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Biomedical subjects

R W Rubin

Publications and source records attributed to R W Rubin.

At least 19 recordsLinked to original sources
PubMed1992

Cardiostimulatory and antiarrhythmic activity of tubulin-binding agents.

Rhythmic, spontaneously pulsating cardiac cells cultured from newborn rats are immediately stimulated to beat faster by addition of a number of tubulin-binding agents but not by their non-tubulin-binding analogues. The tubulin-binding agents tested include vinblastine, vincristine, navelbine, two analogs of vinblastine (S12362 and S12363), nocodazole, colchicine, and podophylotoxin. In addition to binding tubulin, all of the above agents also depolymerize microtubules. In contrast, taxol, a tubulin-binding agent that stabilizes microtubules, does not stimulate cardiac cells. Moreover, the immediate and ensuing cardiac stimulation by vinblastine at 0.05 microgram/ml is completely blocked by pre- and cotreatment with taxol at 1.0 microgram/ml. The time necessary to reverse the cardiostimulatory effect of vinblastine is significantly longer than that required for nocodazole, further implicating depolymerization of microtubules in the cardiac activity of these agents. All of the tubulin-binding agents tested (including taxol) also immediately reverse adriamycin-induced arrhythmias. By using a monoclonal antibody to alpha-tubulin, typical filamentous microtubules are visualized in cardiac muscle and cocultured non-muscle cells by immunofluorescence. When cells are treated for 2 hr with vinblastine at 0.05 microgram/ml, fluorescence is detected in cross-striated patterns in cardiac muscle cells. Overall, these data open the possibility of uncovering an additional relationship between cytoskeletal elements (other than actin and myosin) and the contractility of cardiac muscle. They also suggest an alternative mechanism for affecting cardiac cell function in vitro (namely, by tubulin-binding agents). If these agents are shown to be cardioactive in vivo, they may provide another approach to the treatment and management of cardiac arrhythmias.

Alkaloids↗
PubMed1986

Effects of colchicine on cardiac cell function indicate possible role for membrane surface tubulin.

The effects of the tubulin-binding drug colchicine on cultured neonate cardiac cell function were investigated. Application of low doses of colchicine (but not lumicolchicine) caused an early reversible increase in beating rate with a concomitant decrease in amplitude. Treatment of the cells with trypsin at a dose that removes surface tubulin but does not inhibit spontaneous beating, diminished the colchicine effect. Surface radio-iodination of the live cultures followed by two-dimensional gel electrophoresis and radioautography revealed that two spots were heavily labeled. These spots co-migrated with purified brain tubulin. Fibroblasts derived from the cardiac cultures did not label over the tubulin spots. Trypsin treatment removed the presumptive tubulin from the radioautographs but only removed the most basic portion of the alpha-tubulin spot from the stained gel. These results are consistent with a surface membrane role for an iso-form of tubulin in neonate cardiac cells.

Animals↗
PubMed1985

Cell surface tubulin in leukemic cells: molecular structure, surface binding, turnover, cell cycle expression, and origin.

We report here new characteristics of cell surface tubulin from a human leukemia cell line. These cells (CEM cells) possess tubulin that is readily iodinated on the surface of living cells, turns over at a rate identical to that of other surface proteins, and is present throughout the cell cycle. When removed with trypsin, it rapidly returns to the surface. Peptide mapping of iodinated surface tubulin indicates that it possesses a similar, but not identical, primary structure to total CEM and rat brain tubulin. Living CEM cells are able to bind specifically a subfraction of CEM tubulin from metabolically labeled high speed supernatants of lysed CEM cells. Surface tubulin is more basic than the total tubulin pool. The binding, which is saturable, is inhibited by unlabeled CEM high speed supernatants but not by excess thrice-cycled rat or bovine brain tubulin. Surface tubulin is also shown to bind to living nontransformed normal rat kidney cells but not to normal, circulating, mononuclear white cells. Activated lymphocytes produce a tubulin that binds to CEM cells. Since CEM tubulin was detected in the media of 6-h cultures of CEM cells, we must conclude that at least some of the surface tubulin comes from the media. We further conclude that these leukemic cells produce an unusual tubulin that may bind specifically to any membrane. The presence of iodinatable surface tubulin, however, appears to require both the production of a unique tubulin and the presence of a "receptor-like" surface binding component.

Animals↗
PubMed1984

Regional variations in myosin heavy chain concentration after healing of experimental myocardial infarction in cats.

Densitometric scanning of SDS-polyacrylamide gels was used to measure myosin heavy chain concentration in left ventricular specimens obtained from cat hearts 3 to 12 months after healing of small experimental myocardial infarctions. The study was designed to test the hypothesis that myosin concentration varies as a function of anatomic proximity to the infarct scar. Myosin heavy chain concentration was elevated in non-scarred areas adjacent to a healed infarct and normal in areas remote from the scar. The scar itself had reduced concentrations, reflecting the loss of muscle mass in this area. The increased myosin heavy chain concentration in regions adjacent to the scar may be an attempt to regulate or compensate for the decrease in mechanical function of the scarred area.

Animals↗
PubMed1984

Comparison of the protein content of three different bovine secretory granule membrane types: a search for exocytosis-specific shared proteins.

A two-dimensional polyacrylamide gel analysis of three types of bovine exocytotic granule membranes has been undertaken. Great care was taken to purify the membranes of biochemical homogeneity with minimal contamination from other membrane sources. The goal was to identify proteins that were present in all three membrane types. Although a number of minor components were observed that co-migrated for two membrane types, no proteins were detected that were present in all three granule membranes. We therefore conclude that such exocytosis-specific proteins do not exist or that they represent less than 0.1% of the total membrane protein present in a given isolated membrane preparation.

Animals↗
PubMed1983

Subpicogram analysis of sweat proteins using two-dimensional polyacrylamide gel electrophoresis.

Sweat collected from six normal volunteers was analyzed to determine if reproducible protein patterns could be obtained using two-dimensional polyacrylamide gel electrophoresis of 125I-labeled sweat proteins. This method has the capability of easily detecting picogram quantities of protein. Once the methods of collection of the sweat had been standardized, reproducible patterns were obtained from these volunteers. Over 100 discrete spots were revealed by a combination of fluorography and rare earth screen radioautography of dried two-dimensional gels. This method will allow analysis of sweat for qualitative and quantitative variations in protein content in pathologic conditions such as cystic fibrosis, renal failure, and diabetes.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Identification of a cytoskeleton-associated glycoprotein from isolated microvilli of a mammary ascites tumor.

Microvilli isolated from MAT-C1 13762 ascites tumor cells were extracted with Triton X-100 in phosphate-buffered saline (PBS) to yield cytoskeletal residues. Analysis of the residues by two-dimensional isoelectric focusing-dodecyl sulfate electrophoresis and silver staining suggested that one of the major components is a glycoprotein (CAG). Neuraminidase treatments and glucosamine labeling demonstrated that CAG is a glycoprotein, and lactoperoxidase iodination showed its presence at the microvillar surface. DNase treatments and myosin affinity analysis suggested an association between CAG and the microvillar microfilaments. Thus, CAG has the properties expected of a transmembrane-linking molecule connecting the cell surface to the cytoskeleton.

Animals↗
PubMed1982

Subcellular fractions of the adrenal medulla. Comparison by two-dimensional polyacrylamide gel electrophoresis.

Subfractions of adrenal medullary homogenates were analyzed in two-dimensional polyacrylamide gels to assess the extent of protein homology. Chromaffin granule proteins were highly acidic, with the exception of the soluble form of the enzyme dopamine beta-hydroxylase (EC 1.14.17.1). The purified granule membrane proteins were more heterogeneous, but still predominantly acidic. The soluble and membrane forms of dopamine beta-hydroxylase behaved identically in this gel system. Lactoperoxidase-catalyzed iodination of intact granules revealed that most, but not all, granule membrane proteins are accessible at the cytoplasmic face. Prominent proteins of the purified adrenal medullary mitochondria showed little if any homology with purified granule membranes. The crude microsome fraction showed significant homology with purified granule membranes despite low levels of cross-contamination between the two fractions in marker enzyme analysis. Among proteins that could be identified, dopamine beta-hydroxylase was at a low level in the microsomes, while the granule membrane protein cytochrome b-561 appeared to be in both fractions. The pattern obtained from primary cultures of adrenal chromaffin cells was very complex, but prominent proteins from the subcellular fractions were seen without difficulty. Actin and tubulin were very prominent in the whole cell pattern. Radioiodination of the whole cells resulted in a number of spots being labelled, although the majority of the label appeared to be in only two proteins of molecular weight 70000 and isoelectric point 5.7.

Adrenal Medulla↗
PubMed1982

Lack of tropomyosin correlates with the absence of stress fibers in transformed rat kidney cells.

We have utilized epithelial rat kidney cells and their Kirsten viral transformant (442) to examine the role of actin-binding proteins in cellular morphogenesis. Normal rat kidney cells are well spread while the transformed cells are more spherical, poorly adherent, and lack actin stress fibers (Rubin, R.W., Warren, R.H., Lukeman, D.S. and Clements, E. (1978) J. Cell Biol. 78, 28-35). By immunofluorescence, antitropomyosin prominently stains normal rat kidney cell stress fibers while only a weak, nonspecific fluorescence is observed in 442 cells. Using two-dimensional gel electrophoresis, tropomyosin can be detected in normal rat kidney cells homogenates. The tropomyosin subunits are enriched in Triton-extracted filamentous normal rat kidney cell models, and in extracts of normal rat kidney cell homogenate produced by using a rapid myosin affinity technique to isolate actin and actin-associated proteins. The identity of the tropomyosin subunits has been confirmed by electrophoretic mobility, lack of proline, and the peptide map generated by limited proteolysis. None of these techniques have detected tropomyosin in the corresponding 442 preparations. Our results suggest that the transformation of normal rat kidney cells has led to an overall reduction in tropomyosin content. This may be related to the inability of 442 cells to organize filamentous actin stress fibers.

Actins↗
PubMed1982

Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium.

The significance of intracellular Na+ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K+ for extracellular Na+, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secretory granule numbers appear to be qualitatively reduced after carbachol, multiple carbachol, or Ba2+ stimulation, overall granule distribution remains similar to that in untreated cells. Monensin-stimulated catecholamine release requires extracellular Na+ but not Ca2+ whereas carbachol-stimulated catecholamine release requires extracellular Ca2+ and is partially dependent on extracellular Na+. Despite its high selectivity for monovalent ions, monensin is considerably more effective in promoting catecholamine secretion than the divalent ionophores, A23187 and ionomycin, which mediate a more direct entry of extracellular Ca2+ into the cell. We propose that the monensin-stimulated increase in intracellular Na+ levels causes an increase in the availability of intracellular Ca2+ which, in turn, stimulates exocytosis. This hypothesis is supported by the comparable stimulation of catecholamine release by ouabain which inhibits the outwardly directed Na+ pump and thus permits intracellular Na+ to accumulate. The relative magnitudes of the secretion elicited by monensin, carbachol, and the calcium ionophores, are most consistent with the hypothesis that, under normal physiological conditions, Na+ acts by decreasing the propensity of Ca2+-sequestering sites to bind the Ca2+ that enters the cell as a result of acetylcholine stimulation.

Animals↗
PubMed1982

Tubulin as a major cell surface protein in human lymphoid cells of leukemic origin.

Surface-exposed proteins of vinblastine-sensitive human lymphoid cell line of leukemic origin (CCRF-CEM) were examined by the lactoperoxidase-catalyzed iodination and two-dimensional polyacrylamide gel electrophoresis methods. Spots which comigrate with bovine brain tubulin and rabbit muscle actin were prominently labeled in the whole membrane but not in the high-speed supernatant fraction of the disrupted cells. Mild trypsinization of labeled cells removed the iodinated tubulin and actin without significantly affecting the protein staining pattern. Iodination of normal human lymphocytes resulted in no labeling of the tubulin or actin. The presence of surface-exposed tubulin in this leukemic cell line suggests a possible mechanism for their enhanced sensitivity to the cytotoxic action of vinblastine.

Cell Line↗
PubMed1982

Two-dimensional gel electrophoresis of membrane proteins from the R3327 prostate adenocarcinoma.

The Dunning rat prostate adenocarcinoma (R3327) is a reliable model that shares many similarities with the human tumor. Two sublines of the tumor, G and H, represent opposite extremes in histology and growth rate. Purified membrane fractions from G and H solid tumors were isolated by sucrose gradient. Tumor and normal prostate membrane proteins were labeled with 125I, incubated with G and H antisera, and precipitated by adsorption of antibody-antigen complexes to staphylococcal Protein A. Proteins were resolubilized and electrophoresed on two-dimensional gels, and the gels were autoradiographed. A total of eight labeled proteins were precipitated from the G and H tumors in the presence of G antisera. Of these, seven were homologous. One high-molecular-weight protein (Protein b) present on the G tumor was absent from the H tumor. The H tumor contained another high-molecular-weight protein (i) that was not found on the G tumor or on normal prostate. Normal prostate revealed a pattern similar to the G tumor except that Protein b appeared to be quantitatively reduced. Precipitation in the presence of H antisera showed similar patterns except that Protein b was not detected in the G tumor and was greatly reduced in the normal prostate. Therefore, despite variable growth characteristics, there were few changes in membrane proteins between the solid tumors and between the tumors and normal prostate. Iodination of surface proteins of cultured cells from normal prostate and the G and H sublines also showed a high degree of homology. No consistent differences between cultured cell lines were noted.

Adenocarcinoma↗