Search PubMed⌕ Search

Biomedical subjects

R W Rubin

Publications and source records attributed to R W Rubin.

At least 37 records · Page 2Linked to original sources
PubMed1980

Organizational differences in the membrane proteins of normal and irreversibly sickled erythrocytes.

Using two-dimensional gels, no unique membrane proteins were detected in irreversibly sickled cells. Membranes from irreversibly sickled cells were shown to cross-link much more readily with dithiobis(succinimidyl propionate) than normal erythrocyte membranes. Increased binding of band 4.5 protein and increased intra-chain disulfides were also demonstrated. These changes may correlate to enhanced cellular rigidity.

Anemia, Sickle Cell↗
PubMed1979

A biochemical and ultrastructural comparison of Triton X-100 models of normal and transformed cells.

We have compared the two-dimensional gel profiles of Triton models of normal rat kidney (NRK) cells and their Kirsten viral transformant, 442. Several protein differences were detected. The models of the transformed line lacked five acidic polypeptides and possessed a much higher intermediate filament to actin ratio. Scanning microscopy reveals significant ultrastructural differences in these models, with the NRK line exhibiting a much more filamentous structure. In addition, nuclease treatment of NRK models causes a dramatic change in their scanning image while the 442 models are unaffected. Nuclease treated models lack microfilaments and appear to contain only intermediate filaments, although actin is still a prominent protein constituent.

Actins↗
PubMed1979

Organization of tubulin in normal and transformed rat kidney cells.

We have carried out a quantitative biochemical and ultrastructural study of tubulin and microtubules in a normal rat kidney (NRK) cell line and its viral transformant (442) in culture. Under equivalent culture conditions, both cell lines contain the same amount of tubulin according to a colchicine-binding assay. The normal and transformed cells differ significantly, however, with respect to the state of organization of their tubulin. Counts of microtubules in sectioned cells indicate that NRK cells have almost twice as many microtubules per unit area of cytoplasm as the 442 cells. Centrifugation studies, on the other hand, show that 442 cells have almost twice as much pelletable tubulin as the NRK cells. We propose, therefore, that the transformed cells contain a large amount of tubulin which is in some alternative aggregate form that is not morphologically detectable as microtubles in the cytoplasm

Animals↗
PubMed1978

Over two hundred polypeptides resolved from the human erythrocyte membrane.

A modification of O'Farrell's method of two-dimensional polyacrylamide gel electrophoresis has allowed for the resolution of erythrocyte membranes showing up to 200 individual components. Data is presented which indicates that this protein heterogeneity is not produced by artifactual protein-protein aggregation, ednogenous protease activity of secondary charge modification. Similar patterns are obtained when the samples are added to the unpolymerized isoelectric focusing gel, and isolated and stored in protease inhibitor. Individual spots could be eluted off of stained gels, resolubilized under extreme detergent solubilization conditions and run on one-dimensional gels; these run as sodium dodecyl sulfate in the solubilization procedure. The method chosen for solubilization prior to isoelectric focusing appears to cause selective aggregation of all or most of the spectrin and band 3 proteins. This further allows for excellent resolution of more components.

Electrophoresis, Polyacrylamide Gel↗
PubMed1978

Microtubules and actin in giant nerve fibers of the spiny lobster, Panulirus argus.

Giant axons of the spiny lobster, Panulirus argus, are filled with microtubules that are decorated with fine, irregular filaments. Mitochondria and membrane-limited clear vesicles are the only other distinguishable elements in the axoplasm and are located around the periphery of the axon near the axolemma. Neither 100 A neurofilaments nor 70 A microfilaments are evident in fixed, intact axons or in negatively stained axoplasm. Actin-like microfilaments are a prominent constituent of the glial cells that closely ensheathe the axons, and gel electrophoresis studies suggest that most of the actin in the nerve fibers is located in the glia rather than in the axons. Studies of isolated axoplasm indicate that microtubules are the primary elements stabilizing the axoplasm. The microtubules in the isolated axoplasm are disrupted by Ca2+ in the medium in the presence of protease inhibitors.

Actins↗
PubMed1978

Differences between nucleus and cytoplasm in the degree of actin polymerization.

For purposes of studying the degree of polymerization of actin in nuclei, nuclei from 35S-labeled amoebas (Amoeba proteus) were transplanted into unlabeled cells, which were immediately lysed and extracted under conditions considered to stabilize preexisting fibrous actin. The enucleated 35S-donor cells were similarly treated for analysis of cytoplasmic actin. The extraction conditions permitted separation of soluble (unpolymerized or G) actin from pelletable (polymerized or F) actin, and the radioactivity of each was determined after the actin was separated from other proteins by polyacrylamide gel electrophoresis. We found that about 2/3 of the actin within the nucleus is pelletable, whereas only about 1/3 of the cytoplasmic actin is pelletable. We speculate that polymerized actin in the nucleus is involved in the condensation of chromatin.

Actins↗
PubMed1978

Actin content and organization in normal and transformed cells in culture.

The amount of actin and total protein per cell in normal rat kidney (NRK) cells in culture is initially high in very low density cultures, but rapidly decreases as the cells come into contact in higher density cultures. In a viral transformant of NRK (442), the level of actin and total protein does not change significantly from low to high density cultures. NRK cells, which are flattened against the substrate, have prominent bundles of actinlike microfilaments in the basal cytoplasm adjacent to the substrate. 442 cells, which adhere poorly and are more spherical in shape, lack well-organized basal microfilament bundles, but may display microfilament bundles in cytoplasmic processes extending from the cell body. The percentage of insoluble actin is less than 20% in both cell lines, and 442 cells consistently contain smaller amounts than NRK cells.

Actins↗
PubMed1978

Effects of pronase and concanavalin A upon the freeze-etch morphology of cell membranes of intact human erythrocytes.

Freeze-etch studies reveal that mild pronase treatment with subsequent incubation in concanavalin A induces aggregation of intramembranous particles (IMP) in intact human erythrocytes. This alteration in particle distribution is accompanied by a change in the distribution of the Con A molecules such that they also become clustered on the extracellular etch face. If divalent succinyl Con A is used after pronase instead of tetravalent Con A the IMPs still become clustered. Pronase only, Con A only, or succinyl Con A only does not cause the IMPs to become aggregated. Most surprising is the finding that pronase followed by Con A causes partial haemolysis of the cells whereas pronase only, Con A only, or pronase+succinyl Con A do not cause this haemoglobin loss. These perturbations of the erythrocyte plasma membrane appear to be a result of the pronase+Con A exerting a transmembrane effect on the spectrin. This conclusion is supported by sodium dodecylsulphate polyacrylamide gel electrophoresis of material crosslinked with dimethyl adipimidate dihydrochloride, which indicates that spectrin is more susceptible to being cross-linked after pronase+Con A; i.e. the spectrin is probably aggregated by the enzyme and lectin incubation.

Cell Aggregation↗
PubMed1977

Structural and biochemical aspects of cell motility in amebas of Dictyostelium discoideum.

Amebas of Dictyostelium discoideum contain both microfilaments and microtubules. Microfilaments, found primarily in a cortical filament network, aggregate into bundles when glycerinated cells contract in response to Mg-ATP. These cortical filaments bind heavy meromyosin. Microtubules are sparse in amebas before aggregation. Colchicine, griseofulvin, or cold treatments do not affect cell motility or cell shape. Saltatory movement of cytoplasmic particles is inhibited by these treatments and the particles subsequently accumulate in the posterior of the cell. Cell motility rate changes as Dicytostelium amebas go through different stages of the life cycle. Quantitation of cellular actin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the quantity of cellular actin changes during the life cycle. These changes in actin are directly correlated with changes in motility rate. Addition of cyclic AMP to Dictyostelium cultures at the end of the feeding stage prevents a decline in motility rate during the preaggregation stage. Cyclic AMP also modifies the change in actin content of the cells during preaggregation.

Actins↗
PubMed1976

Changes in glycerol solubility and amino acid incorporation of a protein possessing the characteristics of tubulin during first cleavage in the sea urchin embryo.

A method is described for the quantitation of the pool size of a tubulin like protein during synchronized cell division in the sea urchin Lytechinus variegatus. The method involves the use of a thin SDS slab polyacrylamide gel system in which tubulin can be quantitated in the submicrogram range. Employing a microtubule stabilization buffer, the intact tubule fraction was removed and the soluble tubulin pool was quantitated with this gel system. Amino acid incorporation into this protein was also quantitated. The resulting specific activity values and values for the amount of tubulin-like protein present in the pool fraction suggested that the tubulin pool decreases at fertilization and then remains constant through the first cell cycle. Tubulin synthesis, however, steadily increased after fertilization and then decreased dramatically just prior to mitotic apparatus formation. No change in tubulin pool size was observed at the time of peak mitotic appartus formation. These results are discussed in terms of the regulation of microtubule function.

Animals↗
PubMed1976

Isolation and electrophoretic analysis of nucleoli, phenol-soluble nuclear proteins, and outer cyst walls from Acanthamoeba castellanii during encystation initiation.

A technique is described for isolating nuceoli from Acanthamoeba castellanii. Nuclei isolated by a modification of the technique of F. J. Chlapowski and R. N. Band (1971) are sonicated in a surcrose-Tris-MgSO4-KC1-Triton X-100 buffer and centrifuged on a linear sucrose gradient extending from 1.3 M to 1.5 M with a 2.6 M cushion, at 41000 rpm for 90 min. The only apparent contaminants in the nucleolar preparation are outer cyst walls. A procedure is described for the isolation of chemically pure outer cyst walls, and a comparison of the proteins with the nucleolar preparation reveals that outer cyst walls represent negligible contaminants. The ultrastructure of these isolated nucleoli examined with transmission electron microscopy is found to be identical with that of nucleoli from whole cells, fixed in an identical manner. The 50 nucleolar proteins separated by SDS gel electrophoresis have been examined throughout the growth cycle of Acanthamoeba and into the strat of induced encystment, at which time 10 protein bands disappear, 11 bands are observed to decrease, and 8 are seen to increase in concentration. Phenol-soluble proteins are extracted from the nucleolus which correspond to 29 of the 50 nucleolar proteins, with 17 of these proteins corresponding to nucleolar proteins that change at the onset of encystment. Thes nucleolar proteins are also compared with those of rat liver nucleoli by gel electrophoresis, resulting in the observation that extremely few protein homologies exist between the two. Numerous quantitative and qualitative changes in the gel pattern of phenol-soluble nuclear proteins during early and late log phase growth and the onset of stationary phase were also observed.

Amoeba↗
PubMed1975

Direct biochemical measurements of microtubule assembly and disassembly in Chinese hamster ovary cells. The effect of intercellular contact, cold, D2O, and N6,O2'-dibutyryl cyclic adenosine monophosphate.

A study was undertaken to develop a means of quantitating the amount of tubulin present as a soluble pool and as intact microtubules in cultured Chinese hamster ovary cells. A procedure was developed in which these cells grown on monolayer culture in Petri dishes were placed in a "microtubule stabilizing medium" (MTM) consisting of 50% glycerol, 10% dimethylsulfoxide and sodium phosphate magnesium buffer, as described previously by Filner and Behnke. These cells then were homogenized and the homogenate was spun in the ultracentrifuge. Colchicine binding activity was then determined in the supernates and the pellets. The values, when compared with total colchicine binding activity present in replicate homogenates, were used to determine the percentage of tubulin present as intact microtubules. A statistical analysis of thin sections of cells treated with MTM revealed no statistically significant difference between MTM-treated cells and untreated controls. It was further discovered that the relative amount of colchicine binding activity recovered in the high speed pellet varied dramatically, depending upon the cell number of the culture being studied. Preconfluent cultures showed very low colchicine binding activity averaging less than 5%, while confluent and postconfluent cultures often possessed as high as 25% of their total colchicine binding activity in pelletable material. Although cold and D2O treatment had little or no effect on these values, N6,O2'-dibutyryl cyclic adenosine monophosphate increased them. It is hoped that this study will serve as the basis for a reliable quantitative procedure for measuring microtubule polymerization and depolymerization in vivo.

Animals↗
PubMed1975

Biochemical characterization of crystals from the dermal iridophores of a chameleon Anolis carolinensis.

The biochemical characteristics of dermal iridophore crystals from Anolis carolinensis have been investigated. Iridophores isolated by collangenase-hyaluronidase treatment were sonicated and their contents fractionated through sucrose. Pure iridophore crystals so obtained were examined by chromatography and electron diffraction. They were found to be pure hydrated crystalline form. The suggestion is made that the subcrystalline structure of this guanine does not play a role in color production by the iridophore.

Animals↗
PubMed1974

Cyclic changes in the cell surface. I. Change in thymidine transport and its inhibition by cytochalasin B in Chinese hamster ovary cells.

Cytochalasin B (CB) shows a marked concentration-dependent inhibition of the incorporation of [(3)H]thymidine into Chinese hamster ovary cells. This inhibition was shown to result from an inhibition of thymidine uptake, not from an inhibition of DNA synthesis. Cells normally acquire the capacity to transport thymidine as they move from the G1 stage of the cell cycle into the S phase. If CB is added to cells while they are in G1, they do not acquire the ability to transport thymidine as they enter S. However, the addition of CB to cells that are already in S has no effect on their ability to transport thymidine. These results are discussed in terms of a model in which elements involved in thymidine transport enter the cell surface membrane as the cells move from G1 to S. It is proposed that CB prevents this structural transition by binding to the cell surface.

Animals↗