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J Garnier

Publications and source records attributed to J Garnier.

At least 19 recordsLinked to original sources
PubMed1992

Development of an extended simulated annealing method: application to the modeling of complementary determining regions of immunoglobulins.

An extended simulated annealing process (ESAP) has been developed in order to obtain an ensemble of conformations of a peptide segment from a protein fluctuating at a given temperature. The annealing process was performed with a fast Monte Carlo method using the scaled collective variables developed by Noguti and Go. The system was divided into two parts: one consists of one or more peptide segments and is flexible around the main-chain and side-chain torsional angles; the other represents the rest of the molecule and was maintained fixed at the atomic positions determined by x-ray experiments. The target function included the nonbonding atomic interactions and a distance function to anchor the N and C terminal ends of each segment to the fixed part. Three systems of complementary determining regions (CDR) of antibodies were tested and compared to x-ray data: L2 loop (7 residues) of the light chain of lambda-type Bence-Jones protein, H1 and the H2 loops (14 residues) of McPC603, and H1 and H2 loops (12 residues) of HyHEL-5. Each state of CDR conformations was characterized at room temperature by the average of their coordinates (average conformation) and the internal energy. With a limited number of annealing processes (10), starting from the extended conformation, we have obtained states with conformations close to the observed x-ray structures, from 1.1 to 1.7 A root mean square deviation (rmsd) of main-chain atoms depending on the system. These states were identical or within 0.25 A rmsd of those of lowest internal energy. For unknown CDR structures the criteria of lowest internal energies from ESAP can be used to predict hypervariable loop structures in antibodies with an accuracy comparable to other methods.

Amino Acid Sequence↗
PubMed1992

LH receptor RNA and protein levels after hormonal treatment of porcine granulosa cells in primary culture.

Granulosa cells were prepared from small follicles (less than 3 mm) from the ovaries of 5-month-old gilts. They were cultured in plastic dishes coated with a synthetic adhesion peptide in a chemically defined medium supplemented with 2% serum substitute. After 3 days of culture, the cells reached confluence and expression of the LH receptor could be stimulated in a hormonally defined medium. LH receptor RNAs were estimated by autoradiography using Northern blots and dot blots of total cell RNA. LH receptor RNAs were hybridized with a homologous 32P-labelled random-primed DNA probe. The LH receptor was measured using 125I-labelled human chorionic gonadotrophin (hCG) as tracer. Northern blots of LH receptor RNAs revealed a predominant signal of 4.4 kb and two less-intense hybridization bands of 7.5 and 1.9 kb. The 4.4 kb band was used for quantification of LH receptor RNAs because it was the most intense and may be attributed to the full-length messenger RNA. In these conditions, after 72 h stimulation, FSH (0.6 nM), insulin (5 micrograms/ml), oestradiol (30 nM) and deoxycorticosterone (0.3 nM) yielded high LH receptor RNA levels (eight times unstimulated cell level), while dibutyryl cyclic AMP (1 mM), cortisol (5.4 nM), thyroxine (100 nM) and epidermal growth factor (16 pM) gave low LH receptor RNA levels (one to five times). However, the respective amounts of the receptor RNA did not give yield to the same proportion of LH receptor for every factor, indicating some post-transcriptional regulations. The kinetic study of the production of the LH receptor obtained in a defined medium supplemented with FSH, oestradiol and insulin showed that the receptor appeared after 48 h of stimulation and reached a maximum of about 7000 receptors per cell at 72 h. The three hybridization bands on Northern blots evolved in parallel and appeared as early as 24 h. They were at maximal level from 24 to 48 h of stimulation. When the granulosa cells were pulse-treated for 2 h with cycloheximide (10 micrograms/ml), they exhibited a transient rise in LH receptor RNA content which was followed by a delayed receptor increase especially at 72 h of stimulation. Taken together, these results indicate that the LH receptor in primary culture of granulosa cells seems to be regulated by different physiological factors both at the transcriptional and the translational levels.

Amino Acid Sequence↗
PubMed1991

Influence of the local amino acid sequence upon the zones of the torsional angles phi and psi adopted by residues in proteins.

A set of parameters is derived to express the influence of the local amino acid sequence on the torsional angles phi and psi adopted by each residue in a protein. The formalism used, which is based on information theory, evaluates the probability for a given residue to be in a particular zone of the Ramachandran map. Comparisons with crystallographic structures suggest that the method can extract almost all of the available information from the local sequence and show that the local sequence carries only, on average, about 65% of the information necessary for specifying the conformation of a given residue in a protein. The rest is specified by long-range interactions that are specific for each protein fold. The parameters derived here provide a more detailed description of the prediction than other methods in allowing the allocation of the torsional angles for residues having an aperiodic structure and are intended to be used for directing the conformational search in a subsequent simulation of the three-dimensional structure. This method should also predict segments of the polypeptide chain that are the most stable and thus less sensitive to long-range interactions.

Amino Acid Sequence↗
PubMed1991

Towards understanding the glycoprotein hormone receptors.

Lutropin (LH), follitropin (FSH) and thyrotropin (TSH), as well as choriogonadotropin (CG, which binds to the LH receptor) constitute the glycoprotein hormone family. Their 3 receptors have been cloned during the last few months. They belong to the large group of G-protein coupled membrane proteins, with their specific N-terminal domain likely to bind the hormone and the characteristic 7 membrane-spanning segments in their C-terminal moiety. The present review discusses the main results of amino acid sequence analysis performed on the glycoprotein hormone receptors. The putative extracellular head exhibits less than 45% homology over the 3 receptors, while approximately 70% residue conservation is found in the transmembrane moiety. Here only, limited sequence homologies (approximately 20%) can be found with other G-protein coupled receptors. The secondary structure predictions performed on the 3 receptors revealed that the polypeptide sequence predicted as ordered (either alpha-helix or beta-strand) were repeated evenly throughout the extracellular head with a period of approximately 25 amino acids. This analysis helped to define the intervening loops between this ordered stretches as potential candidates for bearing at least part of the binding site of the hormones. Some of the perspectives opened by the cloning of the receptors are described, like the production of the extracellular head of the porcine LH receptor in baculovirus-infected insect cells, and the exploration of the LH receptor's mechanism of functioning as a dimer.

Amino Acid Sequence↗
PubMed1991

Endocytosis of the lutropin receptor is mediated by a low affinity binding site.

Porcine Leydig cells in primary culture were incubated to equilibrium with increasing doses of either porcine luteinizing hormone (pLH) or human chorionic gonadotropin (hCG). Then, free and total high affinity gonadotropin receptors on the cell surface were dosed with [125I]hCG as tracer. In isotonic high salt medium, the pLH concentration for half receptor occupancy (Kocc) and for half receptor endocytosis (Kendo) were nearly indistinguishable (1-3 x 10(-7) M). But, when the medium was changed to an isotonic low salt buffer, Kocc shifted to 1.2 x 10(-9) M and Kendo to 1.5 x 10(-8) M. However, with hCG, both values were largely independent of the ionic strength (Kocc = 10(-10) M and Kendo = 10(-8) M). The fact that Kendo is higher than Kocc suggests that the endocytosis of the high affinity gonadotropin receptor is controlled by the hormone binding to another lower affinity site.

Animals↗
PubMed1991

The protein structure code: what is its present status?

Current methods of prediction of protein conformation are reviewed and the algorithms on which they rely are presented. For non-homologous proteins and after cross-validation the reported methods exhibit a probability index, i.e. the per cent of correctly predicted residues per predicted residues, of 63-65% with a standard deviation of the order of 7% for three conformational states--helix, beta-strand and coil. This present limitation in the accuracy of predictions that use only the information of the local sequence can be related essentially to the effect of long-range interactions specific for each protein family. The methods based on sequence similarity can improve the accuracy of prediction by expressing explicitly the homology of the protein to be predicted with proteins in the database. In these circumstances the probability index can reach 87% with a standard deviation of 6.6%. This property can be used for modeling homologous proteins by aiding in amino acid sequence alignments. The prediction of the tertiary structure of a protein is still limited to the case of modeling a structure based on the known three-dimensional structure of a homologous protein.

Algorithms↗
PubMed1991

[Adenocarcinoma of the rete testis. Review of the literature apropos of a new case].

The authors report the case of a 66 year old man with a Boden stage I adenocarcinoma of the rete testis treated by orchiectomy and adjuvant chemotherapy. Lung metastases developed 46 months later and were responsible for death despite further chemotherapy. Adenocarcinoma of the rete testis is an exceptional tumour, as only 23 cases satisfying the criteria defined by Feek and Hunter have been reported in the literature. The prognosis is poor even in the apparently localized forms (5 year survival less than 25%) and local recurrences and lung and/or hepatic metastases are frequent. Radiotherapy and chemotherapy appear to have little value.

Aged↗
PubMed1990

Peptide mapping of intersubunit and receptor interactions of human choriogonadotropin.

Seven peptides covering the entire sequence of human choriogonadotropin (hCG) alpha-subunit, eight peptides covering the hCG beta-subunit sequence and two peptides, one of human beta-lutropin and one of beta-thyrotropin were synthesized. We checked their ability to prevent reassociation between hCG alpha- and beta-subunits and between hCG and its receptor. Only the alpha 1-22, alpha 59-92 and beta 1-16 peptides inhibited the reassociation between the alpha- and beta-subunits of hCG with an ED50 of respectively 2 mM, 2 mM and 4 mM. Using porcine Leydig cells in primary culture, we showed that alpha 33-59, alpha 41-59 and beta 1-16 peptides decreased both the specific binding to the cell surface and the internalization of [125I]hCG and [125I]porcine LH with ED50 of 0.3, 0.1 and 0.5 mM, respectively. From these results, the following minimal area may be assigned, (i) to the alpha-beta interaction: alpha 5-16, alpha 52-72 (or alpha 59-70) and beta 8-16, and (ii) to the hormone-receptor association: alpha 41-45 and beta 8-16.

Amino Acid Sequence↗
PubMed1990

Protein structure prediction.

Current methods developed for predicting protein structure are reviewed. The most widely used algorithms of Chou and Fasman and Garnier et al for predicting secondary structure are compared to the most recent ones including sequence similarity methods, neural network, pattern recognition or joint prediction methods. The best of these methods correctly predict 63-65% of the residues in the database with cross-validation for 3 conformations, helix, beta strand and coli with a standard deviation of 6-8% per protein. However, when a homologous protein is already in the database, the accuracy of prediction by the similarity peptide method of Levin and Garnier reaches about 90%. Some conclusions can be drawn on the mechanism of protein folding. As all the prediction methods only use the local sequence for prediction (+/- 8 residues maximum) one can infer that 65% of the conformation of a residue is dictated on average by the local sequence, the rest is brought by the folding. The best predicted proteins or peptide segments are those for which the folding has less effect on the conformation. Presently, prediction of tertiary structure is only of practical use when the structure of a homologous protein is already known. Amino acid alignment to define residues of equivalent spatial position is critical for modelling of the protein. We showed for serine proteases that secondary structure prediction can help to define a better alignment. Non-homologous segments of the polypeptide chain, such as loops, libraries of known loops and/or energy minimization with various force fields, are used without yet giving satisfactory solutions. An example of modelling by homology, aided by secondary structure prediction on 2 regulatory proteins, Fnr and FixK is presented.

Algorithms↗
PubMed1990

Monoclonal antibodies against luteinizing hormone receptor. Immunochemical characterization of the receptor.

Human CG (hCG)-receptor complexes were solubilized from porcine testicular membranes. They were chromatographed on an immunomatrix of Affi-Gel 10-D1E8 anti beta-hCG monoclonal antibody (this antibody has been shown not to interfere with hCG binding to receptor). Elution was performed at alkaline pH, a condition in which hCG-receptor complexes are relatively stable. Immunization of a mouse with these partially (approximately 15%) purified hormone-receptor complexes allowed the preparation of 20 different hybridomas, each secreting antireceptor antibodies. The latter were used for receptor characterization. Immunoblot of testicular membrane extracts or of purified receptor preparations showed the presence of a major band at approximately 85,000 mol wt and minor bands at approximately 68,000 mol wt and approximately 48-45,000 mol wt. The width of all these bands suggested some microheterogeneity possibly due to glycosylation. The same approximately 85,000 mol wt receptor was seen in ovarian membranes, but no detectable antigen was observed in liver, muscle, and kidney membranes. An immunoaffinity method (using antibody LHR 38) was devised to purify the receptor in a single step. This demonstrated that the purified receptor preparation did not contain any protein component other than those detected by immunoblot. Comparison of receptors purified by immunoaffinity chromatography using either antireceptor or antihormone monoclonal antibodies showed in both cases the presence of the 85,000 mol wt and 48-45,000 mol wt species, but the absence, in the latter case, of the 68,000 mol wt species. This suggests that the 68,000 mol wt receptor cannot bind hormone and does not form oligomers with other receptor species.

Animals↗
PubMed1990

Secondary structure prediction and protein design.

For non-homologous proteins, and after cross-validation, the methods reviewed in this article exhibit a probability index (percentage of correctly predicted residues per predicted residues) of 59-65.5% according to the methods employed with a standard deviation of 7% for three conformational states: alpha-helix, beta-strand and coil. These present limitations in the accuracy of prediction are related both to the limited number of known structures and to the effect of long-range interactions. The methods based on sequence similarity can improve the accuracy of prediction by expressing the homology of the protein to be predicted explicitly with proteins in the database. Under these circumstances, the probability index can reach 87% with a standard deviation of 6.6%. This property was used for modelling homologous proteins by assisting in amino acid sequence alignments. Examples will be given for the alignment of serine proteinases.

Amino Acid Sequence↗
PubMed1990

Are receptor-associated nuclear proteins associated with the earliest effects of steroid hormones?

The functional importance of the interaction of hsp90 with receptors for steroid hormones in the action of these hormones has been suggested. This hypothesis, although not yet proven, is supported by new data obtained in our laboratory and in those of others, whereas no conflicting experimental results have been presented. Our recent studies have dealt with the cloning of hsp90, transfection of normal and mutated receptors, the effects of the antihormone RU486 and immunohistochemistry.

Animals↗
PubMed1990

Are receptor-associated nuclear proteins associated with the earliest effects of steroid hormones?

The functional importance of the interaction of hsp90 with receptors for steroid hormones in the action of these hormones has been suggested. This hypothesis, although not yet proven, is supported by new data obtained in our laboratory and in those of others, whereas no conflicting experimental results have been presented. Our recent studies have dealt with the cloning of hsp90, transfection of normal and mutated receptors, the effects of the antihormone RU486 and immunohistochemistry.

Animals↗
PubMed1989

Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain.

Complementary DNA clones, encoding the LH-hCG (luteinizing hormone-human choriogonadotropic hormone) receptor were isolated by screening a lambda gt11 library with monoclonal antibodies. The primary structure of the protein was deduced from the DNA sequence analysis; the protein contains 696 amino acids with a putative signal peptide of 27 amino acids. Hydropathy analysis suggests the existence of seven transmembrane domains that show homology with the corresponding regions of other G protein-coupled receptors. Three other types of clones corresponding to shorter proteins were observed, in which the putative transmembrane domain was absent. These probably arose through alternative splicing. RNA blot analysis showed similar patterns in testis and ovary with a major RNA of 4700 nucleotides and several minor species. The messenger RNA was expressed in COS-7 cells, yielding a protein that bound hCG with the same affinity as the testicular receptor.

Amino Acid Sequence↗
PubMed1989

The cDNA-derived amino acid sequence of chick heat shock protein Mr 90,000 (HSP 90) reveals a "DNA like" structure: potential site of interaction with steroid receptors.

We report cDNA sequence, the complete derived aa sequence, and a predicted secondary structure of the chick hsp 90, a protein which has been found to form complexes with steroid hormone receptors. The modelling of the most negatively charged "region A" indicates that the alpha-helices of this portion of hsp 90 mimick DNA configuration. We propose that this region can, in absence of hormone, interact with and cap the positively charged DNA-binding domain of steroid receptors.

Amino Acid Sequence↗
PubMed1989

A highly charged sequence of chick hsp90: a good candidate for interaction with steroid receptors.

The sequence of the entire chick 90 kDa heat shock protein (hsp90), the non hormone binding component of the heterooligomeric form of steroid receptors, is reported. A comparison of the amino acid sequence of the chick hsp90 to that of the homologous hsp90 from yeast to man, reveals 64-96% identity respectively, and even with E. coli hsp90 an identity of 44% is observed. Analysis of the sequence and a secondary structure prediction of chick hsp90 suggest that two hydrophilic regions A and B, predicted in alpha-helix may play a role in the interaction of hsp90 with other proteins such as steroid hormone receptors. While there are regions of the sequences completely conserved in all hsps90, the most negatively charged hydrophilic region (A) is absent in the E. coli protein.

Algorithms↗
PubMed1989

Dual internalization pathway for lutropin and choriogonadotropin in porcine Leydig cells in primary culture.

The role of the high affinity receptor in the internalization of porcine lutropin (pLH) and human choriogonadotropin (hCG) by porcine Leydig cells in primary culture during short-term stimulation by the two hormones was investigated. The fate of the hormones was followed either by electron microscopy (with colloidal gold-labeled hormones) or by measurement of the cellular distribution of [125I]pLH and [125I]hCG. With both techniques, the internalization of pLH was found to be one order of magnitude greater than hCG, though the recycling rate of the high affinity receptors was the same with both hormones. However, when the cell surface was progressively depleted of its high affinity receptors by preincubation with increasing doses of hCG or pLH, the internalization of [125I]pLH remained high and largely independent of the number of high affinity receptors still available on the cell surface, while that of [125I]hCG was found to be proportional to this number. The endocytosis of [125I]pLH could only be inhibited by the simultaneous presence of micromolar concentrations of unlabeled pLH, hCG or alpha or beta subunits of ovine LH (oLH). The intact alpha-hCG subunit and the deglycosylated alpha-oLH subunit were less potent, while beta-hCG and deglycosylated beta-oLH had no significant effect. These results could be explained by the existence of a "carrier" or "scavenger" receptor for LH, but with a low affinity (congruent to 3.10(6) M-1) and present in excess on the cell surface as compared to the high affinity receptor. The possible physiological significance of this receptor is discussed.

Animals↗