Biomedical subjects
Publications and source records attributed to J I Morgan.
Thymosin beta 4, originally isolated from calf thymus [Low et al., Proc. Nat. Acad. Sci. USA 78, 1162-1166 (1981)] is present in a number of cell lines unrelated to the reticuloendothelium, including myoblasts and fibroblasts. It is also actively synthesized by these cell lines. Its content and rate of synthesis in the cell lines examined appear to be correlated with their ability to adhere and their motility.
A nonganglioside factor(s) present in Sigma types II and III mixed bovine brain ganglioside preparations synergises with suboptimal amounts of serum to induce proliferation specifically in nondividing B 103 neuroblastoma cultures. The active substance is nondialysable and soluble in water as well as in chloroform-methanol mixtures of 1:1-4:1 (vol/vol). It is completely insoluble in ether and acetone at room temperature. Biological activity survives heating to 70 degrees C in the presence of 0.1 M HCl for 1 h as well as boiling at neutral pH. Loss of activity occurs on heating to 70 degrees C for 1 h with 1 M HCl or 1 M NaOH. The activity is insensitive to digestion with neuraminidase, trypsin, pronase, and phospholipases A2 and C. The factor cochromatographs with gangliosides on Dowex AG 50W and Sephadex G100 and is partially recovered with GM1 on DEAE-Sepharose, but may be isolated in a ganglioside-free fraction by sequential chromatography on Sephadex LH20 and silicic acid columns. The substance(s) has the properties of a water-soluble proteolipid protein, the amino acid composition being reported. It is not immunologically cross-reactive with antibodies to GM1 ganglioside or the major proteolipid protein of myelin.
Thymosin beta 4 antibodies have been raised in rabbits by conjugating the peptide to keyhole limpet hemocyanin, and a radioimmunoassay for thymosin beta 4 has been established utilizing tritiated thymosin beta 4 as binding ligand. RIA values for thymosin beta 4 in rat tissues reveal levels ranging between 8 and 448 micrograms/g wet weight. Highest levels are observed in spleen, although the peptide is also relatively abundant in thymus, lung, and nasal epithelium. Brain, kidney, liver and testis contain only 10 to 20% the concentration of thymosin beta 4 seen in spleen, whereas heart and muscle contain only 5 to 10% that of spleen. However, thymosin beta 4 probably has a ubiquitous distribution in the rat, being present in all tissues examined. The specificity of the antiserum used in the RIA has been defined. The thymosin beta 4 molecule seemingly bears two major epitopes that lie between amino acid residues 1-8 and 22-32. The antiserum does not cross-react with other unrelated peptides. The antiserum will precipitate biosynthetically labeled beta 4 from cultured rat peritoneal macrophage.
For many permanent cell lines the transition from a growing (P) to a resting (R) state is reversibly controlled by growth factors present in serum. This P-to-R transition was studied in a neuronal cell line (B104) with respect to the action of serum, dibutyryl cyclic AMP (DBcAMP), gangliosides, and a glioma cell-produced growth factor GGF. In this cell system gangliosides seem to act as differentiation and survival factors. The kinetics of uptake of radioactively labeled gangliosides and survival experiments both support the idea of the stable incorporation of exogenously added gangliosides into the cells. Based on the experimental evidence a new model of cell development is proposed. Thus in addition to the R or Go state, which in this cell system is rather unstable and probably regulated by cyclic nucleotides, we postulate a differentiated D state, which is controlled by gangliosides and which is characterized by its stability (survival time). This D compartment seems to be closer to the in vivo differentiated neuron than does the R or P state. tthe possible mechanisms for the action of gangliosides are discussed.
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There has been much interest in the biochemical and biophysical processes that couple extracellular signals to alterations in gene expression. While many early events associated with the treatment of cells with growth factors have been described (for example, ion flux and protein phosphorylation), it has proved difficult to establish biochemical links to gene expression. Recently, the study of such genomic control signals has been facilitated by the demonstration that the c-fos proto-oncogene is rapidly and transiently induced by treatment of several cell types with polypeptide growth factors and other growth modulating substances. In one particular system it has been shown that nerve growth factor (NGF) causes a transient induction of c-fos in the phaeochromocytoma cell line PC12, within 15 min. Furthermore, the magnitude of this induction can be modulated with pharmacological agents such as peripheral-type benzodiazepines (BZDs). Thus, the study of c-fos expression in PC12 cells could yield valuable clues to the coupling mechanisms linking cell surface activation to genomic events. Here we demonstrate that c-fos is induced in PC12 cells either by receptor-ligand interaction or by agents or conditions that effect voltage-dependent calcium channels.