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Biomedical subjects

D Bellet

Publications and source records attributed to D Bellet.

At least 55 records · Page 3Linked to original sources
PubMed1994

Cloning and sequencing of the equine testicular follitropin receptor.

To investigate the possibility that specific structural determinants within the equine follitropin receptor (eFSHR) are critical to the enhanced specificity of this receptor compared to other FSHRs, we used the RACE-PCR technique to clone the eFSHR from equine testis. Sequence analysis revealed that the eFSHR is highly homologous to other mammal FSHRs, but it presents 10 unique amino acid residue replacements in the extracellular domain. Furthermore, a potential N-glycosylation site was detected at a position not encountered in other receptors. Northern blot analysis identified three transcripts of 4.2 kb, 2.3 kb and 1.0 kb in horse testis.

Amino Acid Sequence↗
PubMed1994

Immunochemical mapping of human lutropin: I. Delineation of a conformational antigenic determinant.

Lutropin (LH), follitropin (FSH), thyrotropin (TSH) and choriogonadotropin (CG) are assembled of two non-covalently alpha (alpha) and beta (beta) subunits. We studied the discontinuous antigenic regions recognized by a monoclonal anti-hLH antibody designated as LH05 which binds to hLH, hCG and hTSH and does not cross-react with either the free subunits or hFSH. LH05 and an antibody designated HT13, recognizing an epitope partly comprizing the alpha64-76 region, did not bind simultaneously to hCG. Furthermore, LH05 was unable to combine with an anti-peptide antibody (LHP03) directed to residues 43-52 of hLHbeta. Thus, LH05 recognizes an epitope partly overlapping with those recognized by HT13 and LHP03. Using various hybrid molecules, we showed that the human alpha-subunit plays a critical role in the assembly of the epitope that, in contrast, contains amino acid residues conserved in the various beta-subunit of several species. Together, our results suggest that the amino acids Leu49-Pro50, Tyr59-Arg60 and Leu86-Ser87 in the hLHbeta and the alpha64-76 region are probably included in the epitope recognized by LH05 which appeared to be not accessible on the CG/LH receptor.

Epitope Mapping↗
PubMed1994

Immunochemical mapping of human lutropin: II. Characterization of two monoclonal antipeptide antibodies reacting with the native beta-subunit.

To investigate the epitopes present on the beta-subunit of the human lutropin (hLHbeta) and their topographical relationship at the surface of the molecule, we produced two monoclonal antipeptide antibodies, designated LHP03 and LHP04, capable of binding to the radiolabeled 125I-hLHbeta and directed to the 43-52 and 110-117 regions of the hLHbeta, respectively. Analysis of the accessibility of the epitopes on hLH and on the beta-subunit of human chorionic gonadotropin (hCGbeta), equine LH (eLHbeta) and ovine LH (oLHbeta) indicated that: (i) LHP03 binds to both the free hLHbeta subunit and dimeric hLH whereas LHP04 binds preferentially to the free hLHbeta, (ii) LHP03 recognizes weakly the hCGbeta and oLHbeta in comparison to hLHbeta and (iii) LHP04 binds oLHbeta as well as hLHbeta but does not bind to hCGbeta and eLHbeta. The topographical relationship of epitopes recognized by LHP03 and monoclonal antibodies recognizing dimer specific epitopes on hLH allowed us to localize discontinuous antigenic sites that overlaps or are located outside the hHLbeta(43-52) region. Together, our results demonstrated that the hHLbeta(43-52) portion is accessible on both the free hLHbeta subunit and hLH whereas the COOH-terminal portion, hHLbeta(110-117), is probably buried at the alpha/beta interface of the hormone.

Antibodies, Monoclonal↗
PubMed1993

Expression of the human follicle-stimulating hormone receptor in the baculovirus system.

Human follitropin receptor (hFSHR) cDNA was inserted into the genome of Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. Spodoptera frugiperda (Sf9) insect cells expressed the hFSHR at the membrane level, 2-3 days after infection. Ligand binding analysis demonstrated saturable, high-affinity binding of 125I-labeled recombinant hFSH (Kd = 0.17 nM) as well as binding specificity for both human and equine FSH. Interestingly, a highly purified preparation of equine choriogonadotropin was found to compete with the binding of 125I-rhFSH. Finally, Western-blot analysis, using a monoclonal antipeptide antibody directed to the 178-206 region of the hFSHR, revealed two bands migrating at M(r) 77 and 130 kDa.

Animals↗
PubMed1993

[Biological markers of cancer. Critical study].

The clinical use of tumor-associated markers still raises several problems, due to the lack of specificity of most biological markers and to insufficient evaluation of their true benefit for the patients. Only two markers, calcitonin and alpha-fetoprotein, markers of medullary thyroid carcinomas and of hepatocellular carcinomas respectively, have been proved useful in screening high risk populations for tumors. The usefulness of the prostate specific antigen in screening for prostatic cancer is still debated. Human chorionic gonadotropin and its free beta subunit are useful in the early detection of testicular cancer. Other biological makers, such as CA 15-3 for breast cancers, CA 19-9 for either gastric or pancreatic cancers, anc CA 125 for ovarian tumors are useful mostly in the follow-up of these tumors. Finally, measurements of tumor markers and analysis of their results must be performed by biologists or physicians who use tumor-associated markers routinely.

Biomarkers, Tumor↗
PubMed1993

Influence of protein-quaternary structure on antigen processing.

T cell recognition of the quaternary structure of human chorionic gonadotropin (hCG), resulting from the association between its alpha (hCG-alpha) and beta (hCG-beta) subunits, was analyzed using hCG-alpha and hCG-beta T cell hybridomas produced in BALB/c mice. First, the fine specificity of these T cell hybridomas was determined, enabling us to divide hCG-alpha-specific T cell hybridomas into two groups. Group I recognized the hCG-alpha(61-81) region, and group II responded to the hCG-alpha(50-70) part of the molecule. Two groups of hCG-beta-specific T cell hybridomas, designated groups III and IV, were analyzed and found to respond to the C- and the N-terminal parts of the hCG-beta(1-22) peptide, respectively. Moreover, we observed that the nature of APC influenced Ag recognition by hCG-beta T cell hybridomas from group IV, but not by other selected T cell hybridomas. We then showed that recognition of the hCG alpha/beta dimer by alpha-specific T cell hybridomas was dramatically reduced compared to both free hCG-alpha and heat-dissociated hCG alpha/beta molecules. In contrast, hCG-beta hybridomas exhibited comparable responses to the free beta subunit and the hCG dimer. Experiments using a dimeric molecule assembled from the alpha-subunit of human follicle-stimulating hormone, which is identical to hCG-alpha, and the beta-subunit of human follicle-stimulating hormone, which is homologous to hCG-beta, confirmed that the three-dimensional structure of the complex rather than the primary structure of the beta-subunit plays a critical role in the processing pathway. Finally, kinetic experiments showed that the presentation of hCG-alpha T cell epitopes differed depending upon whether the alpha-subunit was in its free or combined form. In contrast, the kinetic expression of hCG-beta T cell epitopes appeared to be independent of the quaternary structure of hCG. Thus, conformational alterations resulting from the alpha/beta subunit association mainly influenced processing of the alpha-subunit in its complexed form, rather than processing of the combined beta-subunit. The effect of protein-quaternary structure on T cell recognition may represent a new element in our understanding of the processing and presentation of oligomeric molecules.

Animals↗
PubMed1993

Deficient antigen processing of a protein quaternary structure can be overcome by receptor-mediated uptake.

Human chorionic gonadotropin (hCG) is a dimer of non-covalently associated alpha (hCG-alpha) and beta (hCG-beta) subunits. This molecule was used to study whether receptor-mediated uptake influences the presentation of a protein quaternary structure. Unprimed splenocytes and a B cell lymphoma were capable of presenting only the free (hCG-alpha) but not the combined (hCG) alpha subunit to hCG-alpha T cell hybridomas, while hCG-alpha-primed lymph node cells (LNC) responded to both hCG-alpha and hCG. As antigen (Ag)-specific antigen-presenting cells (APC) present in the hCG-alpha-primed LNC population may be potentially effective for presenting hCG, we investigated the role of specific Ag capture, through mIg and Fc gamma R, in the processing and presentation of hCG and hCG-alpha to HAG5, a T cell hybridoma directed against the immunodominant region (amino acids 61-81) of hCG-alpha. Results showed that only B cells bearing membrane immunoglobulin capable of recognizing hCG-alpha and hCG, and present in hCG-alpha-primed mice, were extremely effective in presenting the free as well as the combined alpha subunit. The effect of FcR-mediated uptake was analyzed using a B cell line transfected with the Fc gamma RII-B2 gene to present immune complexes of either hCG-alpha or hCG. We found that hCG-alpha and hCG were presented equally well, whatever the Ag-binding site of each antibody to hCG or its alpha subunit. Using HBG 6, an hCG-beta T cell hybridoma, we performed similar experiments with the Fc gamma RII-B2 cell line and determined that the potentiation of hCG presentation to HBG 6 was similar to that observed with HAG 5. Then kinetic experiments were performed to examine the effect of Ag uptake through FcR on processing. Results demonstrated that the uptake pathway drastically influenced the expression of alpha T cell determinants in the alpha/beta dimer. In addition, treatment with cycloheximide, a protein synthesis inhibitor, only impaired the ability of APC to present specifically captured Ag. Thus, the processing pathway for specifically captured Ag might be different from the pathway used to process nonspecifically captured Ag. This observation might explain why receptor-enhanced uptake bypasses the inefficient processing of the hCG quaternary structure and enables similar efficiency in the presentation of alpha and beta T cell specificities. These findings provide new insight into the antigenicity of oligomeric molecules, which is modified whether antigen capture is specific or not.

Animals↗
PubMed1993

Immunochemical studies of equine chorionic gonadotropin (eCG), eCG alpha, and eCG beta.

The equine (e) placental glycoprotein hormone eCG plays a critical though not completely understood role during the first trimester of gestation in mares. In the present work, we have developed immunoradiometric assays (m-IRMAs) for detection of eCG, eCG alpha, and eCG beta using combinations of monoclonal antibodies (mAbs) specific for epitopes that reside on free and/or combined subunits. The free eCG alpha m-IRMA was based on AHT20 mAb, specific for the free alpha-subunit of all species, and 125I-labeled ECG01 mAb, which recognizes both free and combined alpha-subunit from equine and primate species. The free eCG beta was measured by the combination of FBT11 mAb, which binds to free beta-subunit from human and equine species, and radiolabeled 518B7 mAb, which detects CG/LH from diverse mammalian species. This assay provided a better sensitivity for eLH beta than for eCG beta. However, after treatment with neuraminidase, the latter molecule was recognized as well as eLH beta, indicating that the carbohydrate structure influenced the binding of mAbs. Detection of eCG was based on the combination of ECG01 mAb (anti-alpha) as capture antibody and 125I-labeled 518B7 mAb (anti-beta). Using such assays, we measured plasma and urinary concentrations of both eCG and free subunits in pregnant mares from mating to day 90 of gestation. eCG was constantly detectable in the serum between days 40-90, as previously reported, but small amounts of the dimeric hormone in the urine were also detectable. Further, m-IRMA specific for the free beta-subunit showed that low levels (5-100 ng/ml) of eCG beta may coexist with eCG in serum and urine during early pregnancy in mares. In contrast, free eCG alpha subunit was undetectable during the first 10 weeks of gestation. These results suggested that eCG and free subunit production in pregnant mares at the beginning of gestation is similar to that observed in pregnant women. These immunoassays, specific for either intact hormone or its free subunits, constitute useful diagnostic tools for investigating reproductive problems in mares.

Animals↗
PubMed1993

Simple sequence repeat polymorphism within the p53 gene.

This report describes a new polymorphism, in intron 3 of the p53 gene, which consists of a single repeat of 16 nucleotides, absent in the published wild-type p53 gene sequence. In the Caucasian population tested (n = 82), 28% of individuals were heterozygotes for this polymorphism. Using PCR-based analysis, we were able to demonstrate p53 allelic losses in three of six breast tumors from heterozygote patients tested.

Base Sequence↗
PubMed1992

[Biology of the trophoblastic tissue and placental tumors].

The trophoblast of early placenta has many attributes of malignant tissue: it displays highly proliferative and invasive properties and expresses hormones, growth factors, growth factor receptors and oncogene products. Moreover, this tissue may have an autocrine control of growth. Collectively, these properties are similar to those of malignant tissues and the normal trophoblast is then considered as a "pseudomalignant" tissue. Moreover, either benign (hydatidiform mole) or malignant (choriocarcinoma) trophoblastic disease may be developed from the trophoblastic cells. In this article, the biological features of both the normal and the tumoral trophoblast will be presented. Finally, the trophoblast is a model and a source of molecules of biological interest.

Female↗
PubMed1992

Monoclonal antibodies to the free beta-subunit of human chorionic gonadotropin define three distinct antigenic domains and distinguish between intact and nicked molecules.

The present study was designed to characterize monoclonal antibodies (mAbs) specific for the free beta-subunit of hCG (free hCG beta), to develop two-site immunoradiometric assays (m-IRMAs) specific for free hCG beta, and to study the reactivities of various molecular forms of hCG beta in these assays. We attempted first to delineate the antigenic regions present specifically on the free hCG beta by studying the binding pattern of seven mAbs directed preferentially to hCG beta, designated FBT11, P8E, P10F, HB2, P5D, P5H, and INN-64. Competitive inhibition experiments performed by RIA demonstrated the specificity of these mAbs for the free hCG beta as noncross-reacting with the beta-subunit of human (h) LH beta. m-IRMAs were used to analyze the arrangement of epitopes on hCG beta. Experiments performed with the seven mAbs used either as capture antibodies or radiolabeled indicators confirmed the specificity of the seven mAbs for the free hCG beta, and that mAbs FBT11, P8E, and P10F bound to equine LH beta (eLH beta), but did not bind to a fragment of hCG beta called the beta-core fragment (beta CF). These antibodies defined an antigenic domain identified as A. In contrast, mAb HB2 bound neither to eLH beta (e beta) nor to beta CF and was directed to domain B (e beta negative, beta CF negative). Finally, mAbs P5D, P5H, and INN-64 bound to beta CF, but did not bind to eLH beta, and defined a third domain identified as C (e beta negative, beta CF positive). Collectively, these results demonstrate that at least six antigenic domains are present on the free hCG beta and that a limited set of amino acids was shared among these domains; domains A, B, and C are present only on the free beta-subunit, while three other domains recognized by mAbs FB19, FBT10, and 518B7 are present on both free hCG beta and hCG. Thus, most of the surface of the hCG beta appears to be antigenic and accessible to antibody binding. Three different m-IRMAs specific for free hCG beta were then constructed using either mAb FBT11 (domain A) or HB2 (domain B). The study of the reactivities of various molecular forms of hCG beta in these assays demonstrated that the recognition of hCG beta forms nicked at position 43 (beta 43), 44, and/or 51 (beta 44/51) varied between assays.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1992

[Stomach adenocarcinomas: comparison between CA 19-9 and carcinoembryonic antigen for the diagnosis of recurrences after surgical treatment].

The aim of this study was to compare carcinoembryonic antigen (CEA) and CA19-9 for the detection of local recurrences and distant metastases after complete resection of gastric carcinoma. At least one postoperative measurement of CEA and CA19-9 was performed in 54 patients. Among these, 32 had recurrence (59%) with a median follow-up of 618 days. Significantly higher sensitivity was observed for CA19-9 in comparison with CEA (68.8% vs 38.2% respectively), but specificity was slightly lower (81.8% vs 95.6% respectively). Increased CEA plasma level never preceded the diagnosis of recurrence while increasing CA19-9 preceded diagnosis in 13 patients (40.6%) from 1 to 22 months (median = 4.5 months). Increasing the normal range of CA19-9 to 80 UI/mL (2.5 x N) raises the specificity to 100% with acceptable sensitivity (53.1%). This study shows that CA19-9, compare with CEA, allows diagnosis of recurrence more often and earlier in the follow-up of resected gastric cancer.

Adenocarcinoma↗