bluuebunny/crossref_metadata_2025 huggingface dataset:
Size and shape of transverse tubule openings in frog twitch muscle fibers.
Authors: Franzini-Armstrong C , Landmesser L , Pilar G
Published: May 2004

The openings of transverse tubules in frog twitch fibers are described. The tubules open to the extracellular space by a narrow neck, with an inner diameter of 20 nm. The most peripheral portion of the tubules is tortuous and has a variable diameter. The similarity in size of the openings of T tubules and caveolae and the meandering path of the tubules are sufficient to account for the paucity of observed openings.
Ultrastructural immunoperoxidase demonstration of autologous albumin in the alveolar capillary membrane and in the alveolar lining material in normal rats.
Authors: Bignon J , Chahinian P , Feldmann G , Sapin C
Published: May 2004

The location of autologous serum albumin within the alveolar-capillary membrane was studied in the rat under physiological conditions using antialbumin antibodies labeled with peroxidase. Albumin was detected in the lung interstitium, and in numerous pinocytic vesicles within endothelial cells and type I alveolar epithelial cells. The immunoreaction was also positive at the level of plasmalemmal membranes of both cell types and in the alveolar lining material.
Hemoglobin switching in sheep and goats. V. Effect of erythropoietin concentration on in vitro erythroid colony growth and globin synthesis.
Authors: Barker J E , Anderson W F , Nienhuis A W
Published: May 2004

Erythroid colonies were generated in response to erythropoietin in plasma clot cultures of sheep and goat bone marrow cells. At low concentration erythropoietin only hemoglobin A (betaA globin) was synthesized in goat cultures, but at high concentrations 50% of the hemoglobin synthesized was hemoglobin C (betaC globin). This effect of erythropoietin on the expression of a specific beta globin gene was manifested only after 72 h in vitro and followed the development of erythroid colonies. Sheep colonies behaved differently from those of goat in that little or no betaC globin synthesis occurred even at high erythropoietin concentration. To investigate this difference, sheep marrow cells were fractionated by unit gravity sedimentation. The erythroid colony-forming cells sedimented more rapidly (3.5-6mm/h) than the hemoglobinized eththroid precursors (1-3.5 mm/h), suggesting that the colonies were formed from an early erythroid precursor, However, the colonies formed from the sheep marrow fractions synthesized only betaA globin even at concentrations of erythropoietin sufficient to stimulate betaC globin synthesis in goat colonies. Morphologically, the goat colonies were larger and more mature than those of the sheep. By 96 h in vitro three-fourths of the goat colonies contained enucleated red cells compared to only 3% of the sheep colonies. Thus, erythropoietin had an equivalent effect in stimulating erythroid colony growth from the marrow of both species although there were both biochemical and morphological differences between the colonies. Hemoglobin switching appeared to require exposure of an early precursor to high erythropoietin concentration, but the results with sheep marrow suggested that the rate of colony growth and cellular maturation might also be important.
Visualization of chromatin substructure: upsilon bodies.
Authors: Olins A L , Carlson R D , Olins D E
Published: May 2004

Spread chromatin fibers, from isolated eucaryotic nuclei, reveal linear arrays of spherical particles (upsilon bodies), about 70 A in diameter, connected by thin filaments about 15 A wide. These particles have been observed in freshly isolated nuclei from rat thymus, rat liver, and chicken erythrocytes. In addition, upsilon bodies can be visualized in preparations of isolated sheared chromatin, and in chromatin reconstructed from dissociating solvent conditions (i.e., high urea-NaCl concentration). As a criterion for perturbation of native chromatin structure low-angle X-ray diffraction patterns were obtained from nuclear pellets at different stages in the preparation of nuclei fro electron microscopy. These results suggest that the particulate (upsilon body) structures observed by electron microscopy may be closely related to the native configuration of chromatin.
Cytoplasmic type 80S ribosomes associated with yeast mitochondria. IV. Attachment of ribosomes to the outer membrane of isolated mitochondria.
Authors: Kellems R E , Allison V F , Butow R A
Published: May 2004

Growing yeast spheroplasts were shown to have, on the average, four times the number of cytoplasmic ribosomes in contact with the outer mitochondrial membrane compared to starved spheroplasts. Ribosomes in contact with mitochondria in the growing spheroplast preparation, like free cytoplasmic ribosomes, exist primarily as polysome structures. In the starved spheroplast preparation, both mitochondria-bound and free cytoplasmic ribosomes exist primarily as monosomes. Mitochondria isolated from growing spheroplasts in a medium containing lmM Mg++ have cytoplasmic ribosomes bound directly to the outer membrane. These ribosomes can be quantitatively removed by washing the mitochondria with 2 mM EDTA. Mitochondria from starved spheroplasts are capable of accepting either free cytoplasmic polysomes or cytoplasmic polysomes extracted from mitochondria. However, the extent of polysome binding to mitochondria was shown to be a direct function of the Mg++ concentration; a smaller percentage of the input polysomes bind as the Mg++ concentration is lowered. At 1 mM Mg++, neither free cytoplasmic nor mitochondria-bound polysomes bind to mitochondria. Nevertheless, when growing spheroplasts are broken and mitochondria isolated in medium containing 1 mM Mg++, the mitochondria are seen to have cytoplasmic ribosomes firmly attached to the outer membrane. This result, in addition to our earlier data (Kellems, R. E., and R. A. Butow. 1974. J. Biol. Chem. 249:3304-3310), support the view that cytoplasmic ribosomes attached to the outer membrane of purified mitochondria were attached in vivo. In preparations of mitochondria isolated from growing spheroplasts, ribosomes appear to be found to specific regions of the outer membrane, namely those regions which are in close association or in contact with the inner mitochondrial membrane. This is particularly evident with mitochondria in a condensed configuration. This finding suggests a mechanism whereby cytoplasmically synthesized mitochondrial protein could be transferred by a process of vectorial translation across both membranes of the organelle.
Concanavalin A receptors, immunoglobulins, and theta antigen of the lymphocyte surface. Interactions with concanavalin A and with Cytoplasmic structures.
Authors: de Petris S
Published: May 2004

The effect of concanavalin A (Con A) on the capping of mouse lymphocyte surface immunoglobulin (surface Ig), cross-linked by rabbit anti-mouse Ig antibody, and on the capping of mouse thymocyte theta antigen, cross-linked by anti-theta alloantibody and rabbit anti-mouse Ig antibody, has been studied by immunofluorescence, using fluorescein conjugated Con A and rhodamine-conjugated anti-mouse Ig antibody, and by electron microscopy, using native or fluorescein-conjugated Con A and ferritin-conjugated anti-mouse Ig antibody. Prior incubation of the cells with Con A inhibited only partially capping os surface Ig, whereas it blocked almost completely capping of theta antigens. Both on cells with rings and on cells with caps the staining for surface Ig or theta antigen was superimposed to the staining for Con A. When Con A receptors on spleen cells were capped by Con A at concentrations of 10 mug/ml or higher, and the distribution of surface Ig was examined under noncapping conditions, all detectable surface Ig were found in the caps. As shown by electron microscopy, surface Ig remained dispersed in a layer of Con A. The ability of Con A to cap surface Ig was not altered by the presence of cohchicine or vinblastine. These results suggest that surface Ig are cross-linked by Con A to other Con A receptors. In these conditions surface Ig behave essentially as Con A receptors, as for example, in their sensitivity to cytochalasin B during inhibition or reversal of capping induced by this drug. The behavior of surface Ig parallels that of Con A receptors also in the presence of vinblastine. It is concluded that in the presence of Con A, antimitotic drugs do not modify directly the interaction between Con A receptors and surface Ig, but probably influence the capping ability of the Con A receptors or, more in general, affect the ability to elicit movements over the cell surface. The role in capping of cytochalasin-sensitive and vinblastine-sensitive structures is discussed. Both types of structures appear to play an active role in the formation of a cap, although the former probably corresponds to the main mechanical system responsible for the active displacement of cytoplasmic and surface material.
Adenosine triphosphate-induced motility and sliding of filaments in mammalian sperm extracted with Triton X-100.
Authors: Lindemann C B , Gibbons I R
Published: May 2004

Bull sperm that had been extracted with 0.2% Triton X-100 could be reactivated with ATP, and their movement closely resembled the motion of intact live sperm. Their motility required the presence of ATP, magnesium, and a medium of suitable salt concentration and pH. When Triton-extracted bull sperm were digested breifly with trypsin at pH 9.0, they appeared to reatin most of their normal structure, but subsequent exposure of the digested sperm to ATP caused a disintegration by light microscopy, using dark-field illumination, combined with an electron microscope study of preparations of the disintegrated sperm, demonstrated the presence of an active sliding mechanism of filament interaction in bull spermatozoa. Human sperm subjected to the same procedures showed similar patterns of reactivation and of disintegration.
Effects of actinomycin D on the association of newly formed ribonucleoproteins with the cistrons of ribosomal RNA in Triturus oocytes.
Authors: Scheer U , Trendelenburg F , Franke W W
Published: May 2004

The effect of actinomycin D(AMD) on the association of the nascent ribonucleo-protein (RNP) fibrils containing the precursors of ribosomal RNA (pre-rRNA) with their template deoxyribonucleoprotein (rDNP) strands has been studied in lampbrush stage oocytes from Triturus alpestris. Ovary pieces were incubated in vitro either in media containing radioactive ribonucleosides and then, for various times, in solutions containing 25 mug/ml AMD, or were directly exposed to the drug. The ultrastructure of the nucleoli and the nuclear periphery was studied by electron microscopy of thin sections and positively stained spread preparations of isolated nuclear contents, and by light and electron microscope autoradiography. The fate of the labeled pre-rRNA was followed by gel electrophoresis of RNA extracted from manually isolated nuclei. Our results show that the growing fibrils which contain the nascent pre-rRNA progressively detach from the DNP strands, the majority being released between 45 and 180 min after application of the drug. The release pattern seems to be random and does not show preference for regions close to the initiator or terminator sites of the transcribed rDNP units. There is a pronounced tendency to removal of groups of adjacent mascent fibrils. The effect of the drug is very heterogeneous. Even after 3 h of treatment with AMD the nucleoli exhibit several individual transcriptional units which appear almost completely covered with lateral fibrils. Autoradiography revealed that most of this released RNP remains within the confinements of the nucleoli which show some foci of aggregation and condensation of fibrillar components but no clear "segregation" phenomenon. In the gel-electrophoretic analysis, a significant but moderate decrease of labeled pre-rRNA was noted only in the first stable pre-rRNA component, whereas pre-rRNA classes of lower molecular weight are very stable under these conditions. The results are discussed in relation to the stability of rDNA transcription complexes and as a basis for an explanation of the ultrastructural changes which are generally observed in nucleoli of AMD-treated cells. It is postulated that inhibition of transcription results in a slow but progressive release of the arrested incomplete RNP fibrils from the template.
Location of procollagen in chick corneal and tendon fibroblasts with ferritin-conjugated antibodies.
Authors: Nist C , Von Der Mark K , Hay E D , Olsen B R , Bornstein P , Ross R , Dehm P
Published: May 2004

Three distinct antiprocollagen preparations were characterized and used in immunocytochemical staining of chick embryo corneal and tendon cells. The several ferritin-conjugated antibody preparations permitted similar location of procollagen in the cisternae of the rough endoplasmic reticulum and in Golgi elements in both cell types. The ability to demonstrate and interpret specific ferritin staining was dependent on the extent of membrane breakage in each of those organelles, coupled with adequate retention of cell morphology. Corneal fibroblasts appeared to suffer more extensive intracellular membrane damage under controlled conditions of homogenization than tendon fibroblasts, facilitating the identification of procollagen in Golgi vacuoles of these cells. None of the labeled material appeared to by cytoplasmic in origin since ferritin was observed in the cytoplasm only in the vicinity of Golgi elements that were extensively broken. This study extends previous immunological evidence for the presence of procollagen in the Golgi complex and calls attention to the problems to be encountered in locating the antigen in small Golgi vesicles and lamellae.
Studies on microperoxisomes. VII. Pigment epithelial cells and other cell types in the retina of rodents.
Authors: Leuenberger P M , Novikoff A B
Published: May 2004

The pigment epithelial cell of the retina actively participates in two aspects of lipid metabolism: (a) the fatty acid esterification of vitamin A and its storage and transport to the photoreceptors, and (b) the phagocytosis and degradation of the lipoprotein membrane disks shed from the photoreceptor cells. Study of the pigment epithelial cells of adult albino and pigmented rodents has revealed the abundance of an organelle, microperoxisomes, not previously known to exist in this cell type. The metabolism, transport, and storage of lipids are major functions of other cell types which possess large numbers of microperoxisomes associated with a highly developed smooth endoplasmic reticulum. Microperoxisomes were encountered, but relatively rarely, in Müller cells and vascular endothelial cells. A tubular system in photoreceptor terminals is reactive in the cytochemical procedure used to visualize microperoxisomes.
Acetylcholine receptor turnover in membranes of developing muscle fibers.
Authors: Devreotes P N , Fambrough D M
Published: May 2004

[125I mono-iodo-alpha-bungarotoxin is used as a specific marker in a description of acetylcholine receptor metabolism. It is concluded that acetylcholine receptors in the surface membranes of chick and rat myotubes developing in cell cultures have a half-life of 22-24 h. Alpha-bungarotoxin (bound to a receptor which is removed from the membrane) is degraded to monoiodotyrosine which appears in the medium. Several observations are consistent with a model in which receptors or alpha-bungarotoxin-receptor complexes are internalized and then degraded: (a) the rate of appearance of iodotyrosine does not reach its maximal rate until 90 min after alpha-bungarotoxin is bound to the surface receptors; (b) 2,4-dinitrophenol, reduced temperature, and cell disruption all inhibit the degradation process. The degradation of surface receptors is not coupled to the process by which receptors are incorporated into the membrane. Evidence suggest that receptors are incorporated into the surface membrane from a presynthesized set of receptors containing about 10% as many alpha-bungarotoxin binding sites as does the surface. Additionally, a third set of acetylcholine receptors is described containing about 30% as amny binding sites as does the surface. These "hidden" recptors are not precursors yet are not readily accessible for binding of extracellular alpha-bungarotoxin. These findings are discussed in relation to both plasma membrane biosynthesis and control of chemosensitivity in developing and denervated skeletal muscle.
Nucleolar necklaces in chick embryo fibroblast cells. II. Microscope observations of the effect of adenosine analogues on nucleolar necklace formation.
Authors: Granick D
Published: May 2004

The round nucleoli of chick embryo fibroblast cells, when exposed to adenosine (2 mM)or to a number of adenosine analogues, lose material and unravel over a period of several hours to become beaded strands, 20 mu M in length, termed nucleolar necklaces (NN). Light microscope observations on this process are described. Biochemical experiments have revealed that most of these analogues interfere with both messenger RNA synthesis and ribosome synthesis, causing extensive degradation of the preribosome species containing 32S RNA although most of the preribosomes containing 18S RNA survive. We suggest that it is the depletion from the nucleolus of the adhesive 32S and 28S RNA preribosomes which allows the remaining nucleolar apparatus to spread apart into the NN configuration. Also required for the maintenance of the NN structure is the synthesis of some ribosomal RNA (rRNA) possibly present as rRNA "feathers" on the DNA. The addition of inhibitors of rRNA synthesis such as actinomycin D to the NN-containing cells causes loss of rRNA. Then a contraction and collapse of the NN structure into small dense spheres is observed.
Selection of mammalian cells resistant to a chloramphenicol analog.
Authors: Wallace R B , Freeman K B
Published: May 2004

This study describes the selection and preliminary characterization of mammalian cells resistant to 100 mug Tevenel/ml. Tevenel, the sulfamoyl analog of chloramphenicol, is a specific inhibitor of mitochondrial protein synthesis. After growth in suspension culture for 5 days in 100 mug Tevenel/ml and subsequent plating in 100 mug Tevenel/ml, LMTK- cells yielded resistant clones. As a control, L cells treated identically yielded no clones. Three resistant clones were chosen for study. Each resistant cell line had an identical growth rate in the presence and absence of 100 mug Tevenel/ml. By plating efficiency analysis, the resistant cells were found to be cross-resistant to D-chloramphenicol. The change responsible for resistance was found to be stable for at least 100 generations in the absence of the drug. Protein synthesis by isolated mitochondria of resistant cells was found to be less inhibited by concentrations of both Tevenel and D-chloramphenicol up to 200 mug/ml than the protein synthesis by LMTK- mitochondria. This resistance in vitro was not changed by incubation of the mitochondria in 0.01% Triton X-100.
Tropomyosin antibody: the specific localization of tropomyosin in nonmuscle cells.
Authors: Lazarides E
Published: May 2004

An antibody against purified chicken skeletal muscle tropomyosin is used in indirect immunofluorescence to visualize the localization of tropomyosin in a variety of nonmuscle cells. The antibody produces a fluorescent pattern which is very similar to that obtained with an actin-specific antibody. This pattern is composed of fluorescent fibers which are shown to be coincident with the fibers seen with phase-contrast optics. High resolution epifluorescent microscopy reveals that fibers stained with the actin antibody show a continuous fluorescence, while fibers reacted with the tropomyosin antibody show a periodic fluorescence. Measurements indicate that the lengths of the fluorescent segments are variable with an average of 1.2 mum while the spacing between segments is approximately 0.4 mum.
Computer analysis of organelle translocation in primary neuronal cultures and continuous cell lines.
Authors: Breuer A C , Christian C N , Henkart M , Nelson P G
Published: May 2004

Organelle translocation in a number of cell types in tissue culture as seen by high-resolution Zeiss-Nomarski differential interference contrast optics was filmed and analyzed by computer. Principal cell types studied included primary chick spinal cord, chick dorsal root ganglion, ratbrain, and various clones of continuous cell lines. Organelle translocations in all cell types studied exhibited frequent, large changes in velocity during any one translocation. The appearance of particles as seen with Nomarski optics was correlated with their fine structures in one dorsal root ganglion neurite by fixing the cell as it was being filmed and obtaining electron micrographs of the region filmed. This revealed the identity of several organelles as well as the presence of abundant neurotubules but no neurofilaments. Primary cell cultures exhibited more high-velocity organelle movements than continuous cell lines. The net progress of an organelle in a given direction was greater in primary neuronal cells than in fibroblasts or continuous cell lines. These findings are correlated with the literature on organelle translocation and axoplasmic transport.
Pressure-induced depolymerization of spindle microtubules. II. Thermodynamics of in vivo spindle assembly.
Authors: Salmon E D
Published: May 2004

The present experiments were designed to test whether the simple equilibrium assembly model proposed by Inoué could predict variations in spindle microtubule assembly in response to changes in hydrostatic pressure as it does for changes in temperature. The results were also analyzed according to a model based on nucleated condensation polymerization since this recently appears to be the mechanism by which purified brain microtubules are assembled in vitro. Equilibrium birefringence (BR) of the meiotic metaphase-arrested spindle was measured in vivo as a function of hydrostatic pressure and temperature in Chaetopterus oocytes using a miniature microscope pressure chamber. Increasing pressure in steps to 3,000 psi at temperatures below 22 degrees C did produce decreases in spindle equilibrium BR predictable directly from the simple equilibrium model of spindle assembly. Thermodynamic analysis of the pressure data yielded a value of delta V congruent to 400 ml/mol of polymerizing unit. Theoretical curves based on the nucleated condensation model can also be made to fit the data, but semilog plots of the dependence of the equilibrium constant versus pressure and versus reciprocal temperature are biphasic, suggesting that either the size of the polymerizing unit changes or more than one equilibrium constant governs the assembly reaction. That the same value of delta V, 90 ml/mol, was estimated from both the majority of the spindle BR data and data for the assembly of neural microtubules in vitro supports the possibility that spindle microtubules are assembled by a nucleated condensation mechanism.
Motility in Echinosphaerium nucleofilum. I. An analysis of particle motions in the axopodia and a direct test of the involvement of the axoneme.
Authors: Edds K T
Published: May 2004

The motion of particles in the axopodia of Echinosphaerium nucleofilum is saltatory. In the present study, photokymograph records of 123 motions from six axopodia have been analyzed. Particles followed rectilinear paths of from 1 to 15 mum while in continuous motion at an average velocity of 0.66 plus or minus 0.32 mum/s. The velocity of the particles was variable in 36% of the cases measured. Some motions were punctuated by pauses either before continuing in the same direction or reversing. Frequently, several particles were moving at the same velocity, but neighboring particles showed no motion or moved in the opposite direction. Two particles occasionally contacted one another and travelled as a unit for varying lengths of time but subsequently moved independently. These motions reflect the underlying mechanism of motive force production. Furthermore, a glass microneedle can be substituted for the microtubular axoneme in the axopodia. In these artificial axopodia, bidirectional particle motions occurred which were similar to those in normal axopodia. Colchicine, at the threshold dose for axonemal dissolution, had no affect on these particel motions. It is concluded that the microtubular axoneme is not responsible for particle motions and also that individual microtubules are unlikely candidates for motive force production in this system.
Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining.
Authors: Krishan A
Published: May 2004

A rapid method for the flow microfluorometric determination of the DNA content per cell is described. Incubation of cells in a hypotonic solution of propidium iodide results in disruption of the cell membrane and rapid staining of nuclear chromatin. DNA distribution histograms generated from cells stained by this method are identical to those generated after fixation and RNase digestion. In contrast to some earlier described methods, the present technique is rapid (5 min of processing), requires a minimal amount of material, and avoids formation of cell clumps.
Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy.
Authors: Mazia D , Schatten G , Sale W
Published: May 2004

Cells of many kinds adhere firmly to glass or plastic surfaces which have been pretreated with polylysine. The attachment takes place as soon as the cells make contact with the surfaces, and the flattening of the cells against the surfaces is quite rapid. Cells which do not normally adhere to solid surfaces, such as sea urchin eggs, attach as well as cells which normally do so, such as amebas or mammalian cells in culture. The adhesion is interpreted simply as the interaction between the polyanionic cell surfaces and the polycationic layer of adsorbed polylysine. The attachment of cells to the polylysine-treated surfaces can be exploited for a variety of experimental manipulations. In the preparation of samples for scanning or transmission electron microscopy, the living material may first be attached to a polylysine-coated plate or grid, subjected to some experimental treatment (fertilization of an egg, for example), then transferred rapidly to fixative and further passed through processing for observation; each step involves only the transfer of the plate or grid from one container to the next. The cells are not detached. The adhesion of the cell may be so firm that the body of the cell may be sheared away, leaving attached a patch of cell surface, face up, for observation of its inner aspect. For example, one may observe secretory vesicles on the inner face of the surface (3) or may study the association of filaments with the inner surface (Fig. 1). Subcellular structures may attach to the polylysine-coated surfaces. So far, we have found this to be the case for nuclei isolated from sea urchin embryos and for the microtubules of flagella, which are well displayed after the membrane has been disrupted by Triton X-100 (Fig. 2).
In vivo recovery of muscle contraction after alpha-bungarotoxin binding.
Authors: Fertuck H C , Woodward W , Salpeter M M
Published: May 2004

Acetylcholine receptors were inactivated in vivo at the mouse neuromuscular junction using alpha-bungarotoxin (alpha-BTX). It was found that neurally produced muscle contraction recovered within 4-8 days (halftime similar to 3 days). Actinomycin D interfered with this recovery, but did not affect normal nerve-stimulated muscle contraction. If the response was initially eliminated by [125-I]alpha-BTX and the end plates examined by EM autoradiography, no evidence of mass internalization of bound radioactivity during recovery was seen. The fine structure of the end plates and muscle was unaltered during the post-alpha-BTX recovery period.
Receptor mobility and the binding of cells to lectin-coated fibers.
Authors: Rutishauser U , Sachs L
Published: May 2004

The ability of cells to bind to nylon fibers coated with lectin molecules interspaced with varying numbers of albumin molecules has been analyzed. The cells used were lymphoma cells, normal lymphocytes, myeloid leukemia cells, and normal and transformed fibroblasts, and the fibers were coated with different densities of concanavalin A or the lectins from soybean or wheat germ. Cells fixed with glutaraldehyde did not bind to lectin-coated fibers. The number of cells bound to fibers could be increased by increasing the density of lectin molecules on the fiber, the density of specific receptors on the cell, or the mobility of the receptors. It is suggested that binding of cells to fibers involves alignment and binding of specific cell surface receptors with lectin molecules immobilized on the fibers, and that this alignment requires short-range rapid lateral mobility (RLM) of the receptors. The titration of cell binding to fibers coated with different densities of lectin and albumin has been used to measure the relative RLM of unoccupied cell surface receptors for the lectin. The results indicate a relationship of RLM to lectin-induced cell-to-cell binding. The RLM or receptors for concanavalin A (Con A) was generally found to be higher than that of receptors for the lectins from wheat germ or soybean. Receptor RLM could be decreased by use of metabolic inhibitors or by lowering the temperature. Receptors for Con A had a lower RLM on normal fibroblasts than on SV40-transformed fibroblasts, and trypsinization of normal fibroblasts increased Con A receptor RLM. Normal lymphocytes, lymphoma cells, and lines of myeloid leukemia cells that can be induced to differentiate had a high receptor RLM, whereas lines of myeloid leukemia cells that could not be induced to differentiate had a low receptor RLM. These results suggest that the RLM of Con A receptors is related to the transformation of fibroblasts and the ability of myeloid leukemia cells to undergo differentiation
Cytoplasmic activation of starfish oocytes by sperm and divalent ionophore A-23187.
Authors: Schuetz A W
Published: May 2004

The relationship between onset of the early cytoplasmic stages of oocyte activation (vitelline membrane separation and elevation) and nuclear meiotic maturation was investigated in starfish oocytes after their exposure to divalent ionophore (A-23187) or sperm. Meiotically mature oocytes, isolated in calcium-free seawater, underwent activation in response to sperm or ionophore as previously reported. Large, immature starfish oocytes, arrested in prophase I of meiosis (germinal vesicle stage), underwent vitelline membrane elevation when treated with divalent ionophore A-23187 or starfish sperm. Histological studies demonstrated that cortical granule breakdown in the oocyte cortex was associated with vitelline membrane elevation after these treatments. Activation of oocytes by sperm occurred only in response to starfish sperm. Sea urchin, sand dollar, surf clam, or marine worm sperm did not induce vitelline membrane elevation of either immature or mature starfish oocytes. Sperm- or ionophore-activated immature oocytes underwent nuclear maturation after addition of the meiosis-inducing hormone, l-methyladenine; however, parthenogenetic development did not occur and embryonic development was markedly inhibited. In contrast to previous studies, the present results indicate that cytoplasmic activation can be initiated before and without hormone induction of the nuclear maturation process. Differentiation of the oocyte cell surface or cortex reactivity therefore appears to occur during oogenesis rather than as a consequence of maturation. The data further support the view that divalent ions mediate certain of the early activation responses initiated by sperm at the time of fertilization and that synchronization of fertilization to the meiotic process in the oocyte is important for the occurrence of normal development.
Ultrastructural localization of lectin-binding sites on the zonae pellucidae and plasma membranes of mammalian eggs.
Authors: Nicolson G L , Yanagimachi R , Yanagimachi H
Published: May 2004

Receptors for Ricinus communis agglutinin I (RCAI), concanavalin A (Con A), and wheat germ agglutinin (WGA) were localized on the zonae pellucidae and plasma membranes of hamster, mouse, and rat eggs with ferritin-lectin conjugates. Intact eggs labeled with the ferritin conjugates showed dense concentrations of RCAI and WGA receptors in the outermost regions of their zonae pellucidae and sparse distributions of Con A receptors throughout the zonae. Ferritin-lectin labeling was specific, since inhibitory saccharides effectively blocked labeling. The asymmetric density of RCAI receptors across the zona was confirmed by ferritin-RCAI and fluorescein-RCAI labeling of mechanically isolated zonae pellucidae, indicating that the RCAI-binding sites are more densely distributed in the exterior zona regions. Plasma membranes of rodent eggs contained RCAI, WGA, and Con A receptors. These receptors were found to be more or less randomly distributed on surfaces of aldehyde-fixed eggs or on eggs labeled near 0 degrees C. However, eggs incubated at 25 degrees C showed aggregated WGA- and Con A-binding site distributions on their plasma membranes. This indicates that lectin-induced receptor redistribution occurs at this temperature. The possibility that plasma membrane receptor mobility is a requirement for sperm-egg fusion is discussed.
Occluding junctions and cell behavior in primary cultures of normal and neoplastic mammary gland cells.
Authors: Pickett P B , Pitelka D R , Hamamoto S T , Misfeldt D S
Published: May 2004

Cells dissociated from normal prelactating mouse mammary glands or from spontaneous mammary adenocarcinomas, when grown at high density on an impermeable substrate, form nonproliferating, confluent, epithelial pavements in which turgid, blister-like domes appear as a result of fluid accumulation beneath the cell layer. To compare the structure of the fluid-segregating cell associations in normal and tumor cell cultures with that of lactating gland in vivo, we have examined such cultures alive and in thick and thin sections and freeze-fracture replicas. Pavement cells in all cases are polarized toward the bulk medium as a lumen equivalent, with microvilli and continuous, well-developed occluding junctions at this surface. Between the pavement and the substrate are other cells, of parenchymal or stromal origin, scattered or in loose piles; these sequestered cells are relatively unpolarized and never possess occluding junctions. Small gap junctions have been found in the pavement layer, and desmosomes may link epithelial cells in any location. Under the culture conditions used, development of the epithelial secretory apparatus is not demonstrable; normal and neoplastic cells do not differ consistently in any property examined. A dome's roof is merely a raised part of the epithelial pavement and does not differ from the latter in either cell or junction structure. We suggest that dome formation demonstrates the persistence of some transport functions and of the capacity to form effective occluding junctions. These basic epithelial properties can survive both neoplastic transformation and transition to culture.
Morphological alterations and ganglioside sialyltransferase activity induced by small fatty acids in HeLa cells.
Authors: Simmons J L , Fishman P H , Freese E , Brady R O
Published: May 2004

Incubation of HeLa cells in the presence of millimolar concentrations of propionate, butyrate, or pentanoate increases the specific activity of CMP-sialic acid:lactosylceramide sialyltransferase 7-20-fold within 24 h. Longer-chain saturated fatty acids or acetate are much less effective, decanoate showing no induction. Unsaturated fatty acid analogs of butyrate and other compounds are ineffective. Only the three most effective compounds also produce characteristic smooth extended cell processes in HeLa cells. Butyrate (5 mM) induces the sialyltransferase after a 4-h lag, producing maximum specific activity by 24 h. The amount of sialyl-lactosylceramide, the glycolipid product of the enzyme, increases during that time 3.5 times more than in control cultures. No other glycosphingolipid enzyme is significantly altered by butyrate exposure. The cellular shape changes occur 2-3 h later than the increase of sialyltransferase activity, and both processes require the continuous presence of inducer and the synthesis of RNA and protein but not the synthesis of DNA or the presence of serum.
Mitogenic effect of fibroblast growth factor on early passage cultures of human and murine fibroblasts.
Authors: Gospodarowicz D , Moran J S
Published: May 2004

Fibroblast growth factor (FGF), a polypeptide that has been shown to stimulate division in 3T3 cells, was tested for mitogenic effects on diploid, early-passage cells from human and murine sources. The quantitative assay of [3H]thymidine incorporation into acid-insoluble material showed that FGF at low concentrations (10 minus 9 M) was more effective than additional serum for provoking the initiation of DNA synthesis in human foreskin fibroblasts or mouse fibroblasts maintained in 5 or 10% serum, respectively. The growth of the human fibroblasts was twice as fast in the presence of FGF plus 10% calf serum as it was in the presence of 10% calf serum or 20% fetal calf serum alone. The addition of FGF to primary cultures of mouse fibroblasts in 0.4% serum resulted in a twofold increase in cell number compared to controls. In contrast to results obtained with 3T3 cells, neither insulin nor a glucocorticoid potentiated the effects of FGF on either human or mouse cells.
The permeability of muscle capillaries to horseradish peroxidase.
Authors: Williams M C , Wissig S L
Published: May 2004

The main objective of this study was to determine the pathways by which horseradish peroxidase (HRP) can cross the endothelium of muscle capillaries. Specimens of mouse diaphragm were fixed for cytochemical analysis at various intervals after intervenous injection of 0.5 mg HRP, at 4 min after intervenous injection of varied amounts of HRP, and at 4 min after intervenous injections in various volumes of isotonic NaCl. Our findings indicate that endothelial junctions serve as a barrier which may allow passage of very limited amounts of HRP. They also suggest that endothelial vesicles transfer HRP from the capillary lumen to the pericapillary interstitium as well as in the reverse direction. Increasing the volume of solution injected to approximately 30% of total blood volume did not increase the amount of HRP that left the capillary lumen. Our results with HRP do not provide clearcut evidence that endothelial junctions are the site of the small pore.
Role of microvilli in surface changes of synchronized P815Y mastocytoma cells.
Authors: Knutton S , Sumner M C , Pasternak C A
Published: May 2004

The surface morphology of synchronized P815Y mastocytoma cells has been examined by scanning electron microscopy. Early G1 cells are comparatively smooth or light villated, whereas at later stages the surface becomes progressively more villated. In G1 cell most microvilli have a uniform diameter, whereas in S and G2 cells, many microvilli show branching and often originate from much larger surface protuberances. Small "blebs" are seen on the surface of many cells but these structures do not appear to be a characteristic feature of cells at any one stage of the cycle. The presence of microvilli increases the total surface of the cell to such an extent that the ratio of volume to surface area remains constant throughout the cell cycle. The mechanism of cytokinesis is thus a physical one, involving the unfolding of previously accumulated microvilli.
Nucleocytoplasmic movement of fluorescent tracers microinjected into living salivary gland cells.
Authors: Paine P L
Published: May 2004

The permeability of the nuclear envelop of a somatic cell, the C. thummi larval salivary gland cell, was studied by intracellular microinjection of fluorescent molecular tracers. As shown previously in oocytes (4,5,15,16), the envelop is permeable to a wide variety of materials, including molecules which are large enough to possess condiderable biological specificities and to play important roles in regulation of cellular activities. The envelop exhibits transport selectivity on the basis of size in the range of naturally occurring intracellular materials and it may thus perform important controlling functions in nucleocytoplasmic exchange. The nucleus to cytoplasm movement of in vivo ribonucleoprotein particulates in these synthetically active cells probably requires conformational changes in the particulates and/or the envelope pore complexes; morphological evidence exists for such processess in these cells (20).
Mouse-human heterokaryon analysis with a 33258 Hoechst-Giemsa technique.
Authors: Moser F G , Dorman B P , Ruddle F H
Published: May 2004

The bibenzimidazol derivative 33258 Hoechst can be used to distinguish microfluorometrically between mouse and human nuclei in heterokaryons. This affords a quick and accurate alternative to autoradiography in the analysis of such heterokaryons. The 33258 Hoechst fluorescence patterns can be converted after irradiation to a Giemsa rendition of the differential staining.
Growth of the nuclear envelope in the vegetative phase of the green alga Acetabularia. Evidence for assembly from membrane components synthesized in the cytoplasm.
Authors: Franke W W , Spring H , Scheer U , Zerban H
Published: May 2004

The primary nucleus of the green alga Acetabularia grows about 25,000-fold in volume while it is separated from the endoplasmic reticulum and the whole cytoplasm by a special paranuclear cisterna of a vacuolar labyrinthum system which shows only very few (two to six per square micrometer) and small (ca. 40-120 nm in diamter) fenestrations. The nuclear envelope does not bear polyribosomes, nor do they occur in the entire zone intermediate between the nuclear envelope and the paranuclear cisterna. It is suggested that this special form of nuclear envelope growth takes place by assembly from cytoplasmically synthesized proteins that are translocated across the paranuclear cisterna in a nonmembrane-structured form.
Aster formation in eggs of Xenopus laevis. Induction by isolated basal bodies.
Authors: Heidemann S R , Kirschner M W
Published: May 2004

We have assayed various materials for their ability to induce aster formation by microinjection into unfertilized eggs of Xenopus laevis. We have found that purified basal bodies from Chlamydomonas reinhardtii and Tetrahymena pyriformis induce the formation of asters and irregular cleavage furrows within 1 h after injection. Other microtubule structures such as flagella, flagellar axonemes, cilia, and brain microtubules are completely ineffective at inducing asters or cleavage furrows in unfertilized eggs. When known amounts of sonicated Tetrahymena and Chlamydomonas preparations are injected into unfertilized eggs, 50% of the injected eggs show a furrowing response at approximately 3 cell equvalents for Chlamydomonas and 0.1 cell equivalent for Tetrahymena. These results are close to those expected if basal bodies were the effective astral-inducing agent in these cells. Other materials effective at inducing asters in unfertilized eggs, such as crude brain nuclei, sperm, and a particulate fraction from brain known to induce parthenogenesis in eggs of Rana pipiens, probably contain centrioles as the effective agent. Our experiments provide the first functional assay to indicate that centrioles play an active role in aster initiation. None of the injected materials effective in unfertilized eggs produced any observable response in fully grown oocytes. Oocytes and eggs were found to have equal tubulin pools as judged by colchicine-binding activity. Therefore, the inability of oocytes to form asters cannot be due to a lack of an organizing center or to a lack of tubulin. Experiments in which D2O was found to stimulate aster-like fibrous areas in eggs but not oocytes suggest that the inability of oocytes to form asters may be due to an inability of tubulin in oocytes to assemble.
Properties of the beta-nerve growth factor receptor in development.
Authors: Herrup K , Shooter E M
Published: May 2004

The cell surface receptor for beta-nerve growth factor was used as a probe to study the development of embryonic chick sensory ganglia. The ganglia were shown to lose their responsiveness to nerve growth factor in vitro between 14 and 16 days of embryonic age. This loss occurred by a decrease in the magnitude of the maximum biological response, not by a shifting of the response to higher concentrations. Binding assays for the beta-nerve growth factor receptor, using 125I-radiolabelled beta-nerve growth factor, were performed with cells from sensory ganglia 8, 12, 14, 16, 18, and 21 days of age. The assays revealed a twofold increase in the number of receptor sites per ganglion between 8 and 14 days and a sixfold drop between 14 and 16 days of embryonic life. Neither increase nor decrease was accompanied by a large change in the affinity of the receptor for the protein. Together with the results of the bioassay, the data show that the loss of biological responsiveness is correlated with and may be due to a loss of the cells' ability to bind beta-nerve growth factor. Correlation of the results of the binding assays with the known ontogeny of the chick embryo provides a hint at the role of nerve growth factor in normal development.
Evidence for biased bidirectional polymerization of actin filaments using heavy meromyosin prepared by an improved method.
Authors: Woodrum D T , Rich S A , Pollard T D
Published: May 2004

Isolated actin filaments decorated with HMM can grow by addition of actin monomers to either end, although there is a bias toward addition at the end which is normally attached to the Z line in striated muscle.
Membrane-bound ribosomes of myeloma cells. III. The role of the messenger RNA and the nascent polypeptide chain in the binding of ribosomes to membranes.
Authors: Mechler B , Vassalli P
Published: May 2004

Mild ribonuclease treatment of the membrane fraction of P3K cells released three types of membrane-bound ribosomal particles: (a) all the newly made native 40S subunits detected after 2 h of [3H]uridine pulse. Since after a 3-min pulse with [35S]methionine these membrane native subunits appear to contain at least sevenfold more Met-tRNA per particle than the free native subunits, they may all be initiation complexes with mRNA molecules which have just become associated with the membranes; (b) about 50% of the ribosomes present in polyribosomes. Evidence is presented that the released ribosomes carry nascent chains about two and a half to three times shorter than those present on the ribosomes remaining bound to the membranes. It is proposed that in the membrane-bound polyribosomes of P3K cells, only the ribosomes closer to the 3' end of the mRNA molecules are directly bound, while the latest ribosomes to enter the polyribosomal structures are indirectly bound through the mRNA molecules; (c) a small number of 40S subunits of polyribosomal origin, presumably initiation complexes attached at the 5' end of mRNA molecules of polyribosomes. When the P3K cells were incubated with inhibitors acting at different steps of protein synthesis, it was found that puromycin and pactamycin decreased by about 40% the proportion of ribosomes in the membrane fraction, while cycloheximide and anisomycin had no such effect. The ribosomes remaining on the membrane fraction of puromycin-treated cells consisted of a few polyribosomes, and of an accumulation of 80S and 60S particles, which were almost entirely released by high salt treatment of the membranes. The membrane-bound ribosomes found after pactamycin treatment consisted of a few polyribosomes, with a striking accumulation of native 60S subunits and an increased number of native 40S subunits. On the basis of the observations made in this and the preceding papers, a model for the binding of ribosomes to membranes and for the ribosomal cycle on the membranes is proposed. It is suggested that ribosomal subunits exchange between free and membrane-bound polyribosomes through the cytoplasmic pool of free native subunits, and that their entry into membrane-bound ribosomes is mediated by mRNA molecules associated with membranes.