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Neuroregulation of the phosphorylation of a cyclic AMP binding protein in rat skeletal muscle cytosol
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Maturation and localization of acetylcholinesterase in primary cultures of fetal mouse brain. Lazar, M. ; Vigny, M. ; Houzet, E. ; Berwald-Netter, Y. Collage de France, Biochimie Cellulaire, 75231 Paris Cedex 05, France. Acetylcholinesterase (ACHE) esists as a family of molecular forms. The mammalian CNS contains mainly the monomeric (GI) and tetrameric (G4) forms ; changes in their relative level, in favour of G4, have been demonstrated in the developing brain (Massouli~ et al., 1982, Ann. Rev. Neurosci. 5, 57). In the present study we have examined the expression of AChE in primary cultures of mouse brain. Dissociated cells derived from 15-day embryos were cultured under conditions allowing for neuronal survival and differentiation (Berwald-Netter et al., 1981, Proc. Natl. Acad. Sci. USA 78, 1245). The AChE activity per dish and the proportion of G! and G4 forms were determined in detergent extracts of cells as a function of time in culture up to 4 weeks. In addition, the cellular localization of the enzyme was assayed by measures of the ectoactivity of whole cells and by use of non-penetrating inhibitors. One of our main conclusions is that the maturation of AChE in long-term cultures of neurons is very similar to that observed in vivo during post-natal development of whole brain. Such an in vitro model system thus may be very useful to explore the molecular mechanisms underlying the evolution pattern of AChE during terminal differentiation of neurons.
Enhanced dopamine uptake in dissociated embryonic mesencephalon co-cultured with metencephalon or astrocytes. Lieth,E., Towle,A.C., Joh*,T., and Lauder,J.M., Dept of Anatomy, UNC Sch. Med., Chapel Hill, NC, and *Dept of Neurol., Cornell Univ. Sch. Med., New York, NY. Brain tissue taken from mes- or metencephalon of 14 day embryos (El4), containing developing substantia nigra or raphe, respectively , was dissociated and co-cultured or grown separately on a substrate of poly-L-lysine. Saturable, high affinity 3H-dopamine (DA) or 3H-serotonin (bHT) uptake was measured after 2, 4 or 6 days in culture. Preliminary results indicate that DA uptake is significantly enhanced in these co-cultures, whereas 5HT uptake is only slightly enhanced. The DA effect is interesting, since axonal projections from the raphe nuclei reach the substantia nigra by E13-14. This raises the possibility that raphe neurons could exert some influence on the differentiation of DA neurons in the intact embryo. In other experiments, an increase in 3H-DA uptake was also observed when mesencephalic cells were cultured on a bed of purified astrocytes, in contrast to fibroblasts. This effect was apparently enhanced with time in culture, and may be due to better survival, growth or differentiation of these neurons on their native gllal substrate, as witnessed in combined anti-TH and anti-GFAP immunocytochemical preparations. Combined autoradiographic-immunocytochemical experiments are presently in progress to evaluate the specificity of the uptake of 3H-monoamines into identified neurons lnthese cultures, as well as to test the hypothesis that 5HT neurons directly influence the differentiation of DA cells in culture.
Neuroregulation of the phosphorylation of a cyclic AMP binding protein in rat skeletal muscle cytosol. McLane, J.A.; Squinto, S.P. and Held, I.R. Hines VA Hospital, Neuroscience Research Lab., Hines, IL and Loyola University Medical Center, Departments of Pharmacology and Biochemistry, May~ood, IL, USA. We have reported that the in vitro phosphorylation (phos.) of a soluble polypeptide by an endogenous protein kinase in cytosolic fractions from rat soleus muscles is increased after the muscle is denervated for a short period (3 hr) and that this change temporally correlates with the length of the distal nerve stump (Trans. Am. Soc. Neurochem. 12:230, 1981). Although autoradiographs of slab gels after SDS-PAGE show that 32po 4 from gamma-32p-ATP is transferred to --I0 soluble eytosolic proteins under our assay conditions, 8~/o of the incorporated 32p-label was bound to a minor protein-staining band of 56 kilodaltons (Kd) (Soc. Neurosci. Abstr. 8:185, 1982). The reduced phos. of the 56Kd protein when the assay included high levels of cAMP (iO -7 to 10 -5 M) or Zn 2+ instead of Mg 2+ , together with the ineffectiveness of a specific inhibitor of cAMP-dependent protein klnase (cAMP-dPK), suggests that the 56Kd subst~ate is the autophos, regulatory subunit o~ cAMP-dPK, type II(R-II). Using a double-label technique in which in vitro 32p-pho~. cytosolic proteina were labeled with a photoaffinity analog of cAMP ~ - ~ - 3 H - c A M P ) , we f c ~ that the highly phos. 56Kd region of the gel contained -'lOTo of the cAMP binding protein. The 49-52Kd region of the gel contained 75-807° of the cAMP binding. These combined results indicate that the autophos, of R-II is modulated by some non-impulse conm~unication of nerve with muscle.
Maturation and localization of acetylcholinesterase in primary cultures of fetal mouse brain. Lazar, M. ; Vigny, M. ; Houzet, E. ; Berwald-Netter, Y. Collage de France, Biochimie Cellulaire, 75231 Paris Cedex 05, France. Acetylcholinesterase (ACHE) esists as a family of molecular forms. The mammalian CNS contains mainly the monomeric (GI) and tetrameric (G4) forms ; changes in their relative level, in favour of G4, have been demonstrated in the developing brain (Massouli~ et al., 1982, Ann. Rev. Neurosci. 5, 57). In the present study we have examined the expression of AChE in primary cultures of mouse brain. Dissociated cells derived from 15-day embryos were cultured under conditions allowing for neuronal survival and differentiation (Berwald-Netter et al., 1981, Proc. Natl. Acad. Sci. USA 78, 1245). The AChE activity per dish and the proportion of G! and G4 forms were determined in detergent extracts of cells as a function of time in culture up to 4 weeks. In addition, the cellular localization of the enzyme was assayed by measures of the ectoactivity of whole cells and by use of non-penetrating inhibitors. One of our main conclusions is that the maturation of AChE in long-term cultures of neurons is very similar to that observed in vivo during post-natal development of whole brain. Such an in vitro model system thus may be very useful to explore the molecular mechanisms underlying the evolution pattern of AChE during terminal differentiation of neurons.
Enhanced dopamine uptake in dissociated embryonic mesencephalon co-cultured with metencephalon or astrocytes. Lieth,E., Towle,A.C., Joh*,T., and Lauder,J.M., Dept of Anatomy, UNC Sch. Med., Chapel Hill, NC, and *Dept of Neurol., Cornell Univ. Sch. Med., New York, NY. Brain tissue taken from mes- or metencephalon of 14 day embryos (El4), containing developing substantia nigra or raphe, respectively , was dissociated and co-cultured or grown separately on a substrate of poly-L-lysine. Saturable, high affinity 3H-dopamine (DA) or 3H-serotonin (bHT) uptake was measured after 2, 4 or 6 days in culture. Preliminary results indicate that DA uptake is significantly enhanced in these co-cultures, whereas 5HT uptake is only slightly enhanced. The DA effect is interesting, since axonal projections from the raphe nuclei reach the substantia nigra by E13-14. This raises the possibility that raphe neurons could exert some influence on the differentiation of DA neurons in the intact embryo. In other experiments, an increase in 3H-DA uptake was also observed when mesencephalic cells were cultured on a bed of purified astrocytes, in contrast to fibroblasts. This effect was apparently enhanced with time in culture, and may be due to better survival, growth or differentiation of these neurons on their native gllal substrate, as witnessed in combined anti-TH and anti-GFAP immunocytochemical preparations. Combined autoradiographic-immunocytochemical experiments are presently in progress to evaluate the specificity of the uptake of 3H-monoamines into identified neurons lnthese cultures, as well as to test the hypothesis that 5HT neurons directly influence the differentiation of DA cells in culture.
Neuroregulation of the phosphorylation of a cyclic AMP binding protein in rat skeletal muscle cytosol. McLane, J.A.; Squinto, S.P. and Held, I.R. Hines VA Hospital, Neuroscience Research Lab., Hines, IL and Loyola University Medical Center, Departments of Pharmacology and Biochemistry, May~ood, IL, USA. We have reported that the in vitro phosphorylation (phos.) of a soluble polypeptide by an endogenous protein kinase in cytosolic fractions from rat soleus muscles is increased after the muscle is denervated for a short period (3 hr) and that this change temporally correlates with the length of the distal nerve stump (Trans. Am. Soc. Neurochem. 12:230, 1981). Although autoradiographs of slab gels after SDS-PAGE show that 32po 4 from gamma-32p-ATP is transferred to --I0 soluble eytosolic proteins under our assay conditions, 8~/o of the incorporated 32p-label was bound to a minor protein-staining band of 56 kilodaltons (Kd) (Soc. Neurosci. Abstr. 8:185, 1982). The reduced phos. of the 56Kd protein when the assay included high levels of cAMP (iO -7 to 10 -5 M) or Zn 2+ instead of Mg 2+ , together with the ineffectiveness of a specific inhibitor of cAMP-dependent protein klnase (cAMP-dPK), suggests that the 56Kd subst~ate is the autophos, regulatory subunit o~ cAMP-dPK, type II(R-II). Using a double-label technique in which in vitro 32p-pho~. cytosolic proteina were labeled with a photoaffinity analog of cAMP ~ - ~ - 3 H - c A M P ) , we f c ~ that the highly phos. 56Kd region of the gel contained -'lOTo of the cAMP binding protein. The 49-52Kd region of the gel contained 75-807° of the cAMP binding. These combined results indicate that the autophos, of R-II is modulated by some non-impulse conm~unication of nerve with muscle.