PubMed Health⌕ Search

Biomedical subjects

D Couchie

Publications and source records attributed to D Couchie.

29 records · Page 2Linked to original sources
PubMed1986

Protein synthesis in astrocytes: 'spontaneous' and cyclic AMP-induced differentiation.

Primary cultures of mouse astrocytes have been used to study astroglial protein synthesis during 'in vitro' differentiation. Spontaneous age-related differentiation was compared to the effect of DBcAMP or forskolin, a drug which directly stimulates the adenylate cyclase and induces 'morphological differentiation' in these cells. Cell differentiation was followed in parallel by phase contrast microscopy and immunofluorescence techniques. Two antisera, one raised against GFA, the other against microtubule-associated protein 2 (MAP2) were used. Anti-GFA serum labelled the cells as early as 7 days in vitro. Anti-MAP2 serum revealed a dense fibrous network at later stages of the culture, whereas the dividing astroblasts appeared poorly stained by this antibody. Both phase contrast microscopy and immunofluorescence techniques suggested that most of the cells spontaneously differentiate after 3 weeks of culture even in the absence of DBcAMP or forskolin. Forskolin, while accelerating differentiation after 7 days of culture, produced smaller cells than DBcAMP and had biphasic effects on cell morphology. Mono- and two-dimensional gel electrophoresis of the 35S-methionine labelled cells also showed that the major changes in protein synthetic activity occur spontaneously during the time course of the culture. Whatever the stage of the culture, DBcAMP or forskolin induced changes in the synthesis of only a few proteins. However, depending on the culture stage the proteins, which were positively or negatively controlled by these drugs, were not the same.

Animals↗
PubMed1985

Immunological characterization of microtubule-associated proteins specific for the immature brain.

Immunoblotting analysis was used to detect the microtubule-associated proteins present at different stages of rat brain development. Polyclonal antibodies were raised against the two main adult brain microtubule-associated proteins: MAP-2 (300 kDa) and TAU (60-70 kDa). Whatever the stage of development, anti-MAP-2 serum detected high molecular mass proteins and at immature stages a protein of 62 kDa. This protein which has previously been referred to as 'young TAU slow' is, therefore, immunologically related to MAP-2. The anti-TAU serum (but not the anti-MAP-2 serum) detected at immature stages of development a 48 kDa protein which also disappears at adulthood. This 48 kDa entity which has been referred to as 'young TAU fast' is progressively replaced by the closely spaced bands (60-70 kDa) of adult TAU proteins. The 62 and 48 kDa proteins appear therefore to be immunologically distinct and represent two microtubule-associated proteins specific to the immature brain.

Animals↗
PubMed1985

Microtubule-associated proteins and in vitro astrocyte differentiation.

Primary cultures of mouse brain astrocytes have been used to identify the microtubule-associated proteins (MAPs) present in this cell type at different stages of in vitro differentiation. The MAPs of the astrocyte have been identified by polyacrylamide gel electrophoresis and immunological detection. Two antisera were raised against two brain MAPs, tau and MAP-2. These antisera were also used to label the microtubular network in the intact astrocytes at different stages of the culture. The mature astrocyte contains a variety of MAP-like proteins. Anti-MAP-2 serum detected several proteins of high molecular weight (380,000, 260,000, 205,000 and 165,000 mol wt) and one microheterogeneous peak of 83,000 mol wt. Anti-tau also detected high molecular weight components (380,000 to approximately 200,000 mol wt) but not the 165,000-mol-wt peak; in addition two microheterogeneous peaks of 83,000 and 62,000 mol wt were detected by the anti-tau serum. The 62,000-mol-wt peak was therefore detected only by the anti-tau serum whereas the 83,000-mol-wt component cross-reacted with both antisera. At early stages of the culture the immature cell contained about two times less immunoreactive material than at mature stages. Qualitative changes of the high molecular weight components were also observed. In the intact cell both antisera revealed a dense fibrous network. At early stages of the culture the astroblasts were stained by the antisera but the reaction was very diffuse in the cytoplasm; few fibrous cells were intensively stained. Morphological differentiation, which began after serum deprivation and which was accelerated by forskolin (a drug that induces cyclic AMP accumulation), led to high labeling of both the cell body and the cellular processes. In the presence of colchicine the staining regressed, the processes shortened, and the cell returned to a less-apparently differentiated state.

Animals↗
PubMed1984

Cyclic nucleotide derivatives as probes of phosphodiesterase catalytic and regulatory sites.

The present data are consistent with the following mechanism of activation of the cGMP-stimulated PDE: cGMP is first bound to an allosteric site of the enzyme; this is followed by a conformational change in protein structure, a shift of kinetic behavior, and sequential activation of cAMP PDE hydrolysis (18). Evidence for the existence of distinct activating and catalytic sites is obtained from the use of several cyclic nucleotide derivatives to elucidate the essential molecular interactions at both of these sites (4). Since the liver PDE under study exhibits positive homotropic cooperativity by cAMP, the stimulatory effect of low concentrations of MIX is consistent with its binding at the substrate-active sites. As shown previously, this enzymatic mechanism is reproduced by an analog of the substrate, c6clPMP, but not by cGMP. Since the order of potency of a series of competitive inhibitors of two PDE (i.e., the cyclic GMP-sensitive enzyme and a calmodulin-sensitive enzyme), is not parallel, it is suggested that the active sites of these enzymes are distinct. The interaction of MIX at the active site of two PDE studied here could reflect a general binding mechanism of the xanthine due to similar chemical forces. Since we propose that the allosteric-activation site is specific for cGMP, the xanthine does not bind to that site. This is also suggested from substrate-velocity relationships measured in the presence of MIX and cGMP (Table 3). Complete characterizations of the cGMP-activating site and the xanthine-sensitive catalytic site are required in order to elucidate the exact chemical interactions at both sites on the cGMP-stimulated PDE.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Characterization of phosphodiesterase catalytic sites by means of cyclic nucleotide derivatives.

Cyclic nucleotide derivatives have been used as a tool to characterize distinct catalytic sites on phosphodiesterase enzyme forms: the cGMP-stimulated enzyme from rat liver and the calmodulin-sensitive enzyme from rat or bovine brain. Under appropriate assay conditions, the analogues showed linear competitive inhibition with respect to cAMP (adenosine 3',5'-monophosphate) as substrate. The inhibition sequence of the fully activated cGMP-stimulated phosphodiesterase was identical to the inhibition sequence of the desensitized enzyme, i.e. the enzyme which has lost its ability to be stimulated by cGMP. The inhibition pattern could, therefore, not be attributed to competition with cGMP at an allosteric-activating site. Also, the inhibition sequence of the calmodulin-sensitive phosphodiesterase was maintained whether activity was basal or fully stimulated by calmodulin. When cAMP and cGMP, with identical chemical ligands substituted at the same position, were compared as inhibitors of the calmodulin-sensitive phosphodiesterase, the cGMP analogues were always the more potent suggesting that, for that enzyme, the catalytic site was sensitive to a guanine-type cyclic nucleotide structure. Comparing the two phosphodiesterases, it was possible to establish both similar and specific inhibitor potencies of cyclic nucleotide derivatives. In particular, the two enzymes exhibited large differences in analogue specificity modified at C-6, 6-chloropurine 3',5'-monophosphate or purine 3',5'-monophosphate.

Animals↗
PubMed1981

Characterization of a rat liver cyclic GMP-activated phosphodiesterase by chromatography on hexyl-agarose. Inhibition of phosphodiesterase activity by hexyl-agarose.

Chromatography on hexyl-agarose resolved a partially purified cyclic GMP-activated phosphodiesterase from rat liver into two peaks of activity: the first was eluted with 0.5 M-KCl and was cyclic AMP-specific. The second was tightly bound to hexyl-agarose and was not eluted with KCl (0--2.0 M), which enhanced the hydrophobic interactions of this form with the matrix. It was eluted with 0.5 M-Tris, hydrolysed cyclic AMP and cyclic GMP and was specifically activated by cyclic GMP. The cyclic GMP-activated phosphodiesterase was immobilized on hexyl-agarose. Enzyme activity, quantitatively bound to hexyl-agarose, was not released from the hydrophobic matrix in the presence of cyclic AMP or cyclic GMP, under our assay conditions. The immobilized form of the enzyme retained catalytic activity, was inhibited by 0.1 mM-cyclic AMP and was activated by micromolar concentrations of cyclic GMP to a lesser extent (7-fold) than the control, i.e. the enzyme mixed with unsubstituted agarose (15-fold). When the enzyme was immobilized, inhibition of cyclic AMP phosphodiesterase activity was only observed in the presence of cyclic GMP (at 3 microM); in its absence, activity remained unchanged. The kinetic behaviour of the immobilized enzyme is consistent with the hypothesis of a binding site distinct from the hydrolytic and activating sites.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1981

Specificity of cyclic GMP activation of a multi-substrate cyclic nucleotide phosphodiesterase from rat liver.

Cyclic nucleotide derivatives have been used as a tool to investigate the existence of distinctive activating and hydrolytic sites on the phosphodiesterase from rat liver activated by cGMP (guanosine 3',5'-monophosphate). This positively cooperative enzyme was stimulated up to 30-fold by 3 microM cGMP when 3 microM cAMP (adenosine 3',5'-monophosphate) was used as substrate. All analogues were less potent activators than cGMP. Most cAMP derivatives were inactive, with two exceptions: 7-deazaadenosine 3',5'-monophosphate and 3'-amino-3'-deoxy-adenosine 3',5'-monophosphate. Benzimidazole ribonucleoside 3',5'-monophosphate, where the two atoms of nitrogen of the pyrimidine ring are missing was a better stimulator than the intact purine-related cyclic derivative. When cAMP and cGMP with identical chemical ligands substituted at the same position were compared, the cGMP analogue was always the more potent activator suggesting that the activating site is sensitive to a guanine-type cyclic nucleotide structure. Degradation of the derivatives by the enzyme was measured by high-performance liquid chromatography: no relation could be established between hydrolysis and effectiveness of activation. In addition, there was no parallelism between inhibitory and activating potency for ten cyclic nucleotide derivatives. Since the chemical interactions between the analogues at the activating site on the one hand and at the catalytic site on the other, are different, it is proposed that the sites are distinct. Consequently, it is suggested that the enzyme operates in steps. In the first activating step, cGMP is fixed by at least two hydrogen bonds at a specific binding site of the enzyme. This is followed by a conformational change of the protein and subsequently a change of the kinetic parameters. In a rather unspecific process and in a second hydrolytic step, several purine-related cyclic nucleotides are converted to the corresponding 5' nucleotides.

3',5'-Cyclic-AMP Phosphodiesterases↗