277 research outputs found
Nerve growth factor inhibits the synthesis of a single-stranded DNA binding protein in pheochromocytoma cells (clone PC12)
No full textArrest of mitosis and neurite outgrowth induced by nerve growth factor (NGF) in rat pheochromocytoma cells (clone PC12) is accompanied by a progressive inhibition of the synthesis of a protein that binds to single-stranded but not to double-stranded DNA. Time course experiments show that this inhibition is already apparent after a 2-day incubation with NGF and is maximum (85-95%) upon achievement of complete PC12 cell differentiation. Inhibition of the synthesis of this single-stranded DNA binding protein after 48 hr of incubation with NGF is potentiated by concomitant treatment of PC12 cells with antimitotic drugs acting at different levels of DNA replication. Purification on a preparative scale of this protein and analysis of its major physicochemical properties show that: (i) it constitutes 0.5% of total soluble proteins of naive PC12 cells; (ii) its molecular weight measured by NaDodSO4/PAGE is Mr 34,000 (sucrose gradient centrifugation under nondenaturing conditions yields a sedimentation coefficient s20,w of 8.1 S, indicating that the native protein is an oligomer); (iii) amino acid analysis demonstrates a preponderance of acidic over basic residues, while electrofocusing experiments show that it has an isoelectric point around 8.0; (iv) approximately 15% of the protein is phosphorylated in vivo. It is postulated that control of the synthesis of this protein is connected with activation of a differentiative program triggered by NGF in the PC12 neoplastic cell line at some step(s) of DNA activity
A macromolecular structure favouring microtubule assembly in NGF-differentiated pheochromocytoma cells (PC12)
No full textCellular extracts derived from pheochromocytoma cells (PC12-) inhibit the assembly of calf brain tubulin, while those derived from nerve growth factor-differentiated cells (PC12+) do not display this effect. Incubation with RNase abolishes the inhibition by PC12- extracts and reveals the presence of an activating effect exerted by PC12+ extracts. Activation of microtubule assembly is enhanced when extracts are prepared from PC12+ cells exposed for 1 day to 1.0 microM taxol and is abolished when PC12+ extracts are: (a) prepared from cells incubated for 1 day with 1 microM colchicine, (b) treated with the non-ionic detergent Nonidet P-40 or (c) centrifuged at 100 000 g instead of 80 000 g. 2D gel electrophoresis of the proteins of the 100 000 g pellet responsible for the activating effect (referred to as 100 K g pellet) reveals the presence of 100 K, 88 K and 32 K proteins which are markedly enriched in PC12+ extracts. The 88 K protein is further enriched in taxol-treated cells and markedly reduced in the same cells incubated with colchicine. A correlation between the differential protein composition of the 100 K g pellets and their effect on microtubule formation is postulated
Intracellular expression of anti-p21ras single chain Fv fragments inhibits meiotic maturation of xenopus oocytes
No full textThe recombinant variable regions of the monoclonal antibody Y13-259, directed against the p21ras protein, have been engineered for expression as intracellular single chain Fv fragments. The activity of the plasmid was confirmed by in vitro and in vivo translation of mRNA showing that the intracellularly expressed single chain fragments are stably and efficiently expressed as cytosolic proteins. The expression of the anti-p21ras single chain antibodies in the cytoplasm of Xenopus laevis oocytes leads to the inhibition of the insulin-induced meiotic maturation. This finding represents the first successful application of the strategy of intracellular antibodies to block a complex biological process in the cytosol of vertebrate cells
Intracellular expression of anti-p21ras single chain Fv fragments inhibits meiotic maturation of xenopus oocytes
No full textThe recombinant variable regions of the monoclonal antibody Y13-259, directed against the p21ras protein, have been engineered for expression as intracellular single chain Fv fragments. The activity of the plasmid was confirmed by in vitro and in vivo translation of mRNA showing that the intracellularly expressed single chain fragments are stably and efficiently expressed as cytosolic proteins. The expression of the anti-p21ras single chain antibodies in the cytoplasm of Xenopus laevis oocytes leads to the inhibition of the insulin-induced meiotic maturation. This finding represents the first successful application of the strategy of intracellular antibodies to block a complex biological process in the cytosol of vertebrate cells
Nerve growth factor inhibits the synthesis of a single-stranded DNA binding protein in pheochromocytoma cells (clone PC12)
No full textArrest of mitosis and neurite outgrowth induced by nerve growth factor (NGF) in rat pheochromocytoma cells (clone PC12) is accompanied by a progressive inhibition of the synthesis of a protein that binds to single-stranded but not to double-stranded DNA. Time course experiments show that this inhibition is already apparent after a 2-day incubation with NGF and is maximum (85-95%) upon achievement of complete PC12 cell differentiation. Inhibition of the synthesis of this single-stranded DNA binding protein after 48 hr of incubation with NGF is potentiated by concomitant treatment of PC12 cells with antimitotic drugs acting at different levels of DNA replication. Purification on a preparative scale of this protein and analysis of its major physicochemical properties show that: (i) it constitutes 0.5% of total soluble proteins of naive PC12 cells; (ii) its molecular weight measured by NaDodSO4/PAGE is Mr 34,000 (sucrose gradient centrifugation under nondenaturing conditions yields a sedimentation coefficient s20,w of 8.1 S, indicating that the native protein is an oligomer); (iii) amino acid analysis demonstrates a preponderance of acidic over basic residues, while electrofocusing experiments show that it has an isoelectric point around 8.0; (iv) approximately 15% of the protein is phosphorylated in vivo. It is postulated that control of the synthesis of this protein is connected with activation of a differentiative program triggered by NGF in the PC12 neoplastic cell line at some step(s) of DNA activit
Neuroantibodies: molecular cloning of a monoclonal antibody against substance P for expression in the central nervous system
No full textWe present a strategy to study functional and/or developmental processes occurring in the nervous system, as well as in other systems, of mice. This strategy is based on the local expression of specific monoclonal antibodies (mAbs) by cells of the nervous system. As an application of this strategy, we report the cloning of the anti-substance P rat mAb NC1/34HL. Functional substance P-binding antibodies were reconstituted from the cloned variable domains by using vectors for expression in myeloma cells. With these and other vectors a general system for the cloning and expression of mAbs under a series of promoters (of the rat VGF8a gene, the neurofilament light-chain gene, and the methallothionein gene) has been created. The activity of these plasmids was confirmed by expressing the recombinant NC1/34HL mAb in GH3 pituitary cells, PC12 pheochromocytoma cells, and COS cells. DNA from the described constructs can be used to target the expression of the NC1/34HL mAb to the central nervous system of transgenic mice. This procedure will allow us to perturb substance P activity in a controlled way in order to dissect its multiple roles
A macromolecular structure favouring microtubule assembly in NGF-differentiated pheochromocytoma cells (PC12)
No full textCellular extracts derived from pheochromocytoma cells (PC12-) inhibit the assembly of calf brain tubulin, while those derived from nerve growth factor-differentiated cells (PC12+) do not display this effect. Incubation with RNase abolishes the inhibition by PC12- extracts and reveals the presence of an activating effect exerted by PC12+ extracts. Activation of microtubule assembly is enhanced when extracts are prepared from PC12+ cells exposed for 1 day to 1.0 microM taxol and is abolished when PC12+ extracts are: (a) prepared from cells incubated for 1 day with 1 microM colchicine, (b) treated with the non-ionic detergent Nonidet P-40 or (c) centrifuged at 100 000 g instead of 80 000 g. 2D gel electrophoresis of the proteins of the 100 000 g pellet responsible for the activating effect (referred to as 100 K g pellet) reveals the presence of 100 K, 88 K and 32 K proteins which are markedly enriched in PC12+ extracts. The 88 K protein is further enriched in taxol-treated cells and markedly reduced in the same cells incubated with colchicine. A correlation between the differential protein composition of the 100 K g pellets and their effect on microtubule formation is postulated
Nuclear localization of a lactic dehydrogenase with single-stranded DNA-binding properties
No full textIn the preceding article [1] we identified the 34 kD single-stranded DNA-binding (ssb) protein, whose synthesis is inhibited in PC12 cells concomitantly with nerve growth factor (NGF)-induced mitotic arrest, with the enzyme lactic dehydrogenase (LDH-ssb protein). Localization studies performed with antibodies raised against the LDH-ssb protein demonstrate the presence of a pool of this protein in the nucleus of several cell types. The nuclear association of this protein is sensitive to DNase treatment of the cells and quantitative electron microscopy confirms that the LDH-ssb protein is located close to chromatin structures. These results point to a possible involvement of the LDH-ssb protein in some nuclear function(s)
The mode of action of nerve growth factor in PC12 cells
No full textThis review deals with the mechanism of nerve growth factor action. In view of the many and diversified effects of this growth factor, and since it could utilize different mechanism(s) in distinct types of cells, we have confined our analysis to the best characterized and more extensively studied target, the clonal cell line PC12. When exposed to NGF in vitro, these neoplastic cells recapitulate the last major steps of neuronal differentiation, i.e., the commitment to become a neuron and the acquisition of the neuronal phenotype. This is characterized by electrically excitable neurites, a display of a highly organized cytoskeleton, and the specific chemical and molecular neuronal properties. These effects are elicited upon the interaction of NGF with a receptor whose gene has been cloned and whose kinetic properties are now relatively well characterized. It is not yet clear, on the contrary, if and which of the several potential second messengers (cAMP, Ca, or phosphoinositides) that undergo marked fluctuations following NGF binding, transduce and amplify the NGF message. Among both the early and late effects of NGF is the modulation of expression of several genes. Some of the products of these genes are mainly restricted to nerve cells and others appear to play a crucial role in regulating the proper assembly of cytoskeletal elements. It is hypothesized that this complex array of chemical, molecular, and ultrastructural changes is triggered by NGF, not through activation of a single pathway, but more likely via combinatorial processes whereby several intracellular signals interplay before the irreversible commitment of becoming a neuron is undertaken
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