bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium release and ionic changes in the sarcoplasmic reticulum of tetanized muscle: an electron-probe study.
Authors: Somlyo A V , Gonzalez-Serratos H G , Shuman H , McClellan G , Somlyo A P
Published: May 2004

Approximately 60-70% of the total fiber calcium was localized in the terminal cisternae (TC) in resting frog muscle as determined by electron-probe analysis of ultrathin cryosections. During a 1.2 s tetanus, 59% (69 mmol/kg dry TC) of the calcium content of the TC was released, enough to raise total cytoplasmic calcium concentration by approximately 1 mM. This is equivalent to the concentration of binding sites on the calcium-binding proteins (troponin and parvalbumin) in frog muscle. Calcium release was associated with a significant uptake of magnesium and potassium into the TC, but the amount of calcium released exceeded the total measured cation accumulation by 62 mEq/kg dry weight. It is suggested that most of the charge deficit is apparent, and charge compensation is achieved by movement of protons into the sarcoplasmic reticulum (SR) and/or by the movement of organic co- or counterions not measured by energy dispersive electron-probe analysis. There was no significant change in the sodium or chlorine content of the TC during tetanus. The unchanged distribution of a permeant anion, chloride, argues against the existence of a large and sustained transSR potential during tetanus, if the chloride permeability of the in situ SR is as high as suggested by measurements on fractionated SR. The calcium content of the longitudinal SR (LSR) during tetanus did not show the LSR to be a major site of calcium storage and delayed return to the TC. The potassium concentration in the LSR was not significantly different from the adjacent cytoplasmic concentration. Analysis of small areas of I-band and large areas, including several sarcomeres, suggested that chloride is anisotropically distributed, with some of it probably bound to myosin. In contrast, the distribution of potassium in the fiber cytoplasm followed the water distribution. The mitochondrial concentration of calcium was low and did not change significantly during a tetanus. The TC of both tetanized and resting freeze-substituted muscles contained electron-lucent circular areas. The appearance of the TC showed no evidence of major volume changes during tetanus, in agreement with the estimates of unchanged (approximately 72%) water content of the TC obtained with electron-probe analysis.
Fodrin: axonally transported polypeptides associated with the internal periphery of many cells.
Authors: Levine J , Willard M
Published: May 2004

Fodrin (formerly designated 26 and 27) comprises two polypeptides (250,000 and 240,000 mol wt) that are axonally transported at a maximum time-averaged velocity of 40 mm/d--slower than the most rapidly moving axonally transported proteins, but faster than at least three additional groups of proteins. In this communication, we report the intracellular distribution of fodrin. Fodrin was purified from guinea pig brain, and a specific antifodrin antibody was produced in rabbit and used to localize fodrin in tissue sections and cultured cells by means of indirect immunofluorescence. Fodrin antigens were highly concentrated in the cortical cytoplasm of neurons and also nonneuronal tissues (e.g., skeletal muscle, uterus, intestinal epithelium). Their disposition resembles a lining of the cell: hence, the designation fodrin (from Greek fodros, lining). In cultured fibroblasts, immunofluorescently labeled fodrin antigens were arranged in parallel arrays of bands in the plane of the plasma membrane, possibly reflecting an exclusion of labeled fodrin from some areas occupied by stress fibers. The distribution of fodrin antigens in mouse 3T3 cells transformed with simian virus 40 was more diffuse, indicating that the disposition of fodrin is responsive to altered physiological states of the cell. When mixtures of fodrin and F-actin were centrifuged, fodrin cosedimented with the actin, indicating that these proteins interact in vitro. We conclude that fodrin is a specific component of the cortical cytoplasm of many cells and consider the possibilities: (a) that fodrin may be indirectly attached to the plasma membrane via cortical actin filaments; (b) that fodrin may be mobile within the cortical cytoplasm and that, in axons, a cortical lining may be in constant motion relative to the internal cytoplasm; and (c) that fodrin could serve to link other proteins and organelles to a submembrane force-generating system.
Postsynaptic density antigens: preparation and characterization of an antiserum against postsynaptic densities
Authors: Sampedro MN , Bussineau CM , Cotman CW
Published: May 2004

Long-term immunization of rabbits with postsynaptic densities (PSD) from bovine brain produced an antiserum specific for PSD as judged by binding to subcellular fractions and immunohistochemical location at the light and electron microscope levels. (a) The major antigens of bovine PSD preparations were three polypeptides of molecular weight 95,000 (PSD-95), 82,000 (PSD-82), and 72,000 (PSD-72), respectively. Antigen PSD-95, also present in mouse and rat PSDs was virtually absent from cytoplasm, myelin, mitochondria, and microsomes from rodent or bovine brain. Antigens PSD-82 and PSD-72 were present in all subcellular fractions from bovine brain, especially in mitochondria, but were almost absent from rodent brain. The antiserum also contained low-affinity antibodies against tubulin. (b)Immunohistochemical studies were performed in mouse and rat brain, where antigen PSD-95 accounted for 90 percent of the antiserum binding after adsorption with purified brain tubulin. At the light microscope level, antibody binding was observed only in those regions of the brain where synapses are known to be present. No reaction was observed in myelinated tracts, in the neuronal cytoplasm, or in nonneuronal cells. Strong reactivity was observed in the molecular layer of the dentate gyrus, stratum oriens and stratum radiatum of the hippocampus, and the molecular layer of the cerebellum. Experimental lesions, such as ablation of the rat entorhinal cortex or intraventricular injection of kainic acid, which led to a major loss of PSD in well- defined areas of the hippocampal formation, caused a correlative decrease in immunoreactivity in these areas. Abnormal patterns of immunohistochemical staining correlated with abnormal synaptic patterns in the cerebella of reeler and staggerer mouse mutants. (c) At the electron microscopic level, immunoreactivity was detectable only in PSD. The antibody did not bind to myelin, mitochondria or plasma membranes. (d) The results indicate that antigen PSD-95 is located predominantly or exclusively in PSD and can be used as a marker during subcellular fractionation. Other potential uses include the study of synaptogenesis, and the detection of changes in synapse number after experimental perturbations of the nervous system.
Transmembrane linkage between surface glycoproteins and components of the cytoplasm in neutrophil leukocytes.
Authors: Sheterline P , Hopkins C R
Published: May 2004

An experimental approach is described that enables the analysis of interactions between exogenous surface ligands and components of the cytoplasm in neutrophil leukocytes. Neutrophils treated with the nonionic detergent Lubrol PX, under controlled conditions, yield intact detergent-insoluble ghosts. Morphological analysis of neutrophil ghosts shows that they retain the original dimensions of the cell and consist almost entirely of a peripheral filamentous network, representing the submembranous cortical web, concentric to nuclear remnants. All intracellular membrane-bounded organelles, plasma membrane, and background cytoplasmic electron density are absent. Biochemical analysis of the ghosts shows that less than 10% of enzyme markers for the soluble and granule fractions remain, and that greater than 90% of total cell phospholipid is removed during detergent extraction. The major proteins remaining in the ghosts comigrate, on polyacrylamide gels in the presence of SDS, with chicken gizzard actin, myosin, filamin, and a 110-kdalton protein. Patches and caps induced on neutrophils with either fluorescein isothiocyanate-concanavalin A or ferritin-concanavalin A retain their original location and morphology on ghosts after lysis, as determined by both fluorescence and electron microscopy. In similar experiments, but using 125I-labeled lectins, 37% of total cell bound concanavalin A (Con A) and 25% succinylated Con A remain attached to the ghosts. A major 125I-labeled membrane glycoprotein (80 kdaltons) is associated with ghosts prepared from intact neutrophils iodinated in the presence of exogenous lactoperoxidase. Further 125I-labeled membrane glycoproteins (217, 170, and 147 kdaltons) become associated with ghosts prepared from iodinated cells treated before lysis with Con A, but not with succinylated Con A. These data taken together suggest that linkages exist in neutrophils between proteins exposed on the outer surface of the plasma membrane and the peripheral filamentous network independent of the presence of lipid bilayer. The implications of these findings for surface motile phenomena will be discussed.
F-actin aggregates in transformed cells.
Authors: Carley W W , Barak L S , Webb W W
Published: May 2004

Polymerized actin has been found aggregated into distinctive patches inside transformed cells in culture. The F-actin-specific fluorescent probe, nitrobenzoxadiazole-phallacidin, labels these F-actin aggregates near the ventral cell surface of cells transformed by RNA or DNA tumor viruses, or by chemical mutagens, or spontaneously. Their appearance in all eight transformed cell types studied suggests their ubiquity and involvement in transformation morphology. Actin patches developed in normal rat kidney (NRK) cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (LA23-NRK) within 30 min after a shift from the nonpermissive (39 degrees C) to the permissive temperature (32 degrees C). Patch appearance paralleling viral src gene expression tends to implicate pp60src kinase activity in destabilizing the cytoskeleton. However, appearance of the actin aggregates in cells not transformed by retrovirus calls for alternative mechanisms, perhaps involving an endogenous kinase, for this apparently common trait.
Addition of lipid to the photosynthetic membrane: effects on membrane structure and energy transfer.
Authors: Siegel C O , Jordan A E , Miller K R
Published: May 2004

We have carried out a series of experiments in which the lipid composition of the photosynthetic membrane has been altered by the addition of lipid from a defined source under experimental conditions. Liposomes prepared by sonication are mixed with purified photosynthetic membranes obtained from spinach chloroplasts and are taken through cycles of freezing and thawing. Several lines of evidence, including gel electrophoresis and freeze-fracture electron microscopy, indicate that an actual addition of lipid has taken place. Structural analysis by freeze-fracture shows that intramembrane particles are widely separated after the addition of large amounts of lipid, with one exception: large hexagonal lattices of particles appear in some regions of the membrane. These lattices are identical in appearance with lattices formed from a single purified component of the membrane known as chlorophyll-protein complex II. The suggestion that the presence of such lattices in lipid-enriched membranes reflects a profound rearrangement of photosynthetic structures has been confirmed by analysis of the fluorescence emission spectra of natural and lipid-enriched membranes. Specifically, lipid addition in each of the cases we have studied results in the apparent detachment of chlorophyll-protein complex II from photosynthetic reaction centers. It is concluded that specific arrangements of components in the photosynthetic membrane, necessary for the normal functioning of the membrane in the light reaction of photosynthesis, can be regulated to a large extent by the lipid content of the membrane.
Thylakoid membrane biogenesis in Chlamydomonas reinhardtii 137+: cell cycle variations in the synthesis and assembly of polar glycerolipid.
Authors: Janero D R , Barrnett R
Published: May 2004

The synthesis and assembly of thylakoid membrane polar glycerolipid (glycolipid, phospholipid, and ether lipid) have been monitored in synchronous cultures of the green alga Chlamydomonas reinhardtii 137+. A "pulse" protocol using radioactive acetate as the lipogenic precursor was devised to allow assessment of both processes during the 24-h (12-h light/12-h dark) vegetative cell cycle. Under these conditions, acetate incorporation into each chromatographically resolved lipid at the cellular level reliably reflects lipid synthesis, and the appearance of radiolabeled lipid in purified photosynthetic membrane is indicative of the lipid assembly attendant to thylakoid biogenesis. Our results demonstrate that polar glycerolipid is synthesized by the alga and is assembled into its thylakoid membrane continuously, but differentially, with respect to cell cycle time. Synthesis and assembly are most rapid during the photoperiod (mid-to-late G1), reach maximum rates at mid-photoperiod, and are comparatively negligible in the dark (S, M, and early-to-mid G1). The extent to which synthesis and assembly vary within this general kinetic pattern, though, is characteristic of each thylakoid lipid, suggesting that the processes take place in a multistep manner with some temporal coordination among the different lipid types. Parallelism between the cyclic patterns of polar lipid synthesis at the cellular level and of polar lipid assembly into photosynthetic membrane at the subcellular level indicates that lipid production is not only essential to continuing thylakoid biogenesis but is also the critical determinant of the kinetics of thylakoid lipid assembly.
Influence of collagen gel on the orientation of epithelial cell polarity: follicle formation from isolated thyroid cells and from preformed monolayers.
Authors: Chambard M , Gabrion J , Mauchamp J
Published: May 2004

The influence of collagen gels on the orientation of the polarity of epithelial thyroid cells in culture was studied under four different conditions. (a) Isolated cells cultured on the surface of a collagen gel formed a monolayer. The apical pole was in contact with the culture medium and the basal membrane was attached to the substratum. (b) Isolated cells embedded inside the gel organized within 8 into follicles. The basal pole was in contact with collagen and the apical pole was oriented towards the interior of the follicular lumen. (c) Cells were first organized into floating vesicles, structures in which the apical surface is in contact with the culture medium, and the vesicles were embedded inside the collagen gel. After 3 d, cell polarity was inverted, the apical pole being oriented towards the cavity encompassed by cells. Vesicles had been transformed into follicles. (d) Monolayers formed on collagen gels as in a were overlaid with a second layer of collagen, which was polymerized in contact with the apical cell surface. A disorganization of the continuous pavement occurred within 24 h; cells attached to the upper layer of collagen and reorganized into follicles in the collagen sandwich within 4-8 d. A similar process occurred when the monolayer was grown on plastic and overlaid with collagen, or grown on collagen and covered with small pieces of glass cover slips. No reorganization was observed between two glass surfaces. In conclusion, first, a basal pole was always formed in the area of contact between the cell membrane and an adhesive surface and, second, the interaction of a preformed apical pole with an adhesive surface was not compatible with the stability of this domain of the plasma membrane. The interaction of the cell membrane with extracellular components having adhesive properties appears to be a determinant factor in the orientation and stabilization of epithelial cell polarity.
Two Drosophila melanogaster proteins related to intermediate filament proteins of vertebrate cells.
Authors: Falkner F G , Saumweber H , Biessmann H
Published: May 2004

Monoclonal antibodies were prepared against a 46,000 mol wt major cytoplasmic protein from Drosophila melanogaster Kc cells. These antibodies reacted with the 46,000 and a 40,000 mol wt protein from Kc cells. Some antibodies showed cross-reaction with 55,000 (vimentin) and 52,000 mol wt (desmin) proteins from baby hamster kidney (BHK) cells that form intermediate sized filaments in vertebrate cells. In indirect immunofluorescence, the group of cross reacting antibodies stained a filamentous meshwork in the cytoplasm of vertebrate cells. In Kc cells the fluorescence seemed to be localized in a filamentous meshwork that became more obvious after the cells had flattened out on a surface. These cytoskeletal structures are heat-labile; the proteins in Kc or BHK cells rearrange after a brief heat shock, forming juxtanuclear cap structures.
Degradation of proteins microinjected into IMR-90 human diploid fibroblasts.
Authors: Neff N T , Bourret L , Miao P , Dice J F
Published: May 2004

Erythrocyte ghosts loaded with 125I-labeled proteins were fused with confluent monolayers of IMR-90 fibroblasts using polyethylene glycol. Erythrocyte-mediated microinjection of 125I-proteins did not seriously perturb the metabolism of the recipient fibroblasts as assessed by measurements of rates of protein synthesis, rates of protein degradation, or rates of cellular growth after addition of fresh serum. A mixture of cytosolic proteins was degraded after microinjection according to expected characteristics established for catabolism of endogenous cytosolic proteins. Furthermore, withdrawal of serum, insulin, fibroblast growth factor, and dexamethasone from the culture medium increased the degradative rates of microinjected cytosolic proteins, and catabolism of long-lived proteins was preferentially enhanced with little or no effect on degradation of short-lived proteins. Six specific polypeptides were degraded after microinjection with markedly different half-lives ranging from 20 to 320 h. Degradative rates of certain purified proteins (but not others) were also increased in the absence of serum, insulin, fibroblast growth factor, and dexamethasone. The results suggest that erythrocyte-mediated microinjection is a valid approach for analysis of intracellular protein degradation. However, one potential limitation is that some microinjected proteins are structurally altered by the procedures required for labeling proteins to high specific radioactivities. Of the four purified proteins examined in this regard, only ribonuclease A consistently showed unaltered enzymatic activity and unaltered susceptibility to proteolytic attack in vitro after iodination.
cis-Unsaturated fatty acids induce the fusion of chromaffin granules aggregated by synexin.
Authors: Creutz C E
Published: May 2004

When isolated chromaffin granules were aggregated by synexin (a Ca2+-binding protein present in chromaffin and other secretory tissues) and then exposed to cis-unsaturated fatty acids at 37 degrees C, they fused together to form large vesicles. The fusion was monitored by phase and electron microscopy and by turbidity measurements on the granule suspension. Arachidonic acid was the most effective fusogen, whereas trans-unsaturated fatty acids, saturated fatty acids, detergents or lysolecithin were inactive. During fusion some of the epinephrine of the granules was released but the soluble core proteins remained trapped in the resulting vesicles. These vesicles swelled to enclose the maximum volume. Although this swelling could be inhibited by increasing the osmotic strength of the medium, it did not appear to depend on the chemiosmotic properties of the granule membranes as it was not influenced by ATP, a proton ionophore, or an anion transport inhibitor. The regulators of this in vitro fusion--Ca2+, synexin, and free, cis-unsaturated fatty acids--may be present in the cytoplasm of the chromaffin cell when it is stimulated to release epinephrine and granule proteins by exocytosis. Therefore, this fusion event may be the same that occurs between chromaffin granules undergoing compound exocytosis.
Appearance of functional insulin receptors during the differentiation of embryonal carcinoma cells.
Authors: Heath J , Bell S , Rees A R
Published: May 2004

The insulin-receptor binding activity and insulin-stimulated growth response of PC13 clone 5 cells were investigated for both the embryo carcinoma (EC) and retinoic acid-induced differentiated derivatives of this cell line. Whereas the EC cell was found to have very few, if any, receptors and showed no demonstrable dependence on insulin for growth, the differentiated derivative cell expressed a large number of insulin receptors and, when challenged with the hormone, showed stimulation of both DNA synthesis and cell division. The same data were obtained for five independent PC13 clones. These results, coupled with previous observations, lend weight to the suggestion that the appearance of specific receptors for growth regulatory substances may be a manifestation of a general change in growth-regulatory mechanisms accompanying EC cell differentiation and loss of malignancy.
Cytoskeletal organization at the postsynaptic complex.
Authors: Gulley R L , Reese T S
Published: May 2004

Postsynaptic densities and the adjacent cytoskeleton were examined in deep-etched, unfixed slices of guinea pig anteroventral cochlear nucleus. The postsynaptic density seen in conventional thin sections corresponds to a meshwork of 4-nm filaments associated with intramembrane particles at the postsynaptic active zone of inhibitory as well as excitatory synapses. These filaments intermesh with a lattice of 8- to 9-nm microfilaments, tentatively identified as F-actin, that is concentrated under the postsynaptic density. We postulate that the meshwork of 4-nm filaments anchors receptors to the adjacent microfilament lattice; this extended postsynaptic complex may limit the mobility of receptors and help maintain the curvature of the postsynaptic membrane.
Protein tightly bound near the termini of the Physarum extrachromosomal rDNA palindrome.
Authors: Cheung M K , Drivas D T , Littau V C , Johnson E M
Published: May 2004

The genes coding for ribosomal RNa in plasmodia of Physarum polycephalum are arranged palindromically on extrachromosomal rDNA molecules of 61 kb (kilobasepairs). Incubation of mildly extracted rDNA with the 125I Bolton-Hunter reagent results in incorporation of label not removed by SDS, CsCl, or various organic solvents. Labeled protein is preferentially associated with terminal rDNA restriction fragments, as detected after gel electrophoresis of the DNA. Antibody reaction with dinitrophenylated protein-rDNA complexes allows visualization of protein located from 1 to 2 kb from the termini, in a region containing multiple inverted repeat sequences and single-strand gaps. DNase I treatment of either rDNA or rDNA termini releases primarily two labeled protein bands of 5,000 and 13,000 daltons as well as less prominent bands of higher molecular weight. We discuss mechanisms for involvement of terminal protein in replication of 3' ends and chromosomal integration of the rDNA.
Interaction of the retinol/cellular retinol-binding protein complex with isolated nuclei and nuclear components.
Authors: Liau G , Ong D E , Chytil F
Published: May 2004

Retinol (vitamin A alcohol) is involved in the proper differentiation of epithelia. The mechanism of this involvement is unknown. We have previously reported that purified cellular retinol-binding (CRBP) will mediate specific binding of retinol to nuclei isolated from rat liver. We now report that pure CRBP delivers retinol to the specific nuclear binding sites without itself remaining bound. Triton X-100-treated nuclei retain the majority of these binding sites. CRBP is also capable of delivering retinol specifically to isolated chromatin with no apparent loss of binding sites, as compared to whole nuclei. CRBP again does not remain bound after transferring retinol to the chromatin binding sites. When isolated nuclei are incubated with [3H]retinol-CRBP, sectioned, and autoradiographed, specifically bound retinol is found distributed throughout the nuclei. Thus, CRBP delivers retinol to the interior of the nucleus, to specific binding sites which are primarily, if not solely, on the chromatin. The binding of retinol to these sites may affect gene expression.
Kinetochore structure, duplication, and distribution in mammalian cells: analysis by human autoantibodies from scleroderma patients.
Authors: Brenner S , Pepper D , Berns M W , Tan E , Brinkley B R
Published: May 2004

The specificity of the staining of CREST scleroderma patient serum was investigated by immunofluorescence and immunoelectron microscopy. The serum was found to stain the centromere region of mitotic chromosomes in many mammalian cell types by immunofluorescence. It also localized discrete spots in interphase nuclei which we have termed "presumptive kinetochores." The number of presumptive kinetochores per cell corresponds to the chromosome number in the cell lines observed. Use of the immunoperoxidase technique to localize the antisera on PtK2 cells at the electron microscopic level revealed the specificity of the sera for the trilaminar kinetochore disks on metaphase and anaphase chromosomes. Presumptive kinetochores in the interphase nuclei were also visible in the electron microscope as randomly arranged, darkly stained spheres averaging 0.22 micrometers in diameter. Preabsorption of the antisera was attended using microtubule protein, purified tubulin, actin, and microtubule-associated proteins. None of these proteins diminished the immunofluorescence staining of the sera, indicating that the antibody-specific antigen(s) is a previously unrecognized component of the kinetochore region. In some interphase cells observed by both immunofluorescence and immunoelectron microscopy, the presumptive kinetochores appeared as double rather than single spots. Analysis of results obtained using a microspectrophotometer to quantify DNA in individual cells double stained with scleroderma serum and the DNA fluorescent dye, propidium iodide, led to the conclusion that the presumptive kinetochores duplicate in G2 of the cell cycle.
Isolation of a glycopeptide fraction from the surface of the sea urchin egg that inhibits sperm-egg binding and fertilization.
Authors: Kinsey W H , Lennarz W J
Published: May 2004

The role of cell surface glycoproteins of the sea urchin egg in binding sperm has been examined by studying the biological activity of glycopeptides derived from these glycoproteins. Glycopeptides were produced from egg surface glycoproteins by Pronase digestion. After fractionation by gel filtration the glycopeptides were tested for their ability to inhibit the binding of sperm to eggs, presumably by competing with the egg surface glycoproteins for binding sites on the sperm. One glycopeptide fraction with an apparent molecular weight of approximately 6,000 was found to be a potent inhibitor of sperm-egg binding, as well as fertilization, even at nanomolar concentrations. This activity was heat stable and exerted its effect against the sperm and not the egg. Experiments with a radiolabeled form of the glycopeptide fraction directly demonstrated that at least one component of it bound to sperm. Specific binding of the radiolabeled glycopeptide occurred only to acrosome-reacted sperm. Because the isolated glycopeptide fraction has many of the characteristics that one would expect of a biologically active fragment of an egg surface receptor for sperm, these findings are consistent with the idea that one or more glycoconjugates on the surface of the egg are involved in sperm binding.
Cerebroside sulfotransferase: preparation of antibody and localization of antigen in kidney.
Authors: Tennekoon G I , Frangia J , Aitchison S , Price D L
Published: May 2004

This immunohistochemical study describes the localization of the enzyme cerebroside sulfotransferase (phosphoadenosine phosphosulfate: galactosylceramide sulfotransferase, EC 2.8.2.11) in rat kidney. The enzyme was purified from kidney and the preparation was used to raise antibodies for immunocytochemical investigations. In the kidney, the antigen was present only on the brush border of the epithelial cells of the proximal tubules, suggesting that sulfation of glycolipids occurs in the cytoplasm and plasma membranes of these specific cells. Moreover, biochemical and immunocytochemical studies of cerebroside sulfotransferase during development indicate that catalytic activity is correlated with the appearance of enzyme protein.
Identification of minor components of coated vesicles by use of permeation chromatography.
Authors: Pfeffer S R , Kelly R B
Published: May 2004

Coated vesicles are thought to be vehicles for the intracellular transport of membranes. Clathrin is the major protein component of coated vesicles. Minor components of these organelles can be identified in highly purified preparations if they can be shown to copurify with clathrin. To show copurification we have made use of the relatively uniform diameter of coated vesicles (50-150 nm) to fractionate conventionally purified coated vesicles according to size in glass bead columns of 200-nm pore size. We have found that bovine brain coated vesicles prepared by the standard procedure of Pearse can be contaminated with large membrane fragments that are removed by permeation chromatography on such glass bead columns. Gel electrophoretic analysis of column fractions shows that only three major polypeptide chains, and a family of polypeptides with molecular weights close to 100,000 are always in constant ratio to clathrin, and are unique to fractions containing coated vesicles. Two other major polypeptides that appear to be components of coated vesicles are also present in other membrane fractions. We have also used permeation chromatography to monitor artifactual membrane trapping during vesicle isolation. Pure radiolabeled synaptic vesicle membranes were added to bovine brain tissue before homogenization. Considerable amounts of the added radioactivity could be recovered in the fractions conventionally pooled in the preparation of coated vesicles. After permeation chromatography, the radioactivity in the coated vesicle peak was reduced essentially to background.
Status of mitochondria in living human fibroblasts during growth and senescence in vitro: use of the laser dye rhodamine 123.
Authors: Goldstein S , Korczack L B
Published: May 2004

Rhodamine 123, a fluorescent laser dye that is selectively taken up into mitochondria of living cells, was used to examine mitochondrial morphology in early-passage (young), late-passage (old), and progeric human fibroblasts. Mitochondria were readily visualized in all cell types during growth (mid-log) and confluent stages. In all cell strains at confluence, mitochondria became shorter, more randomly aligned, and developed a higher proportion of bead-like forms. Treatment of cells for six days with Tevenel, a chloramphenicol analog that inhibits mitochondrial protein synthesis, brought about a marked depletion of mitochondria and a diffuse background fluorescence. Cyanide produced a rapid release of preloaded mitochondrial fluorescence followed by detachment and killing of cells. Colcemid caused a random coiling and fragmentation of mitochondria particularly in the confluent stage. No gross differences were discernible in mitochondria of the three cell strains in mid-log and confluent states or after these treatments. Butanol-extractable fluorescence after loading with rhodamine 123 was lower in all cell strains in confluent compared to mid-log stages. At confluence all three cell strains had similar rhodamine contents at zero-time and after washout up to 24 h. At the mid-log stage, young cells contained more rhodamine initially and lost it more rapidly than old or progeria cells, in that order. The data indicate no gross derangement in the morphology or number of mitochondria in old and progeria fibroblasts but there is a reduction of protonmotive force evident in these cells at the mid-log stage that may be growth limiting.
Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells.
Authors: Hirokawa N , Heuser J E
Published: May 2004

The cytoskeleton that supports microvilli in intestinal epithelial cells was visualized by the quick-freeze, deep-etch, rotary-replication technique (Heuser and Salpeter. 1979. J. Cell Biol. 82: 150). Before quick freezing, cells were exposed to detergents or broken open physically to clear away the granular material in their cytoplasm that would otherwise obscure the view. After such extraction, cells still displayed a characteristic organization of cytoskeletal filaments in their interiors. Platinum replicas of these cytoskeletons had sufficient resolution to allow us to identify the filament types present, and to determine their characteristic patterns of interaction. The most important new finding was that the apical "terminal web" in these cells, which supports the microvilli via their core bundles of actin filaments, does not itself contain very much actin but instead is comprised largely of narrow strands that interconnect adjacent actin bundles with one another and with the underlying base of intermediate filaments. These strands are slightly thinner than actin, do not display actin's 53A periodicity, and do not decorate with myosin subfragment S1. On the contrary, two lines of evidence suggested that these strands, could include myosin molecules. First, other investigators have shown that myosin is present in the terminal web (Mooseker et al. 1978. J. Cell Biol. 79: 444-453), yet we could find no thick filaments in this area. Second, we found that the strands were removed completely in the process of decorating the core filament bundles with the myosin subfragment S1, suggesting that they had been competitively displaced by exogenous myosin. We conclude that myosin may play a structural role in these cells, via its cross-linking distribution, in addition to whatever role it plays in microvillar motility.
Mechanics of chromosome separation during mitosis in Fusarium (Fungi imperfecti): new evidence from ultrastructural and laser microbeam experiments.
Authors: Aist J R , Berns M W
Published: May 2004

The anaphase-telophase spindle usually elongates, and it has been assumed that the spindle pushes the incipient daughter nuclei apart. To test this assumption, we used a laser microbeam to sever the central spindle of the fungus, Fusarium solani, and measured the rate of separation of incipient daughter nuclei. When the microbeam was aimed beside the spindle separation occurred at a rate (8.6 micrometer/min) that did not differ significantly from the rate (7.6 micrometer/m) in unirradiated cells. But when the spindle was irradiated, it broke, and the separation was much faster (22.4 micrometer/min). Irradiation of cytoplasm lateral to one spindle pole resulted in a 1.5 micrometer/min reduction in the rate (6.1 micrometer/min) of separation. From these and other data, we infer that extranuclear forces, presumably involving astral microtubules, pull on the incipient daughter nuclei and that the central spindle limits the separation rate. Astral microtubules are associated with the plasma membrane or, sometimes, with the rough endoplasmic reticulum. Most of the spindle microtubules that are present at metaphase are depolymerized during anaphase and early telophase.
Comparative studies on microinjected high-mobility-group chromosomal proteins, HMG1 and HMG2.
Authors: Wu L , Rechsteiner M , Kuehl L
Published: May 2004

The nonhistone chromosomal proteins, HMG1 and HMG2, were iodinated and introduced into HeLa cells, bovine fibroblasts, or mouse 3T3 cells by erythrocyte-mediated microinjection. Autoradiographic analysis of injected cells fixed with glutaraldehyde consistently showed both molecules concentrated within nuclei. Fixation with methanol, on the other hand, resulted in some leakage of the microinjected proteins from the nuclei so that more autoradiographic grains appeared over the cytoplasm or outside the cells. Both injected and endogenous HMG1 and HMG2 partitioned unexpectedly upon fractionation of bovine fibroblasts, HeLa, or 3T3 cells, appearing in the cytoplasmic fractions. However, in calf thymus, HMG1 and HMG2 molecules appeared in the 0.35 M NaCl extract of isolated nuclei, as expected. These observations show that the binding of HMG1 and HMG2 to chromatin differs among cell types or that other tissue-specific components can influence their binding. Coinjection of [125I]HMG1 and [131I]HMG2 into HeLa cells revealed that the two molecules display virtually equivalent distributions upon cell fractionation, identical stability, identical intracellular distributions, and equal rates of equilibration between nuclei. In addition, HMG1 and HMG2 did not differ in their partitioning upon fractionation nor in their stability in growing vs. nongrowing 3T3 cells. Thus, we have not detected any significant differences in the intracellular behavior of HMG1 and HMG2 after microinjection into human, bovine, or murine cells.
Intercellular communication in normal and regenerating rat liver: a quantitative analysis.
Authors: Meyer D J , Yancey S B , Revel J P
Published: May 2004

We have compared intercellular communication in the regenerating and normal livers of weanling rats. The electrophysiological studies were conducted at the edge of the liver, and we have found that here as elsewhere in the liver there is a dramatic decrease in the number and size of gap junctions during regeneration. The area of hepatocyte membrane occupied by gap junctions is reduced 100-fold 29-35 h after hepatectomy. By combining observations made with the scanning electron microscope with our freeze fracture data we have estimated the number of "communicating interfaces" (areas of contact between hepatocytes that include at least one gap junction) formed by hepatocytes in normal and regenerating liver. In normal liver a hepatocyte forms gap junctions with every hepatocyte it contacts (approximately 6). In regenerating liver a hepatocyte forms detectable gap junctions with, on average, only one other hepatocyte. Intercellular spread of fluorescent dye and electric current is reduced in regenerating as compared with normal liver. The incidence of electric coupling is reduced from 100% of hepatocyte pairs tested in control liver to 92% in regenerating liver. Analysis of the spatial dependence of electronic potentials indicates a substantial increase in intercellular resistance in regenerating liver. A quantitative comparison of our morphological and physiological data is complicated by tortuous pattern of current flow and by inhomogeneities in the liver during regeneration. Nevertheless we believe that our results are consistent with the hypothesis that gap junctions are aggregates of channels between cell interiors.
Motion of particles adhering to the leading lamella of crawling cells.
Authors: Dembo M , Harris A K
Published: May 2004

Time-lapsed films of particle motion on the leading lamella of chick heart fibroblasts and mouse peritoneal macrophages were analyzed. The particles were composed of powdered glass or powdered aminated polystyrene and were 0.5-1.0 micrometer in radius. Particle motions were described by steps in position from one frame to the time-lapse movies to the next. The statistics of the step-size distribution of the particles were consistent with a particle in Brownian motion subject to a constant force. From the Brownian movement, we have calculated the two-dimensional diffusion coefficient of different particles. These vary by more than an order of magnitude (10(-11)-10(-10) cm2/s) even for particles composed of the same material and located very close to each other on the surface of the cell. This variation was not correlated with particle size but is interpretable as a result of different numbers of adhesive bonds holding the particles to the cells. The constant component of particle movement can be interpreted as a result of a constant force acting on each particle (0.1-1.0 x 10(-8) dyn). Variations in the fractional coefficient for particles close to each other on the cell surface do not yield corresponding differences in velocity, suggesting that the frictional coefficient and the driving force vary together. This is consistent with the hypothesis that the particles are carried by flow of the membrane as a whole or by flow of some submembrane material. The utility of our methods for monitoring cell motile behavior in biologically interesting situations, such as a chemotactic gradient, is discussed.
Translocation of proteins across the endoplasmic reticulum III. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes.
Authors: Walter P , Blobel G
Published: May 2004

The previously observed (Walter, et al. 1981 J. Cell Biol. 91:545-550) inhibitory effect of SRP selectively on the cell-free translation of mRNA for secretory protein (preprolactin) was shown here to be caused by a signal sequence-induced and site-specific arrest in polypeptide chain elongation. The Mr of the SRP-arrested nascent preprolactin chain was estimated to be 8,000 corresponding to approximately 70 amino acid residues. Because the signal sequence of preprolactin comprises 30 residues and because approximately 40 residues of the nascent chain are buried (protected from protease) in the large ribosomal subunit, we conclude that it is the interaction of SRP with the amino-terminal signal peptide of the nascent chain (emerged from the large ribosomal subunit) that modulates translation and thereby causes an arrest in chain elongation. This arrest is released upon SRP-mediated binding of the elongation-arrested ribosomes to the microsomal membrane, resulting in chain completion and translocation into the microsomal vesicle.
Direct measurement of intracellular pH changes in Xenopus eggs at fertilization and cleavage.
Authors: Webb D J , Nuccitelli R
Published: May 2004

We have used Thomas-type recessed-tip pH-sensitive microelectrodes to measure the intracellular pH (pHi) in Xenopus eggs during both fertilization and ionophore activation. The average pHi in unfertilized eggs is 7.33 +/- 0.11 (SD; n = 21) with a resting membrane potential of -10.1 +/- 3.5 (SD; n = 38) mV. Within 2 min after the onset of the fertilization potential, there is a slight, transient pHi decrease of 0.03 +/- (SD, n = 8), followed by a distinct, permanent pHi increase of 0.31 +/- 0.11 (SD; n = 7) beginning approximately 10 min after the start of the fertilization potential and becoming complete approximately 1 h later. The pHi remains near this level of 7.67 +/- 0.13 (SD, n = 10) through at least 10 cleavage cycles, but it is possible to discern pHi oscillations with a mean amplitude of 0.03 +/- 0.02 (SD, n = 38). Eggs perfused for at least 2 h in Na+-free solution with 1 mM amiloride exhibited all of these pHi changes, so these changes do not require extracellular Na+. Similar cytoplasmic alkalinizations that accompany the activation of metabolism and the cell cycle in a wide variety of cell types are discussed.
Role of osmotic forces in exocytosis: studies of ADH-induced fusion in toad urinary bladder.
Authors: Kachadorian W A , Muller J , Finkelstein A
Published: May 2004

Antidiuretic hormone (ADH) treatment of toad urinary bladder activates an exocytotic-like process by which intramembrane particle aggregates are transferred from membranes of elongated cytoplasmic tubules to the luminal-facing plasma membrane. We find that the number of these ADH-induced fusion events, and the number of aggregates appearing in the luminal membrane, are reduced when the luminal bathing medium is made hyperosmotic. As an apparent consequence of the inhibition of their fusion with the luminal membrane, the elongated cytoplasmic tubules become enormously swollen into large, rounded vesicles. These results are consistent with the view that osmotic forces are essential to the basic mechanism of exocytosis.
Distribution of microtubule organizing centers in migrating sheets of endothelial cells.
Authors: Gotlieb A I , May L M , Subrahmanyan L , Kalnins V I
Published: May 2004

This study was designed to investigate the relationship between the position of the microtubule organizing center (MTOC) and the direction of migration of a sheet of endothelial cells (EC). Using immunofluorescence and phase microscopy the MTOC's of migrating EC were visualized as the cells moved into an in vitro experimental wound produced by mechanical denudation of part of a confluent monolayer culture. Although the MTOC's in nonmigrating EC were randomly positioned in relation to the nucleus, in migrating cells the position of the MTOC's changed so that 80% of the cells had the MTOC positioned in front of the nucleus toward the direction of movement of the endothelial sheet. This repositioning of the MTOC occurred within the first 4 h after wounding and was associated with the beginning of migration of EC's into the wounded area as seen by time-lapse cinemicrophotography. These studies focus attention on the MTOC as a cytoskeletal structure that may play a role in determining the direction of cell movement.
Fibronectin-independent adhesion of fibroblasts to the extracellular matrix: mediation by a high molecular weight membrane glycoprotein.
Authors: Harper P A , Juliano R L
Published: May 2004

Fibroblastic CHO cells readily adhere to fibronectin (Fn) coated substrata. From the parental cell population we have recently selected a series of adhesion variants (ADV cells) that cannot adhere to Fn substrata (Harper and Juliano. 1980. J. Cell. Biol. 87:755-763). However, ADV cells readily adhere to substrata coated with extracellular matrix material (ECM) derived from human diploid fibroblasts by a mechanism that does not involve fibronectin (Harper and Juliano. 1981. Nature (Lond.). 290:136-138). Te Fn-dependent adhesion mechanism of parental cells (type 1 adhesion) and the ECM-dependent adhesion of ADV cells (type II adhesion) can also be discriminated on the basis of their differential sensitivity to proteolysis, with the type II mechanism being far more sensitive. In this communication we report that parental CHO cells possess both type I and type II mechanisms whereas ADV cells possess only the type II mechanism. We also identify a high molecular weight membrane glycoprotein (gp 265) that seems to play a role in type II adhesion. This component is detected by [125I]lactoperoxidase of [3H]borohydride-galactose oxidase labeling of surface proteins in WT and AD cells. Cleavage of gp 265 with low doses of proteases correlates completely with the loss of type II adhesion capacity. Thus CHO cells possess two functionally and biochemically distinct adhesion mechanisms, one involving exogenous Fn and the other mediated by the membrane component gp 265.
Fibronectin molecule visualized in electron microscopy: a long, thin, flexible strand.
Authors: Erickson H P , Carrell N , McDonagh J
Published: May 2004

We have determined the structure of plasma fibronectin by electron microscopy of shadowed specimens. the 440,000 molecular weight, dimeric molecule appears to be a long, thin, highly flexible strand. The contour length of the most extended molecules is 160 nm, but a distribution of lengths down to 120 nm was observed, indicating flexibility in extension as well as in bending. The average diameter of the strand is 2 nm and there are no large globular domains. the large fragments produced by limited digestion with plasmin are not globular domains but are segments of the strand, whose length corresponds to the molecular weight of the polypeptide chain. We conclude that each polypeptide chain of the dimeric molecule spans half the length of the strand, with their carboxyl termini joined at the center of the strand and their amino termini at the ends. This model is supported by images of fibronectin-fibrinogen complexes, in which the fibrinogen is always attached to an end of the fibronectin strand.