bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Method for quantifying F‐Actin in chemotactic peptide activated neutrophils: Study of the effect of tBOC peptide
Authors: Howard Thomas H. , Oresajo C. O. 
Published: February 2005

AbstractThe 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.
A new model of reticulopodial motility and shape: Evidence for a microtubule‐based motor and an actin skeleton
Authors: Travis Jeffrey L. , Bowser Samuel S. 
Published: February 2005

AbstractCytoskeletal inhibitors were used as probes to test the involvement of microtubules and actin microfilaments in the development, motility, and shape maintenance of the pseudopodial networks (i e, reticulopodia) of the foraminifers Allogromia sp strain NF and Allogromia laticcllaris. Agents that disassemble cytoplasmic microtubules (cold, colchicine, and nocodazole) arrest all movement but have variable effects on reticulopodial shape. Electron microscopy reveals a granulofibrillar matrix but few, if any, microtubules in these motility‐arrested reticulopods. Allogromiids treated with cytochalasin B or D lose substrate adhesion and undergo dramatic changes in shape and motile behavior, highlighted by the coalescence of reticulopodial cytoplasm into irregularly shaped bodies with chaotic motility. Serial semithick sections of such preparations, viewed by high‐voltage electron microscopy, document a striking rearrangement of microtubules within these cytochalasin‐induced bodies. All aspects of cytochalasin‐altered motility are completely inhibited by colchicine. Actin is present in reticulopodia, as determined by staining with rhodamine‐phalloidin; this staining is not observed in cytochalasin‐treated organisms. These data provide compelling evidence that microtubules are required for reticulopodial motility. An actin‐based cytoskeleton is thought to play a role in maintaining shape, mediating pseudopod/substrate adhesion, and coordinating the various microtubule‐dependent processes.
Isolation of cilia from porcine tracheal epithelium and extraction of dynein arms
Authors: Hastie Annette T. , Dicker David T. , Hingley Susan T. , Kueppers Friedrich , Higgins Michael L. , Weinbaurn George 
Published: February 2005

AbstractMilligram amounts of mammalian ciliary axonemes were isolated from porcine tracheas. These were reactivated upon addition of ATP, indicating intact functional capability with a mean beat frequency at 37°C of 8.2 Hz. Electron microscopy showed typical ultrastructure of the isolated demembranated axonemes. Electrophoresis into polyacrylamide gradient gels containing sodium dodecyl sulfate revealed reproducible protein profiles from ten different tracheal preparations. Four major protein bands were observed in the 300–330 K molecular weight region, as well as tubulin at 51–54K. Extraction of the isolated tracheal axonemes with 0.6M KCl removed the outer dynein arms seen in electron microscopic cross‐section of axonemes, preferentially solubilized two of the high molecular weight proteins at 320 and 330 K, and resulted in a three‐ to four‐fold increase in ATPase specific activity. Sedimentation of the dialyzed salt extract on a 5–30% sucrose density gradient and subsequent fractionation yielded two peaks of ATPase activity. The faster migrating, 19S major ATPase peak correlated with the 320 and 330 K proteins, and two other proteins at 81 and 67 K. The slower sedimenting, 12S minor ATPase peak corresponded to a 308 K protein and two smaller proteins at 33 and 48 K. Thus, the outer dynein arm of tracheal cilia appeared to be associated with at least two high molecular weight proteins. These results demonstrate that adequate quantities of functionally intact axonemes can be reproducibly isolated from porcine tracheas, allowing further fractionation and analysis of mammalian cilia.
Abnormal distribution of the periaxonemal structures in a human sperm flagellar dyskinesia
Authors: Serres Catherine , Feneux Danielle , Jouannet Pierre 
Published: February 2005

AbstractThe spermatozoa from four infertile patients showing a flagellar dyskinesia due to abnormal flagellar wave development have been studied by light and transmission electron microscopy (TEM) for flagellar morphology. No axonemal anomalies were found but modification of the periaxonemal structures was observed. The results of a stereological analysis revealed abnormal extension of the individual dense fibres along the axoneme in the four cases as compared with a control group. The order of termination of those structures was therefore altered. However, the overall fibre extension was the same in both groups (ie, 60% of the principal piece). The number and the location of the longitudinal columns were also modified, the predominant anomaly being the presence of a single column. The possible influence of those structural anomalies on the pattern of sperm movement is discussed. Our observations seem to agree with a previous hypothesis of the literature, that the dense fibres might play a role in flagellar flexibility. More particularly, we suggest that the abnormal extension of dense fibres No. 2, 3, and 4 and the symmetric distribution of the dense fibres on both sides of the plane of beating may alter the flagellar curvature amplitude and the cell rotation frequency.
Parameters of the ciliary cycle under membrane voltage control
Authors: Machemer H. , Sugino K. 
Published: February 2005

AbstractAxial views of depolarization‐ and hyperpolarization‐dependent activation of the frontal cirri of Stylonychia were cinematically recorded at high rate (250 frames/s) under voltage‐clamp. Images of a cirrus performing the cycle were processed by using computer assistance. In responding to the polarity and amplitude of the voltage signal, a cirrus inclines proximally with a particular angle and orientation. The ciliary cycle–always counterclockwise–is superimposing upon steady inclination. Correction for inclination allowed the assessment of the directional change rate and, after inclusion of the amplitude data, the determination of the ciliary angular velocity during the cycle. The method serves to isolate a new ciliary parameter: inclination, and to register precisely parameters of the cycle which may be meaningful for the understanding of the sliding mechanism.
Towards a new classification of intracellular particle movements based on quantitative analyses
Authors: Weiss Dieter G. , Keller Franz , Gulden Josef , Maile Willi 
Published: February 2005

AbstractA survey study of organelle movements in a variety of cell types of plant and animal origin was made with the aid of video‐enhanced contrast, differential interference contrast (AVEC‐DIC) microscopy followed by fine analysis of the motile behavior of the individual organelles. We found that there exists besides Brownian motion a wide spectrum of active motions in cells, i.e. motion that is directionally biased through the expenditure of metabolic energy. The types of active motion seen range from a continuous motion (sometimes appearing as streaming) in plant cells and neurons to various types of less ordered and less well directed motion. We did not see any clear‐cut qualitative difference between plant and animal cells or between systems presumed to be actin‐ and microtubule‐based. A preliminary classification of the types of active motion is presented. The ongoing research activities, which aim at a more precise definition of the different types of motion by a set of quantitative parameters, are described, and the progress made so far is reported.
Polarized cytoplasmic movement and inhibition of saltations induced by calcium‐mediated effects of microbeams in fungal hyphae
Authors: McKerracher L. J. , Heath I. B. 
Published: February 2005

AbstractWe have investigated the mechanisms that hyphae of the fungus Basidiobolus magnus use to accomplish bulk movement of their cytoplasm and saltatory organelle movements. When cells were irradiated with an ultraviolet microbeam, cytoplasmic contraction occurred. The posterior cytoplasm (toward the septum) always moved forward toward the irradiated area, whereas anterior cytoplasm (between the cell tip and target) never contracted back toward the site of irradiation. Thus, there is an intrinsic polarity in the cytoplasm. Irradiations also arrested saltatory movements. The effects of irradiation on both saltations and cytoplasmic movement appear to be mediated by Ca++. Chelating exogenous Ca++ before irradiation eliminated contractions and prevented the inhibition of saltations. Furthermore, the effects of irradiation could be duplicated by using the Ca++ ionophore A23187. We relate the present results to our previous report on the effects of irradiation on the cytoskeleton [McKerracher and Heath, 1986]. We conclude that two separate cytoskeletal networks exist in fungal cells, and that an actin‐containing network generates bulk cytoplasmic movement.
Structural evidence for contractile units in the giant smooth muscle cell of Beröe
Authors: Hernandez‐Nicaise M. L. , Nicaise G. 
Published: February 2005

AbstractBundles of giant smooth muscle fibers of the ctenophore Beröe have been stretched up to four times their initial length and then examined by transmission electron microscopy. Stretching induces the segregation of actin and thick (myosinlike) filaments into separate domains. The thick filaments domains are made of 20–30 filaments, with a regular spacing identical to that of nonstretched fibers. A moderate stretching permits the visualization of microtendons associating actin filament bundles to hyaluronidase‐resistant condensations of the extracellular matrix. It is deduced from these observations that Beröe giant smooth muscle fibers possess myofibrils which attach at both ends upon the sarcolemmal membrane. Each myofibril is probably made of two long actin filament bundles (of approximately 150 filaments) and short (2–3 μm) myosinlike bundles (of approximately 30 filaments).
The mechanical behavior of doublet microtubules simulated by helical models
Authors: Jarosch Robert 
Published: February 2005

AbstractThe structural and mechanical properties of the helix‐shaped isolated doublet microtubules described by Costello [Biol. Bull. 145:279–291, 1973], Zobel [J. Cell Biol. 59:573–94, 1973], and Miki‐Noumura and Kamiya [Exp. Cell Res. 97:451–53, 1976, J. Cell Biol 81:355–60, 1979] are simulated by a left‐handed superhelix model that consists of two intertwined springlike helices with a slight difference in their pitches. It is shown by combinations of two and more superhelices of this kind that the straight shape of the doublets in the axoneme is the consequence of a position‐dependent mechanical coil‐coil interaction between interconnected doublets whose curvatures bend against one another. A counterclockwise torsion changes the sense of the superhelix from the left‐handed form with a smaller pitch (L2) to the right‐handed form with a larger pitch (R3). The coil‐coil interaction of L helices with R helices results in uniplanar, meanderlike shapes or in flattened helices. The one‐sided distribution of L and R doublets as described for sperm axonemes by Costello [Biol. Bull. 145:279–91, 1973] may therefore be responsible for the uniplanar. meanderlike shape of these flagella.
Independence of centriole formation and initiation of DNA synthesis in Chinese hamster ovary cells
Authors: Kuriyama Ryoko , Dasgupta Santanu , Borisy Gary G. 
Published: February 2005

AbstractThe relationship between centriole formation and DNA synthesis was investigated by examining the effect of taxol on the centriole cycle and the initiation of DNA synthesis in synchronized cells. The centriole cycle was monitored by electron microscopy of whole‐mount preparations [Kuriyama and Borisy, J. Cell Biol., 1981, 91:814–821]. A short daughter centriole appeared in perpendicular orientation to each parent during late G1 or early S and elongated slowly during S to G2. Addition of 5–20 μg/ml taxol to a synchronous population of cells in S phase did not inhibit centriole elongation; rather, elongation was accelerated. In contrast, when taxol was added to M phase or early G1 cells, centriole duplication was completely inhibited. The taxol block was reversible since nucleation and elongation of centrioles resumed as soon as the drug was removed. Cells exposed to taxol progressed through the cell cycle and became blocked in mitosis, as indicated by an increase in the mitotic index, but eventually the mitotic arrest was overcome, resulting in formation of multinucleated cells. A peak in mitotic index was seen in the following generation, indicating that chromosomes duplicated in the presence of taxol. Incorporation of 3H‐thymidine followed by autoradiography confirmed that DNA synthesis was initiated in the presence of taxol even though formation of daughter centrioles was inhibited. It seems, therefore, that centriole duplication is not a prerequisite for entry into S phase. Since DNA synthesis has already been demonstrated not to be necessary for centriole duplication, these two events, normally coordinated in time, appear to be independent of each other.
Structure and composition of the cytoskeleton of nucleated erythrocytes: III. Organization of the cytoskeleton of Bufo marinus erythrocytes as revealed by freeze‐dried platinum‐carbon replicas and immunofluorescence microscopy
Authors: Centonze Victoria E. , Ruben George C. , Sloboda Roger D. 
Published: February 2005

AbstractPlatinum‐carbon (Pt‐C) replicas of freeze‐dried erythrocyte cytoskeletons of the toad, Bufo marinus, were prepared using a modified Balzers 300 system. Examination in stereo of replicas of the microtubule‐containing marginal band revealed filaments projecting from the microtubule walls to form links between adjacent microtubules. These cross‐bridging proteins may bundle the microtubules into the configuration of the marginal band (MB) and may also serve to stabilize the structure. The MB appears to have linkages to components of the surface‐associated cytoskeleton (SAC). The SAC forms a continuous matrix that spreads across the upper and lower surfaces of the cell adjacent to the plasma membrane and extends around the outer perimeter of the MB. Thus, the SAC encapsulates the MB and the central nucleus. After lysis, the elements of the cytoskeleton remain in a configuration similar to that found in the whole cell. Spectrin (fodrin) and actin were identified by immunofluorescence in the region of the SAC. When labeled with antibodies specific for vimentin and synemin, a network of intermediate filaments can be detected in the region between the nucleus and the MB. These vimentin filaments are also enclosed within the SAC and appear in Pt‐C replicas to emerge from the area of the nuclear envelope. As the filaments extend toward the periphery of the cell, they form attachments to the SAC. Attachments of intermediate filaments to both the nucleus and the SAC thus appear to anchor the nucleus in its central position within the cytoskeleton.
Polarized microtubule gliding and particle saltations produced by soluble factors from sea urchin eggs and embryos
Authors: Pryer Nancy K. , Wadsworth Patricia , Salmon E. D. 
Published: February 2005

AbstractIn this report, we describe an in vitro system for analyzing microtubule‐based movements in supernatants of sea urchin egg and embryo homogenates. Using video enhanced DIC microscopy, we have observed bidirectional saltatory particle movements on native taxol‐stabilized microtubules assembled in low speed supernatants of Lytechinus egg homogenates, and gliding of these microtubules across a glass surface. A high speed supernatant of soluble proteins, depleted of organelles, microtubules, and their associated proteins supports the gliding of exogenous microtubules and translocation of polystyrene beads along these microtubules. The direction of microtubule gliding has been determined directly by observation of the gliding of flagellar axonemes in which the (+) and (–) ends could be distinguished by biased polar growth of microtubules off the ends. Microtubule gliding is toward the (−) end of the microtubule, is ATP sensitive, and inhibited only by high concentrations of vanadate. These characteristics suggest that the transport complex responsible for microtubule gliding in S2 is kinesin‐like. The implications of these molecular interactions for mitosis and other motile events are discussed.
Calmodulin and caimodulin‐binding proteins in the morphological transformation of sea urchin coelomocytes
Authors: Venuti Judith M. , Edds Kenneth T. 
Published: February 2005

AbstractSea urchin coelomocytes contain an actin‐based cytoskeleton that undergoes major organizational changes as the cells transform from one morphology (petaloid) to another (filopodial). The molecular mechanisms directing and regulating this cytoskeletal reorganization are not well understood; Ca2+ has been implicated, but the specific targets of its action have not been identified. Since the effect of Ca2+ on a variety of cellular processes has been shown to be mediated by the Ca2+‐binding protein calmodulin, we investigated the role of this protein in coelomocyte morphological transformation. The calmodulin inhibitory drugs trifluoperazine, chlorpromazine, and calmidazolium were shown to inhibit coelomocyte morphological transformation in response to hypotonic shock in a dosedependent fashion and at concentrations consistent with their reported potencies as anti‐calmodulin agents. Calmodulin isolated from coelomocytes using trifluorophenothiazine affinity chromatography co‐migrates with bovine brain calmodulin on 15% SDS‐polyacrylamide gels and demonstrates a characteristic shift in electrophoretic mobility in the presence of Ca2+. Another diagnostic for calmodulin, Ca2+‐dependent activation of exogenous 3':5' cyclic AMP phosphodiesterase, was demonstrated by whole coelomocyte homogenates, heat‐treated homogenates, and the affinity purified coelomocyte protein. Localization of calmodulin in coelomocytes by indirect immunofluorescence reveals an association of calmodulin, at least in part, with the actin‐based cytoskeleton. Calmodulin‐binding polypeptides with estimated relative mobilities of 240,000, 195,000, 170,000, 70,000, 60,000, 30,000, and 20,000 daltons were identified using 125I‐calmodulin overlay procedures. Ca2+‐dependent calmodulin‐binding in these preparations was demonstrated for all but the Mr 30,000 and 20,000 coelomocyte polypeptides. The majority of the calmodulin‐binding proteins identified in whole petaloid coelomocyte preparations are also found in Triton X‐100 insoluble cytoskeletal fractions. Immunoblotting with antiserum raised against chicken erythrocyte alpha‐spectrin suggests that the 240,000 Mr calmodulin‐binding polypeptide corresponds to coelomocyte alpha‐spectrin. This protein was enriched in isolated coelomocyte filopodia where, we propose, it serves an analogous function to its counterpart in erythrocytes, in linking the actin‐cytoskeleton to the plasma membrane. Thus, calmodulin is present in coelomocytes and possibly participates in the morphological transformation of these cells through regulation of cytoskeletal and/or membrane‐cytoskeletal interactions.
Antigenic interrelationship between the 40‐kilodalton cytokeratin polypeptide and desmoplakins
Authors: Gigi‐Leitner Orith , Geiger Benjamin 
Published: February 2005

AbstractWe describe here antigenic cross‐reactivity between the human 40‐kilodalton cytokeratin polypeptide [Moll et al] and components of bovine desmosomal plaque, namely desmoplakins I and II. This relationship was revealed by an antibody (KM 4.62), raised against cytoskeletal preparation of cultured human breast adenocarcinoma cells (MCF‐7) and selected by immunoblotting and immunofluorescent labeling. In cultured human cells that contain the 40‐kD cytokeratin, antibody KM 4.62 labeled arrays of filaments throughout the cytoplasm. This antibody labeled the basal layer of nonkeratinizing squamous epithelia as well as various simple (normal and malignant) epithelia and epithelial elements of the thymus. In liver tissue, labeling was obtained only in bile ducts and canaliculi but not in the hepatocytes.In bovine cells and tissues, on the other hand, immunofluorescent labeling with antibody KM 4.62 was strictly confined to desmosomes. This was verified by double immunolabeling with both antibody KM 4.62 and specific cytokeratin or desmosomal antibodies. Immunoblotting analysis indicated that the former antibody reacts specifically with the high molecular weight components of the bovine desmosomal plaque, namely desmoplakins I and II. These immunochemical results suggest that bovine desmoplakins share same structural relationship with the human acidic, 40‐kD cytokeratin.