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L Glaser

Publications and source records attributed to L Glaser.

At least 55 records · Page 3Linked to original sources
PubMed1984

Expression of acetylcholine receptor alpha-subunit mRNA during differentiation of the BC3H1 muscle cell line.

The accumulation of translatable acetylcholine receptor alpha-subunit mRNA was examined in the BC3H1 muscle cell line in response to serum and cell growth. Relative amounts of alpha-subunit mRNA were quantitated during differentiation by cell-free translation and immunoprecipitation with an alpha-subunit-specific monoclonal antibody. Logarithmically growing cells do not possess cell surface acetylcholine receptors; however, a significant amount of alpha-subunit mRNA is detectable in cells under these conditions. Furthermore, alpha-subunit is synthesized in growing undifferentiated cells at a rate similar to that of differentiated cultures. Following growth arrest of BC3H1 cells, surface receptors are induced to levels greater than 100-fold above that of growing cells. The relative level of translatable alpha-subunit mRNA in differentiated cells, however, is only approximately 4-fold greater than in growing cultures. Induction of alpha-subunit mRNA appears to be reversible since reinitiation of growth in quiescent differentiated BC3H1 cells results in a reduction in relative abundance of this mRNA species to levels comparable to that of undifferentiated cells and the concomitant loss of surface receptors. These results indicate that receptor expression during differentiation is regulated both post-translationally and at the level of receptor subunit mRNA accumulation.

Animals↗
PubMed1984

Vanadate stimulates Na+/H+ exchange activity in A431 cells.

Recent studies have established that polypeptide growth factors cause an elevation of the cytoplasmic pH (pHi) in cultured mammalian cells by stimulating Na+/H+ exchange. We show that vanadate, previously found to act as a mitogen for a number of cells, reversibly activates Na+/H+ exchange at micromolar concentrations in A431 cells, leading to a large increase of pHi. The stimulation of Na+/H+ exchange by vanadate is not due to inhibition of the Na+/K+ ATPase and is unrelated to possible effects of vanadate on cAMP levels. Elevation of pHi by vanadate and by epidermal growth factor (EGF) both display similar kinetics, and both EGF and vanadate stimulate the rate of pHi recovery following an acute acid load, suggesting that vanadate stimulates Na+/H+ exchange by a mechanism similar to that of polypeptide growth factor stimulation. Thus, stimulation of Na+/H+ exchange may be a common property not only of polypeptide growth factors but also of other, chemically unrelated mitogens.

Carcinoma, Squamous Cell↗
PubMed1984

Identification of novel neural- and neural retina-specific antigens with a monoclonal antibody.

A fluorescence-activated cell sorter screening method has been used to identify hybridomas that secrete monoclonal antibodies that can bind to viable subpopulations of embryonic chicken neural retina cells. One monoclonal antibody, C1H3, recognizes two nervous tissue-specific polypeptides that exhibit distinct developmental patterns. The monoclonal antibody reacts with a 140-kilodalton (kDa) polypeptide that is present at early stages of development (day 7) but is detected by immunoblotting in only negligible amounts at later times (day 17). In contrast, a 170-kDa polypeptide is first detectable by immunoblotting at day 10 and is the predominant C1H3 antigen at day 17. Analysis of proteolytic fragments of the two proteins indicates that the polypeptides are distinct molecules that share a common antigenetic determinant. Both polypeptides are neural-specific; the 140-kDa polypeptide appears to be retina-specific, while the 170-kDa polypeptide is also present in other areas of the nervous system. Metabolic labeling of retina cells in situ at early embryonic stages reveals only the synthesis of the 140-kDa polypeptide. When such cells are dissociated and labeled in vitro, they synthesize primarily the 170-kDa polypeptide. Thus, the differential rate of synthesis of these two polypeptides is controlled by environmental factors that possibly include cell-cell contacts or an unknown systemic factor. The 140-kDa polypeptide is a unique marker for early neural retina cells.

Animals↗
PubMed1984

PC12 cells as a source of neurite-derived cell surface mitogen, which stimulates Schwann cell division.

Primary cultures of rat dorsal root ganglion Schwann cells were used to assay the efficacy of PC12 cells in stimulating Schwann cell proliferation. Co-cultures of PC12 cells and Schwann cells assayed by [3H]thymidine labeling followed by autoradiography showed proliferation of Schwann cells only where contact occurred between PC12 neurites and Schwann cells. Membranes derived from PC12 cells were shown to have many characteristics similar to membranes derived from sensory neurons; both could mimic whole cells in stimulating Schwann cell division; both were inactivated by mild heat treatment and by trypsinization, and both elevated intracellular cyclic AMP concentrations in Schwann cells 16 h after addition of membranes. We conclude that PC12 cells will be a valuable source for the isolation of the neuronal cell surface component which controls proliferation of Schwann cells during development of the peripheral nervous system.

Animals↗
PubMed1984

Tumor promoter phorbol 12-myristate 13-acetate inhibits mitogen-stimulated Na+/H+ exchange in human epidermoid carcinoma A431 cells.

Addition of polypeptide growth factors to cultured cells results in a rapid stimulation of Na+/H+ exchange, which leads to cytoplasmic alkalinization. We studied the effects of the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) on the Na+/H+ exchange system of A431 cells. Stimulation of Na+/H+ exchange by epidermal growth factor (EGF) and serum as well as by vanadate ions is strongly inhibited after treatment of cells with nanomolar concentrations of PMA. Phorbol esters that have no activity as tumor promoters also do not modulate the activation of Na+/H+ exchange. By contrast, the stimulation of Na+/H+ exchange that is produced upon exposure of cells to hypertonic solution is only slightly inhibited by PMA treatment, indicating that PMA treatment does not directly block the activity of the Na+/H+ antiporter. Furthermore, incubation of cells with PMA causes a weak stimulation of Na+/H+ exchange, although this effect is mostly observed at relatively high PMA concentrations and appears to require external Ca2+. The inhibition BY PMA of EGF-promoted Na+/H+ exchange is not due to inhibition of EGF-binding to the EGF receptor. Since PMA activates protein kinase C, our observations are consistent with the hypothesis that protein kinase C functions to attenuate the stimulation of Na+/H+ exchange by polypeptide growth factors.

Carcinoma, Squamous Cell↗
PubMed1984

Cell-substratum adhesion in embryonic chick central nervous system is mediated by a 170,000-mol-wt neural-specific polypeptide.

Embryonal chick neural retina cells release into the culture medium a complex of proteins and glycosaminoglycans, termed adherons, that promote cell to substratum adhesion. A monoclonal antibody (C1H3) blocks adheron-mediated cell to substratum adhesion and specifically binds to a 170,000-mol-wt protein present in retinal adherons (Cole, G.J., and L. Glaser, 1984, J. Biol. Chem., 259:4031-4034). The 170,000-mol-wt protein also can be identified in embryonic chick brain and peripheral nervous tissue. In the neural retina, C1H3 also binds to a second antigen with a molecular weight of 140,000 that is absent in the brain. Embryonic brain, therefore, provides a source for the immunopurification of the 170,000-mol-wt protein. Brain adherons also contain the 170,000-mol-wt protein, and cell to substratum adhesion mediated by these adherons is blocked by the C1H3 monoclonal antibody. The 170,000-mol-wt protein in the brain is therefore functionally identical to that in the retina. To demonstrate that adheron-mediated cell to substratum adhesion is caused by cell binding to the 170,000-mol-wt protein, we showed that (a) protease digestion, but not glycosaminoglycan hydrolase digestion of adherons, blocked their ability to bind cells to substratum; (b) the immunopurified 170,000-mol-wt protein blocks adheron-mediated cell to substratum adhesion; and (c) cells can bind to immunopurified 170,000-mol-wt protein bound to glass surfaces.

Animals↗
PubMed1983

Regulation of surface expression of acetylcholine receptors in response to serum and cell growth in the BC3H1 muscle cell line.

The regulation of acetylcholine receptor synthesis, assembly, and cell surface expression in response to serum and cell growth in the BC3H1 muscle-like cell line have been examined. In the presence of media containing low concentrations of serum, BC3H1 cells withdraw from the cell cycle and express high levels of cell surface acetylcholine receptors. Exposure of quiescent, differentiated BC3H1 cells to high concentrations of serum results in re-entry of cells into the cell cycle and a loss of surface acetylcholine receptors by a first order process (t1/2 = 8.6 h) which appears to be initiated within 1 h following serum addition. Under these conditions, the rate of synthesis of acetylcholine receptor alpha-subunits is not significantly reduced; however, the efficiency of assembly of alpha-subunits into functional acetylcholine receptors is significantly less than that observed in quiescent cells. The receptors which are assembled following serum stimulation of quiescent cells are not transported to the cell surface, nor do they accumulate within the cells in a functional form (defined by the ability to bind alpha-bungarotoxin) in the cells. We conclude that reinitiation of cell division in quiescent cultures of BC3H1 cells results in alterations in several post-translational processing steps required for the surface expression of acetylcholine receptors.

Animals↗
PubMed1983

Activation of Na+/H+ exchange by epidermal growth factor elevates intracellular pH in A431 cells.

Epidermal growth factor (EGF) increases Na+ uptake in several cell types through an electroneutral, amiloride-sensitive pathway putatively identified as Na+/H+ countertransport. We have previously shown (Rothenberg, P., Glaser, L., Schlesinger, P., and Cassel, D. (1983) J. Biol. Chem. 257, 4883-4889) that EGF rapidly activates amiloride-sensitive net Na+ influx in the A431 human epidermoid carcinoma cell line. We also described the presence of transmembrane, amiloride-sensitive Na+/H+ exchange in A431 cells using a new fluorescence technique for the measurement of intracellular pH (pHi) based on the incorporation of fluorescein-dextran into the cell cytoplasm. The low pKa of fluorescein (approximately 6.4) prevented the direct assessment of the inferred, EGF-induced cytoplasmic alkalinization, mediated by stimulated Na+/H+ exchange. In this paper, 4',5'-dimethylfluorescein (pKa 6.75) was coupled to dextran, allowing increased pH sensitivity of the fluorescence assay in the physiological range. Using this improved assay, basic features of pHi regulation in A431 cells are documented, including the role of Na+/H+ exchange and Na+-linked C1-/HCO3-exchange in acid extrusion. We directly demonstrate a rapid elevation of pHi by addition of EGF as well as by serum in A431 cells. The pHi increase is half-maximal at 5-10 ng/ml of EGF, is dependent on external Na+, independent of external Ca2+, and inhibited by millimolar amiloride. EGF and serum also enhance Na+/H+ exchange-mediated cytosolic acidification when the transmembrane Na+ concentration gradient favors Na+ efflux from the cells. An alkaline pHi shift, caused by activation of Na+/H+ exchange, may be an important primary event in the mechanism of EGF action.

Amiloride↗
PubMed1983

Epidermal growth factor stimulates amiloride-sensitive 22Na+ uptake in A431 cells. Evidence for Na+/H+ exchange.

Epidermal growth factor (EGF) increases Na+ uptake in several cell types through an electroneutral, amiloride-sensitive pathway putatively identified as Na+/H+ countertransport. The inferred cytosolic alkalinization resulting from this process has been proposed to be an important component of mitogenic stimulation. We studied the effect of EGF on the Na+/H+ exchange system of A431 cells, a cell line having a very high EGF receptor density but which is not mitogenically stimulated by EGF. We demonstrate that EGF rapidly activates net Na+ influx in A431 cells. Amiloride inhibits the EGF-dependent Na+ uptake (65% inhibition at 3 mM, ID50 approximately 0.3 mM) and inhibits much less the EGF-independent uptake. EGF is known to enhance 45Ca+ accumulation in A431 cells (Sawyer, S. T., and Cohen, S. (1981) Biochemistry 20, 6280-6286). The following findings indicate that EGF-dependent 22Na+ and 45Ca2+ uptake are two independent processes. 1) EGF effectively stimulates an amiloride-sensitive 22Na+ uptake in the absence of external Ca2+. 2) EGF-dependent 45Ca2+ uptake is not inhibited by amiloride. A new fluorescence technique is described for intracellular pH determination based on the introduction of fluorescein-labeled dextran into the cell cytoplasm. Using this method, the presence of amiloride-sensitive Na+/H+ exchange in A431 cells is documented. Although the lack of pH sensitivity of fluorescein fluorescence above pH 7.3-7.4 prevents a direct assessment of an EGF-induced increase of intracellular pH, the combined results of 22Na+ flux and intracellular pH measurements suggest that EGF activates Na+/H+ exchange in A431 cells. We conclude that enhanced Na+/H+ exchange may not necessarily be coupled to mitogenic triggering in different cell types, although the stimulation of Na+/H+ exchange may constitute a primary event in the mechanism of EGF action.

Amiloride↗
PubMed1983

Regulation of creatine phosphokinase expression during differentiation of BC3H1 cells.

The intracellular mechanisms involved in the regulation of creatine phosphokinase expression in the BC3H1 muscle-like cell line have been examined under conditions of enzyme induction and repression. In the presence of low serum concentrations, BC3H1 cells cease to grow and synthesize high levels of creatine phosphokinase. When differentiated BC3H1 cultures are exposed to media containing high serum concentrations, cell division is reinitiated and further induction of creatine phosphokinase is inhibited. Accumulation of creatine phosphokinase-mRNA appears to be intimately coupled to the state of growth of BC3H1 cells. Log phase cells do not contain detectable levels of translatable creatine phosphokinase-mRNA; however, following cessation of growth, creatine phosphokinase-mRNA accumulates in approximate proportion to the increase in creatine phosphokinase activity. Reinitiation of cell division in quiescent differentiated cultures results in the arrest of further accumulation of creatine phosphokinase-mRNA but does not inhibit the translation of pre-existing creatine phosphokinase-mRNA. Under conditions of enzyme repression, however, the newly synthesized creatine phosphokinase appears to be enzymatically inactive. These results indicate that the expression of the muscle phenotype in BC3H1 cells is regulated by components present in serum and that myogenic differentiation is at least partially reversible following re-entry of quiescent cells into the cell cycle.

Animals↗
PubMed1983

Visuospatial analysis of the printed word.

Aphasic individuals often lose the ability to analyze written information phonetically because of left hemisphere damage experienced through cerebrovascular accident (CVA) or head trauma. In this study, aphasic and normal adults demonstrated use of a right hemisphere visuospatial strategy to analyze printed whole works and word parts such as prefixes and suffixes. The performances of the two groups were similar, suggesting that the hypothesized strategy could be useful as a reading approach for aphasics.

Adult↗
PubMed1983

Identification of sub-populations of chick neural retinal cells by monoclonal antibodies: a fluorescence activated cell sorter screening technique.

An assay was developed which identifies monoclonal antibodies recognizing the cell surfaces of sub-populations of chick retinal cells which survive in culture. Antibodies from hybridoma culture supernatants were bound to monolayers of retina cells followed by a fluorescent secondary antibody. The quantitative fluorescence analysis ability of the fluorescence-activated cell sorter (FACS) was used to determine the fluorescence intensity associated with viable single cells and the frequency with which these cells appear in the total population. Hybridomas were generated which define both overlapping and non-overlapping retina cell populations. The cytotoxic activity of many of the monoclonal antibodies was determined on the FACS by propidium iodide exclusion, and the identity of various sub-populations was demonstrated by immunohistochemical staining of retina sections.

Animals↗
PubMed1983

Monoclonal antibodies against a differentiated retinal cell population.

Two monoclonal antibodies have been isolated which bind preferentially to the plexiform layers of embryonic chick neural retina and to 50-60% of dissociated neural retinal cells in culture as determined by surface binding to cells followed by analysis in a fluorescent activated cell shorter. Each antibody appears to recognize a distinct antigenic determinant on a common cell surface antigen, a protein of approximately 230 kdalton. This antigen increases dramatically in concentration between embryonic days 7 and 11 concomitant with the elaboration of the retinal plexiform layers. The antigen appears first in the central portion of the neural retina and at later times in the periphery, an appearance consistent with the normal pattern of differentiation of the retina.

Animals↗
PubMed1983

Platelet-derived growth factor stimulates Na+/H+ exchange and induces cytoplasmic alkalinization in NR6 cells.

Stimulation of Na+/H+ exchange by growth factors has been implicated as a mechanism allowing quiescent cells to resume growth because of a predicted elevation of intracellular pH(pHi). We tested this prediction in NR6 cells by using a further development of our technique for pHi measurement, based on introduction of the fluorescent pH indicator 4',5'-dimethylfluorescein (pKa = 6.75) coupled to dextran into the cytoplasm. Addition of the potent mitogens platelet-derived growth factor (PDGF) or serum to NR6 cells stimulates an amiloride-sensitive 22Na+ uptake and causes an elevation of pHi. The PDGF-dependent pHi increase follows a lag period of approximately equal to 2 min, reaches a maximal level within 10 min (delta pHi approximately equal to 0.1 at an external pH of 7.18), and remains at this level for at least 1 hr. Serum addition initially produces a large elevation of pHi, which later declines to a level similar to that obtained with PDGF. The effects of PDGF and serum are partially additive (delta pHi approximately equal to 0.14). The magnitude of pHi elevation by PDGF decreases with increasing extracellular pH. Serum- and PDGF-dependent elevations of pHi are inhibited by amiloride and by eliminating Na+ from the medium. Under conditions in which Na+/H+ exchange is inhibited, PDGF and serum induce an initial cytoplasmic acidification that does not show a lag period. The results show that a single purified growth factor, as well as serum, can promote a sustained elevation of pHi by stimulating Na+/H+ exchange. The extent of pHi elevation may be modulated by the concomitant stimulation by the growth factor of a process generating H+ within the cell.

Amiloride↗
PubMed1982

Proteolytic cleavage of epidermal growth factor receptor. A Ca2+-dependent, sulfhydryl-sensitive proteolytic system in A431 cells.

The Mr = 160,000 epidermal growth factor (EGF) receptor in A431 cells is partially cleaved during membrane isolation to a Mr = 145,000 polypeptide containing both EGF binding and phosphate acceptor sites. We show that the proteolytic degradation of the EGF receptor depends upon the presence of Ca2+ in the medium used to scrape the cells from the substratum. Only the high molecular weight form of the receptor is detected in membranes prepared in the absence of Ca2+. Ca2+-dependent proteolysis occurs rapidly (t1/2 approximately 5 min) following cell scraping. Proteolysis results in a decrease in EGF-dependent phosphorylation of the receptor while retaining EGF binding capacity. In addition, membranes containing the uncleaved form of the receptor reveal a substantial increase in EGF-dependent phosphorylation of proteins with Mr approximately 80, 89, and 185 X 10(3). In the presence of Ca2+, addition of iodoacetic acid to the scraping medium strongly inhibits receptor fragmentation, whereas other inhibitors (phenylmethylsulfonyl fluoride, leupeptin, and pepstatin) have no effect. The results implicate a role for a Ca2+-dependent, SH-sensitive protease in EGF receptor degradation. Prevention of proteolysis yields membrane preparations with highly active EGF-dependent kinase system.

Calcium↗