Search PubMed⌕ Search

Biomedical subjects

C Rougeot

Publications and source records attributed to C Rougeot.

At least 55 records · Page 3Linked to original sources
PubMed1988

In vitro degradation of extracellular matrix with Mr 52,000 cathepsin D secreted by breast cancer cells.

It has been proposed that proteases secreted by cancer cells facilitate metastasis by degrading extracellular matrix. Estrogen receptor-positive breast cancer cells secrete a Mr 52,000 pro-cath-D under estrogen stimulation, whereas this protease is produced constitutively by estrogen receptor-negative cancer cells. We report on the degradation in vitro of extracellular matrix by purified Mr 52,000 cathepsin D (cath-D) and by conditioned media prepared from different cell lines. The purified Mr 52,000 pro-cath-D was autoactivated at pH 4.5 into a Mr 51,000 cath-D and found to digest the extracellular matrix of endothelial bovine corneal cells labeled with [3H]proline or [35S]methionine. Culture medium conditioned by estrogen-treated MCF7 cells had a similar effect at pH 4.5 but not at pH 7.4. Matrix degradation was totally inhibited by pepstatin. Other breast cancer cells (BT20, MDA-MB231, T47D cells, etc.) and other cancer cells also secreted a pepstatin-sensitive proteinase able to degrade extracellular matrix. By contrast, the U2 variant of MCF7 cells, which lacks the Mr 52,000 cath-D gene, and the nontumoral epithelial mammary cells secreted a negligible amount of this proteinase. In all conditioned media, the pepstatin-dependent extracellular matrix degrading activity was highly correlated to the Mr 52,000 cath-D concentration measured by immunoenzymatic assay. We conclude that the Mr 52,000 cath-D is the major acidic protease secreted by mammary cancer cells. We suggest that this protease may degrade basement membrane and consequently facilitate tumor invasion when it is released in an acidic microenvironment.

Breast Neoplasms↗
PubMed1988

Inhibitory effect of platelet-activating factor (PAF) on luteinizing hormone-releasing hormone and somatostatin release from rat median eminence in vitro correlated with the characterization of specific PAF receptor sites in rat hypothalamus.

Platelet-activating factor (PAF) exhibits a wide range of biological activities, including the stimulation of secretory processes in various cell types. However, little is known regarding its possible influence on the release of brain neuropeptides. In the present study we have examined the effect of PAF on the release of three hypothalamic releasing hormones in adult male rats, and have characterized the presence of specific PAF binding sites in rat hypothalamic membranes. PAF decreased LHRH and somatostatin (SRIF) release from the median eminence with a maximal inhibition at 10(-14) M for both neuropeptides, whereas GRF release was not significantly altered. Moreover, PAF strongly counteracted the Ca2+ ionophore A 23187-stimulated release of LHRH and SRIF from median eminence and medial basal hypothalamus (greater than 50% inhibition). These results suggest an involvement of Ca2+ dependent events in PAF action. This inhibitory effect was specifically exerted at a hypothalamic site because PAF failed to depress LH and GH release from the anterior pituitary. A specific, reversible and saturable binding of [3H]PAF to membrane preparations of rat hypothalamus was demonstrated and two classes of binding sites were characterized. The affinity (KD) of each binding class was 2.14 +/- 0.32 nM and 61.63 +/- 16.4 nM, respectively, and the corresponding maximal number of each binding class was 25.41 +/- 3.2 fmol/mg protein and 146.2 +/- 47.5 fmol/mg protein. In the same conditions no specific binding was observed using rat pituitary membranes. The specificity of PAF analogs for these binding sites was well correlated to their relative effectiveness in altering LHRH and SRIF release (order of potency: L-652,731, kadsurenone greater than BN 52021 greater than Lyso-PAF). These data suggest that the binding sites identified in the hypothalamus have the characteristics expected of a specific PAF receptor and that PAF effect on neuropeptides release is a receptor-mediated process.

Animals↗
PubMed1987

Binding in vitro of pipequaline (45319 RP) onto plasma proteins and blood cells in man.

Serum binding of pipequaline, a new anxiolytic drug, was studied in vitro by equilibrium dialysis. The percent binding in serum is high, 96.3%, and remains constant within the range of therapeutic concentrations. Investigations performed on isolated proteins with a wide range of concentrations showed one site with a high affinity constant (Ka = 450,000 M-1) for alpha 1-acid glycoprotein and two sites with a lower affinity constant (Ka = 58,000 M-1) for human serum albumin. Binding to lipoproteins was saturable, with an affinity constant of 22,000 less than or equal to Ka less than or equal to 35,000 M-1. Over the range of therapeutic concentrations, the ratio of pipequaline concentrations in serum and red blood cells remained constant (14.4%) and was shown to be dependent on the free fraction of pipequaline in serum.

Adult↗
PubMed1987

Pulsatile secretion of growth hormone and insulin in relation to feeding in rats.

In unrestrained male Wistar rats chronically implanted with intracardiac catheters, blood samples were taken every 20 min throughout the 24 h of the diurnal cycle. Plasma concentrations of growth hormone (GH), insulin, and glucose were measured. The pattern of food intake was continuously monitored. The existence of 3-h pulsatile cycles of GH secretion was confirmed. In addition, short bursts of insulin secretion were observed in the middle of every second GH peak-to-peak interval. Food intake appeared to be enhanced during short periods that corresponded with GH release into the blood and was reduced during the GH peak-to-peak periods in which the bursts of insulin secretion were observed. From these observations this study draws a schematic relationship between the rhythmicity of the secretion of GH and insulin and the probability of occurrence of feeding. We speculate that the rhythmic endocrine activity may be causally related to feeding.

Animals↗
PubMed1986

Purification and first characterization of the secreted and cellular 52-kDa proteins regulated by estrogens in human-breast cancer cells.

An estrogen-regulated 52-kDa glycoprotein secreted by MCF7 breast cancer cells was first purified from serum-free conditioned medium by concanavalin-A--Sepharose (ConA--Sepharose). The 13% pure protein was then used to obtain monoclonal antibodies to the 52-kDa protein [Garcia et al. (1985) Cancer Res. 45, 709-716]. Using ConA--Sepharose and monoclonal antibody affinity chromatographies, the secreted 52-kDa protein was finally purified to homogeneity as verified by silver staining of sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and one single N-terminal amino acid. The purification factor was approximately 1400 and the yield 40%. The same two-step procedure, applied to MCF7 cell extracts, yielded four immunologically related proteins of 52 kDa, 48 kDa, 34 kDa and 17 kDa, which were purified 1250-fold with a yield of 30%. These components were further separated by high-performance liquid chromatography gel filtration under denaturing conditions. The final products were homogeneous on the basis of silver-stained SDS-PAGE and gel filtration. However, isoelectrofocusing showed that the pI of the secreted 52-kDa protein and the cellular 34-kDa protein varied from 5.5 to 6.5. Amino acid analysis of the secreted and the related cellular 34-kDa protein is given. Western immunoblotting, pulse chase studies and post-translational studies indicate that the 52-kDa protein is the precursors of a lysosomal enzyme which is partially secreted and partially processed into smaller cellular forms.

Amino Acids↗
PubMed1986

ELISA for detection of human antibodies to Chlamydiae.

An enzyme-linked immunosorbent assay for the detection of human antibodies to Chlamydiae is described which exploits the cross-react properties between the genus-specific antigen of Chlamydiae and the ReLPS constituent of the outer membrane of a Salmonella minnesota mutant. Of 100 random sera tested by ELISA-ReLPS and immunofluorescence 78% showed an absolute correlation, 15% were positive in immunofluorescence and negative in ELISA and 7% were positive in ELISA and negative in immunofluorescence. Furthermore results obtained by the ELISA-ReLPS on 55 sera from patients with clinical evidence of Chlamydiae infection correlated well with the values obtained by an ELISA using Chlamydia-coated microtitration plates and by two immunofluorescence tests using Chlamydia trachomatis and Chlamydia psittaci as antigens. The method described here is sensitive, simple, reproducible and may be employed for epidemiological and pathogenetic studies of chlamydial infections.

Antibodies, Bacterial↗
PubMed1986

Transhybridomas from fusion gene transgenic lymphocytes can be used to produce foreign protein of biological interest.

A method is described for achieving the efficient production of proteins of biological interest by the establishment of hybridomas from lymphocytes of transgenic animals carrying a fusion gene having promoter-regulatory sequences functional in lymphocytes fused to the coding sequences of the protein to be produced. While possible improvements in the method are discussed, it is anticipated that the method will ultimately be applicable to the production of any protein of interest.

Animals↗
PubMed1986

Radioimmunoassay of [D-Trp6]-luteinizing hormone-releasing hormone: its application to animal pharmacokinetic studies after single injection and long-acting formulation administration.

A sensitive radioimmunoassay (RIA) for [D-Trp6]-luteinizing hormone-releasing hormone (LHRH) has been developed. This assay allowed measurement of the LHRH analog in unextracted plasma with a minimum detectable concentration of 10 pg/ml. Validation of plasma assays was performed through Sep-Pak and HPLC purification. The in vivo fate of the peptide was investigated in dogs after subcutaneous or intravenous injections. In both cases, the LHRH analog showed longer plasma half-life than native LHRH with an elimination half-life superior to 80 min. Long-acting formulations were tested in dogs and rats: the day following administration, [D-Trp6]-LHRH plasma level rose to 2.9-4.6 ng/ml in dogs and 0.8-3.8 ng/ml in rats. From day 4 to day 30, [D-Trp6]-LHRH plasma level followed a plateau with concentrations of 0.3-0.8 ng/ml in dogs and 0.2-0.4 ng/ml in rats. In parallel, testosterone plasma concentration was reduced to castrate level between day 4 and day 7 in dogs and was significantly lowered in rats. This sensitive [D-Trp6]-LHRH RIA will be particularly useful for the evaluation of long-acting formulations in patients with advanced prostate cancer.

Animals↗
PubMed1984

Decreased corticotropin-releasing factor-like immunoreactivity in rat intermediate and posterior pituitary after stalk section.

We determined the corticotropin-releasing factor(CRF)-like immunoreactivity by radioimmunoassay in intermediate and posterior pituitary lobes of stalk-sectioned and sham-operated control rats. The antigenic determinant read by the CRF antibody used was contained within the region of amino acids 26 (Gln) to 37 (Leu) of the molecule. Intermediate and posterior lobes of control rats contained similar amounts of CRF (591 +/- 78 and 487 +/- 34 pg/mg protein, respectively). The section of the pituitary stalk produced a marked decrease in CRF-like immunoreactivity in both structures. The CRF content on the intermediate lobe after stalk section was 62 +/- 17 pg/mg protein, a decrease of 90%, and that of the posterior lobe was 90 +/- 13 pg/mg protein, an 83% decrease. Our results suggest that most of the CRF-like immunoreactivity in the rat intermediate and posterior pituitary lobes is contained in nerve fibers of brain origin and support the hypothesis of a role of CRF in the release of intermediate and posterior lobe peptides.

Animals↗
PubMed1983

High, vasopressin-reversible, immunoreactive somatostatin in specific hypothalamic nuclei of rats with diabetes insipidus (Brattleboro rats).

Somatostatin content was studied in brain nuclei and pituitary lobes of homozygous Brattleboro rats lacking vasopressin (DI rats) with and without vasopressin replacement, and their corresponding controls, heterozygous Brattleboro (HZ) rats and Long-Evans (LE) rats. High, vasopressin-reversible, somatostatin levels occurred in DI rats. The changes in somatostatin were localized to the nucleus periventricularis and to brain areas receiving somatostatin projections from this nucleus, including the eminentia mediana. In contrast, vasopressin administration failed to alter somatostatin levels in HZ rats. In brain areas not belonging to the somatostatin periventricular system, the peptide concentration in DI rats was not higher than that of LE controls. High somatostatin levels were present in the intermediate lobe of the pituitary gland. Somatostatin was higher in DI and HZ rats when compared to LE rats. In the posterior pituitary lobe, HZ rats had higher, and DI rats lower, somatostatin than control LE rats. These results suggest that rat brain and pituitary vasopressin and somatostatin systems are interrelated.

Animals↗
PubMed1983

Presence of somatostatin, enkephalins, and substance P-like peptides in cultured neurons from embryonic chick cerebral hemispheres.

The presence of peptides in pure cultures of neurons from 8-day-old chick embryo cerebral hemispheres has been investigated by means of specific radioimmunoassays and chromatographic purification. Somatostatin, Met-enkephalin, Leu-enkephalin, and substance P immunoreactive substances have been detected in 8-day-old cultures grown in serum-free culture medium. The peptides were present in the cellular extracts, as well as in the culture medium extracts. beta-Endorphin, thyroliberin, luteinizing hormone-releasing hormone, and ACTH could not be detected. The largest amount was accounted by somatostatin (48 +/- 2 ng/mg protein). Some 60% of the somatostatin-immunoreactive material was found in the culture medium. Met-enkephalin, Leu-enkephalin, and substance P were present at lower concentrations: 1.61 +/- 0.27, 0.24 +/- 0.02, and 0.14 +/- 0.005 ng/mg protein, respectively. The identities of somatostatin- and enkephalin-immunoreactive materials were confirmed by high pressure liquid chromatography. The findings suggest that cultured neurons that express dopaminergic and GABAergic properties contain peptides similar, if not identical, to somatostatin, Met-enkephalin, Leu-enkephalin, and substance P.

Animals↗