bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of cDNA clones for Acanthamoeba profilin‐I and profilin‐II shows end to end homology with vertebrate profilins and a small family of profilin genes
Authors: Pollard Thomas D. , Rimm David L. 
Published: February 2005

AbstractWe have cloned and sequenced full length cDNAs for Acanthamoeba profilin‐I and profilin‐II. The genes and the encoded proteins are nearly identical except for the region between bp 121 and 210 where 35% of the nucleotides and 47% of amino acids differ. Most of these substitutions are conservative, although three of them are responsible for the differences in the isoelectric points of the isoforms [Kaiser et al., Cell Biol., 102:221–226, 1986]. The DNA sequence revealed six corrections in the previously published protein sequence of profilin‐I [Ampe et al., J. Biol. Chem. 260:834–840, 1985] and for the first time resolved the ambiguities at the five positions where profilin‐IA and ‐IB differ. The DNA sequence of profilin‐II also allowed us to make two corrections in the protein sequence [Ampeet al., FEBS Lett. 228:17–21, 1988a]. Probes prepared from the cDNAs revealed 1 profilin‐IA gene. one strongly cross‐hybridizing profilin‐I gene and one strongly reacting profilin‐II gene on Southern blots of Acanthamoeba DNA. Weaker reactions with other genomic DNA fragments leave open the possibility of one additional gene each for profilin‐I and profilin‐II. Four different profilin RNAs were resolved on Northern blots. It possible to align the sequences of the three Acanthamoeba profilins with the sequences of nine other profilins from five different phyla. There are only two invariant residues in these profilin sequences, but many pairwise identities and conservative substitutions that indicate considerable divergence of this family of proteins from its ancestral precursor.
In vitro migration of Hydra nematocytes: The influence of the natural extracellular matrix (the mesoglea), of collagen type IV and type I, laminin, and fibronectin on cell attachment, migration parameters, and on patterns of cytoskeletal proteins
Authors: Agosti Charo González , Stidwill Robert P. 
Published: February 2005

AbstractWe have established an in vitro migration system for nematocytes of the fresh water cnidarian Hydra. Nematocytes display a migratory behavior on isolated sheets of the naturally occurring extracellular matrix, the mesoglea, as well as on surfaces coated with collagen type IV or laminin. Cell behavior was analyzed using video microscopic techniques. Average migration speeds of nematocytes on the mesoglea (140 μm/hr) were lower than values reported from in vivo studies (500 μm/hr). Cells on collagen IV moved at about the same average speed (115 μm/hr) as nematocytes on the natural extracellular matrix; those on laminin were considerably slower (20 μm/hr). Attachment but no movement of cells was found on glass or on surfaces coated with collagen type I and fibronectin. In addition to the differential migration speeds, nematocytes displayed distinct morphologies depending on the substratum. In order to elucidate the causes of the observed cell shape and behavior modulations induced by the offered substratum, the arrangement of major cytoskeletal proteins in Hydra nematocytes during the in vitro migration or attachment was investigated. The pattern of F‐actin, myosin, and tubulin was determined by immunocytochemical techniques and confocal laser scanning microscopy in nematocytes moving on the mesoglea, on collagen IV, and on laminin, or in cells attaching to fibronectin. We found that the distribution of the cytoskeletal proteins was strikingly different in moving and in stationary cells. The patterns of cytoskeletal proteins in all nematocytes moving on the different substrata, however, was quite similar.
Compartmentalization and actin binding properties of ABP‐50: The elongation factor‐1 alpha of Dictyostelium
Authors: Dharmawardhane S. , Demma M. , Yang F. , Condeelis J. 
Published: February 2005

AbstractABP‐50 is the elongation factor‐1 alpha (EF‐1 alpha) of Dictyostelium discoideum (Yang et al.: Nature 347:494–496, 1990). ABP‐50 is also an actin filament binding and bundling protein (Demma et al.: J. Biol. Chem. 265:2286–2291, 1990). In the present study we have investigated the compartmentalization of ABP‐50 in both resting and stimulated cells. Immunofluorescence microscopy shows that in addition to being colocalized with F‐actin in surface extensions in unstimulated cells, ABP‐50 exhibits a diffuse distribution throughout the cytosol. Upon addition of cAMP, a chemoattractant, ABP‐50 becomes localized in the filopodia that are extended as a response to stimulation. Quantification of ABP‐50 in Triton‐insoluble and‐soluble fractions of resting cells indicates that 10% of the total ABP‐50 is recovered in the Triton cytoskeleton, while the remainder is in the soluble cytosolic fraction. Stimulation with cAMP increases the incorporation of ABP‐50 into the Triton cytoskeleton. The peak of incorporation of ABP‐50 at 90 sec is concomitant with filopod extension. Immunoprecipitation of the cytosolic ABP‐50 from unstimulated cells using affinity‐purified polyclonal anti ABP‐50 results in the coprecipitation of non‐filamentous actin with ABP‐50. Purified ABP‐50 binds to G‐actin with a Kd of approximately 0.09 μM. The interaction between ABP‐50 and G‐actin is inhibited by GTP but not by GDP, while the bundling of F‐actin by ABP‐50 is unaffected by guanine nucleotides. We conclude that a significant amount of ABP‐50 is bound to either G‐ or F‐actin in vivo and that the interaction between ABP‐50 and F‐actin in the cytoskeleton is regulated by cheniotactic stimulation.
Disruption of the golgi apparatus with brefeldin a does not destabilize the associated detyrosinated microtubule network
Authors: Burgess Teresa L. , Skoufias Dimitrios A. , Wilson Leslie 
Published: February 2005

AbstractStable subsets of microtubules (MTs) are often enriched in detyrosinated α‐tubulin. Recently it has been found that the Golgi apparatus is associated with a subset of relatively stable MTs and that detyrosinated MTs colocalize spatially and temporally with the Golgi apparatus in several cell lines. To determine whether the Golgi apparatus actively stabilizes associated MTs and thus allows their time‐dependent detyrosination, we have used the drug brefeldin A (BFA) to disrupt the Golgi apparatus and have monitored changes in the Golgi apparatus and MT populations using simultaneous immunofluorescence and fluorescent lectin microscopy. We found that although BFA caused the Golgi apparatus to completely redistribute to the endoplasmic reticulum (ER), the detyrosinated MTs were not disrupted and remained in a juxtanuclear region. By Western blot analysis we found that even after 6 h of continuous exposure of cells to BFA, there was no detectable reduction in the level of detyrosinated α‐tubulin. Simultaneous treatment with nocodazole and BFA led to a complete disruption of all MTs and normal Golgi structure/organization. Upon removal of nocodazole in the continued presence of BFA, we found that the detyrosinated MTs reformed in a compact juxtanuclear location in the absence of an intact Golgi complex. Finally, we found that the detyrosinated MTs colocalized precisely with a BFA‐resistant structure that binds to the lectin, wheat germ agglutinin. We conclude that the juxtanuclear detyrosinated MTs are not actively stabilized by association with BFA‐sensitive Golgi membranes. However, another closely associated structure which binds wheat germ agglutinin may serve to stabilize the juxtanuclear MTs. Alternatively, the MT organizing center (MTOC) and/or MT‐associated proteins (MAPs) may organize and stabilize the juxtanuclear detyrosinated MTs.
The calcium‐induced curvature reversal of rat sperm is potentiated by cAMP and inhibited by anti‐calmodulin
Authors: Lindemann Charles B. , Gardner Tressa K. , Westbrook Evonne , Kanous Kathleen S. 
Published: February 2005

AbstractRat sperm, demembranated with 0.1% Triton X‐100, were used to explore the reversal in flagellar curvature induced by calcium ion. As reported earlier (Lindemann and Goltz, Cell Motil. Cytoskeleton, 10:420–431, 1988), the radius of curvature of the flagellar midpiece of rat sperm is controlled by the free Ca2+ concentration. A reversal of the direction of curvature (judged by the asymmetric sperm head) takes place at ≈ 2.5 + 10−6 M free Ca2+.In our current study, the time course of the curvature change, after elevating free Ca2+ to 3.5 ± 10−4 M, was utilized to assess the effects of the cAMP‐kinase A pathway on the calcium response. In addition, calmodulin's involvement in this response was explored using anti‐calmodulin and Cd2+. The activity state of the sperm models (which could be directly influenced through cAMP) was found to control the rate of curvature change in response to increased free Ca2+. In the most extreme case, fully quiescent sperm did not respond to Ca2+ at all, and cAMP‐primed sperm models completed the response to Ca2+ in two minutes or less.Anti‐calmodulin demonstrated strong inhibitory effects on the curvature reversal. Cadmium ion was also extremely potent at blocking the response to Ca2+, completely eliminating the curvature reversal at 2 × 10−10 M free Cd2+.Based on these findings, it appears that the Ca2+‐activated curvature reversal of rat sperm is potentiated by cAMP‐dependent kinase and may be mediated through calmodulin.
Mechanical perturbation of webbed edges in 3T3 cells
Authors: Zand Martin S. , Albrecht‐Buehler Guenter 
Published: February 2005

AbstractWe have previously described actin edge‐bundles (AEBs) as cables of microfil‐aments lining the webbed edges of 3T3 cells (Zand and Albrecht‐Buehler: Cell Motil. Cytoskeleton 13:195–211, 1989). We have suggested that AEBs, along with their cell‐substratum adhesions, resist cortical tension and prevent the collapse of cytoplasm towards the nucleus. In this paper, we report several stages of AEB disassembly and re‐formation induced by the following micro‐manipulations
Scoring of the webbed edge of a 3T3 cells with a microneedle. As a result the sides of the score retracted and the severed AEB appeared to disassemble down to its terminal adhesion points. The retraction stopped after 20–40 seconds and the cells formed a webbed edge with large curvature. Over a period of 20–80 minutes, the new web decreased in length and depth, until it regained its approximate original shape.
Bending of cell processes at acute angles. As a result the processes moved until they projected at right angles to the side of the cell and formed new webs gradually expanded their area. In both cases, the nascent webs were lined by actin edge‐bundles.
Evidence for a gelsolin‐rich, labile F‐actin pool in human polymorphonuclear leukocytes
Authors: Watts Raymond G. , Howard Thomas H. 
Published: February 2005

AbstractFilamentous (F) actin is a major cytoskeletal element in polymorphonuclear leukocytes (PMNs) and other non‐muscle cells. Exposure of PMNs to agonists causes polymerization of monomeric (G) actin to F‐actin and activates motile responses. In vitro, all purified F‐actin is identical. However, in vivo, the presence of multiple, diverse actin regulatory and binding proteins suggests that all F‐actin within cells may not be identical. Typically, F‐actin in cells is measured by either NBDphallacidin binding or as cytoskeletal associated actin in Triton‐extracted cells. To determine whether the two measures of F‐actin in PMNs, NBDphallacidin binding and cytoskeletal associated actin, are equivalent, a qualitative and quantitative comparison of the F‐actin in basal, non‐adherent endo‐toxin‐free PMNs measured by both techniques was performed. F‐actin as NBD‐phallacidin binding and cytoskeletal associated actin was measured in cells fixed with formaldehyde prior to cell lysis and fluorescent staining (PreFix), or in cells lysed with Triton prior to fixation (PostFix). By both techniques, F‐actin in PreFix cells is higher than in PostFix cells (54.25 ± 3.77 vs. 23.5 ± 3.7 measured as mean fluorescent channel by NBDphallacidin binding and 70.3 ± 3.5% vs. 47.2 ± 3.6% of total cellular actin measured as cytoskeletal associated actin). These results show that in PMNs, Triton exposure releases a labile F‐actin pool from basal cells while a stable F‐actin pool is resistant to Triton exposure. Further characterizations of the distinct labile and stable F‐actin pools utilizing NBDphallacidin binding, ultracentrifugation, and electron microscopy demonstrate the actin released with the labile pool is lost as filament. The subcellular localization of F‐actin in the two pools is documented by fluorescent microscopy, while the distribution of the actin regulatory protein gelsolin is characterized by immunoblots with antigelsolin. Our studies show that at least two distinct F‐actin pools coexist in endotoxin‐free, basal PMNs in suspension: (1) a stable F‐actin pool which is a minority of total cellular F‐actin, Triton insoluble, resistant to depolymerization at 4°C, gelsolin‐poor, and localized to submembranous areas of the cell; and (2) a labile F‐actin pool which is the majority of total cellular F‐actin, Triton soluble, depolymerizes at 4°C, is gelsolin‐rich, and distributed diffusely throughout the cell. The results suggest that the two pools may subserve unique cytoskeletal functions within PMNs, and should be carefully considered in efforts to elucidate the mechanisms which regulate actin polymerization and depolymerization in non‐muscle cells.
Increased calmodulin affects cell morphology and mRNA levels of cytoskeletal protein genes
Authors: Rasmussen Colin D. , Means Anthony R. 
Published: February 2005

AbstractWe have previously described stable mouse C127 cell lines in which a CaM mini‐gene has been expressed in a bovine papilloma virus‐based expression vector (Rasmussen and Means: EMBO J. 6:3961–3968. 1987). Elevation of CaM to levels five‐fold higher than in control cells caused an acceleration in cell cycle progression by reducing the length of the G1 period. When these cell lines were originally isolated it was observed that cells in which CaM levels were increased had a flattened morphology. In this study we have examined the localization of actin, vimentin, and tubulin in these cells as compared to the BPV‐transformed control cell line in order to determine if changes in shape were accompanied by differences in the cytoskeletal organization. Cell‐cycle‐dependent changes in the levels of mRNAs for histone H4, glyceraldehyde‐3‐phosphate dehydrogenase, β‐actin, vimentin, and β‐tubulin have also been examined. Our results indicate that increased CaM causes differences in the organization of microfilaments, intermediate filaments, and microtubules and that these changes are accompanied by selective differences in the cell‐cycle‐dependent expression of some mRNAs. Elevated CaM was also correlated with a reduced stability of β‐tubulin mRNA. These studies indicate that CaM has pleiotropic effects on cell function and suggest that stable cell lines with altered CaM levels may provide a useful model system for understanding the moiecular basis of CaM‐dependent regulation of cellular processes.
Trout sperm swimming patterns and role of intracellular Ca++
Authors: Boitano Scott , Omoto Charlotte K. 
Published: February 2005

AbstractWe investigated the swimming patterns of trout sperm using computer‐assisted analyses of video microscopy. Under full activation conditions, in which 80–100% of sperm activate their motility, sperm swim in circular paths for 2–5 sec, followed by 30–60 sec of a more linear swimming, and, finally, cessation of movement, with a straightening of the flagella. Threshold activation, in which 50% of the sperm activate, is characterized by circular patterns of swimming for less than 20 sec, with straightened flagella upon cessation. Full activation and threshold activation are observed in low‐K+ solution or in an Mg++ ‐supplemented K+ solution. Similarities in swimming patterns in low‐K+ solution and in a Mg++ ‐supplemented K+ solution suggest a common underlying mechanism of activation. Initiation of movement in solutions with high Ca++ to K+ ratio is similar to activation in K+ ‐free solution. However, sperm in Ca++ ‐supplemented media resume circular swimming within 20–25 sec after activation, and, upon cessation of movement, the flagella are frequently cane shaped or bent. Differences in swimming patterns upon activation by high Ca++ concentration suggest additional effects of Ca++ on regulating swimming patterns. We used the fluorescent Ca++ indicator Fluo‐3 to measure changes in intracellular Ca++ concentration upon activation. Intracellular Ca++ concentration transiently increases upon activation, with peak Ca++ concentration coinciding with the period of circular swimming. This transient increase in Ca++ concentration is seen in the absence of external Ca++, providing strong evidence for the released of Ca++ from intracellular stores upon activation.
Reactivation of newt lung cilia: Evidence for a possible temperature‐ and MgATP‐induced activation mechanism
Authors: Hard Robert , Cypher Christopher 
Published: February 2005

AbstractOptimal conditions have been developed for the isolation and reactivation of highly coupled, demembranated ciliary axonemes from newt lungs [Hard, Cypher, and Schabtach, 1988, Cell Motil. Cytoskeleton 10:271–284]. In the present study, the motility of these cilia was further characterized by examining the effects of nucleotides, divalent cations, and temperature on beat frequency. When exposed to a reactivating solution containing Mg2+ and ATP, nearly 100% of the axonemes were motile and beat at frequencies of 0–50 Hz, depending on [MgATP] and temperature. Divalent cations were required for movement, with Mg2+ 2–3 times more effective than Ca2+. There was no absolute requirement for Ca2+ for motility. The beat frequencies obtained with fixed ATP and varying Mg2+ concentrations indicate that MgATP serves as the actual substrate. The effects of MgATP on beat frequency depended on the degree of mechanochemical coupling and temperature and MgATP‐induced transition between two distinct states whose maximum beat frequencies differ by 200–300%.
Colchicine‐binding sites of brain tubulins from an Antarctic fish and from a mammal are functionally similar, but not identical: Implications for microtubule assembly at low temperature
Authors: Skoufias Dimitrios A. , Wilson Leslie , Detrich H. William 
Published: February 2005

AbstractThe tubulins of Antarctic fishes possess adaptations that favor microtubule for mation at low body temperatures (Detrich et al.: Biochemistry 28:10085–10093, 1989). To determine whether some of these adaptations may be present in a domain of tubulin that participates directly or indirectly in lateral contact between microtubule protofilaments, we have examined the energetics of the binding of colchicine, a drug thought to bind to such a site, to pure brain tubulins from an Antarctic fish (Notothenia gibberifrons) and from a mammal (the cow, Bos taurus), At temperatures between 0 and 200C, the affinity constants for colchicine binding to the fish tubulin were slightly smaller (1.5–2.6‐fold) than those for bovine tubulin. van't Hoff analysis showed that the standard enthalpy changes for colchicine binding to the two tubulins were comparable (δH° = + 10.6 and + 7.4 kcal mol‐1 for piscine and bovine tubulins, respectively), as were the standard entropy changes (δS° = +61.3 eu for N. gibberifrons tubulin, +51.2 eu for bovine tubulin). At saturating concentrations of the ligand, the maximal binding stoichiometry for each tubulin was ∼ 1 mol colchicine/mol tubulin dimer. The data indicate that the colchicine‐binding sites of the two tubulins are similar, but probably not identical, in structure. The apparent absence of major structural modifications at the colchicine site suggests that this region of tubulin is not involved in functional adaptation for low‐temperature polymerization. Rather, the colchicine site of tubulin may have been conserved evolutionarily to serve in vivo as a receptor for endogenous molecules (i.e., “colchicine‐like” molecules or MAPs) that regulate microtubule assembly.
Human cardiac and skeletal muscle spectrins: Differential expression and localization
Authors: Vybiral Tomas , Winkelmann John C. , Roberts Robert , Joe Eun‐Hye , Casey Douglas L. , Williams Joseph K. , Epstein Henry F. 
Published: February 2005

AbstractWe describe multiple human cardiac and skeletal muscle spectrin isoforms. Cardiac muscle expresses five erythroid α,β spectrin‐reactive isoforms with estimated MR's of 280, 274, 270, 255, and 246 kD, respectively At least one nonerythroid α‐spectrin of MR 284 kD is expressed in heart. While skeletal muscle shares the 280, 270, and 246 kD erythroid spectrins, it expresses an immunologically distinct 284 kD nonerythroid α‐spectrin isoform. The 255 kD erythroid β‐spectrin isoform is specific for cardiac tissue. By immunocytochemistry, both erythroid β‐ and nonerythroid α‐spectrins are localized to costameres, the plasma membrane, and the neuromuscular junctional region.
Different assembly properties of cod, bovine, and rat brain microtubules
Authors: Fridén Bo , Stromberg Elisabeth , Wallin Margareta 
Published: February 2005

AbstractAssembly properties of cod, bovine, and rat brain microtubules were compared. Estramustine phosphate, heparin, poly‐L‐aspartic acid, as well as NaCl, inhibited the assembly and disassembled both bovine and rat microtubules by inhibition of the binding between tubulin and MAPs. The assembly of cod brain microtubules was in contrast only marginally affected by these agents, in spite of a release of the MAPs. The results suggest that cod tubulin has a high intrinsic ability to assemble. This was confirmed by studies on phosphocellulose‐purified cod tubulin, since the critical concentration for assembly was independent of the presence or absence of MAPs. The results show therefore that cod brain tubulin has, in contrast to bovine and rat brain tubulins, a high propensity to assemble under conditions which normally require the presence of MAPs.Even if cod MAPs, which have an unusual protein composition, were not needed for the assembly of cod microtubules, they were able to induce assembly of bovine brain tubulin. Both cod and bovine MAPs bound to cod microtubules, and bovine MAPI and MAP2 bound to, and substituted at least the 400 kDa cod protein. This suggests that the tubulin‐binding sites and the assembly‐stimulatory ability of MAPs are common properties of MAPs from different species, independent of the tubulin assembly propensity.