bluuebunny/crossref_metadata_2025_split huggingface dataset:
Changes in cell surface and cortical cytoplasmic organization during early embryogenesis in the preimplantation mouse embryo
Authors: Ducibella T , Ukena T , Karnovsky M , Anderson E 
Published: May 2004

Membrane topography and organization of cortical cytoskeletal elements and organelles during early embryogenesis of the mouse have been studied by transmission and scanning electron microscopy with improved cellular preservation. At the four- and early eight-cell stages, blastomeres are round, and scanning electron microscopy shows a uniform distribution of microvilli over the cell surface. At the onset of morphogenesis, a reorganization of the blastomere surface is observed in which microvilli becomes restricted to an apical region and the basal zone of intercellular contact. As the blastomeres spread on each other during compaction, many microvilli remain in the basal region of imminent cell-cell contacts, but few are present where the cells have completed spreading on each other. Microvilli on the surface of these embryos contain linear arrays of microfilaments with lateral cross bridges.
Microtubules and mitochondria become localized beneath the apposed cell membranes during compaction. Arrays of cortical microtubules are aligned parallel to regions of apposed membranes. During cytokinesis, microtubules become redistributed in the region of the mitotic spindle, and fewer microvilli are present on most of the cell surface. The cell surface and cortical changes initiated during compaction are the first manifestations of cell polarity in embryogenesis. These and previous findings are interpreted as evidence that cell surface changes associated with trophoblast development appear as early as the eight-cell stage. Our observations suggest that morphogenesis involves the activation of a developmental program which coordinately controls cortical cytoplasmic and cell surface organization.
Choline acetyltransferase activity of spinal cord cell cultures increased by co-culture with muscle and by muscle-conditioned medium
Authors: Giller EL , Neale JH , Bullock PN , Schrier BK , Nelson PG 
Published: May 2004

Activity of the enzyme choline acetyltransferase (CAT), which mediates the synthesis of the neurotransmitter, acetylcholine, was increased up to 20- fold in spinal cord (SC) cells grown in culture with muscle cells for 2 wk. This increase was directly related to the duration of co-culture as well as to the cell density of both the SC and muscle involved and was not affected by the presence of the acetylcholine receptor blocking agent, α-bungarotoxin. Glutamic acid decarboxylase (GAD) activity was often markedly decreased in SC-muscle cultures while the activities of acetylcholinesterase and several other enzymes were little changed.
Increased CAT activity was also observed when SC cultures were maintained in medium which had been conditioned by muscle cells or by undifferentiated cells from embryonic muscle. Muscle-conditioned medium (CM) did not affect the activities of SC cell GAD or acetylcholinesterase. Dilution or concentration of the CM directly affected its ability to increase SC CAT activity , as did the duration and timing of exposure of the SC cells to the CM. The medium could be conditioned by muscle cells in the presence or absence of serum, and remained effective after dialysis or heating to 58 degrees C. Membrane filtration data were consistent with the conclusion that the active material(s) in CM had a molecular weight in excess of 50,000 daltons. We conclude that large molecular weight material that is released by muscle cells is capable of producing a specific increase in CAT activity of SC cells.
Immunological and ultrastructural studies of neurofilaments isolated from rat peripheral nerve
Authors: Schlaepfer WW 
Published: May 2004

Neurofilaments were isolated from desheathed and minced segments of rat peripheral nerve by osmotic shock into 0.01 M Tris-HCI buffer, pH 7.2. Freshly isolated neurofilaments were observed to undergo disassembly by progressive fragmentation upon exposure of dilute tissue extracts to this buffer. Low- and high-speed centrifugations of these tissue extracts separated membranous and particulate constituents and produced a progressive enrichment of 68,000-dalton polypeptide band in successive supernates, as determined by analyses of soluble proteins by SDS-polyacrylamide electrophoresis. The final high-speed supernatant fractions (S3) of nerve extracts, which were predominantly composed of 68,000-dalton polypeptide, were used to raise a specific experimental antisera in rabbits.
Utilizing techniques of immune electron microscopy, experimental rabbit antisear was shown to contain antibodies against neurofilaments. Intact neurofilaments isolated from rat nerves and attached to carbon-coated grids became decorated when exposed to experimental rabbit antisera or purified gamma globulin (IgG) derivatives. The decoration of neurofilaments closely resembled the IgG coating seen in immune electron microscopy. Antibody absorption techniques were used to identify the biochemical constituency of neurofilamentous antigenic determinants. The decoration of neurofilament by experimental IgG was not altered by additions of tubulin or bovine serum albumin, but was prevented by additions of S3 fractions as well as the 68,000-dalton polypeptide of this fraction which was eluted and recovered from polyacrylamide gels.
These findings are indicative that a 68,000-dalton polypeptide is a constituent subunit of rat peripheral nerve neurofilaments.
Assembly of the sarcoplasmic reticulum proteins in differentiating rat skeletal muscle cell cultures: localization by immunofluorescence of sarcoplasmic reticulum proteins in differentiating rat skeletal muscle cell cultures
Authors: Jorgensen AO , Kalnins VI , Zubrzyca E , MacLennan DH 
Published: May 2004

Immunofluorescent staining techniques were used to study the distribution of the Ca(2) + Mg(2+)-dependent ATPase and calsequestrin in primary cultures of differentiating rat skeletal muscle cells, grown for different periods of time under various culture conditions.
In mononucleated myoblasts calsequestrin was detected after 45 h in culture whereas the ATPase was not detected until 60 h. After cell fusion began, both proteins could be identified in all multinucleated cells. Myoblasts grown for longer than 60 h in low Ca(2+) medium contained calsequestrin and the ATPase, even though they were unable to fuse. These studies at the cellular level confirm biochemical findings on the biosynthesis of calsequestrin and the ATPase.
Immunofluorescent staining of myoblasts showed that calsequestrin first appears in a well-defined region of the cell near one end of the nucleus. At later times, the staining occupied progressively larger regions adjacent to the nucleus and took on a fibrous appearance. This suggests that calsequestrin first accumulates in the Golgi region and then gradually spreads throughout the cell. In contrast, the ATPase appeared to be concentrated in many small patches or foci throughout the cytoplasm and was never confined to one particular region, although some parts of the cell often stained more intensely than others. In multinucleated cells, alternating dark and fluorescent strands parallel to the longitudinal axis of the cells were evident.
Temporal expression of membrane antigens during mouse spermatogenesis
Authors: Millete CF , Bellve AR 
Published: May 2004

The temporal expression of cell surface antigens during mammalian spermatogenesis has been investigated using isolated populations of mouse germ cells. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids, and residual bodies of Regaud and mature spermatozoa, contain common antigenic membrane components which are not detected before the pachytene stage of the first meiotic prophase. These surface constituents are not detected on isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, or leptotene and zygotene primary spermatocytes.
These results have been demonstrated by immunofluorescence microscopy, by complement-mediated cytotoxicity, and by quantitative measurements of immunoglobulin (Ig) receptors on the plasma membrane of all cell populations examined. The cell surface antigens detected on germ cells are not found on mouse thymocytes, erythrocytes, or peripheral blood lymphocytes as determined by immunofluorescence and by cytotoxicity assays. Furthermore, absorption of antisera with kidney and liver tissue does not reduce the reactivity of the antibody preparations with spermatogenic cells, indicating that these antigenic determinants are specific to germ cells.
This represents the first direct evidence for the ordered temporal appearance of plasma membrane antigens specific to particular classes of mouse spermatogenic cells. It appears that at late meiotic prophase, coincident with the production of pachytene primary spermatocytes, a variety of new components are inserted into the surface membranes of developing germ cells. The further identification and biochemical characterization of these constituents should facilitate an understanding of mammalian spermatogenesis at the molecular level.
Changes in the distribution of a major fibroblast protein, fibronectin, during mitosis and interphase
Authors: Stenman S , Wartiovaara J , Vaheri A 
Published: May 2004

The distribution of a major fibroblast protein, fibronectin, was studied by immunofluorescence and immunoscanning electron microscopy in cultures of human and chicken fibroblasts during different phases of the cell cycle. The main findings were: (a) In interphase cells, the intensity of surface-associated fibronectin fluorescence correlated with that of intracellular fibronectin fluorescence. (b) The intensity of the fluorescence of both surface-associated and intracellular fibronectins was not changed in cells that were synthesizing DNA. (c) Mitotic cells had reduced amounts of surface-associated but not of intracellular fibronectin. The surface fibronectin that remained on meta-, ana-, or telophase cells had a distinct punctate distribution and was also localized to strands attaching the cells to the substratum. Fibronectin strands first reappeared on the surface of flattening cytoplasmic parts of telophase cells. (d) Fibronectin was also detected in extracellular fibrillar material on the growth substratum, particularly around dividing cells.
Thus, surface-associated fibrillar fibronectin was present during G(1), S, and G(2) but in cells undergoing mitosis the distribution was altered and the amount appeared to be reduced. The observations on the distribution of surface-associated fibronectin suggest that rather than being involved in growth control this fibronectin plays a structural role in interactions of cells with the environment.
Polymorphism of myosin among skeletal muscle fiber types
Authors: Gauthier GF , Lowey S 
Published: May 2004

An immunocytochemical approach was used to localize myosin with respect to individual fibers in rat skeletal muscle. Transverse cryostat sections of rat diaphragm, a fast-twitch muscle, were exposed to fluorescein-labeled immunoglobulin against purified chicken pectoralis myosin. Fluorescence microscopy revealed a differential response among fiber types, identified on the basis of mitochondrial content. All white and intermediate fiber but only about half of the red fiber reacted with his antimyosin. In addition, an alkali-stable ATPase had the same pattern of distribution among fibers, which is consistent with the existence of two categories of red fibers.
The positive response of certain red fibers indicates either that their myosin has antigenic determinants in common with "white" myosin, or that the immunogen contained a "red" myosin. Myosin, extracted from a small region of the pectorlis which consists entirely of white fibers, was used to prepare an immunoadsorbent column to isolate antibodies specific for white myosin. This purified anti-white myosin reacted with the same fibers of the rat diaphragm that had reacted with the white, intermediate, and some red fibers are sufficiently homologous to share antigenic determinants.
In a slow-twitch muscle, the soleus, only a minority of the fiber reacted with antipectoralis myosin. The majority failed to respond; hence, they are not equivalent to intermediate fibers of the diaphragm; despite their intermediate mitochondrial content.
Immunocytochemical analysis of two different musles of the rat has demonstrated that more than one isoenzyme of myosin can exist in a single muscle, and that individual fiber types can be recognized by immunological differences in their myosin. We conclude that, in the rat diaphragm, there are at least two immunochemically distinct types of myosin and four types of muscle fibers: white, intermediate, and two red. We suggest that these fibers correspond to the four types of motor units described by Burke et al. (Burke, R. E., D. N. Levine, P. Tsairis, and F. E. Zajac, III 1973. J. Physiol. (Lond) 234:723-748.)in the cat gastrocnemius.`
Localization of thyroglobulin antigenicity in rat thyroid sections using antibodies labled with peroxidase or (125)I-radioiodine
Authors: Paiement J , Leblond CP 
Published: May 2004

In the hope of localizing thyroglobulin within focullar cells of the thyroid gland, antibodies raised against rat thyroglobulin were labeled with the enzyme horseradish peroxidase or with (125)I-radioiodine. Sections of rat thyroids fixed in glutaraldehyde and embedded in glycol methacrylate or Araldite were placed in contact with the labeled antibodies. The sites of antibody binding were detected by diaminobenzidine staining in the case of peroxidase labeling, and radioautography in the case of 125(I) labeling.
Peroxidase labeling revealed that the antibodies were bound by the luminal colloid of the thyroid follicles and, within focullar cells, by colloid droplets, condensing vacuoles, and apical vesicles. (125)I labeling confirmed these findings, and revealed some binding of antibodies within Golgi saccules and rough endoplasmic reticulum. This method provides a visually less distinct distribution than peroxidase labeling, but it allowed ready quantitation of the reactions by counts of silver grains in the radioautographs. The counts revealed that the concentration of label was similar in the luminal colloid of different follicles, but that it varied within the compartments of follicular cells. A moderate concentration was detected in rough endoplasmic reticulum and Golgi saccules, whereas a high concentration was found in condensing vacuoles, apical vesicles, and in the luminal colloid. Varying amounts of label were observed over the different types of colloid droplets, and this was attributed to various degrees of lysosomal degradation of thyroglobulin.
It is concluded that the concentration of thyroglobulin antigenicity increases during transport from the ribosomal site of synthesis to the follicular colloid, and then decreases during the digestion of colloid droplets which leads to the release of the thyoid hormone.
Changes of the cell surface and of the digestive apparatus of dictyostelium discoideum during the starvation period triggering aggregation
Authors: De Chastellier C , Ryter A 
Published: May 2004

The effects of starvation on the cell morphology of Dictyostelium discoideum were studied with different cytochemical techniques, and with a morphometric method by which the surface areas of the cell membrane and of the digestive system can be determined.
During the first 2 h, the cell membrane becomes very wrinkled and many phagocytic cups and filopods are formed. These changes are in accord with the 40 percent increase in the cell surface area to cytoplasmic volume ratio observed, which is mainly due to a strong decrease in the cytoplasmic volume. At this time of starvation, cells are able to ingest twice as many yeast as during growth. Afterwards, while the phagocytic ability decreases, the phagocytic cups disappear, and all the cells become bristled with many thin filopods. In spite of these morphological changes, no quantitative or topological differences have been observed concerning the polysaccharide content of the plasma membrane, whether it was stained with phosphotungstic acid, silver proteinate, or ruthenium red.
During this time, the digestive vacuoles imbricate one into the other. Part of the vacuoles are degraded by this process, thus leading to an atrophy of the digestive apparatus. The digestive apparatus is progressively replaced by an autophagic system. Polysaccharide stainings and morphological observations show that the cytosegresomes seem to originate from the food vacuoles which flatten and sequester portions of cytoplasm. After 5 h of starvation, the digestive system is entirely transformed into an autophagic apparatus. The cell population appears to be homogeneous with respect to these changes. Therefore, potential precursors of prestalk and prespore cells were not observed.
Structural analysis of human neutrophil migration: Centriole, microtubule, and microfilament orientation and function during chemotaxis
Authors: Malech HL , Root RK , Gallin JI 
Published: May 2004

Orientation of nucleus, centriole, microtubules, and microfilaments within human neutrophils in a gradient of chemoattractant (5 percent Escherichia coli endotoxin-activated serum) was evaluated by electron microscopy. Purified neutropils (hypaque-Ficoll) were placed in the upper compartment of chemotactic chambers. Use of small pore (0.45 μm) micropore filters permitted pseudopod penetration, but impeded migration. Under conditions of chemotaxis with activated serum beneath the filter, the neutrophil population oriented at the filter surface with nuclei located away from the stimulus, centrioles and associated radial array of microtubules beneath the nuclei, and microfilament-rich pseudopods penetrating the filter pores. Reversal of the direction of the gradient of the stimulus (activated serum above cells) resulted in a reorientation of internal structure which preceded pseudopod formation toward the activated serum and migration off the filter. Coordinated orientation of the entire neutrophil population did not occur in buffer (random migration) or in a uniform concentration of activated serum (activated random migration). Conditions of activated random migration resulted in increased numbers of cells with locomotory morphology, i.e. cellular asymmetry with linear alignment of nucleus, centriole, microtubule array, and pseudopods. Thus, activated serum increased the number of neutrophils exhibiting locomotory morphology, and a gradient of activated serum induced the alignment of neutrophils such that this locomotory morphology was uniform in the observed neutrophil populayion.
In related studies, cytochalasin B and colchicines were used to explore the role of microfilaments and microtubules in the neutrophil orientation and migration response to activated serum. Cytochalasin B (3.0 μg/ml) prevented migration and decreased the microfilaments seen, but allowed normal orientation of neutrophil structures. In an activated serum gradient, colchicines, but not lumicolchicine, decreased the orientation of nuclei and centrioles, and caused a decrease in centriole-associated microtubules in concentrations as low as 10(-8) to 10(-7) M. These colchicines effects were associated with the rounding of cells and impairment of pseudopod formation. The impaired pseudopod formation was characterized by an inability to form pseudopods in the absence of a solid substrate, a formation of narrow pseudopods within a substrate, and a defect in pseudopod orientation in an activated serum gradient. Functional studies of migration showed that colchicines, but not lumicolchicine, minimally decreased activated random migration and markedly inhibited directed migration, but had not effect on random migration. These studies show that, although functioning microfilaments are probably necessary for neutrophil migration, intact microtubules are essential for normal pseudopod formation and orientation, and maximal unidirectional migration during chemotaxis.
Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs
Authors: Detering NK , Decker GL , Schmell ED , Lennarz WJ 
Published: May 2004

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent.
Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.