bluuebunny/crossref_metadata_2025 huggingface dataset:
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.`
Myosin subfragment binding for the localization of actin-like microfilaments in cultured cells. A light and electron microscope study.
Authors: Schloss J A , Milsted A , Goldman R D
Published: May 2004

Fluorescein-labeled heavy meromyosin subfragment-1 (F-S-1) has been purified by ion exchange chromatography and characterized in terms of its ability to bind specifically to actin. F-S-1 activates the Mg++-adenosine triphosphatase activity of rabbit skeletal muscle actin and decorates actin as shown by negative stains and thin sections of rabbit actin and rat embryo cell microfilament bundles, respectively. Binding of F-S-1 to cellular structures is prevented by pyrophosphate and by competition with excess unlabeled S-1. The F-S-1 is used in light microscope studies to determine the distribution of actin-containing structures in wnterphase and mitotic rat embryo and rat kangaroo cells. Interphase cells display the familiar pattern of fluorescent stress fibers. Chromosome-to-pole fibers are fluorescent in mitotic cells. The glycerol extraction procedures employed provide an opportunity to examine cells prepared in an identical manner by light and electron microscopy. The latter technique reveals that actin-like microfilaments are identifiable in spindles of glycerinated cells before and after addition of S-1 or HMM. In some cases, microfilaments appear to be closely associated with spindle microtubles. Comparison of the light and electron microscope results aids in the evaluation of the fluorescent myosin fragment technique and provides further evidence for possible structural and functional roles of actin in the mitotic apparatus.
Adult rat hepatocytes in primary monolayer culture. Ultrastructural characteristics of intercellular contacts and cell membrane differentiations.
Authors: Wanson J C , Drochmans P , Mosselmans R , Ronveaux M F
Published: May 2004

Primary monolayer cultures were obtained in 60-mm petri dishes by incubating 3 X 10(6) isolated hepatocytes at 37 degrees C in Dulbecco's medium supplemented with 17% fetal calf serum. The ultrastructure of monolayer cells was examined after various incubation periods. Within 4 h of plating, the isolated spherical cells adhere to the plastic surface, establish their first contacts by numerous intertwined microvilli, and form new hemidesmosomes. After 12 h of culture, wide branched trabeculae of flattened polyhedral cells extend in all directions. Finally, after 24 h of culture, bile canaliculi are reconstituted, and a biliary polarity is recovered: the Golgi elements, which are scattered throughout the cytoplasm in the isolated cells, are reassembled in front of the newly formed bile canalculi, symmetrically in the adjacent cells; lysosomes are concentrated in that region, and microtubules reappear. Concomitantly, plasma membrane differentiations, namely desmosomes and tight junctions, develop. Tight junctions sealing the bile ducts constitute a barrier to the passage of ruthenium red and horseradish peroxidase. De novo formation of these junctions was studied by the freeze-etching technique: 10-nm particles compose a network of anastomosed linear arrays in the vicinity of the bile canaliculi; in the next step of differentiation, the particles fuse, form short ridge segments and finally continuous branched smooth strands, characteristic of the mature tight junction.
Characterization of the myosin-phosphorylating system in normal murine astrocytes and derivative sv40 wild-type and A-mutant transformant.
Authors: Scrodilis S P , Anderson J L , Pollack R , Adelstein R S
Published: May 2004

Myosin and myosin light-chain kinase have been isolated and characterized from small quantities of normal and SV40-transformed, murine astrocytic neuroglial cells in culture and from intact normal mouse brain. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the astrocyte myosins revealed a heavy chain of 200,000 daltons and two light chains of 20,000 and 15,000 daltons. These myosins are similar to other cytyplasmic myosins. The astrocyte 20,000-dalton light chain can be phosphorylated by an endogenous myosin light-chain kinase which has properties similar to those of the myosin light-chain kinase found in human platelets. No differences were detected in either the astrocyte myosins or myosin light-chain kinases between (a) the normal and transformed cells, (b) the transformed cells grown at the permissive and nonpermissive temperatures, or (c) the SV40 wild-type and A-mutant transformants.
Lectin-binding sites on the plasma membranes of rabbit spermatozoa: Changes in surface receptors during epididymal maturation and after ejaculation
Authors: Nicolson GL , Usui N , Yanagimachi R , Yanagimachi H , JR Smith
Published: May 2004

Modifications in rabbit sperm plasma membranes during epididymal passage and after ejaculation were investigated by used of three lectins: concanavalin A (Con A); Ricinus communis I (RCA(I)); and wheat germ agglutinin (WGA). During sperm passage from caput to cauda epididymis, agglutination by WGA drastically decreased, and agglutination by RCA(I) slightly decreased, although agglutination by Con A remained approximately unchanged. After ejaculation, spermatozoa were agglutinated to a similar degree or slightly less by Con A, WGA, and RCA(I), compared to cauda epididymal spermatozoa. Ultrastructural examination of sperm lectin-binding sites with ferritin- lectin conjugates revealed differences in the densities of lectin receptors in various sperm regions, and changes in the same regions during epididymal passage and after ejaculation. Ferritin-RCA(I) showed abrupt changes in lectin site densities between acrosomal and postacrosomal regions of sperm heads. The relative amounts of ferritin-RCA(I) bound to heads of caput epididymal or ejaculated spermatozoa. Tail regions were labeled by ferritin RCA(I) almost equally on caput and cauda epididymal spermatozoa, but the middle-piece region of ejaculated spermatozoa was slightly more densely labeled than the principal-piece region, and these two regions on ejaculated spermatozoa were labeled less than on caput and cuada epididymal spermatozoa. Ferritin-WGA densely labeled the acrosomal region of caput epididymal spermatozoa, although labeling of cauda epidiymal spermatozoa was relatively sparse except in the apical area of the acrosomal region. Ejaculated spermatozoa bound only a few molecules of ferritin-WGA, even at the highest conjugate concentrations used. Caput epididymal, but not cauda epididymal or ejaculated spermatozoa, bound ferritin-WGA in the tail regions. Dramatic differences in labeling densities during epididymal passage and after ejaculation were not found with ferritin-Con A.
Locomotory behavior, contact inhibition, and pattern formation of 3T3 and polyoma virus-transformed 3T3 cells in culture
Authors: Bell PB
Published: May 2004

The social behavior of 3T3 cells and their polynoma virus-transformed derivative (Py3T3 cells) was examined by time-lapse cinemicrography in order to determine what factors are responsible for the marked differences in the patterns formed by the two cell lines in culture. Contrary to expectations, both cell types have been found to exhibit contact inhibition of cell locomotion. Therefore, the tendency of 3T3 cells to form monolayers and of Py3T3 cells to form crisscrossed multilayers cannot be explained on the basis of the presence versus the absence of contact inhibition. Morevover, with the exception of cell division control, the social behavior of the two cell types is qualitively similar. Both exhibit cell underlapping and, after contact between lamelliopodia, both show inhibition of locomotory activity and adhesion formation. Neither cell type was observed to migrate over the surface of another cell. The two cell types do show quantitative differences in the frequency of underlapping, the frequency with which contact results in inhibition of locomotion, and the proportion of the cell margin that adheres to the substratum. The increased frequency pf Py3T3 underlapping is correlated with the reduced frequency of substratum adhesions, which in turn favors underlapping. On the basis of these observations, it is concluded that the differences in culture patterns are the result of differences in the shapes of the individual cells, such that underlapping, and hence crisscrossing, is favored in Py3T3 cell interactions and discouraged in 3T3 cells.
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.
Evidence that coated vesicles isolated from brain are calcium-sequestering organelles resembling sarcoplasmic reticulum.
Authors: Blitz A L , Fine R E , Toselli P A
Published: May 2004

Coated vesicles from the brain have been purified to near morphological homogeneity by a modification of the method of Pearse. These vesicles resemble sarcoplasmic reticulum fragments isolated from skeletal muscle. They contain proteins with 100,000- and 55,000-dalton mol wt which co-migrate on polyacrylamide gels, in the presence of sodium dodecyl sulfate, with the two major proteins of the sarcoplasmic reticulum fragment. These vesicles contain adenosine triphosphatase (ATPase) activity which is stimulated by calcium ions in the presence of Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.), displaying maximal activity at 8 x 10(-7) M Ca ++. They take up calcium ions from the medium, and this uptake is stimulated by ATP and by potassium oxalate, a calcium-trapping agent. The 100,000-dalton protein of the coated vesicles displays immunological reactivity with an antiserum directed against the 100,000-dalton, calcium-stimulated ATPase of the sarcoplasmic reticulum. As with the sarcoplasmic reticulum fragment, this protein becomes radiolabeled when coated vesicles are briefly incubated with gamma-labeled [32P]ATP. The possible functions of coated vesicles as calcium-sequestering organelles are discussed.
Morphometric and cytochemical studies of Dictyostelium discoideum in vegetative phase. Digestive system and membrane turnover.
Authors: Ryter A , de Chastellier C
Published: May 2004

The morphometric analysis of growing cells shows that the membranes of the digestive apparatus have a surface area equal to the cell surface area. After yeast phagocytosis, the surface area of the membrane surrounding the ingested yeast is equal to 40% of the surface area of the cell membrane. In spite of this internalization, the cell surface remains constant. Its renewal is insured by the translocation of the membrane of the digestive system, the surface area that concomitantly decreases by 40%. This means that the influx of plasma membrane is continually compensated for by the same outflow of internal membranes. During this turnover, the characteristic polysaccharide stainability (two different stains were used) of the plasma membrane is maintained after internalization, at the level of the digestive system, despite the presence of hydrolases in the digestive vacuoles. The cytochemical demonstration of acid phosphatase shows that this enzyme penetrates into phagosomes by fusion between phagosomes and vacloles of various sizes. The debris of digested yeast are released into the culture medium after 2 h. This process of defecation is accompanied by the appearance of new pinocytotic vacuoles, which indicates that the uptake of axenic medium has resumed. A model of membrane turnover is proposed to explain these observations.
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.
Freeze-fracture of membrane fusions during exocytosis in pancreatic B-cells.
Authors: Orci L , Perrelet A , Friend D S
Published: May 2004

To examine the freeze-fracture appearance of membrane alterations at sites of exocytosis in mammalian cells, we studied the secretory granule and plasma membrane of rat pancreatic B-cells during glucose-stimulated insulin secretion. Constant features observed were the scarcity of particles in secretory-granule P-fracture faces and the almost total clearance of intramembranous particles in P-and E fracture faces of the plasma membrane in areas of close apposition of these two membranes preceding fusion; also observed was the temporary persistence of particle-cleared regions after the fusion was completed. Our observations thus support the concept that membranes fuse at sites of closely apposed, particle-free regions and that the physiologically created clear areas found in freeze-fracture replicas of the plasma membrane are the hallmarks of incipient or recent membrane fusion.
Biochemical investigations of retinotectal adhesive specificity
Authors: Marchase RB
Published: May 2004

The preferential adhesion of chick neural retina cells to surfaces of intact optic tecta has been investigated biochemically. The study uses a collection assay in which single cells from either dorsal or ventral halves of neural retain adhere preferentially to ventral or dorsal halves of optic tecta respectively. The data presented support the following conclusions: (a) The adhesion of ventral retina to dorsal tecta seems to depend on proteins located on ventral retina and on terminal β-N-acetylgalactosamine residues on dorsal tecta. (b) The adhesion of dorsal retina to ventral tecta seems to depend on proteins located on ventral tecta and on terminal β- N-acetylgalactosamine residues on dorsal retina. (c) A double gradient model for retinotectal adhesion along the dorsoventral axis is consistent with the data presented. The model utilizes only two complementary molecules. The molecule suggested to be concentrated dorsally in both retina and tectum seems to require terminal β-N-acetylgalactosamine residues for adhesion. Its activity is not affected by protease. A molecule fitting these qualifications, the ganglioside GM(2), could not be detected in a gradient, but lecithin vesicles containing GM(2) adhered preferentially to ventral tectal surfaces. The second molecule, concentrated ventrally in both retina and tectum, is a protein and seems capable of binding terminal β-N- acetylgalactosamine residues. One enzyme, UDP-galactose:GM(2) galactosyltransferase, has been found to be more concentrated in ventral retina than dorsal, but only by 30 percent.
Patterns of plasminogen activator production in cultured normal embryonic cells.
Authors: Rohrlich S T , Rifkin D B
Published: May 2004

Cultured normal low-passage embryo fibroblasts, from a number of species, and two untransformed clones of a Balb/3T3 line elaborate increasing amounts of plasminogen activator (PA) as they approach confluence; the low-passage cells then lose this PA activity after reaching confluence, while the 3T3 cells retain it indefinitely. Even at their peaks, however, the PA activities of the low-passage cells remain well below those of the corresponding virally or spontaneously transformed cells. The PA increases in normal cells are probably a result of PA production rather than of adsorption of secreted PA to the cell surface, or of changes in cell-associated protease inhibitors. The elaboration of PA by normal cells is dependent upon their metabolic activity, such that the level of serum supplementation and the growth phase of the culture directly influence the level of cell-associated PA observed. In addition, there may be a component of serum which exerts a negative control on PA production and which is not an acid-labile protease inhibitor.
Effects of myosin and heavy meromyosin on actin-related gelation of HeLa cell extracts.
Authors: Weihing R R
Published: May 2004

The gelation induced by warming (to 25 degrees C) the 100,000 g supernatant fraction (extract) of HeLa cells lysed in a buffer containing sucrose, ATP, DTE, EGTA, imidazole, and Triton X-100 was studied in the presence of myosin and heavy meromyosin (HMM). Myosin mixed with extract induces shrinkage of the gel, but jelled extract or myosin alone does not shrink. In the concentration range, 0.14-1.04 mg/ml of myosin, the degree of shrinkage is roughly proportional to the concentration of myosin. Supplementa MgCl2 also promotes shrinkage. HMM (0.4-0.8 mg/ml) can inhibit gel formation by extract in tubes or floated on a sucrose cushion. Gel electrophoresis of gels shrunken by added myosin or electrophoresis of the proteins which can be sedimented from extract after incubation in the presence of HMM indicate that both myosin and HMM interfere with the changes in sedimentability of the high molecular weight protein (HMWP) thought to participate (together with actin) in gel formation in HeLa cell extracts (R. R. Weihing, 1976. J. Cell Biol. 71:303-307). These results, together with previous results showing that actin is present and that HMWP is enriched in the plasma membrane fraction of HeLa cells (R. R. Weihing, 1976. Cold Spring Harbor Conf. Cell Proliferation. 3:671-684), point to the possibility of dynamic changes in the interactions of HMWP or myosin with actin in processes of movement occurring at the cell surface.
Localization of creatine kinase isoenzymes in myofibrils. I. Chicken skeletal muscle.
Authors: Wallimann T , Turner D C , Eppenberger H M
Published: May 2004

Purified, repeatedly washed, skeletal muscle myofibrils contain approx. 0.2 U of creatine kinase (CK) activity (equivalent to 2.5 micrograms CK) per milligram dry weight; this firmly bound CK activity is estimated to represent 3-5% of the total cellular CK. It had been shown previously that the myofibrillar CK, which can be quantitatively extracted at low ionic strength and purified to homogeneity, is very similar, if not identical, to the bulk MM-CK. It is shown that the two protein preparations also have the same peptide pattern after cyanogen bromide fractionation and very similar specific activities, confirming their identity. The earlier demonstration that the bound CK is specifically located at the M-lines of isolated myofibrils has been confirmed by immunofluorescence. Antibodies directed against purified MM- and BB-CK were used in the indirect fluorescent antibody technique to study the specificity of myofibril binding sites for different forms of CK. With myofibrils from adult muscle, which has only MM-CK, as well as from early developmental stages in which BB-CK is the predominant isoenzyme, M-type CK was localized exclusively at the M-line, while greater or lesser amounts of B-type CK were found at the Z-line. The data provide strong evidence that the MM-CK at the M-lines in skeletal myofibrils is not adventitiously bound but is rather an integral element in the M-line structure. The amount of CK bound is reasonably consistent with the earlier proposal that the CK molecules might be the transverse M-bridges and appears to be sufficient to regenerate all of the ATP hydrolyzed during muscle contraction.
Incorporation of glycoproteins into peripheral nerve myelin.
Authors: Gould R M
Published: May 2004

Peripheral nerve myelin contains a dominant low molecular weight glycoprotein called Po. To study the metabolism of this glycoprotein, tritiated fucose was injected into the peripheral nerves of adult mice and developing rats, and the temporal distribution of label was examined by autoradiography and gel electrophoresis. Mice and rat pups, injected with fucose, were sacrificed from 1 h to 98 days later. Series of autoradiographs were prepared. At the shortest labeling periods, newly formed product was confined to juxtanuclear Schwann cell cytoplasm, in association with regions rich in Golgi apparatus. After longer labeling periods, silver grain levels in Schwann cell cytoplasm decreased; concomitantly, there was an increase of silver grains associated with myelin. In adult animals, label associated with myelin was concentrated over outer layers of thickly myelinated fibers. Even at the longest time intervals examined (72 and 98 days), this distribution of label was largely retained. In contrast, in developing animals, label became associated with inner layers of the thicker sheaths. At no time was label observed over axons. Gel electrophoresis revealed that tritiated fucose was a suitable precursor for the faster migrating peripheral nerve glycoprotein(s). At all times examined, there was a single major peak of radioactivity that co-migrated on sodium dodecyl sulfate (SDS) acrylamide gels with the Po protein. Sometimes, a faster migrating shoulder of radioactivity was noted. With increased labeling periods, there was an enrichment of radioactivity associated with Po, indicative of a relatively slow turnover rate.
Actin filaments in sensory hairs of inner ear receptor cells.
Authors: Flock A , Cheung H C
Published: May 2004

Receptor cells in the ear are excited through the bending of sensory hairs which project in a bundle from their surface. The individual stereocilia of a bundle contain filaments about 5 nm in diameter. The identity of these filaments has been investigated in the crista ampullaris of the frog and guinea pig by a technique of decoration with subfragment-1 of myosin (S-1). After demembranation with Triton X-100 and incubation with S-1, "arrowhead" formation was observed along the filaments of the stereocilia and their rootlets and also along filaments in the cuticular plate inside the receptor cell. The distance between attached S-1 was 35 nm and arrowheads pointed in towards the cell soma. It is concluded that the filaments of stereocilia are composed of actin.
Changes in thick filament length in Limulus striated muscle.
Authors: Dewey M M , Walcott B , Colflesh D E , Terry H , Levine R J
Published: May 2004

Here we describe the change in thick filament length in striated muscle of Limulus, the horseshoe crab. Long thick filaments (4.0 microns) are isolated from living, unstimulated Limulus striated muscle while those isolated from either electrically or K+-stimulated fibers are significantly shorter (3.1 microns) (P less than 0.001). Filaments isolated from muscle glycerinated at long sarcomere lengths are long (4.4 microns) while those isolated from muscle glycerinated at short sarcomere lengths are short (2.9 microns) and the difference is significant (P less than 0.001). Thin filaments are 2.4 microns in length. The shortening of thick filaments is related to the wide range of sarcomere lengths exhibited by Limulus telson striated muscle.
Correlation between movement of concanavalin A membrane receptors and cytolysis. A scanning electron microscopy study.
Authors: Lustig S , Ascher O , Fishman P , Djaldetti M , Pluznik D H
Published: May 2004

The present study was undertaken to test whether cytolysis induced by Concanavalin A (Con A) requires lateral mobility of membranal lectin receptor sites into caps. Treatment of interphase murine mastocytoma cells with 10(-4) M colchicine promoted cap formation by Con A in about 30% of the cells, followed by cytolysis. Pretreatment of the cells with NaN3, low temperature, or glutaraldehyde decreased the degree of capping and, to the same extent, the degree of cytolysis. The addition of antibodies to cells bound with Con A increased the appearance of capping and cytolysis. A linear relationship with a high correlation coefficient exists between the degree of capping and cytolysis, suggesting that lateral mobility of membrane Con A receptors is required for cytolysis by the lectin. The process of cap formation by Con A up to the stage of cytolysis was followed by scanning electron microscopy.
Glucocorticoids modulate the in vitro development of the embryonic rat pancreas.
Authors: Rall L , Pictet R , Githens S , Rutter W J
Published: May 2004

The effect of the glucocorticoid analogue, dexamethasone, on the development of the embryonic pancreas was studied in tissue culture. It specifically enhances the accumulation of exocrine enzymes without altering the level of general cell proteins. The enhancement, however, is not symmetrical: the cellular levels of the two major exocrine products, amylase and chymotrypsinogen, are increased about 10- and 2-fold, respectively. Two other zymogens that are present in minor quantities, procarboxypeptidases A and B, are also increased, whereas no effect is seen on lipase A. Coordinate with these effects on synthesis, there is a dramatic change in the morphology of dexamethasone-stimulated acinar cells. Their number of zymogen granules is higher and crystalline arrays are found in the rough endoplasmic reticulum. Dexamethasone also inhibits cell replication, perhaps by selectively inhibiting the last cell divisions of the culture period. At the same time, there is a disproportionate reduction in the insulin content of cultured rudiments. We find that pancreatic development is normal in the absence of dexamethasone and that this glucocorticoid does not precociously induce the appearance of the specific secretory products, but rather enhances by a constant degree their synthesis and accumulation. Therefore, we conclude that glucocorticoids may play a modulatory but not an inductive role in pancreatic development.
Unequal division in Saccharomyces cerevisiae and its implications for the control of cell division.
Authors: Hartwell L H , Unger M W
Published: May 2004

The budding yeast, Saccharomyces cerevisiae, was grown exponentially at different rates in the presence of growth rate-limiting concentrations of a protein synthesis inhibitor, cycloheximide. The volumes of the parent cell and the bud were determined as were the intervals of the cell cycle devoted to the unbudded and budded periods. We found that S. cerevisiae cells divide unequally. The daughter cell (the cell produced at division by the bud of the previous cycle) is smaller and has a longer subsequent cell cycle than the parent cell which produced it. During the budded period most of the volume increase occurs in the bud and very little in the parent cell, while during the unbudded period both the daughter and the parent cell increase significantly in volume. The length of the budded interval of the cell cycle varies little as a function of population doubling time; the unbudded interval of the parent cell varies moderately; and the unbudded interval for the daughter cell varies greatly (in the latter case an increase of 100 min in population doubling time results in an increase of 124 min in the daughter cell's unbudded interval). All of the increase in the unbudded period occurs in that interval of G1 that precedes the point of cell cycle arrest by the S. cerevisiae alpha-mating factor. These results are qualitatively consistent with and support the model for the coordination of growth and division (Johnston, G. C., J. R. Pringle, and L. H. Hartwell. 1977. Exp. Cell. Res. 105:79-98.) This model states that growth and not the events of the DNA division cycle are rate limiting for cellular proliferation and that the attainment of a critical cell size is a necessary prerequisite for the "start" event in the DNA-division cycle, the event that requires the cdc 28 gene product, is inhibited by mating factor and results in duplication of the spindle pole body.
Functional correlation between cell adhesive properties and some cell surface proteins.
Authors: Takeichi M
Published: May 2004

The adhesive properties of Chinese hamster V79 cells were analyzed and characterized by various cell dissociation treatments. The comparisons of aggregability among cells dissociated with EDTA, trypsin + Ca2+, and trypsin + EDTA, revealed that these cells have two adhesion mechanisms, a Ca2+-independent and a Ca2+-dependent one. The former did not depend on temperature, whereas the latter occurred only at physiological temperatures. Both mechanisms were trypsin sensitive, but the Ca2+-dependent one was protected by Ca2+ against trypsinization. In morphological studies, the Ca2+-independent adhesion appeared to be a simple agglutination or flocculation of cells, whereas the Ca2+-dependent adhesion seemed to be more physiological, being accompanied by cell deformation resulting in the increase of contact area between adjacent cells. Lactoperoxidase-catalyzed iodination of cell surface proteins revealed that several proteins are more intensely labeled in cells with Ca2+-independent adhesiveness than in cells without that property. It was also found that a cell surface protein with a molecular weight of approximately 150,000 is present only in cells with Ca2+-dependent adhesiveness. The iodination and trypsinization of this protein were protected by Ca2+, suggesting its reactivity to Ca2+. Possible mechanisms for each adhesion property are discussed, taking into account the correlation of these proteins with cell adhesiveness.
Diffusion rates of cell surface antigens of mouse-human heterokaryons. I. Analysis of the population.
Authors: Edidin M , Wei T Y
Published: May 2004

The rate of appearance, in a newly formed heterokaryon population, of cells bearing completely intermixed mouse and human surface antigens may be used to estimate diffusion constants for antigens on individual cells. From this estimate, it appears that the surface antigens in most cells do not diffuse at the rate expected, but rather move more slowly, by a factor of ten or more, than expected from either measured or calculated diffusion constants for proteins freely mobile in the plane of a lipid membrane. Differences in diffusion rates between cells are not due to effects of Sendai virus, or of trypsin. Restrictions on diffusion are apparently not due to cytochalasin B- or Colcemid-sensitive elements.
Transformations in the structure of the cytoplasmic ground substance in erythrophores during pigment aggregation and dispersion. I. A study using whole-cell preparations in stereo high voltage electron microscopy.
Authors: Byers H R , Porter K R
Published: May 2004

Pigment migration in cultured erythrophores of the squirrel fish Holocentrus ascensionis, after manipulation with K+, epinephrine, 3',5'-dibutyryl cyclic adenosine monophosphate, theophylline, and caffeine, is essentially identical to that observed in this chromatophore in situ. For such observations, the erythrophores are dissociated from the scales with hyaluronidase and collagenase, and allowed to spread on an amorphous collagen substrate, where they resemble the discoid erythrophore in situ. In this state, they are readily fixed by glutaraldehyde and osmium tetroxide, and are then critical-point dried for whole-cell viewing in the high voltage electron microscope. The organization and fine structure of the erythrophore cytoplast was stereoscopically examined after fixation of the pigment granules in four experimental states: pigment dispersed, pigment aggregated, pigment aggregating, and pigment dispersing. In the dispersed cell, granules are contained in an extensive three-dimensional lattice composed of radially oriented microtubules and a network of fine filaments 3-6 nm in diameter (microtrabeculae), whereas in the aggregated cell, the microtrabecular system is absent, and the majority of the microtubules appear displaced into the cortices on the cytoplasmic surface of the plasma membrane. In cells fixed while aggregating, few microtrabeculae are observed, although formless thickenings are observed in the cortices, on granules, and between clumped granules. In dispersing cells, the microtrabecular system is reformed from material stored in the cortices and with the granules in the centrosphere. These observations suggest that the granules are suspended in a dynamic microtrabecular system that withdraws during pigment aggregation and is restructured during pigment dispersion. The microtubules guide linear granule motion not by defining physical channels, but by a recognizable affinity of microtubules, microtrabeculae, and granules for one another.
Morphometry of the renal corpuscle during normal postnatal growth and compensatory hypertrophy. A light microscope study.
Authors: Olivetti G , Anversa P , Rigamonti W , Vitali-Mazza L , Loud A V
Published: May 2004

Renal corpuscles from the juxtamedullary and subcapsular regions of the renal cortex were morphometrically analyzed in young rats and in adult rats that had been unilaterally nephrectomized or sham-operated at an early age. Mean corpuscular volumes increased 4.5-fold during normal development, and 7.7-fold as a result of compensatory hypertrophy in both cortical regions. Relative and absolute volumes were determined for Bowman's space, the glomerular tuft, and five glomerular components: epithelial, endothelial, and mesangial cells, capillaries, and the filtration membrane. Normal and hypertrophic enlargement of Bowman's space was slightly greater than glomerular growth, and the growth response of subcapsular glomeruli was greater than that of juxtamedullary glomeruli. The ratio of mean glomerular volumes between outer and inner glomeruli was 1:2 in both adult groups. Both adult groups also developed nearly identical proportions of all glomerular component structures, representing a relative decrease of epithelial cells and increase of capillaries compared to the young animals. Normal and hypertrophic maturation involved absolute increases in all glomerular cell populations, the length of capillary loops and the surface area of the filtration membrane, all nearly in proportion to the respective four- and seven-fold increases in glomerular volume. Changes in the filtration surface area are consistent with published data for glomerular filtration rates in normal and hypertrophied kidneys. The mean cell size in epithelial and mesangial populations doubled during growth, but was not greater than normal in mononephrectomized rats. Hyperplasia among all populations of glomerular cells is indicated in normal growth, and to a greater extent in compensatory renal hypertrophy.
Ultrastructure of the sodium pump: Comparison of thin sectioning, negative staining, and freeze-fracture of purified, membrane-bound (Na+, K+)-ATPase
Authors: Deguchi N , Jorgensen PL , Maunsbach AB
Published: May 2004

Purified (Na+, K+)-ATPase was studied by electron microscopy after thin sectioning, negative staining, and freeze-fracturing, particular emphasis being paid to the dimensions and frequencies of substructures in the membranes. Ultrathin sections show exclusively flat or cup-shaped membrane fragments which are triple-layered along much of their length and have diameters of 0.1-0.6 μm. Negative staining revealed a distinct substructure of particles with diameters between 30 and 50 A and with a frequency of 12,500 +/- 2,400 (SD) per μm(2). Comparisons with sizes of the protein components suggest that each surface particle contains as its major component one large catalytic chain with mol wt close to 100,000 and that two surface particles unite to form the unit of (Na+,K+)-ATPase which binds one molecule of ATP or ouabain. The further observations that the surface particles protrude from the membrane surface and are observed on both membrane surfaces in different patterns and degrees of clustering suggest that protein units span the membrane and are capable of lateral mobility. Freeze-fracturing shows intramembranous particles with diameters of 90-110 A and distributed on both concave and convex fracture faces with a frequency of 3,410 +/- 370 per μm(2) and 390 +/- 170 per μm(2), respectively. The larger diameters and three to fourfold smaller frequency of the intramembranous particles as compared to the surface particles seen after negative staining may reflect technical differences between methods, but it is more likely that the intramembranous particle is an oliogomer composed of two or even more of the protein units which form the surface particles.
Changes in ionic movements across rabbit polymorphonuclear leukocyte membranes during lysosomal enzyme release. Possible ionic basis for lysosomal enzyme release.
Authors: Naccache P H , Showell H J , Becker E L , Sha'afi R I
Published: May 2004

Changes in the movements of Na+, K+, and Ca+2 across rabbit neutrophils under conditions of lysosomal enzyme release have been studied. We have found that in the presence of cytochalasin B, the chemotactic factor formyl methionyl leucyl phenylalanine (FMLP) induces within 30 s large enhancements in the influxes of both 22Na+ and 45Ca+2 and an increase in the cellular pool of exchangeable calcium. The magnitude of the changes induced by cytochalasin B and FMLP exceeds that induced by FMLP or cytochalasin B alone, and cannot be explained on the basis of an additive effect of the two agents. However, these compounds either separately or together produce much smaller enhancements in 45Ca efflux. The divalent cation ionophore A23187 also produces a rapid and large increase in the influxes of both 22Na and 45Ca+2 in the presence and absence of cytochalasin B. We have also found an excellent correlation between calcium influx and lysosomal enzyme release. 42K influx is not significantly affected by any of these compounds. On the other hand, a large and rapid increase of 42K efflux is observed under conditions which give rise to lysosomal enzyme release. A flow diagram of the events that are thought to accompany the stimulation of polymorphonuclear leukocytes (PMNs) by chemotactic or degranulating stimuli is presented.
Motility of triton-demembranated sea urchin sperm flagella during digestion by trypsin.
Authors: Brokaw C J , Simonick T F
Published: May 2004

The survival curves for a population of reactivated spermatozoa exposed to digestion by trypsin indicate that a large number of trypsin-sensitive targets must be digested before the flagellum disintegrates. Changes in flagellar movement during trypsin digestion can be very small, especially when the spermatozoa are reactivated at 0.25 M KCl. They are not the changes which would be expected if elastic resistance of the trypsin-sensitive structures responsible for maintaining the integrity of the axoneme is a significant determinant of flagellar bend amplitude. By carrying out trypsin digestion under a variety of conditions, at least six distinct effects of trypsin digestion on parameters of flagellar movement have been detected. These include a gradual increase in the rate of sliding between tubules, gradual and abrupt changes in beat frequency accompanied by reciprocal decreases in bend angle, changes in the symmetry and planarity of bending, and selective interference with mechanisms for bend initiation and bend propagation.
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.