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R Lecocq

Publications and source records attributed to R Lecocq.

At least 37 records · Page 2Linked to original sources
PubMed1986

Differential protein synthesis in the induction of thyroid cell proliferation by thyrotropin, epidermal growth factor or serum.

Protein synthesis in the G1 period of the cell cycle has been investigated using two-dimensional gel electrophoresis in primary cultures of dog quiescent thyroid cells, incubated in defined medium and induced to proliferate by the combined action of thyrotropin (TSH), epidermal growth factor (EGF) and serum or by each of these agents, acting alone. The analysis of the proteins, pulse-labeled for 3 h with [35S]methionine, in quiescent cells deprived of serum and in cells that had been stimulated for various periods of time by the addition of TSH, EGF and serum showed maximal modifications before entry into S phase: the labeling of at least ten proteins was enhanced while that of at least six proteins was decreased. The synthesis of one of these proteins (protein 1; Mr approximately equal to 81 000) was maximal 9-12 h after stimulation by the proliferative agents but began to decrease at 15-18 h and was still decreased at 29-32 h. The study of the effect of each of the proliferation agents alone on the labeling of these sixteen proteins showed that TSH specifically stimulated the labeling of eight polypeptides (proteins 2-9) and that, in contrast, EGF and serum specifically increased the labeling of two other proteins (proteins 1 and 10). The labeling of one protein was decreased by each of the different agents (protein 6') while TSH specifically decreased the labeling of four polypeptides (proteins 1'-4') and increased the labeling of one polypeptide (protein 5') whose synthesis was decreased by EGF and serum. The specific effect of TSH on one protein labeling (protein 7; Mr approximately equal to 39 000) was potentiated by EGF and serum while the specific effect of EGF and serum on another protein labeling (protein 1) was potentiated by TSH. There is thus a correlation between the level of synthesis of these two proteins and the proliferative state of the cells, which is much greater when the stimulating agents are acting together. The induction of protein 1 synthesis by EGF was no longer observed when the cells were no longer proliferating. In the same way, TSH no longer stimulated the synthesis of protein 7 in thyroid cells at confluence. In conclusion, the present study has identified some proteins (proteins 1 and 7) which, as judged by the peculiar stimulation and the kinetics of their synthesis, could be part of the final key events triggering DNA replication in thyroid cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Thyrotropin modifies the synthesis of actin and other proteins during thyroid cell culture.

Primary cultures of dog thyroid cells have been used to study the effects of thyrotropin on the synthesis of proteins. The cells were cultured for 4 days in serum-free and thyrotropin-free conditions. Thyrotropin was then added for varying periods of time (6-96 h). In the absence of thyrotropin, the cells have an elongated flattened aspect. Exposure to thyrotropin for 6-24 h produces retraction and rounding up of cells whereas cells incubated with thyrotropin for longer periods of time have an epithelial cuboidal shape. After varying periods of culture the cells were labelled with [35S]methionine for 6 h and then analyzed by one- and two-dimensional gel electrophoresis, followed by autoradiography. The results were as follows. After exposure to thyrotropin for 32 h and 48 h, the synthesis of about 18 proteins was increased while that of about 14 others was decreased. After 6 h the labelling of three and five of these proteins was already increased or decreased, respectively. Some of the proteins whose synthesis is modified in the presence of thyrotropin were identified. Actin synthesis was markedly decreased with a maximum 24-48 h after the addition of thyrotropin. A modification in the ratio between alpha and beta tubulins was also observed together with very large changes in a group of proteins having both the relative molecular mass (30 000-40 000) and the isoelectric points of tropomyosins. Forskolin and cholera toxin caused the same qualitative and quantitative changes as thyrotropin; this suggests that the regulation by thyrotropin of the synthesis of several thyroid cell proteins is mediated by cAMP. In conclusion, the data obtained in this work might help to explain the molecular mechanisms by which thyrotropin (and cAMP) triggers the changes in cell shape which occur during thyroid cell culture. They also indicate that one of the main effects of thyrotropin takes place at the level of several proteins which belong to the cytoskeleton and which are involved in the definition of the cytostructure of the thyroid cells.

Actins↗
PubMed1985

Analysis of proteins synthesized by fibroblasts from patients with cystic fibrosis by two-dimensional gel electrophoresis and double label autoradiography.

Mucoviscidosis, the most frequently lethal genetic syndrome of Caucasian population, is a recessive disease with multiple tissue involvement. Although the major pathological changes are observed in lungs and pancreas, abnormalities have also been detected in several other exocrine glands. For many reasons, such as the ready availability of tissue material, the absence of secondary changes and the potential for prenatal diagnosis, cultured skin fibroblasts could be the tissue of choice to search for the primary defect. Several abnormalities have been reported in CF fibroblasts, suggesting that the genetic abnormality is expressed in these cells. To search for potentially mutant protein(s) we have compared the protein composition of normal and CF fibroblasts by two dimensional gel electrophoresis and double-labeling autoradiography using 35S and 75Se methionine as tracer. The results demonstrate the power of the method; however, we have not found one protein spot consistently missing in CF cells. Possible reasons for the absence of a single common identifiable defect are discussed.

Adolescent↗
PubMed1985

Characterization of thyroxine-binding globulin secreted by a human hepatoma cell line.

T4-binding globulin (TBG) is a glycoprotein synthesized by the liver and is the principal carrier of T4 and T3 in serum. In this report, we demonstrate that the Hep G2 cell line, derived from a human hepatoblastoma, synthesizes and secretes TBG, the properties of which were characterized. Hep G2 cells secreted TBG into the medium after more than 100 transfers in tissue culture conditions. At confluency and after changing to serum-free culture conditions, TBG accumulation into the medium was linear for 3 days and constituted approximately 0.16% of the proteins synthesized over 24 h. Its abundance relative to albumin is 10-fold greater than that found in normal human serum. TBG secreted by the Hep G2 cells was indistinguishable from native normal human serum TBG, as determined immunologically, by electrophoresis on polyacrylamide gel in denaturing and nondenaturing conditions, and by isoelectric focusing. It also specifically bound T4 and T3, albeit with slightly reduced affinity, and had increased heat lability. Although slightly different from normal serum TBG in caucasians, the physical and biological properties of the Hep G2-derived TBG are similar to those of the variant TBG found in the serum of some healthy Australian Aborigines.

Carcinoma, Hepatocellular↗
PubMed1985

Patterns of protein synthesis in endometrial tissues from ovariectomized rats treated with oestradiol and progesterone.

Groups of ovariectomized rats were taken as controls or given hormonal treatment mimicking the successive steps in the sequence of ovarian secretions leading to implantation. Total endometrium or separated epithelium and stroma were incubated in vitro with [35S]methionine. Dissolved proteins were submitted to two-dimensional polyacrylamide gel electrophoresis (pH range 5-7), followed by autoradiography. Priming with oestradiol (2 days) and subsequent treatment with progesterone (3 days) enhanced the synthesis of 12 and 14 polypeptides, respectively, which are specific for each of these treatments. Progesterone also suppressed the production of 10 oestrogen-dependent proteins both in the epithelium and the stroma. When an oestrogen-progesterone-oestrogen treatment was given, synthesis of all but 4 of the progesterone-induced polypeptides in the epithelium was inhibited while 5 of the proteins abolished by progesterone in this tissue compartment reappeared. These results are compatible with a mechanism of implantation acting at the epithelial level by lifting of intrauterine inhibition and stimulation by embryotrophic substances.

Animals↗
PubMed1982

Pattern of protein phosphorylation in rat hepatocytes stimulated by glucagon or by the Ca2+-linked hormones.

We have adapted the high-resolution electrophoretic technique of O'Farrell to analyze phosphorylated proteins from rat hepatocytes. Total proteins were extracted from rat hepatocytes which had been incubated in the presence of [32P]phosphate and with two types of stimuli: glucagon on the one hand and the Ca2+-linked hormones on the other hand. About 200 phosphorylated polypeptides have been separated. Glucagon modifies the incorporation of [32P]phosphate in at least 17 polypeptides and dibutyryladenosine 3',5'-monophosphate mimics this hormonal effect, implying a common mechanism of action. Phenylephrine (in the presence of the beta-antagonist propranolol), vasopressin and angiotensin all modify the incorporation of [32P]phosphate in about 13 polypeptides; since the Ca2+ ionophore A23 187 reproduces the effect of these agents it may be concluded that Ca2+ mediates their effect. Not all the substrates affected by the two types of hormones are identical. Both types of stimuli increase the phosphorylation of a same set of seven proteins and decrease the phosphorylation of a same set of three proteins but seven proteins have their phosphorylation uniquely enhanced by glucagon whereas three other specific proteins get more phosphorylated by the Ca2+ -linked hormones. The clear differences between the patterns of protein phosphorylation observed in the presence of glucagon and dibutyryladenosine 3',5'-monophosphate on the one hand and by the Ca2+-linked hormones on the other hand strongly suggest different mechanisms of action for these two types of stimuli.

Angiotensin II↗
PubMed1982

Changes in the pattern of endometrial protein synthesis during decidualization in the rat.

Ovariectomized rats were given hormonal treatment mimicking progestational ovarian secretions. At maximal sensitivity a decidual reaction was induced by scratching the endometrium. After incubation of tissue with [35S]methionine, proteins were extracted from decidualizing and control endometrium and submitted to two-dimensional polyacrylamide gel electrophoresis (pH range 5--7), followed by staining or autoradiography. A total of about 800 different peptides could be distinguished on the gels. By 24 h after the decidual stimulus the most prominent changes were the appearance of 4 new peptides (mol. wt 12 000, 25 000, 42 000 and 56 000) and the disappearance of 5 others (mol. wt 31 000, 35 000, 100 000, 110 000 and 140 000). This new pattern remained grossly unchanged up to 72 h after decidual induction.

Animals↗
PubMed1979

Nature of the phosphorylated 26 000 molecular weight protein extracted from dog thyroid with contractile proteins.

Dog thyroid contractile proteins are characterized by their ATPase activity at high KCl concentration. In the presence of Ca(2+), 80 nmol ATP are hydrolyzed per min per mg protein. This Ca(2+) -ATPase activity is inhibited by Mg(2+) but not influenced by sodium azide. The 26 000 molecular weight protein which is present in thyroid contractile protein preparations and the phosphorylation of which is stimulated by thyroid stimulating hormone (TSH) is suggested to be identical to the lysine-rich histones (H1). Indeed, radioactive thyroid H1 histones added to unlabelled thyroid slices copurify with the contractile proteins and migrate at the same level as the 26 000 molecular weight when submitted to electrophoresis in polyacrylamide sodium dodecyl sulfate gels of different acrylamide concentrations.

Animals↗
PubMed1979

Pattern of protein phosphorylation in intact stimulated cells: thyrotropin and dog thyroid.

Two-dimensional, high-resolution electrophoretic technique of O'Farrell has been adapted to the analysis of thyroid phosphorylated proteins. Proteins were extracted from dog thyroid slices which had been incubated in the presence of [32P]phosphate with thyrotropin or with different agents which enhance the intracellular accumulation of cyclic AMP. About 350 phosphorylated polypeptides have been separated. Thyrotropin stimulates the phosphorylation of at least eight of these polypeptides. An increase in the phosphorylation of the same polypeptides was observed was observed when dog thyroid slices were incubated with dibutyryl adenosine 3':5'-monophosphate, cholera toxin or prostaglandin E1 instead of thyrotropin. Our results confirm that most of dog thyroid protein phosphorylation is independent of cyclic AMP. They offer a first link between the action of cyclic AMP on protein kinase and the physiological effects of thyrotropin. They strongly substantiate the hypothesis that most thyrotropin effects are mediated by cyclic AMP.

Animals↗
PubMed1978

Action of thyrotropin on phosphate incorporation into thyroid proteins in vitro.

It is now well established that cAMP is the intracellular mediator of many effects of thyrotropin on the thyroid. Greengard has postulated that all the effects of cAMP inside the cell are secondary to the phosphorylation of proteins by cAMP activated protein kinase(s). The purpose of our work is to define, in an intact cell system, the nature of the thyroid proteins, the phosphorylation of which is stimulated by cAMP and TSH.

Animals↗
PubMed1977

Thyrotropin stimulation of a thyroid contractile protein phosphorylation.

Contractile proteins were isolated from preincubated dog thyroid slices by a procedure identical to the one used for the preparation of muscle actomyosin. At least two of these proteins (molecular weight = 26000 and 15000) were phosphorylated in the intact cell system. After one hour of incubation of the slices with thrytropin (10 mU/ml) and [32P]phosphate, the specific activity of the 26000-Mr protein was increased by a factor of three while no significant change in specific activity was observed in the 1500-Mr contractile protein. This effect of thyrotropin was already observed after 30 min of action, was elicited at hormone concentrations of the same order as those required to induce secretion and has been reproduced by dibutyryl adenosine 3':5'-monophosphate. Cycloheximide (0.35 mM) which almost totally inhibited protein synthesis in thyroid slices, did not affect the thyrotropin stimulation of the phosphorylation of the 26000-Mr protein. The phosphorylation of serine residue(s) has been demonstrated in this protein but the presence of radioactive phosphothreonine could not be detected. On the basis of its molecular weight, the 26000-Mr protein could be similar to the inhibitory component of muscle troponin complex (TN-I).

Animals↗
PubMed1977

Thyrotropin stimulation of the phosphorylation of serine in the N-terminal of thyroid H1 histones.

The analysis of thyroid nuclear proteins by polyacrylamide gel electrophoresis has demonstrated that thyrotropin and dibutyryl adenosine 3':5'-monophosphate stimulate specifically the phosphorylation of H1 histones in an intact cell system. This effect does not require new protein synthesis and implicates the phosphorylation of secrine residue(s) situated in the N-terminal part of H1 histones.

Animals↗