of microtubule stabilization by taxol depended on both the concentration and the duration of exposure to taxol. Taxol stabilization was reversible. After a 15 min preincubation with 1 μM or 10 μM taxol then washout, stability of spindle BR to 100 μM Ca2+ decreased exponentially with a time constant of 30–60 min. Thus taxol dissociates from spindle microtubules at significant rates; taxol‐stabilized microtubules are not “fixed.”
Interaction of bimane‐labeled fluorescent tubulin with the isolated mitotic apparatus
Authors: Wadsworth Patricia , Sloboda Roger D. 
Published: February 2005

AbstractFluorescent derivatives of cellular proteins that retain their native characteristics have become useful probes to investigate the dynamics of specific cytoskeletal proteins. In the experiments reported here, a previously characterized fluorescent derivative of tubulin, bimane‐tubulin [Wadsworth and Sloboda, 1982a], was used to investigate microtubule assembly in vitro. The results demonstrate that bimanetubulin was competent to assemble onto a variety of organizing centers in vitro, including microtubule organizing centers (MTOCs) present in homogenates of sea urchin eggs, isolated mitotic apparatuses (MAs), and lysed mitotic cells. When homogenates of fertilized sea urchin eggs containing MTOCs were incubated with bimane‐tubulin at 37°C, discrete areas of linear fluorescence were observed. Only diffuse fluorescence was observed when calcium or colchicine was added to the homogenate or if the temperature was maintained at 0°C. Negative‐stain electron microscopy of the fluorescent arrays revealed morphologically normal microtubules radiating from electron dense regions. When mitotic spindles, isolated in glycerol containing buffers and therefore cold stable, were incubated with bimane‐tubulin, linear fluorescence was observed emanating from the spindle poles but not from the region occupied by the kinetochores. MAs incubated with bimane‐labeled bovine serum albumin or bimane‐labeled microtubule‐associated proteins showed only diffuse fluorescence. However, when mitotic cells which were hypotonically lysed in the absence of detergents or microtubule stabilizing solvents, were perfused with bimane‐tubulin intense fluorescence was observed in the asters and throughout the spindle. Two experiments suggested that the fluorescence observed in the results outlined above was due to the assembly of normal microtubules from the fluorescent subunits. First, the observed fluorescence was sensitive to cold temperataure, which is known to disassemble microtubules. Second, when the isolated, fluorescent MAs were examined by thin section electron microscopy, microtubules of normal diameter were seen. No aggregated material appeared associated with the walls of the microtubules, which might have been expected if the fluorescent protein was nonspecifically adsorbed to the microtubules. The results of these experiments demonstrate that isolated, stabilized MAs support the growth of new microtubules from the spindle poles while labile spindles, present in lysed cells, incorporate fluorescent tubulin throughout the spindle and asters. The significance of these results for hypotheses concerning microtubule assembly and disassembly during mitosis is discussed.
A comparison of methods used to characterize gelation of actin in vitro
Authors: Rockwell Mark A. , Fechheimer Marcus , Taylor D. Lansing 
Published: February 2005

AbstractWe have compared the meniscus depletion assay and falling ball viscometry, two means of assessing the extent of gelation in actin‐based systems using mixtures of actin and the actin‐binding protein filamin. We examined the effect of varying the concentrations of actin and filamin in both assays. The interaction of actin and filamin was detected only above a threshold concentration of filamin. This threshold concentration was lower for falling ball viscometry than for the meniscus depletion assay at equal actin concentrations. At constant concentrations of filamin, an increase in actin concentration caused an increase in apparent viscosity measured by the falling ball assay, but a decrease in sedimentability detected by the meniscus depletion assay. The rate of sedimentation of actin was dependent on the molar ratio of actin to filamin. At each molar ratio, the sedimentation of actin was not dependent on the specific concentrations of actin and filamin used. The apparent viscosity was dependent on both the molar ratio and the specific concentrations of actin and filamin. To relate the present results to earlier studies, we examined mixtures of actin and filamin using a macroscopic assay of gelation (tube tipping assay), and polarized light microscopy. The effect of increasing filamin concentration in the four assays was compared at three actin concentrations. Mixtures of actin and filamin whose apparent viscosities were low enough to be estimated by falling ball viscometry were optically isotropic fluids that flowed out of inverted test tubes. Mixtures of actin and filamin in the range of sensitivity of the meniscus depletion assay were either viscous fluids or gels, and were either optically isotropic or anisotropic. Thus, the four assays provide different estimates of gelation. Both the meniscus depletion assay and falling ball viscometry can be used to determine relative gelation activity, but neither can be used as a quantitative assay of gelation.
Direct visualization of particle velocity distribution by pseudostereoscopic viewing of time‐lapsed sequential images: Application to fast axonal transport
Authors: Hodge Alan J. , Adelman William J. 
Published: February 2005

AbstractWe describe a simple method for direct visualization of the velocity distribution of particles moving against an immobile background. The technique involves pseudostereoscopic viewing of image pairs separated by an appropriate time interval in a sequential recording of the subject. Under these conditions, the positive or negative parallax arising from particle motion results in the binocular image of a particle being perceived as raised or lowered relative to an immobile background plane depending on its direction of movement, and with the degree of perceived elevation being proportional to its speed. In effect, the binocular optic axis becomes a velocity (speed) axis under these conditions. The technique is illustrated with examples of image pair sequences showing fast axonal transport in lobster and squid axons using video‐enhanced differential interference contrast microscopy. However, the pseudostereoscopic method is quite generally applicable to both microscopic and macroscopic time‐dependent phenomena. Particle speeds can be quantitated using standard procedures for measuring frame‐to‐frame particle displacements, or alternatively, by determination of parallax using stereogrammatic methods. It should be also readily adaptable for on‐line monitoring of particle velocity distribution, particularly in video systems where frame buffers can be utilized to extract and present serial image pairs having any desired time separation from video‐taped sequences.
Localization of the centriole and keratin intermediate filaments in PtK1 cells by double immunofluorescence
Authors: Eckert Barry S. , Caputi Susan E. , Brinkley B. R. 
Published: February 2005

AbstractWe present observations on the relative location of the centriole and keratin filament cap in motile PtK1 cells. Subconfluent cells were double labeled with anticentriole and antikeratin sera. These preparations revealed that the centriole is separate from, but neighboring, the keratin filament cap. Serial ultrathin sections confirm this observation. These observations are consistent with the idea that the microtubule organizing center and intermediate filament distribution center are not identical or concentric in PtK1 cells.
Actin microfilaments in paramecium: Localization and role in intracellular movements
Authors: Cohen Jean , de Loubresse Nicole Garreau , Beisson Janine 
Published: February 2005

AbstractUsing heavy meromyosin (HMM) or the fragment S1 of myosin as probes for actin microfilaments, we studied their organization in Paramecium both by fluorescence and electron microscopy.In interphasic cells, HMM decorates (a) most prominently the periphery of nascent and young food vacuoles and their route during the early phase of their intracellular transit; (b) a thin meshwork radiating from the gullet throughout the cytoplasm; (c) a small area beneath the pore of contractile vacuoles and beneath the cytoproct when open to release food residues. Most of these HMM‐decorated structures are in close contact with microtubular arrays. All HMM decoration disappears in dividing cells and in cytochalasin‐treated cells. In vivo, the drug immediately blocks food vacuole formation but does not affect cytokinesis, cyclosis, contractile vacuole pulsation, defecation, or nuclear movements.The data show that, as in the cells of other organisms, actin microfilaments form defined arrays that undergo physiologically controlled cycles of assembly/disassembly. These arrays contribute (at least in the phagocytotic process) to diverse types of movement: constriction, membrane fusion, and migration of food vacuoles. However, aside from their massive concentration along the phagocytotic tractus, actin microfilaments are neither major structural components of Paramecium cytoplasm nor the only cytoskeletal components ensuring motility or contractility processes.
Flow birefringence of microtubules and its relation to birefringence measurements in cells
Authors: Hard Robert , Allen Robert D. 
Published: February 2005

AbstractUnderstanding 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.
Bending patterns of Chlamydomonas flagella: II. Calcium effects on reactivated Chlamydomonas flagella
Authors: Omoto C. K. , Brokaw C. J. 
Published: February 2005

AbstractCa2+ 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.
Video microscopy of fast axonal transport in extruded axoplasm: A new model for study of molecular mechanisms
Authors: Brady Scott T. , Lasek Raymond J. , Allen Robert D. 
Published: February 2005

AbstractThe 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.
Actin assembly and filament cross‐linking in the presence of TW 260/240, the tissue‐specific spectrin of the chicken intestinal brush border
Authors: Fishkind Douglas J. , Mooseker Mark S. , Bonder Edward M. 
Published: February 2005

AbstractTW 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.
Live and reactivated motility in the 9 + 0 flagellum of Anguilla sperm
Authors: Gibbons Barbara H. , Baccetti B. , Gibbons I. R. 
Published: February 2005

AbstractThe 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.
Newt lung ciliated cell models: Effect of MgATP on beat frequency and waveforms
Authors: Weaver Alice , Hard Robert 
Published: February 2005

AbstractHighly 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.