bluuebunny/crossref_metadata_2025_split huggingface dataset:
Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP
Authors: Melmed RN , Karanian PJ , Berlin RD 
Published: May 2004

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis.
The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4'acetamido, 4-isothiocyano 2,2' disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation.
Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.
Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of polytomella
Authors: McKeithan TW , Rosenbaum JL 
Published: May 2004

The alga polytomella contains several organelles composed of microtubules, including four flagella and hundreds of cytoskeletal microtubules. Brown and co-workers have shown (1976. J. Cell Biol. 69:6-125; 1978, Exp. Cell Res. 117: 313-324) that the flagella could be removed and the cytoskeletans dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell.
In order to define the different species of tubulin in polytonella cytoplasm, a (35)S- labeled cytoplasmic fraction was subjected to two cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated polytomella flagella by two-dimensional gel electrophoresis showed that, whereas the β-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the two α-tubulins had distinctly different isoelectic points. As a second method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were found to be highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant α-tubulin of this in vitro- assembled cytoskeletal tubulin corresponded to the major cytoplasmic α-tubulin obtained by coassembly of labeled polytomella cytoplasmic extract with brain tubulin and was quite distinct from the α-tubulin of purified flagella. These results clearly show that two different microtubule-containing organelles from the same cell are composed of distinct tubulins.
Immunological dissimilarity in protein component (dynein 1) between outer and inner arms within sea urchin sperm axonemes
Authors: Ogawa K , Negishi S , Obika M 
Published: May 2004

The 0.5 M KCl-treatment solubilizes the outer arms from sea urchin sperm axonemes. Approximately 30 percent of A-polypeptide, corresponding to dynein 1 in SDS- polyacrylamide gel, was solubilized by this treatment (as SEA-dynein 1). Electron microscopic observation indicated that the extracted axonemes lacked the outer arms in various degrees. The DEA-dynein 1 was that the extracted axonemes lacked the outer arms in various degrees. The SEA-dyenin 1 was purified and an antiserum against it was prepared in rabbits. The specificity of antiserum to dynein 1 was determined by immunoelectrophoresis and ouchterlony's double-diffusion test. The anti-dynein 1 serum inhibited ATPase activity of purified SEA-dynein 1 by 95 percent. By the indirect peroxidase-conjugated antibody method, the loci of SEA-dynein 1 within the intact, salt- extracted and mechanically disrupted axonemes were determined to be the outer arms: deposition of electron-dense materials which represents their localization was detected at the distal ends of the outer arms, in the case of intact axonemes. The 5-6 cross- bridge was hardly decorated. No decoration was seen in the salt-extracted axonemes lacking all the outer arms. In disrupted axonemes, which consist of single to several peripheral doublets, electron-dense materials were deposited only on the outer arms.
Approximately 73 percent of axonemal ATPase activity sensitive to antiserum was solubilized by repeated salt-extractions. One-half of A-polypeptide (SEA-dynein 1 located at the outer arms) was contained in the pooled extracts. The extracted axonemes contained another half of A-polypeptide (SUA-dynein 1 supposed to locate at the inner arms) and retained 31 percent of axonemal ATPase activity that was almost resistant to antiserum. Solubilized SUA-dynein 1 was immunologically the same as SEA-dynein 1. This result indicates that in situ SUA-dynein 1 did not receive anti-dynein 1 antibodies, coinciding with the result obtained for salt-extracted axonemes lacking all the outer arms by the enzyme-antibody method mentioned above. These observations suggest that immunological dissimilarity in dynein 1 between outer and inner arms but do not tell us that the inner arms do not contain dynein 1.
Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane
Authors: Lange Y , Hadesman RA , Steck TL 
Published: May 2004

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity.
The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network.
The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.
Assembly of storage protein oligomers in the endoplasmic reticulum and processing of the polypeptides in the protein bodies of developing pea cotyledons
Authors: Chrispeels MJ , Higgins TJV , Spencer D 
Published: May 2004

Cotyledons of developing pea seeds (pisum sativum L.) were labeled with radioactive amino acids and glucosamine, and extracts were prepared and separated into fractions rich in endoplasmic reticulum (ER) or protein bodies, The time-course of synthesis of the polypeptides of legumin and vicilin and the site of their assembly into protein oligomers were studied using immunoaffinity gels and sucrose density gradients. When cotyledons were pulse-labeled (1-2 h), newly synthesized vicilin was present as a series of polypeptides with M(r) 60,000-65,000, and newly synthesized vicilin was present as series of polypeptides with M(r) 75,000, 70,000, 50,000, and 49,000. These radioactive polypeptides were found primarily in the ER (Chrispeels et al., 1982, J Cell Biol., 93:5- 14). During a subsequent chase period, newly synthesized reserve proteins were initially present in the protein bodies in the above-named polypeptides. Between 1 and 20 h later, radioactive legumin subunits (M(r) 40,000 and 19,000) and smaller vicilin polypeptides (M(r) 34,000, 30,000, 25,000, 18,000, 14,000, 13,000, and 12,000) appeared in the protein bodies. The appearance of these labeled polypeptides in the protein bodies was not the result of a slow transport from the ER (or cytoplasm).
Newly synthesized legumin and vicilin polypeptides were assembled into oligomers of 8S and 7S, respectively, in the ER. They appeared in the protein bodies in these oligomeric forms before the appearance of the smaller polypeptides (M(r) less than 49,000). These results indicate that the smaller vicilin polypeptides (M(r) less than 49,000) arise delayed posttranslational processing of some or all of the larger vicilin polypeptides. The precursors of legumin are completely processed in the protein bodies 2-3 h after their synthesis. The processing of the vicilin precursors is much slower (6-20 h) and only a fraction of the precursor molecules are processed. As a result both large (M(r) more than 49,000) and small polypeptides of vicilin accumulate in the protein bodies, whereas legumin accumulates only as polypeptides of M(r) 40,000 and 19,000.
Changes in contractile proteins during differentiation of myeloid leukemia cells. II. Purification and characterization of actin
Authors: Nagata K , Sagara J , Ichikawa Y 
Published: May 2004

A myeloid leukemia cell line, M1, differentiates to macrophage and gains locomotive and phagocytic activity when incubated with conditioned medium (CM) from a fibroblast culture and bacterial endotoxin. To characterize the actin molecules before and after differentiation, the actin was purified through three sequential steps: DEAE-sephadex A- 50, polymerization/depolymerization, and sephadex G-150 chromatography.
There were no essential differences between the inhibitory activity of actins from control M1 cells and CM-treated M1 cells on both DNase I and heavy meromyosin (HMMM) K(+)-EDTA-ATPase; the same dose response as with skeletal muscle actin took place. After the treatment with CM, however, the specific activity for the activation of HMMM Mg(2+)- ATPase by actin became two-fold that of untreated M1 actin, which was one third of the value for skeletal muscle actin. The V(max) for the control and the CM-treated M1 cell, as well as the skeletal muscle actins, proved to be the same. By contrast, the K(app) values for the control and CM-treated M1-cell actins were 3- and 1.5-fold the value for skeletal-muscle actin. This means that CM treatment of the M1 actin produced a twofold affinity for the Mg(2+)-ATPase of skeletal-muscle myosin.
The critical concentrations for polymerization were compared under different salt concentrations and temperatures. Although no marked difference was found for the presence of 2 mM MgCl(2), 0.1 M KCl in place of MgCl(2) at 5 degrees C gave the following values: 0.1 mg/ml for skeletal-muscle actin, 0.7 mg/ml for control M1 actin, 0,5 mg/ml for CM- treated M1 actin, and 1.0 mg/ml for the D(-) subline that is insensitive to CM. Although the critical concentration of D(-) actin is extraordinarily high, this actin showed normal polymerization above the critical concentration. This together with the data presented in our previous paper, that the D(-) actin in the crude extract did not polymerize, suggests that an inhibitor for actin polymerization is present in the subline.
The kinetics experiment at 0.1 M KCl and 25 degrees C revealed a slower polymerization of untreated M1- and D(-)-cell actins as compared with CM-treated M1 actin. This delayed polymerization was due to a delay during the nucleation stage, not during the elongation stage.
By isoelectric focusing, the ratios of β- to γ-actin showed a marked difference depending on the states of cells: about 4.9 for control M1, 2.8 for CM-treated M1, and 7.6 for D(-)-subline actins. Tryptic peptide maps also revealed the presence of different peptides. Thus, the functional differences of actin before and after the differentiation was accompanied by some chemical changes in actin molecules.
Cross-linker system between neurofilaments, microtubules and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method
Authors: Hirokawa N 
Published: May 2004

The elaborate cross-connections among membranous organelles (MO), microtubules (MT), and neurofilaments (NF) were demonstrated in unifixed axons by the quick-freeze, deep-etch, and rotary-shadowing method. They were categorized into three groups: NF-associated cross-linker, MT-associated cross-bridges, and long cross-links in the subaxolemmal space. Other methods were also employed to make sure that the observed cross-connections in the unfixed axons were not a result of artifactual condensation or precipitation of soluble components or salt during deep-etching. Axolemma were permeablized either chemically (0.1% saponin) or physically (gentle homogenization), to allow egress of their soluble components from the axon; or else the axons were washed with distilled water after fixation. After physical rupture of the axolemma or saponin treatment, most of the MO remained intact. MT were stabilized by adding taxol in the incubation medium.
Axons prepared by these methods contained many longitudinally oriented NF connected to each other by numerous fine cross-linkers (4-6 nm in diameter, 20-50 nm in length). Two specialized regions were apparent within the axons: one composed of fascicles of MT linked with each other by fine cross-bridges; the other was in the subaxolemmal space and consisted of actinlike filaments and a network of long cross-links (50-150 nm) which connected axolemma and actinlike filaments with NF and MT. F-actin was localized to the subaxolemmal space by the nitrobenzooxadiazol phallacidin method. MO were located mainly in these two specialized regions and were intimately associated with MT via fine short (10-20 nm in length) cross-bridges. Cross-links from NF to MO and MT were also common. All these cross-connections were observed after chemical extraction or physical rupture of the axon; however, these procedures removed granular materials which were attached to the filaments in the fresh unextracted axons. The cross-connections were also found in the axons washed with distilled water after fixation. I conclude that the cross- connections are real structures while the granular material is composed of soluble material, probably protein in nature.
The cytokineplast: purified, stable, and functional motile machinery from human blood polymorphonuclear leukocytes
Authors: Malawista SE , De Boisfleury Chevance A 
Published: May 2004

We examined the formation of motile, chemotactically active, anucleate fragments from human blood polymorphonuclear leukocytes (PMN, granulocytes), induced by the brief application of heat. These granule-poor fragments are former protopods (leading fronts, lamellipodia) that become uncoupled from the main body of the cell and leave it, at first with a connecting filament that breaks and seals itself. The usual random orientation of such filaments can be controlled by preorientation of cells in a gradient of the chemotactic peptide, N-formylmethionylleucylphenylalanine (F-Met-Leu-Phe) (2x10(-9) M- 1x10(-8)). Cytochalsin B, 2.5-5 μg/ml, prevents fragment formation; colchicine, 10(-5) M, does not. In scanning electron micrographs, fragments are ruffled and the cell body rounded up and rather smooth. In transmission electron micrographs, fragments contain microfilaments but lack centrioles and microtubules. Like intact cells, both bound and free fragments can respond chemotactically to an erythrocyte destroyed by laser microirradiation (necrotaxis); the free, anucleate fragments may do so repeatedly, even after having been held overnight at ambient temperatures. We propse the name cytokineplast for the result of this self-purification of motile apparatus.
The exodus of the motile machinery from the granulocyte requires anchoring of the bulk of the cell to glass and uncoupling, which may involve heat-induced dysfunction of the centrosome. In ultrastructural studies of the centrosomal region after heat, centriolar structure remains intact, but pericentriolar osmiophilic material appears condensed, and microtubules are sparse. These changes are found in all three blood cell types examined: PMN, eosinophil, and monocyte. Of these, the first two make fragments under our conditions; the more sluggish monocyte does not. Uncoupling is further linked to centrosomal dysfunction by the observation that colchicines-treated granulocytes (10(-5)M, to destroy the centrosome's efferent arm) make fragments after less heat than controls.
If motive force and orientation are specified mainly from the organelle-excluding leading front, then endoplasmic streaming in PMN is a catch-up phenomenon, and microtubules do not provide the vector of locomotion but rather stabilize and orient the "baggage" (nucleus, granuloplasm) - i.e., they prevent fishtailing. Moreover, constraints emanating from the centrosome may now be extended to include, maintenance of the motile machinery as an integral part of the cell.
Export of major cell surface proteins is blocked in yeast secretory mutants
Authors: Novick P , Schekman R 
Published: May 2004

The transport of newly synthesized proteins to the yeast cell surface has been analyzed by a modification of the technique developed by Kaplan et al. (Kaplan, G., C. Unkeless, and Z.A. Cohn, 1979, Proc. Natl. Acad. Sci. USA, 76:3824-3828). Cells metabolically labeled with (35)SO(4)(2-) are treated with trinitrobenzenesulfonic acid (TNBS) at 0 degrees C under conditions where cell-surface proteins are tagged with trinitrophenol (TNP) but cytoplasmic proteins are not. After fractionation of cells into cell wall, membrane and cytoplasmic samples, and solubilization with SDS, the tagged proteins are immunoprecipitated with anti-TNP antibody and fixed staphylococcus aureus cells. Analysis of the precipitates by SDS gel electrophoresis and fluorography reveals four major protein species in the cell wall (S(1)-S(4)), seven species in the membrane fraction (M(1)-M(7)), and no tagged proteins in the cytoplasmic fraction.
Temperature-sensitive mutants defective in secretion of invertase and acid phosphatase (sec mutants; Novick, P., C. Field, and R. Schekman, 1980, Cell, 21:204-215) are also defective in transport of the 11 major cell surface proteins at the nonpermissive temperature (37 degrees C). Export of accumulated proteins is restored in an energy- dependent fashion when secl cells are returned to a permissive temperature (24 degrees C). In wild-type cells the transit time for different surface proteins varies from less than 8 min to about 30 min. The asynchrony is developed at an early stage in the secretory pathway.
All of the major cell wall proteins and many of the externally exposed plasma membrane proteins bind to concanavalin A. Inhibition of asparagine-linked glycosylation with tunicamycin does not prevent transport of several surface proteins.
Participation of plasma membrane proteins in the formation of tight junction by cultured epithelial cells
Authors: Griepp EB , Dolan WJ , Robbins ES , Sabatini DD 
Published: May 2004

Measurements of the transepithelial electrical resistance correlated with freeze-fracture observations have been used to study the process of tight junction formation under various experimental conditions in monolayers of the canine kidney epithelial cell line MDCK.
Cells derived from previously confluent cultures and plated immediately after trypsin- EDTA dissociation develop a resistance that reaches its maximum value of several hundred ohms-cm(2) after approximately 24 h and falls to a steady-state value of 80-150 ohms- cm(2) by 48 h. The rise in resistance and the development of tight junctions can be completely and reversibly prevented by the addition of 10 μg/ml cycloheximide at the time of plating, but not when this inhibitor is added more than 10 h after planting. Thus tight junction formation consists of separable synthetic and assembly phases. These two phases can also be dissociated and the requirement for protein synthesis after plating eliminated if, following trypsinization, the cells are maintained in spinner culture for 24 h before plating. The requirement for protein synthesis is restored, however, if cells maintained in spinner culture are treated with trypsin before plating. Actinomycin D prevents development of resistance only in monolayers formed from cells derived from sparse rather than confluent cultures, but new mRNA synthesis is not required if cells obtained from sparse cultures are maintained for 24 h in spinner culture before plating. Once a steady-state resistance has been reached, its maintenance does not require either mRNA or protein synthesis; in fact, inhibition of protein synthesis causes a rise in the resistance over a 30-h period. Following treatments that disrupt the junctions in steady- state monolayers recovery of resistance also does not require protein synthesis.
These observations suggest that proteins are involved in tight junction formation. Such proteins, which do not turn over rapidly under steady-state conditions, are destroyed by trypsinization and can be resynthesized in the absence of stable cell-cell or cell-substratum contact. Messenger RNA coding for proteins involved in tight junction formation is stable except when cells are sparsely plated, and can also be synthesized without intercellular contacts or cell-substratum attachment.