Eleni Dicou

Serum and thyroid hormones T3 and T4 regulate nerve growth factor mRNA levels in mouse L cells

Mouse L cells synthesize and secrete a neurotrophic factor related to the β subunit of the submaxillary gland nerve growth factor (NGF) of male mice. Use of a cDNA probe which encodes the β‐NGF mRNA demonstrated that L cells produce a transcript identical in size to that of the submaxillary gland. Moreover, target sites of restriction enzymes EcoRI, PstI and BamHI were not significantly rearranged in the β‐NGF gene locus of these cells. The abundance of the β‐NGF transcript was found to depend on culture conditions. Removal of serum depressed the cellular content of polyadenylated RNA by a factor of 1.7, and decreased specifically the pool of β‐NGF transcript by an additional factor of 4. The presence of 10−7 M testosterone in the serum‐free medium did not modify the level of β‐NGF mRNA, while addition of 10−7 M T3 (or T4) increased this level by a factor of 1.5. These data provide the first evidence that the β‐NGF mRNA of L cells is subjected to regulation, but in a way apparently different from that described for the submaxillary gland.

L cells potentiate the effect of the extracellular NGF activity in co-cultures with PC12 pheochromocytoma cells.

Two mouse cell lines, 3T3 and L, reported to secrete an NGF-like activity in the culture medium were co-cultured with the pheochromocytoma cell line PC12 which responds to NGF in vitro. In these co-cultures mitomycin-treated L or 3T3 cells were employed at low cell density (1 000 cells/cm2). L cells, but not 3T3, promoted efficiently neurite outgrowth of PC12. The response of the PC12 cells was blocked by an antiserum to male mouse submaxillary gland beta NGF. The NGF secreted by the L cells and immunoprecipitated by this antiserum co-migrated with the submaxillary gland beta NGF monomer in SDS-polyacrylamide gels. Surprisingly the neurite-promoting activity of media conditioned by L or by L-PC12 co-cultures was at most one-tenth of that expected on the basis of the response of PC12 cells in the co-cultures. This was not due to proteolytic degradation of the NGF-like factor or to losses by manipulation of the media. It seems therefore that co-cultures provide conditions which enhance the effect of the factor. Possible mechanisms responsible for this effect are discussed.

Inhibition of cell differentation in a phosphodiesterase defective mutant of Dictyostelium discoideum

Abstract A biochemical analysis of a mutant, HP X235, which is dependent upon an exogenous source of a soluble phosphodiesterase in order to aggregate, is presented. The mutant is shown to undertake the developmental changes in its adenylate cyclase activity and to excrete cAMP. Only if the mutant is starved in the presence of an extracellular source of phosphodiesterase are these above changes in adenylate cyclase and cAMP excretion accompanied by increases in cell surface cAMP binding sites, contact A sites and periodic oscillations in light scattering. The data is discussed in terms of the relationship between the generation and perception of the chemotactic signal and cell differentiation to aggregation competence.

Synthesis and secretion of β-nerve growth factor by mouse teratocarcinoma cell lines

Using a cDNA probe and a two-site enzyme immunoassay, beta-nerve growth factor (beta NGF) synthesis was monitored in several mouse teratocarcinoma cell lines. Trace amounts of NGF mRNA were detected in the embryonal carcinoma (EC) PCC4, F9 and 1003 clones, whereas the myocardial (PCD1), myogenic (1168) and adipogenic (1246) clones contained significantly higher levels of NGF mRNA and secreted mature beta NGF peptide in the culture medium. The 1003, 1168 and 1246 strains were derived from the same teratocarcinoma cell line and their ability or inability to synthesize the neurotrophic factor may reflect a developmental decision for divergent differentiation programs. Induction of NGF mRNA and protein synthesis was observed in a differentiated derivative of an SV40-transformed F9 clone which expresses the viral T antigen. Southern blot analysis of the genomic DNAs revealed no structural alterations of the NGF locus between teratocarcinoma cells that express the NGF gene and those that do not. Similar analysis of the DNA methylation pattern in C-C-G-G sequences using the Hpa II and Msp I isoschizomers indicated no methylation changes of the NGF gene in the teratocarcinoma DNAs. At least two, and probably all four, of the already mapped Msp I sites within the NGF gene are methylated in all teratocarcinoma DNAs examined, as well as in the male mouse submaxillary gland DNA, the organ richest in this factor.

Synthesis and partial maturation of the α- and γ-subunits of the mouse submaxillary gland nerve growth factor in Xenopus laevis oocytes

Sera raised against the α‐, β‐ and γ‐subunits of the mouse 7 S NGF were used to characterize translation products coded by submaxillary gland mRNAs microinjected into Xenopus oocytes. Anti‐βNGF sera did not cross‐react with any material. In contrast, the precursors of the α‐ and γ‐subunits, as well as that of renin were identified. Use of tunicamycin, and a comparison of the translation products obtained in oocytes or in the reticulocyte lysate indicated that oocytes achieved the cleavage of signal sequences, the glycosylation of the α‐ and γ‐precursors, and the subsequent secretion of the 3 proteins. In the submaxillary gland, however, the mature forms of αNGF, γNGF and renin are composed of peptides of smaller size than those produced by the oocytes. These latter appear to lack specific proteases involved in the terminal processing of the submaxillary gland proteins.

Synthesis of Dictyostelium discoideum secretory proteins in Xenopus laevis oocytes.

The cellular slime mould Dictyostelium discoideum presents a model system for the study of the developmental regulation of gene expression. Development is initiated by nutrient deprivation. After a few hours of starvation, cells develop new properties which allow them to emit and respond to pulses of cyclic AMP, the chemotactic agent, and to move toward aggregation centers [ 11. During this stage of their development toward aggregation competence, several proteins are secreted in the extracellular medium. Among them a cyclic AMP phosphodiesterase and its specific inhibitor play a regulatory role in the aggregation process [2,3]. In the present work, we have used an antiserum against Dictyostelium secretory proteins to monitor the expression of the genes coding for proteins released into the extracellular medium until the amoebae reach the aggregation phase. For this purpose, poly(A)containing RNAs from growth-phase cells and from aggregation competent cells were microinjected into Xenopus oocytes and the translation products were analysed for proteins crossreacting with this antiserum.

Purification of the inhibitor of the 3′–5′ cyclic AMP phosphodiesterase of Dictyostelium discoideum by affinity chromatography

Summary A method is described to purify in a single step the inhibitor of the 3′–5′ cyclic AMP phosphodiesterase of D. discoideum by affinity chromatography to phosphodiesterase immobilized to Sepharose. Biologically active, pure inhibitor is eluted with 5M salt at high yields. Application of this technique to the study of mutants which overproduce or are deficient in the synthesis of the inhibitor is presented.

Interaction of the inhibitor of the 3′,5′ cyclic amp phosphodiesterase of Dictyosteliumdiscoideum with the affi-gel blue

Summary A large scale purification of the inhibitor of the cAMP phosphodiesterase of Dictyostelium discoideum is achieved in one step by chromatography on Affi-Gel blue. The chromophoric component of the resin is responsible for the affinity. Caffeine and trifluoperazine partly inhibit the binding of the inhibitor while cAMP and AMP have no effect. The purified inhibitor does not bind any of the several adenine and guanine nucleotides tested. The free blue chromophore does not affect the formation of the inhibitor-phosphodiesterase complex.

RNA involvement in T4 DNA synthesis in toluene-treated cells

In T4-infected cells made permeable with toluene, pulses with [(alpha-32P deoxyribonucleoside triphosphates demonstrated covalent linkage of RNA to DNA of the Okazaki fragments. Analysis of the transfer of the 32P label to the 2(3) ribonucleoside monophosphates indicated that the 3-end of the RNA primer is heterogeneous. The most frequently encountered ribonucleotide was rCMP, but also transfer to rUMP, rAMP and rGMP occurred at different frequencies. In contrast, no heterogeneity was observed for the deoxyribonucleoside at the RNA-DNA junction. Of all the [to-32P] deoxyribonucleoside triphosphates tested, transfer of the 32P label to 2(3) rNMPs was predominant when [alpha32P] dGTP was the substrate, indicating that the deoxyribonucleoside most frequently encountered at the RNA-DNA linkage is dG. These observations suggest that the starts for the Okazaki fragments may occur at unique sites of the T4 genome.