bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunocytochemical studies of spectrin in hamster cardiac tissue
Authors: Messina Dino A. , Lemanski Larry F. 
Published: February 2005

AbstractThe spectrins are a family of cytoskeletal‐membrane proteins that have a wide tissue distribution. In the present study, we employed polyclonal antibodies made against mammalian and avian erythroid spectrins as well as mammalian brain spectrin to assess their presence and distributions in the mammalian heart. Western blot analyses revealed that all three antibodies were specific for a 240,000 molecular weight α‐spectrin subunit found in hamster erythrocyte ghost homogenates, whole hamster heart, and isolated hamster cardiac myofibril homogenates. Spectrin staining was absent from the Triton X‐100‐extracted supernatant fraction of myofibril preparations, suggesting that the protein is linked to the myofibril precipitate after exposure to the detergent. Frozen, unfixed, 2‐μm‐thick; sections of adult, Syrian golden hamster cardiac tissue exhibited strong immunofluorescent staining of intercalated discs and Z‐bands using all three antibodies. In addition, the mammalian erythroid spectrin antibodies showed staining of the sarcolemma, and in cross section, revealed a delicate internal network of staining that appears to surround individual myofibrils. This may be T‐tubule‐associated staining. Myofibrils isolated from cardiac myocytes using Triton X‐100 show positive Z‐band staining using all three antibodies. Double staining with Texas Red‐labeled monoclonal desmin and FITC‐labeled polyclonal spectrin antibodies revealed that both stained the myofibrillar Z‐line regions. These results demonstrate that spectrin is closely associated with the membranes, myofibrils, and intermediate filaments in the mammalian heart.
Establishment of a stable, acetylated microtubule bundle during neuronal commitment
Authors: Falconer Marcia M. , Vielkind Ursula , Brown David L. 
Published: February 2005

AbstractTwo posttranslational modifications of alpha‐tubulin, acetylation and detyrosination, are associated with stable microtubule (MT) populations, including those of neuronal processes. We have used a pluripotent embryonal carcinoma cell line, P19, to investigate changes in MT isotype and stability found in MT arrays during neurogenesis. This cell line has an advantage in that both commitment‐ and differentiation‐related events can be observed. Uncommitted P19 cells have minimal arrays of acetylated and detyrosinated MTs. Following neuronal induction with retinoic acid (RA), indirect immunofluorescence microscopy shows that the first MT modifications occur during commitment and before any morphological change is observed. RA‐induced cells initially polymerize a temporarily enlarged population of MTs. Included in this population is a new array of acetylated MTs arranged in a bundle of parallel MTs. This bundle is colchicine‐stable, although no MT‐associated proteins (MAPs) are detectable using a battery of anti‐MAP antibodies. Observation of MT arrays with patterns that are intermediate between the early bundles and short neurites suggests that the acetylated MT bundle subsequently extends to form a neurite. MAP 2 is first detected at about the time of neurite extension. However, at this early stage of differentiation, MAP 2 is not yet limited to dendritic processes. This report provides the first evidence that the stable MTs of mature neurons may be initiated during neuronal commitment.
Copurification of kinesin polypeptides with microtubule‐stimulated Mg‐ATPase activity and kinetic analysis of enzymatic properties
Authors: Wagner Mark C. , Pfister K. Kevin , Bloom George S. , Brady Scott T. 
Published: February 2005

AbstractDetermination of kinetic properties for kinesin adenosine triphosphatase (ATPase), a proposed motor for transport of membranous organelles, requires adequate amounts of kinesin with a consistent level of enzymatic activity. A purification procedure is detailed that produces approximately 2 mg of kinesin at up to 96% purity from 800 g of bovine brain. This protocol consists of a microtubule affinity step using 5′‐adenylylimidodiphosphate (AMP‐PNP); followed by gel filtration, ion exchange, and hydroxylapatite chromatography; and then sucrose density gradient centrifugation. The microtubule‐activated ATPase activity of kinesin coeluted with kinesin polypeptides throughout the purification. Highly purified kinesin had a Vmax of 0.31 μmol/min/mg in the presence of microtubules, with a Km for ATP of 0.20 mM. The kinetic constants obtained in these studies compare favorably with physiological levels of ATP and microtubules. Variations in buffer conditions for the assay were found to affect ATPase activity significantly. A study of the ability of kinesin to utilize a variety of cation‐ATP complexes indicated that kinesin is a microtubule‐stimulated Mg‐ATPase, but kinesin is able to hydrolyze Ca‐ATP, Mn‐ATP, and Co‐ATP as well as Mg‐ATP in the presence of microtubules. In the absence of microtubules, Ca‐ATP appears to be the best substrate. Studies with several inhibitors of ATPases determined that vanadate inhibited kinesin ATPase at the lowest concentrations of inhibitor, but significant inhibition of the ATPase also occurred with submillimolar concentrations of AMP‐PNP. Other inhibitors of kinesin include N‐ethylmaleimide, adenosine diphosphate (ADP), pyrophosphate, and tripolyphosphate. Further characterization of the kinetic properties of the kinesin ATPase is important for understanding the molecular mechanisms for transport of membranous organelles along microtubules.
Fluorescence microscopic localization of actin in pollen tubes: Comparison of actin antibody and phalloidin staining
Authors: Tang Xiaojing , Lancelle Susan A. , Hepler Peter K. 
Published: February 2005

AbstractA comparison of actin localization in pollen tubes of Nicotiana has been made using a monoclonal actin antibody and rhodamine‐phalloidin (RP). The monoclonal antiactin, based on Western blotting of pollen tube extract, labels a polypeptide at 45 kD that comigrates with muscle actin. A 51‐kD unknown protein and three bands less than 45 kD, presumed to be proteolytic fragments of actin, are also observed. Structural observaations using this antibody reveal a network of axially oriented strands of microfilaments (MFs). The MFs are distributed throughout the length of the pollen tube except at the very tip, where diffuse staining is usually observed. A similar pattern of MFs is evident after RP staining. When pollen tubes are treated with cytochalasins (CB or CD) cytoplasmic streaming is inhibited, as is tube elongation. Microscopic analysis reveals that the microfilament (MF) pattern is markedly altered; however, the antibody and RP produce different staining patterns. The antibody reveals many MF strands that distribute throughout the tube length and extend into the very tip. In contrast, RP shows mostly a diffuse staining pattern with only a few short clumps of filamentous material. Immunogold labelling of sections of pollen tubes prepared by rapid‐freeze fixation and freeze substitution reveals that actin MF bundles are indeed present after cytochalasin treatment. Our results thus question reports in the literature, based on phalloidin staining, asserting that cytochalasin fragments or destroys actin MFs.
The molecular structure of adrenal medulla kinesin
Authors: Hisanaga Shin‐ichi , Murofushi Hiromu , Okuhara Koji , Sato Reiko , Masuda Yayoi , Sakai Hikoichi , Hirokawa Nobutaka 
Published: February 2005

AbstractThe molecular structure of bovine adrenal kinesin was studied by electron microscopy using the low‐angle rotary shadowing technique. Adrenal kinesin exhibited either a folded or an extended configuration; the ratio of the two is dependent on the salt concentration. Almost all adrenal kinesin molecules were folded in a low‐ionic solution, and the ratio of extended molecules increased to 40–50% in a solution containing 1 M ammonium acetate. Kinesin in the extended configuration displayed a rod‐shaped structure with a mean length of about 80 nm. The morphologies of the ends were different; one end was composed of two globular particles, similar to the two‐headed structure of myosin, while the other end had a more ill‐defined structure, appearing either as a globular particle, an aggregate of two to four small granules, or a frayed, fan‐like structure. The folded kinesin molecule possessed a hinge region in the middle of the rod, at about 32 nm from the neck of the two heads. In our preparations, the majority of adrenal kinesin molecules were folded at physiological salt concentrations. Adrenal kinesin bound to microtubules in the presence of adenylyl imidodiphosphate (AMP‐PNP) also displayed a folded morphology.
Rearrangements of pterinosomes and cytoskeleton accompanying pigment dispersion in goldfish xanthophores
Authors: Palazzo Robert E. , Lynch Thomas J. , Lo Szecheng J. , Taylor John D. , Tchen T. T. 
Published: February 2005

AbstractThe cytoskeleton of goldfish xanthophores contains an abundance of unique dense structures (400 nm in diameter) that are absent in goldfish nonpigment cells and are probably remnants of pterinosomes. No major difference in protein composition between xanthophores and nonpigment cells (without these structures) was found that could account for these structures. In xanthophores, these structures are foci of radiating filaments. The addition or withdrawal of ACTH causes a radical rearrangement of the xanthophore Cytoskeleton accompanying redistribution of carotenoid droplets, namely, the virtual exclusion of these dense bodies with associated filaments from the space occupied by the carotenoid droplet aggregate vs. a relatively even cytoplasmic distribution of these structures when the carotenoid droplets are dispersed. These changes in cytoskeletal morphology are not accompanied by any major changes in the protein or phosphoprotein composition of the cytoskeleton.
Cytoskeletal organization of migrating retinal pigment epithelial cells during wound healing in organ culture
Authors: Hergott Greg J. , Sandig Martin , Kalnins Vitauts I. 
Published: February 2005

AbstractRetinal pigment epithelial (RPE) cells maintained in organ culture on Bruch's membrane and the associated choroid spread and migrate into a linear wound along the exposed basal lamina. Changes in cell shape, in the organization of microfilaments, and in cell‐cell and cell‐substratum interactions during this time were examined by epifluorescence and transmission electron microscopy. In contrast to cuboidal stationary cells distant from the wound edge, which display well‐developed apical circumferential microfilament bundles (CMBs) associated with zonulae adhaerentes junctions, the migrating RPE cells near the wound edge instead are flat, and, in addition to microfilament bundles near junctions between adjacent cells, display prominent stress fibers. Furthermore, monoclonal antibodies to vinculin labeled regions at the terminal ends of these stress fibers indicating that the RPE cells form focal contacts with the basal lamina at these sites. Electron microscopy of these regions of cell‐substratum interaction confirmed the presence of microfilament bundles that terminate on the cell membrane. Folds present in the basal lamina near these sites suggest that tension is being generated by the microfilaments in the stress fibers as the migrating cells pull on the underlying basal lamina through these adhesion points.
Latrunculins—novel marine macrolides that disrupt microfilament organization and affect cell growth: I. Comparison with cytochalasin D
Authors: Spector Ilan , Shochet Nava R. , Blasberger Dina , Kashman Yoel 
Published: February 2005

AbstractThe latrunculins are architecturally novel marine compounds isolated from the Red Sea sponge Latrunculia magnifica. In vivo, they alter cell shape, disrupt microfilament organization, and inhibit the microfilament‐mediated processes of fertilization and early development. In vitro, latrunculin A was recently found to affect the polymerization of pure actin in a manner consistent with the formation of a 1:1 molar complex with G‐actin. These in vitro effects as well as previous indications that the latrunculins are more potent than the cytochalasins suggest differences in the in vivo mode of action of the two clases of drugs. To elucidate these differences we have compared the short‐ and long‐term effects of latrunculins on cell shape and actin organization to those of cytochalasin D. Exposure of hamster fibroblast NIL8 cells for 1–3 hr to latrunculin A, latrunculin B, and cytochalasin D causes concentration‐dependent changes in cell shape and actin organization. However, the latrunculin‐induced changes were strikingly different from those induced by cytochalasin D. Furthermore, while initial effects were manifest with both latrunculin A and cytochalasin D already at concentrations of about 0.03 μg/ml, latrunculin A caused complete rounding up of all cells at 0.2 μg/ml, whereas with cytochalasin D maximum contraction was reached at concentrations 10–20 times higher. The short‐term effects of latrunculin B were similar to those of latrunculin A although latrunculin B was slightly less potent. All three drugs inhibited cytokinesis in synchronized cells, but their long‐term effects were markedly different. NIL8 cells treated with latrunculin A maintained their altered state for extended periods. In contrast, the effects of cytochalasin D progressed with time in culture, and the latrunculin B‐induced changes were transient in the continued presence of the drug. These transient effects were found to be due to a gradual inactivation of latrunculin B by serum and were used to compare recovery patterns of cell shape and actin organization in two different cell lines. This comparison showed that the transient effects of latrunculin B were fully reversible for the NIL8 cells and not for the mouse neuroblastoma N1E‐115 cells.
Distribution of desmoplakin in normal cultured human keratinocytes and in basal cell carcinoma cells
Authors: Jones Jonathan C. R. , Grelling Kent A. 
Published: February 2005

AbstractIn cultured human keratinocytes (NHEK) maintained in medium containing low levels of Ca2+ (0.04 mM) desmoplakin is a component of certain electron‐dense bodies in the cytoplasm. These bodies are associated with bundles of intermediate filaments. Upon elevation of the level of Ca2+ in the culture medium to 1.2 mM, desmoplakin first appears at sites of cell—cell contact in association with bundles of intermediate filaments. Subsequently, desmoplakin becomes incorporated into desmosomes in a manner comparable to that seen in mouse keratinocytes (Jones and Goldman: Journal of Cell Biology 101:506–517, 1985). NHEK cells maintained for 24 hr at Ca2+ concentrations between 0.04 mM and 0.18 mM were processed for immunofluorescence, immunoelectron, and conventional electron microscopical analysis. In NHEK cells grown at Ca2+ concentrations of 0.11 mM, desmoplakin appears to be localized in electron‐dense bodies associated with intermediate filaments at sites of cell—cell contact in the absence of formed desmosomes. At a Ca2+ concentration of 0.13 mM desmoplakin is arrayed like beads on a “string” of intermediate filaments at areas of cell—cell association. At 0.15 mM, desmosome formation occurs, and desmoplakin is associated with the desmosomal plaque. In basal cell carcinoma cells desmoplakin is not restricted to desmosomes but also occurs in certain electron‐dense bodies morphologically similar to those seen in NHEK maintained in low levels of Ca2+ and during early stages of desmosome assembly. We discuss the possibility of “cycling” of desmoplakin through these bodies in proliferative cells.
What structures, besides adhesions, prevent spread cells from rounding up?
Authors: Zand Martin S. , Albrecht‐Buehler Guenter 
Published: February 2005

AbstractThe outline of cells in sparse cultures consists prediminantly of concave and convex segments; straight segments are rare and ephemeral. The convex segments are areas of active cell expansion. The concave segments are stationary and web‐shaped, similar in profile to the cables of a suspension bridge. In 3T3 fibroblasts, we have found a single microfilament bundle following the outline of every webbed edge and have called it the actin edge‐bundle (AEB). While the AEB is composed predominantly of actin, α‐actinin and myosin are also present. In contrast to normal stress fibers, AEBs are more resistant to several treatments that depolymerize F‐actin. Once an AEB disassembles, however, the webbed edge collapses and retracts, suggesting that the actin edge‐bundle is a specialized cytoskeletal structure that supports the webbed edges of interphase 3T3 fibroblasts. The stability of AEBs is independent of microtubules. We suggest that the microfilament bundles that frequently line the lateral contacts between epithelial cells in vivo may be related to the actin edge‐bundle.
Rapid kinetochore movements in Mesostoma ehrenbergii spermatocytes: Action of antagonistic chromosome fibres
Authors: Fuge Harald 
Published: February 2005

AbstractChromosome movements in Mesostoma ehrenbergii spermatocytes were studied using conventional video light microscopy. Kinetochore regions of the three bipolarly oriented bivalents displayed periodic back and forth movements directed to both poles at metaphase I, leading to periodic lenght changes of the bivalents. Velocity was 8–10 μm/min (maximum 17 μ/min), about one order of magnitude higher than the normal meiotic or mitotic chromosome movements of other species. One cycle of movement lasted for about 100 seconds. The movement of kinetochore regions implies that the antagonistic chromosome fibres periodically grow (assemble) and shorten (disassemble) at comparable rates. Poleward movements must be caused by forces generated in disassembling fibres, whereas movements away from the poles, accompanied by fibre growth, are probably brought about by the internal elastic force of the chromosomes. Antagonistic fibres of a bivalent can operate in or out of phase. The movements of the three kinetochore regions are coordinated insofar as growing and shortening fibres coexist in a half‐spindle at almost any time [Fuge, H. (1987): European Journal of Cell Biology 44:294–298]. These observations are discussed in terms of microtubule dynamics.
Dynamics of the endoplasmic reticulum in living non‐muscle and muscle cells
Authors: Sanger Jean M. , Dome Jeffrey S. , Mittal Balraj , Somlyo Avril V. , Sanger Joseph W. 
Published: February 2005

AbstractThe dynamic changes of the endoplasmic reticulum (ER) in interphase and mitotic cells was detected by the vital fluorescent dye 3,3′‐dihexyloxacarbocyanine iodide. Two types of arrays characterize the continuous ER system in the non‐muscle PtK2 cell: (1) a lacy network of irregular polygons and (2) long strands of ER that are found aligned along stress fibers. In cross‐striated myotubes there was a periodic localization of fluorescence over each I‐band corresponding to the positions of the terminal cisternae of the sarcoplasmic reticulum (SR). In contrast to the arrangement in muscle cells, the aligment of the long strands of ER along stress fibers showed no strict periodicity that could be correlated with the sarcomeric units of the stress fibers. The ER and SR arrays seen in living cells were also detected in fixed cells stained with antibodies directed against proteins of the endoplasmic reticulum and sarcoplasmic reticulum, respectively. Observations of vitally stained PtK2 cells at 1 to 2 minute intervals using low light level video cameras and image processing techniques enabled us to see the polygonal ER units form and undergo changes in their shapes. During cell division, the ER, rhodamine 123‐stained mitochondria, and phagocytosed fluorescent beads were excluded from the mitotic spindle while soluble proteins were not. No obvious concentration or alignment of membranes could be found associated with the contractile proteins in the cleavage furrow. After completion of cell division there was a redeployment of the ER network in each daughter cell.
Synthesis of mammalian profilin in Escherichia coli and Its characterization
Authors: Babcock Gary , Rubenstein Peter A. 
Published: February 2005

AbstractProfilin is a G‐actin binding protein that may have a role in controlling the ratio of G/F actin within the cells To devise a way for obtaining large amounts of mammalian profilin in an active state, we transfected Escherichia coli with a plasmid containing a full‐length rat spleen profilin cDNA adjacent to a promoter inducible by isopropyl thiogalactoside (IPTG). Upon induction, they synthesized a new protein of 15,000 MW constituting approximately 5% of the total cell protein. This protein bound to poly‐L‐proline Sepharose and could be eluted with 7 M urea, behavior similar to that exhibited by authentic profilin. The protein could be released from the bacteria in soluble form following sonication, and the profilin could then be purified to homogeneity following chromatography on Sephadex G‐75 and DEAE A‐50 Sephadex. The protein began with an unblocked Ala, indicating that the initiating formyl and methionine residues had been removed. The dissociation of the recombinant profilin from chicken skeletal muscle actin was characterized by a Kd of approximately 2 μM based on gel filtration analysis and actin polymerization assays. These results show that purified active mammalian profilin can be made conveniently in large quantities. This study also demonstrates the feasibility of using bacterially synthesized profilin in structure‐function studies involving mutant profilins altered by site‐directed mutagenesis.
Acetylated α‐tubulin in spermatogenic cells of the crane fly Nephrotoma suturalis: Kinetochore microtubules are selectively acetylated
Authors: Wilson P. J. , Forer A. 
Published: February 2005

AbstractWe studied the distribution of acetylated α‐tubulin in the microtubules of spermatogenic cells from the crane fly Nephrotoma suturalis (Loew) using a mono‐clonal antibody specific for acetylated α‐tubulin (6‐11B‐1). We found that cells in all stages of spermatogenesis contained acetylated microtubules including primary spermatocytes, meiotic cells, spermatids, and sperm. A subset of the acety‐lated microtubules (those in midbodies and flagella) were resistant to cold depolymerization. Newly polymerized microtubules in nondividing cells were not acetylated for up to 15 min. indicating that acetylation lagged behind polymerization. In spindles, newly polymerized microtubules were acetylated after 5 min. Antibodies to acetylated α‐tubulin selectively stained chromosome‐to‐pole fibers in dividing cells, but the staining appeared to decrease and taper of at the kinetochores. This observation supports the hypothesis that tubulin subunits add at the kinetochore in metaphase and that acetylation occurs subsequent to addition. Further, this taper may be useful as a marker in anaphase, to distinguish between different hypotheses of chromosome motion.