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Biomedical subjects

C Cochet

Publications and source records attributed to C Cochet.

At least 91 records · Page 5Linked to original sources
PubMed1988

Twofold CuZnSOD activity suggesting homozygous submicroscopic duplication of chromosome 21 in the orangutan.

A twofold CuZnSOD activity was observed in an orangutan as compared to man and a chimpanzee. This suggests that a submicroscopic duplication of chromosome 21 (similar to those demonstrated in patients with the trisomy 21 phenotype but with a normal karyotype) has occurred in the homozygous state during evolution of the orangutan phylum. Such duplications could be important evolutionary mechanisms, together with visible chromosome rearrangements and classical gene mutations.

Animals↗
PubMed1987

Type beta transforming growth factor affects adrenocortical cell-differentiated functions.

Type beta transforming growth factor (TGF-beta) had no detectable effect on mitogenic activities of bovine adrenocortical cells in culture. However, the presence of TGF-beta (1 ng/ml) in the medium resulted in a striking alteration of adrenocortical cell steroidogenic activities, maximally expressed after 18-20 h of treatment. TGF-treated cells exhibited a basal as well as an adrenocorticotropin-stimulated cortisol production inhibition by an average 50-60%, while cAMP accumulation in response to the hormone was not modified. Detailed study of the adrenocortical steroid biosynthetic pathway by high performance liquid chromatography analysis and supply of representative steroid substrates revealed a drastic loss (average 50%) of the steroid 17 alpha-hydroxylase activity following TGF treatment. TGF-beta thus appeared as a potent negative modulator of adrenocortical 17 alpha-hydroxylase activity. This TGF-induced loss in the activity of a key steroidogenic enzyme resulted in a shift of the adrenocortical cell secretion pattern at the expense of the 17 alpha-hydroxysteroid end products. This 17 alpha-hydroxylation alteration was also expressed when TGF-treated cells were challenged by angiotensin II. However, in this case, an additional lesion was suggested by a 70-90% inhibition in angiotensin II-activated cortisol production. This could be explained by the observation that TGF-beta exposure induced an average 50% decrease in the adrenocortical cell angiotensin II receptor number without any detectable change in receptor affinity (Ka approximately 10(9) M-1). In addition, a parallel alteration in the angiotensin II-activated phosphoinositide breakdown was observed in TGF-treated cells, indicating that TGF-beta appears as a negative effector of the adrenocortical cell transmembrane signaling system in the case of angiotensin II. It is concluded that, in vitro, TGF-beta is a potent modulator of differentiated adrenocortical cell functions, in which at least two major negatively regulated specific targets were characterized. The mechanism(s) of action and the possible physiological significance of TGF-beta in the control of the development and the differentiated functions of the adrenocortical gland in vivo remain to be established.

Adrenal Cortex↗
PubMed1987

Maternal origin of a de novo balanced t(21q21q) identified by ets-2 polymorphism.

Molecular investigations were done in a woman with a de novo balanced t(21q21q) discovered because of the birth of a trisomic 21 baby. Polymorphisms detected with probe ets-2 after Msp I digestion showed that both chromosomes 21 involved in the rearrangement were of maternal origin. The most likely hypothesis is that of a disomic 21 oocyte fertilized by a nullisomic 21 sperm.

Chromosomes, Human, Pair 21↗
PubMed1986

Type beta transforming growth factor is a potent modulator of differentiated adrenocortical cell functions.

Whereas TGF-beta exhibited no detectable effect on DNA synthesis, it was found to exert a striking inhibitory effect on the steroidogenic activities of bovine adrenocortical cells in culture. Basal, as well as ACTH- and angiotensin II- activated adrenocortical cortisol productions were inhibited in a time and dose-dependent manner following TGF-beta treatment. Half-maximum inhibition of ACTH- and AII-activated steroidogenesis was observed with TGF-beta concentrations of 0.40 and 0.12 ng/ml, respectively. This effect was half maximal after 6 hours of cell exposure to optimally effective TGF-beta concentrations (1 ng/ml) and reached a plateau after 12-15 hours, resulting in an average 60% inhibition in the steroidogenic response to ACTH and 90% in the case of AII. Supply of different exogenous steroid substrates to support steroidogenesis in adrenocortical cells pointed to a marked loss in steroid-17 alpha hydroxylase activity as a major alteration following TGF-beta treatment. TGF-beta thus appears as a potent modulator of differentiated adrenocortical cell functions in vitro; in this regard it may play a significant role in the development and the regulation of adrenal cortex in vivo.

17-alpha-Hydroxypregnenolone↗
PubMed1986

Phorbol ester-induced alteration of protein kinase C catalytic properties occurs at the membrane level and is not reproduced by physiological stimuli.

Potent tumor promoter TPA (1-100 nM) has previously been shown to induce a striking alteration of protein kinase C catalytic properties in target cells (C. Cochet et al., 1986, Biochem. Biophys. Res. Comm. 134, 1031-1037). This alteration contributes to the apparent loss of cellular protein kinase C, secondary to TPA treatment, when the enzyme is probed by its phospholipid-dependent histone kinase activity. This effect was observed as well when rat-1 cells were treated by other tumor promoters such as mezerein, teleocidin, aplysiatoxin and palytoxin, whereas inactive phorbol ester structures were ineffective. On the other hand, 1,2-dioctanoyl glycerol did not induce that effect. This protein kinase C alteration was shown to occur at the cellular membrane level. It is suggested that membrane translocation and activation of protein kinase C induced by potent tumor promoter structures are not functionally equivalent to that secondary to physiological stimuli. Although the mechanisms underlying this phenomenon remains to be understood at the molecular level, it may be of significance in the process of tumor promotion.

Animals↗
PubMed1986

Catalytic properties of a purified phosphatidylinositol-4-phosphate kinase from rat brain.

A phosphatidylinositol-4-phosphate (PIP) kinase activity was purified from rat brain extract through several chromatographic steps to yield an active preparation (specific activity 1 mumol of 32P incorporated into phosphatidylinositol 4,5-bisphosphate/min per mg of protein) with an apparent molecular size of 100-110 kDa in the native form. The isolated PIP kinase required Mg2+ (optimally 20-30 mM) for its activity and was not influenced by Ca2+. The enzyme used ATP (Km 25 microM) and GTP (Km 133 microM) as phosphate sources and appeared specific for PIP (Km 3.3 micrograms/ml) as the lipid substrate. The PIP-phosphorylation reaction was inhibited by micromolar concentrations of heparin [ID50 (concn. giving 50% inhibition) 2 micrograms/ml] and the flavonoid quercetin (ID50 0.2 microM). Whereas heparin behaves as a competitive inhibitor to PIP, quercetin was competitive towards ATP (or GTP). Phosphorylation of the preparation by a highly active purified protein kinase C did not detectably alter PIP kinase activity. Whereas 12-O-tetradecanoylphorbol acetate and various phospholipids had no effect, phosphatidylserine elicited a dose-dependent activation of PIP activity. This suggests that a phosphatidylserine-PIP kinase interaction may be considered as a possible regulatory process at the cell-membrane level.

Animals↗
PubMed1986

Altered catalytic properties of protein kinase C in phorbol ester treated cells.

Exposure of various cell types (rat-1 fibroblasts, bovine adrenocortical cells, human lymphoid cells) to nanomolar concentrations of TPA, resulted in a rapid, apparent loss of cellular protein kinase C content, when the enzyme was assayed by its phospholipid and Ca2+-dependent histone (H1)-kinase activity, following solubilization and DEAE-cellulose chromatography isolation. By contrast, no loss of protein kinase C was detected when the enzyme was probed by its high affinity PDBu binding capacity nor when the kinase activity was assayed with protein substrates other than histones, such as vinculin and a cytochrome P-450. It is concluded that, in addition to the previously reported enzyme subcellular redistribution, following TPA treatment, the phorbol ester induces striking alterations of the cellular protein kinase C catalytic activities. The molecular mechanisms of these changes and their implication in the tumor promotion process remain to be clarified.

Animals↗
PubMed1986

Panel of twenty-five independent man-rodent hybrids for human genetic marker mapping.

To increase the efficiency of the human gene mapping, a panel of 25 man-rodent hybrids was selected out of 200 independent man-rodent hybrids produced in our laboratory since 1969. The hybrid panel was selected in such a fashion as to allow the asignment of a human marker M by respecting the two following complementary criteria: correlation between M and a chromosome and exclusion of the other chromosomes. The panel characteristics allowing the use of these two criteria were presented and discussed. A first series of enzyme markers were analysed to test the validity of the hybrid panel for the human gene mapping. The different possibilities and limits of the hybrid panel were also discussed, especially for the assignment of markers with DNA probes.

Animals↗
PubMed1985

Separation and characterization of a phosphatidylinositol kinase activity that co-purifies with the epidermal growth factor receptor.

Two retroviral protein-tyrosine kinases, v-src and v-ros, have been reported to possess phosphatidylinositol (PtdIns) kinase activity. Because the epidermal growth factor (EGF) receptor is a protein-tyrosine kinase with structural homology to p60v-src and because EGF stimulates PtdIns turnover in A431 cells, the EGF receptor has been examined for PtdIns kinase activity. Preparations of the EGF receptor, isolated from A431 cells and purified by two different methods of affinity chromatography, possessed an associated PtdIns kinase activity. This activity which co-purified with the EGF receptor represented only about 2% of the total PtdIns kinase activity of A431 membranes, and there was no correlation between the number of EGF receptors and the amount of PtdIns kinase activity in membranes from various cell types. A peptide substrate, angiotensin II, and PtdIns did not compete with each other as substrates for the protein-tyrosine and PtdIns kinase activities of the EGF receptor. When self-phosphorylated EGF receptor was fractionated by Sephacryl S-300 gel permeation chromatography, the peak of PtdIns kinase activity was separated from the comigrating peak of protein-tyrosine kinase activity and the self-phosphorylated EGF receptor. These results indicate that the protein-tyrosine kinase and PtdIns kinase activities which co-purify with the EGF receptor reside on different molecules. Angiotensin II and PtdIns did not compete as substrates for p60v-src isolated by immunoabsorption with a monoclonal antibody, suggesting that PtdIns kinase activity may also not be intrinsic to p60v-src.

1-Phosphatidylinositol 4-Kinase↗
PubMed1985

Regulation of the epidermal growth factor receptor by phosphorylation.

The receptor for epidermal growth factor (EGF) is a glycosylated transmembrane phosphoprotein that exhibits EGF-stimulable protein tyrosine kinase activity. On EGF stimulation, the receptor undergoes a self-phosphorylation reaction at tyrosine residues located primarily in the extreme carboxyl-terminal region of the protein. Using enzymatically active EGF receptor purified by immunoaffinity chromatography from A431 human epidermoid carcinoma cells, the self-phosphorylation reaction has been characterized as a rapid, intramolecular process which is maximal at 30-37 degrees C and exhibits a very low Km for ATP (0.2 microM). When phosphorylation of exogenous peptide substrates was measured as a function of receptor self-phosphorylation, tyrosine kinase activity was found to be enhanced two to threefold at 1-2 mol of phosphate per mol of receptor. Analysis of the dependence of the tyrosine kinase activity on ATP concentration yielded hyperbolic kinetics when plotted in double-reciprocal fashion, indicating that ATP can serve as an activator of the enzyme. Higher concentrations of peptide substrates were found to inhibit both the self- and peptide phosphorylation, but this inhibition could be overcome by first self-phosphorylating the enzyme. These results suggest that self-phosphorylation can remove a competitive/inhibitory constraint so that certain exogenous substrates can have greater access to the enzyme active site. In addition to self-phosphorylation, the EGF receptor can be phosphorylated on threonine residues by the calcium- and phospholipid-dependent protein kinase C. The sites on the EGF receptor phosphorylated in vitro by protein kinase C are identical to the sites phosphorylated on the receptor isolated from A431 cells exposed to the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. This phosphorylation of the EGF receptor results in a suppression of its tyrosine kinase and EGF binding activities both in vivo and in vitro. The EGF receptor can thus be variably regulated by phosphorylation: self-phosphorylation can enhance tyrosine kinase activity whereas protein kinase C-catalyzed phosphorylation can depress enzyme activity. Because these two phosphorylations account for only a fraction of the phosphate present in the EGF receptor in vivo, other protein kinases can apparently phosphorylate the receptor and these may exert additional controls on EGF receptor/kinase function.

Adenosine Triphosphate↗
PubMed1984

Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor binding and antagonists of epidermal growth factor-stimulated tyrosine protein kinase activity.

Four mouse monoclonal antibodies specific for human epidermal growth factor (EGF) receptors have been prepared using EGF receptor protein from human A431 epidermoid carcinoma cells as immunogen. We have determined the effect of these antibodies on two known functions of the EGF receptor: EGF binding and tyrosine kinase. Three of these antibodies (225, 528, and 579) are inhibitors of EGF binding, whereas the fourth (455) does not compete for binding but immunoprecipitates the EGF receptor. Inhibition is of the mixed competitive and noncompetitive type. The three competing monoclonal antibodies are antagonists of EGF-stimulated tyrosine protein kinase activity assayed both in intact cells and using an exogenous peptide substrate in solubilized membranes. These immunoglobulins are partial agonists in self-phosphorylation of the EGF receptor in solubilized membranes but exhibit only antagonist activity for this reaction in intact cells. The three competing monoclonal immunoglobulins recognize receptors in variant A431 cells with the same efficiency as in parental A431 cells. Such antagonist monoclonal antibodies can be used to control the concentration of receptors which can be activated by EGF.

Animals↗
PubMed1984

Hormonal regulation of a calcium-activated, phospholipid-dependent protein kinase in bovine adrenal cortex.

The calcium-activated, phospholipid-dependent protein kinase (C kinase) and its proteolytic product (M kinase), originally discovered in central nervous tissue (Takai, Y., Kishimoto, A., Inoue, M., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7603-7610) were characterized in bovine adrenal cortex cytosol. An endogenous calcium-dependent protease able to generate M kinase from the isolated C kinase in vitro was also present in adrenocortical extracts. Bovine adrenocortical cells in suspension as well as in primary culture contain the C and the M kinase activities. Treatment of these cells by steroidogenic concentrations (nM to microM) of ACTH resulted in a time and dose-dependent increase of cytosolic C kinase activity, whereas no change in M kinase activity was detected. This apparent activation appears to result mostly from an intracellular shift of the membrane-associated C kinase to a soluble cytosolic form of the enzyme. These observations open the question of the possible implication of the calcium, phospholipid-dependent protein phosphorylation system in hormone-dependent cellular regulatory processes.

Adrenal Cortex↗
PubMed1984

C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity.

The Ca2+- and phospholipid-dependent protein kinase (C-kinase) binds tightly in the presence of Ca2+ to purified membranes of A431 human epidermoid carcinoma cells. The major membrane substrate for C-kinase is the epidermal growth factor (EGF) receptor. Phosphorylation of the EGF receptor is Ca2+-dependent and occurs at threonine and serine residues. After tryptic digestion of the receptor, three major phosphothreonine-containing peptides were identified. These are identical with three new phosphopeptides present in the EGF receptor isolated from A431 cells treated with either of the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. C-kinase catalyzes phosphorylation at these same sites in purified EGF receptor protein. These results indicate that, in A431 cells exposed to tumor promoters, C-kinase catalyzes phosphorylation of a significant population of EGF receptor molecules. This phosphorylation of EGF receptors results in decreased self-phosphorylation of the EGF receptor at tyrosine residues both in vivo and in vitro and in decreased EGF-stimulated tyrosine kinase activity in vivo.

Animals↗
PubMed1984

Conservation of the human COL1A1-TK-GAA synteny and homoeologous assignment in the African green monkey and the baboon (Cercopithecoidae).

The pro alpha 1 (I) collagen structural gene (COL1A1), the acid alpha-glucosidase (GAA), and the thymidine kinase (TK) genes, known to be closely linked in man (HSA) and mapped to HSA17, were found syntenic in two Cercopithecoidae species, the baboon (Papio papio, PPA) and the African green monkey (Cercopithecus aethiops, CAE) and assigned to homoeologous chromosomes, PPA16 and CAE19, respectively. The assignment of COL1A1 was obtained using two human cDNA probes. Hind III restriction sites found in man were present in the two species. The particular CAE individual used in the experiment showed a polymorphism for one DNA fragment.

Animals↗
PubMed1983

Reversibility of the phosphate transfer between ATP and phosphoproteins catalysed by a cyclic nucleotide independent (G type) casein kinase.

Purified casein kinase G was found able to catalyse the synthesis of [gamma-32P]ATP in the presence of ADP, phosphocasein (previously 32P-labeled by the forward kinase reaction) and magnesium. Apparent Km values of approx. 0.5 mM for phosphocasein and 7.5 mM for ADP were calculated, these values indicating low affinities for the substrates as compared to those exhibited for casein and ATP in the forward reaction. The reverse casein kinase G activity appeared to prefer ADP and GDP as phosphate acceptors. Whereas the casein kinase G reverse reaction could be supported by casein, phosvitin and histone previously phosphorylated by the enzyme, the same proteins could not serve as a phosphate source when previously phosphorylated by the cAMP-dependent protein kinase. Forward and reverse casein kinase G reactions exhibited different optimal pH values (8.5 and 7.2, respectively) and a different sensitivity to Mg2+. Spermine, which activated the kinase activity, blocked the reverse reaction at millimolar concentrations. Although the biological significance of the casein kinase G reverse activity remains to be assessed in intact cell, the process may be useful as a tool in the characterization of phosphorylatable sites in phosphoproteins.

Adenosine Diphosphate↗
PubMed1983

Identification of the catalytic subunit of an oligomeric casein kinase (G type). Affinity labeling of the nucleotide site using 5'-[p-(fluorosulfonyl)benzoyl]adenosine.

Identification of the catalytic subunit of a G type [using guanosine 5'-triphosphate (GTP) as well as adenosine 5'-triphosphate (ATP) as phosphate donor], oligomeric, cyclic nucleotide independent casein kinase purified from bovine lung was carried out after reaction with 5'-[p-(fluorosulfonyl)-benzoyl]adenosine (FSBA) and isolation of the subunit components of the enzyme. FSBA exhibited the major characteristics of an affinity label reacting at the nucleotide (ATP, GTP) site of the casein kinase. FSBA acted as a competitive inhibitor of ATP (and GTP), led to complete inactivation of the enzyme in a reaction showing two kinetic steps, and became irreversibly bound to the protein. After being labeled with FSBA, the casein kinase (apparent molecular weight of 140 000) was separated into its two monomeric components of apparent molecular weights 38 000 (alpha) and 27 000 (beta), respectively, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Use of radioactive FSBA showed that specific affinity labeling was limited to the alpha casein kinase subunit. This result was in agreement with the fact that casein kinase activity was found associated with the alpha monomer after electrophoretic separation of the alpha and beta subunits. It may thus be concluded that the largest (alpha) subunit contains the catalytic site of the casein kinase G. Electrophoretic analysis of purified protein kinase under denaturing conditions suggested an alpha 3 beta 2 combination for an apparent molecular weight of 130 000-140 000. However, a maximum of 2 mol of FSBA could be specifically bound to the alpha subunit per mol of enzyme, with a concomitant complete inactivation. These data would be in agreement with an alpha 2 beta 2 subunit composition for casein kinase G, as proposed by other research groups for a similar type of protein kinase of different sources. These observations suggest that the alpha subunits are functionally similar, each of them containing a nucleotide (ATP, GTP) binding site. The possible role of the beta subunit in the enzyme activity remains to be established.

Adenosine↗
PubMed1983

Catalytic and molecular properties of a highly purified G type casein kinase from bovine lung tissue.

A preparation procedure has been worked out to obtain a highly purified G type (using GTP as well as ATP) casein kinase from large quantities of bovine lung tissue. It included ion-exchange (DEAE and phosphocellulose) and affinity (casein and ATP-Sepharose) chromatography combined with a flocculation step, and yielded an apparently homogeneous preparation with a 16% yield and a purification factor of more than 1400. The purified lung casein kinase used GTP (Km 16 microM) almost as well as ATP (Km 6.7 microM) and exhibited the major catalytic properties of the casein kinase G previously described in bovine adrenal cortex (Cochet, C., Job, D., Pirollet, F. and Chambaz, E.M. (1981) Biochim. Biophys. Acta 658, 191-201). Mg2+ (30-50 mM) and spermine (2 mM) were potent activators of lung casein kinase G activity, whereas the enzyme was inhibited by heparin and quercetin. The purified enzyme underwent self-phosphorylation in the presence of ATP or GTP, serine being the only target amino acid under these conditions, whereas both serine and threonine were phosphorylated by the enzyme in casein. Lung casein kinase G exhibited an apparent molecular weight between 140 000-160 000 upon gel filtration and appeared formed by the association of two different subunits upon SDS-polyacrylamide gel electrophoresis. The two subunits of Mr 38 000 (alpha) and 27 000 (beta) exhibited a 2:1 ratio upon quantitative scanning, suggesting an alpha 3 beta 2 combination in the oligomeric native enzyme structure. Peptide mapping of the two isolated subunits following 125I-labeling and papain digestion did not disclose any common fragment. The casein kinase catalytic activity was found associated with the alpha (38 kDa) enzyme subunit after recovery from gel electrophoresis in the presence of SDS, whereas the 27 kDa (beta) subunit was the major target of the enzyme self-phosphorylation reaction. alpha and beta subunits appeared strongly associated in the oligomeric enzyme and the possible role of the beta subunit in the casein kinase G activity remains to be examined. The purified casein kinase G, which can be obtained by the present procedure, should facilitate the study of the biological significance of this phosphorylation system in the intact cell.

Animals↗