bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of trypsin and low Ca2+ on zonulae adhaerentes between chick retinal pigment epithelial cells in organ culture
Authors: Sandig Martin , Hergott Greg J. , Kalnins Vitauts I. 
Published: February 2005

AbstractThe junctional complexes in chick retinal pigment epithelial (RPE) cells in situ contain unusually large zonulae adhaerentes (ZAs) composed of subunits termed zonula adhaerens complexes (ZACs). To determine whether the properties of the ZAs differ between RPE cells which contain ZACs, and MDCK cells which lack ZACs, we investi‐gated the effects of treatment with trypsin and/or low Ca2+ by transmission electron microscopy and staining for F‐actin. Treatment of RPE cells for 1 h with trypsin alone has no apparent effect on the morphology of the ZA in either MDCK or RPE cells. In contrast to the ZAs in MDCK cells, which split after 3 min in low Ca2+, the ZAs in chick RPE cells stay intact even after 2 h, although the intermembrane discs, i.e., the extracellular components of the ZACs, are no longer visible. After 30 min of treatment with trypsin and low Ca2+, the ZAs split in both cell types. The CMBs start to contract, translocate toward the cell interior, and eventually disappear. This process continues even when the RPE cells are returned to normal medium. New ZAs, composed of ZACs, form between RPE cells 3 h after return to normal medium. These findings suggest that the ZACs in the ZAs of RPE cells are not directly responsible for the increase in resistance to low Ca2+. They also show that the ZA‐junctions in RPE cells are not only structurally different from those previously examined, but also behave differently in response to experimental manipulation.
Behavior of C‐shaped microtubule endings in the cell
Authors: Wolf Klaus Werner 
Published: February 2005

AbstractThe association of incomplete microtubule assemblies with either another incom‐plete structure or complete microtubules was studied in two organisms, the phytoflag‐ellate Polytoma papillatum and the phorid fly Megaselia scalaris, using transmission electron microscopy. In the alga, hook‐shaped appendages on cytoplasmic and spindle microtubules were detected. These resulted from the lateral association of a curved ribbon of protofilaments with the surface of a complete microtubular wall. In the fly, an S‐shaped protofilament sheet was found embedded in the kinetochore plate of a prometaphase I spematocyte. Tracing of the S‐shaped element towards the spindle pole revealed that it was formed by the lateral junction of two curved protofilament sheets. In all cases, the C‐shaped protofilament sheets represented the endings of complete micro‐tubules. Incomplete microtubules are generally considered as representing intermediates of microtubule assembly and disassembly. Since high molecular weight proteins are believed to be responsible for maintaining microtubule‐microtubule spacing, it is hypo‐thesized that the endings of growing and shrinking microtubules are sparsely studded with these proteins; their depletion allows lateral microtubule contacts. In addition, the microtubule‐microtubule contacts may be rendered possible by the flexibility of the slender elongated microtubule‐associated molecules. Relatively long C‐shaped proto‐filament appendages (0.6‐1.4 μm) were detected in this study. Therefore, it is plausible to assume that the protofilament sheets are stabilized by contact with one another or with an intact tubule wall.
Transmembrane cytoskeletal modulation in preterminal growing axons: I. Arrest of bulk and organelle transport in goldfish retinal ganglion cell axons regenerating in vitro by lectins binding to sialoglycoconjugates
Authors: Edmonds Brian T. , Koenig Edward 
Published: February 2005

AbstractGoldfish retinal ganglion cell (RGC) axons regenerating in vitro exhibit a novel mode of axoplasmic transport that entails a rapid bidirectional bulk redistribution of axoplasm, “packaged” as protruding varicosities and non‐protruding phase‐dense inclusions (Koenig et al.: J. Neurosci. 5:715–729, 1985; Edmonds and Koenig Brain Res. 406:288–293, 1987). We have used phase‐contrast video microscopy to study transmembrane effects of surface‐binding lectins on bulk transport and transport of single visible organelles in RGC axons. Our findings show that certain lectins which crosslink sialoglycoconjugates, such as wheat germ agglutinin (WGA) and the more specific sialic acid‐binding lectin Limax flavus agglutinin (LFA), induce a rapid inhibition of transport activity. The LFA‐induced inhibition of transport can be reversed by appropriate simple sugar haptens, and can also be antagonized by pretreatment with cytochalasin D. One of the consequences of LFA binding is an increase in RITC‐conjugated phalloidin fluorescence staining of preterminal axons. The latter observation in conjunction with the antagonistic action of cytochalasin D suggests that one possible explanation for the transmembrane arrest of transport induced by crosslinking of surface sialoglycoconjugates may involve a polymerization and/or reorganization of the actin filament network which hinders translocation of mobile axoplasmic components.
Reorganization of circumferential microfilament bundles in retinal epithelial cells during mitosis
Authors: Sandig Martin , Kalnins Vitauts I. 
Published: February 2005

AbstractTo examine the behaviour of the apical circumferential microfilament bundles (CMBs) associated with the zonula adhaerens (ZA)‐junctions during mitosis, retinal pigment epithelial cells were labelled for F‐actin, and retinas were serially sectioned for TEM. The results show that the ZA‐CMB‐complex persists throughout all stages of mitosis. At metaphase, the cells round up, but stay joined apically to adjacent cells by ZA‐junctions. At telophase, the cleavage furrow forms asymmetrically from the basal end progressively toward the apical end, where the daughter cells remain connected by an intercellular bridge (IB). As the cleavage furrow with the contractile ring (CR) approaches the CMB, the two microfilament (MF) systems are oriented perpendicularly to each other. At the level of the CMB, the MFs of the CR connect the opposite sides of the CMB and bisect it into two CMBs, one for each of the two daughter cells. Subsequently, the CR in the IB splits into two, one on either side of the midbody. The two daughter cells, having acquired a complete CMB of their own, do not become direct neighbours, since adjacent cells, which remain joined to the apical ZA‐junction of the dividing cell, are observed in the cleavage furrow, where they meet and form a ZA‐junction between themselves, just below the IB. Separation of the daughter cells without losing contact with neighbouring cells at the level of the apical ZA‐junction thus maintains the integrity of the epithelial sheet during mitosis.
Dynamic properties of intermediate filaments: Disassembly and reassembly during mitosis in baby hamster kidney cells
Authors: Rosevear Ellen Rae , McReynolds Manette , Goldman Robert D. 
Published: February 2005

AbstractA morphological analysis of the organizational changes in the type III intermediate filament (IF) system in dividing baby hamster kidney (BHK‐21) cells was carried out by immunofluorescence and immunoelectron microscopy. The most dramatic change occurred during prometaphase, when the typical network of long 10‐nm‐diameter IF characteristic of interphase cells disassembled into aggregates containing short 4–6 nm filaments. During anaphase‐telophase, arrays of short IF reappeared throughout the cytoplasm, and, in cytokinesis, the majority of IF were longer and concentrated in a juxtanuclear cap. These results demonstrate that the relatively stable IF cytoskeletal system of interphase cells is partitioned into daughter cells during mitosis by a process of disassembly and reassembly. This latter process occurs in a series of morphologically distinct steps at different stages of the mitotic process.
Myosin II antibodies inhibit the resorption activity of isolated rat osteoclasts
Authors: Sato Masahiko , Grasser William 
Published: February 2005

AbstractWe present microinjection data in support of an indirect approach by which cytoplasmic protein interactions important in the processes of bone resorption can be elucidated. Three polyclonal antibodies (M1, M3, M5) raised against myosin II from perfused rat liver differently affected the actin‐activated Mg ATPase of myosin II. These antibodies microinjected into isolated rat osteoclasts affected osteoclast morphology and activity in bone resorption. M1, which completely inhibited myosin ATPase activity at a antibody:myosin ratio of 10:1, initially promoted the extension/retraction motility of lamellipodia but eventually reduced the spread area of osteoclasts along the substrate after 20 hr. M3, which inhibited ATPase activity by 70%, had similar effects; however, M5, which weakly inhibited ATPase activity, neither promoted extension/retraction nor reduced spread area of osteoclasts. Immunofluorescence showed that these antibodies removed myosin II from the majority of actin filaments in injected osteoclasts. Because antibodies that did not bind to a myosin II column had little effect on the extension/retraction of lamellipodia or the osteoclast spread area, these data suggest that myosin II participates in the stabilization of osteoclast lamellipodia along the substrate. M1 injection strongly inhibited injected osteoclasts from excavating resorption lacunae in bone slices, compared to control antibody. M3 and M5 were less effective but also inhibited bone resorption. These data show that myosin II is functionally important in bone resorption and that the osteoclast‐differentiated activity of bone resorption is a more sensitive assay for myosin activity than lamellipodia motility or cell morphology.
Modulation of keratin intermediate filament distribution in vivo by induced changes in cyclic AMP‐dependent phosphorylation
Authors: Eckert Barry S. , Yeagle Philip L. 
Published: February 2005

AbstractTreatment of PtK1 cells with 5 mM acrylamide for 4 hr induces reversible de‐phosphorylation of keratin in concert with reversible aggregation of intermediate filaments (Eckert and Yeagle, Cell Motil. Cytoskeleton 11:24–30, 1988). We have examined this phenomenon by 1) in vitro phosphorylation of isolated PtK1 keratin filaments and 2) combined treatments of PtK1 cells with both acrylamide and agents which elevate intracellular cAMP levels. PtK1 keratins were incubated in gamma‐32P‐ATP in the presence or absence of cAMP‐dependent kinase (A‐kinase) and cAMP. Levels of phosphorylation were analyzed by electrophoresis and autoradiography. Phosphorylation of keratin polypeptides (56 kD, 53 kD, 45 kD, 40 kD) occurred without added kinase, suggesting the presence of an endogenous kinase which remains with intermediate filaments in residues of Triton X‐100 extracted cells. Phosphorylation levels were increased by A‐kinase but not by cAMP alone, indicating the presence of cAMP‐dependent phosphorylation sites in addition to sites phosphorylated by the endogenous kinase. To study the possible role of cAMP‐dependent phosphorylation in acrylamide‐induced aggregation of keratin filaments, we treated cells with acrylamide in the presence of 8‐bromo‐cAMP (brcAMP), pertussis toxin (PT), isobutylmethylxanthine (IBMX), or forskolin, which increase intracellular cAMP levels. The distribution and phosphorylation levels of keratin filaments, as well as intracellular cAMP levels, were determined for each of these treatments. In addition to aggregation and dephosphorylation of keratin filaments reported previously, treatment of cells with acrylamide alone also results in reduced levels of intracellular cAMP. 8‐bromo‐cAMP, IBMX, and forskolin prevent acrylamide‐induced aggregation of keratin filaments and result in both normal levels of keratin phosphorylation and normal intracellular cAMP levels. PT was apparently ineffective. These observations suggest that 1) PtK1 keratins are phosphorylated by cAMP‐dependent kinase and an endogenous, cAMP‐independent kinase and 2) alteration of levels of cAMP‐dependent phosphorylation may be involved in aggregation of keratin filaments in response to acrylamide.
Localization of the centrin‐related 165,000‐Mr protein of PtK2 cells during the cell cycle
Authors: Baron Andre T. , Greenwood Tammy M. , Salisbury Jeffrey L. 
Published: February 2005

AbstractIn this study, we follow changes in localization of the centrin‐related 165,000‐Mr protein of PtK2 cells during the cell cycle. This protein is a component of a pericentriolar lattice that consists of pericentriolar satellites, pericentriolar matrix, and basal feet (Baron A.T., and J.L. Salisbury, J. Cell Biol. 107:2669–2678, 1988). By immunofluorescence microscopy, the 165,000‐Mr protein is seen as a constellation of pericentrosomal spots. We observe that cells in late G1 and S are characterized by a dense centrosomal focus of spots with additional spots dispersed throughout the cytoplasm. In G2, one bright centrosomal focus of clustered spots is observed. As the cells proceed through prophase this single focus divides, forming two foci that move toward opposite sides of the nucleus. During prometaphase, each polar focus of spots disperses. At metaphase, the spots are distributed throughout each half‐cytoplast from the poles to the chromosomes. During anaphase chromosome movement, some spots are seen beside and behind the trailing chromosome arms while others are clustered at the poles. At telo‐phase, pericentrosomal spots radiate from the poles to surround each mass of chromatin. In early G1, pericentrosomal spots surround each newly formed nucleus. We conclude that the 165,000‐Mr protein is a dynamic component of both the centrosome (pericentriolar matrix) and the mitotic apparatus (spindle matrix).
Microtubule reorganization during the cell cycle in synchronized BY‐2 tobacco suspensions
Authors: Hasezawa Seiichiro , Marc Jan , Palevitz Barry A. 
Published: February 2005

AbstractTobacco BY‐2 suspension cultures were synchronized with aphidicolin in order to assess the relationship between microtubules (MTs), microfilaments (MFs), and the nuclear envelope (NE) at different stages of the cell cycle. Using immunofluorescence techniques, ordered MT arrays were found in the cortex in G1; few MTs are evident deeper in the cytoplasm or near the nucleus. However, MTs radiate from the surface of the nucleus during S and G2 as the interphase cortical array is replaced by the preprophase band. Perinuclear fluorescence is also visible at the end of cytokinesis but does not overlap with new ordered cortical arrays early in G1. When isolated nuclei are examined, associated MTs are again evident in S and G2, but not in G1. Microfilaments are colocalized with the MTs in the radiating arrays, as ascertained by dual staining of cells with rhodamine phalloidin. Propyzamide treatment leads to the loss of MTs at all stages, while cytoplasmic and perinuclear MF networks persist. Conversely, cytochalasin D disrupts MFs, including those radiating from the nucleus during S and G2, without any apparent effect on MTs. The results cast doubt on a proposed role for the NE in the generation of cortical MTs in plants. A universal role for MFs in the deployment of MTs is also in question.
Effect of cytochalasins on actin in dividing root tip cells of Allium and Triticum: A comparative immunocytochemical study
Authors: McCurdy David W. , Palevitz Barry A. , Gunning Brian E. S. 
Published: February 2005

AbstractA collaborative effort was initiated to resolve differences in two recent papers on the effects of cytochalasins in root cells. While both studies reported similar effects on interphase cells (i.e., replacement of microfilaments by many small specks and rods), Palevitz (Cell Motil. Cytoskeleton 9:283‐298, 1988) maintained that cytochalasins B and D induce actin aggregation at the poles of dividing Allium root cells at a concentration of 10 μM with rhodamine phalloidin as a reporter probe, whereas McCurdy and Gunning (Cell Motil. Cytoskeleton 15:76‐87, 1990) could not find these aggregates following antiactin immunocytochemistry in Triticum roots treated with CB at 50 μM. Employing identical methods and materials in the same laboratory, we found that CD induces polar actin aggregates in dividing cells of both species. However, the aggregates in Triticum are smaller and occur less frequently than those in Allium. A similar pattern is seen with CB.
Microvascular endothelial‐derived autacoids regulate pericyte contractility
Authors: Dodge Andrea B. , Hechtman Herbert B. , Shepro David 
Published: February 2005

AbstractA silicone rubber assay is used in conjunction with morphometric measurements to characterize in vitro the contractile properties of retinal pericytes in response to endothelial secreted factors. Factor(s) present in conditioned media derived from pulmonary and retinal microvascular endothelial cells and pulmonary artery endothelial cells promote pericyte contractions. Using a radioimmunoassay significant levels of endothelin immunoreactivity are measured in conditioned media obtained from all three cell lines. Thrombin treatment enhanced endothelin‐like secretions by pulmonary microvascular endothelial cells, but significantly reduced levels of endothelin‐like immunoreactivity secreted by retinal microvascular endothelial cells. Synthetic endothelin and thromboxane A2 (TxA2) stimulate pericyte contractions, whereas prostaglandin I2 (PGI2) promotes pericyte relaxation. Thrombin and angiotensin II (ang II) have no effect on pericyte contractility. However, using cocultures of pericytes and endothelial cells we observe endothelial‐dependent pericyte contractions in response to thrombin and ang II. Thrombin and ang II stimulate the release of endothelial‐derived contracting factors, with characteristics similar to endothelin. These data suggest microvascular endothelial cell‐pericyte interactions may regulate, at least in part, microvessel contractility.
Variant cDNAs encoding proteins similar to the α subunit of chicken CapZ
Authors: Cooper John A. , Caldwell Jane E. , Gattermeir David J. , Torres Michelle A. , Amatruda James F. , Casella James F. 
Published: February 2005

AbstractChicken adult muscle and liver cDNA libraries were screened with a cDNA, α1, previously isolated from a chicken embryo library by screening with antibodies against the α subunit of chicken CapZ. cDNAs with a new coding region, called α2, were found in addition to ones with the α1 coding region. α2 predicts a protein sequence that matches exactly the N‐terminal sequence of 5 peptides prepared from CapZ α purified from chicken muscle, while the protein sequence predicted by α1 matches the peptides well, but not exactly. The predicted protein sequences of α1 and α2 are very similar to each other, and they are similar to those of the α subunit of capping protein from Dictyostelium [Hartmann et al., J. Biol. Chem. 163:5254–5254, 1989] and an actin‐binding protein from Xenopus [Ankenbauer et al., Nature 342:822–824, 1989]. Other conserved features of the predicted primary and secondary structures are noted. Chicken α1 and α2 are transcribed in all of 7 adult chicken muscle and non‐muscle tissues in comparable amounts by Northern analysis. α2 has four poly(A)+ RNA transcripts, one of which is rare in liver. α1 has two transcripts. α1 and α2 are encoded by different single‐copy genes by Southern analysis of chicken genomic DNA.
Decrease of internal free calcium and human sperm movement
Authors: Serres Catherine , Feneux Danielle , Berthon Brigitte 
Published: February 2005

AbstractIn order to elucidate the effects of calcium on the movement of human spermatozoa, studies were conducted using motile cells selected by swim‐up migration at 37°C in 5% CO2 in air in a synthetic BWW medium containing 1.7 × 10−3 M CaCl2 or BWW without added calcium (BWW‐Ca). Preliminary experiments have confirmed that the addition of EGTA (5 × 10−3; 10−2 M) to BWW medium decreased the intracellular calcium concentration ((Ca++)i) of spermatozoa, as measured in cells loaded with a fluorescent Ca++ indicator, Quin‐2. Concomitant measurements of (Ca++)i and sperm movement (analysed by videomicrography at 200 f/s at room temperature) were carried out on Quin‐2 loaded cells incubated in BWW‐Ca medium plus EGTA (10−5 M; 10−4 M; 10−3 M). Under these conditions a decrease in (Ca++)i was observed and associated with a decrease in mean amplitude of lateral head displacement (ALH). Analysis using an automatic analyser (Hamilton Thorn at 37°C) confirmed these results: the percentage of spermatozoa swimming with ALH ≤ 6 μm is decreased when the external free calcium in BWW‐Ca is decreased by the addition of 10−5 M, 10−4 M, or 10−3 M EGTA. Flagellar analysis of the sperm population characterized by ALH ≤ 6 μm showed a large proximal curvature of the tail associated with a low propagation wave velocity and a low beat frequency as compared to the spermatozoa with ALH ≤ 6 μm with similar progressive velocities. These characteristics result in a high flagellar beat efficiency (in terms of head displacement per beat). The disappearance of this pattern of movement when intracellular calcium is lowered indicates that calcium plays a complex role in the relationship between curvature and wave propagation. The ability of spermatozoa to modulate their movement in response to an alteration in the intracellular calcium level confirms the role of calcium in controlling flagellar movement in intact cells.
Relation of nucleolar structure and position to the cytoplasmic microtubule system in Dictyostelium
Authors: Sameshima Masazumi , Fujimoto Hirotaka , Imai Yoshihisa , Tsukita Shoichirou , Hashimoto Yohichi 
Published: February 2005

AbstractThe cytoplasmic microtubule system seems to influence the position and structure of nucleoli in Dictyostelium discoideum amoebae in several growing and migrating states. For example, nucleoli were usually excluded from the nuclear periphery near the microtubule‐organizing center (MTOC) in all cases; and in migrating adherent cells, more than half the nucleoli were located opposite the MTOC. This localization was disrupted by nocodazole treatment, after which the nucleoli were largely dispersed except near the MTOC. More extensive effects of microtubules on nucleolar structure were seen in aggregating cells. In contrast to the normal oval structure in growing cells, nucleoli took on a different morphology: they protruded from the leading edge of nuclei and elongated to form nozzle‐like structures. Analysis by rapid‐freeze substitution and indirect immunofluorescence showed each nozzle surrounded by more than 10 microtubules; and in the presence of nocodazole, the microtubules shortened as expected and the nozzles disappeared. Between microtubules and the outer nuclear envelope, various‐sized cross‐bridges were seen. The implication that microtubules were associated with the nucleoli in aggregating cells was verified in vitro: nuclei isolated from growing cells contained the MTOC but few if any detectable microtubules; but nuclei from aggregating cells were surrounded by them. These data are consistent with the notion the microtubule system may help regulate the position and conformation of nucleoli during early development of Dictyostelium.