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R Rousseaux-Prevost

Publications and source records attributed to R Rousseaux-Prevost.

At least 19 recordsLinked to original sources
PubMed1998

Y-autosome translocation and infertility: usefulness of molecular, cytogenetic and meiotic studies.

An apparently balanced reciprocal translocation 46,X,t(Y;6) (q11.23 approximately q12;p11.1) was observed in an infertile man with severe oligozooteratozoospermia. Different mitotic chromosome banding patterns were performed and fluorescence in situ hybridization indicated a breakpoint in the fluorescent Yq heterochromatin. Molecular genetic deletion experiments for the azoospermia factor region in distal Yq11 showed the retention of the DAZ gene and meiotic pairing configurations suggested that the man's infertility could be due to the pairing behaviour of the Y;6 translocation chromosome with the X chromosome visualised by synaptonemal complex analysis at the electron microscopy level. The morphological appearance of the normal chromosome 6 and the Y;6 translocated chromosome included in the compartment of the sex vesicle may allow an explanation of the degeneration of most spermatocytes after the pachytene stage.

Adult↗
PubMed1997

Distribution of gelsolin in human testis.

Gelsolin, an actin-binding and severing protein present in many mammalian cells, was characterized in human testis. Although abundant in testicular extracts, gelsolin was not detected in purified spermatogenic cells by immunoblot analysis. Immunofluorescence studies of testis sections showed that gelsolin has two main localizations: peritubular cells and the seminiferous epithelium. In peritubular cells, gelsolin was present together with alpha-SM actin, in agreement with the myoid cell characteristics of these cells. In a large proportion of the tubules, gelsolin was found mainly, together with actin, in the apical part of the seminiferous epithelium. This localization of gelsolin also was observed in seminiferous tubules with a partial or complete absence of germinal cells, which evokes a presence of gelsolin at the apex of Sertoli cells. However, in normal testis, a complex pattern of gelsolin labeling was also present, mostly in the apical third of the epithelium, around cells or groups of cells, mainly spermatids, and, less frequently, in various other localizations from the apical to the basal part of the seminiferous epithelium. Taken together, these observations suggest that gelsolin may play different functions in the seminiferous epithelium: (1) regulation of the dynamic alterations of the actin cytoskeleton in the apical cytoplasm of Sertoli cells, and (2) modification of actin filaments assemblies in specific structures at germ cell-Sertoli cell contacts. Thereby, the actin-modulating properties of gelsolin are probably involved in reorganization of the seminiferous epithelium related to germ cell differentiation.

Animals↗
PubMed1995

Molecular localization of free thiols in human sperm chromatin.

The presence in human sperm nuclear proteins of a limited amount of unoxidized thiol groups, stabilized by reversible binding to zinc ions, has been presumed to play a role in the decondensation of sperm within the oocyte. In the present study, the number and molecular localization of free sulfhydryls in the major proteins of human sperm chromatin, protamines P1 and P2, were determined by alkylation of reactive thiols with 14C-iodoacetamide, isolation of protamines, and peptide mapping. Less than 1.5% of the cysteines of protamines were found as reactive thiols, a proportion strikingly lower than that reported previously for whole human sperm proteins. The amount of sulfhydryls was unaffected by the zinc chelating agent EDTA. Labeling was evenly distributed on every cysteine of protamines P1 and P2. The results confirm the extensive stabilization of sperm chromatin by disulfide bridges and show that the unoxidized cysteines remaining at the end of epididymal transit in some protamine molecules may be one of the six (protamine P1) or five (protamine P2) cysteines present in the sequence of each class of protamines. This even distribution of the reactive cysteines could facilitate decondensation of sperm nuclei initiated by a thiol-disulfide exchange.

Alkylation↗
PubMed1994

Interaction of human P1 and P2 protamines with DNA.

The interaction of two classes of human sperm protamines, P1 (HP1) and P2 (HP2, HP3, HP4), with DNA was investigated. Gel mobility shift assays with a range of DNA fragments of defined sizes show that, whatever its length, all the DNA is complexed with protamines at arginine to phosphate ratio of 0.1. Further addition of protamine molecules leads to a precipitation of the protamine-DNA complexes for arginine-phosphate ratio > or = 1.2. Fluorescence studies using the dye Hoescht 33258 as a fluorophore and DNAase I footprinting experiments suggest that P1 and P2 protamines bind at the DNA surface without apparent location in the minor or major groove of DNA. No differences in protamine-DNA interaction were observed between the two classes of human protamines P1 and P2. Moreover the binding to DNA was not influenced by the presence of zinc which induces the formation of a zinc-finger structure in protamine P2.

Binding, Competitive↗
PubMed1994

Interaction of mammalian sperm nuclear protamines and peptides derived thereof with immobilized zinc.

The interaction of mammalian and human protamines with zinc was studied by immobilized metal ion affinity chromatography (IMAC). The affinity of protamines containing blocked cysteine residues was found to correlate in part with the presence and number of histidine residues in the protamine structure: absence or low affinity of P1 protamines containing 0 or 1 histidine residue; high affinity of human P2 protamine containing 9 histidines. Nevertheless a fraction strongly retained on an IDA-Zn(II) column was observed for P1 protamines with one histidine in the N-terminal sequence (ram and boar protamines). The strong binding was found to be related to the presence of tyrosine, serine and threonine closely spaced to the histidyl side chain. In the case of human protamine P2, the strong retention on the IDA-Zn(II) column seems to result from the additive contribution of all the histidine residues of the molecule. Thus, strong retention of protamines in IMAC seems to depend on an additive contribution of amino-acid side chains: histidine, tyrosine, serine, threonine and perhaps arginine. The high affinity of protamines, more especially P2 protamines, for zinc suggests that this metal ion could play a role for their correct folding and binding to DNA.

Amino Acid Sequence↗
PubMed1992

P2 protamines from human sperm are zinc -finger proteins with one CYS2/HIS2 motif.

P1 (HP1) and P2 (HP2, HP3, HP4) protamines were isolated from human sperm nuclei in the reduced form and their interaction with zinc and cobalt was studied. One zinc atom per molecule of P2 protamines but not of P1 protamine was found. Absorption spectra of P2 protamines with cobalt were characteristic of a tetrahedral complex involving two histidine and two cysteine residues and with one cobalt per molecule. A tetrahedral complex was found neither in P1 protamines nor in P2 protamines alkylated at cysteine or at histidine residues. The zinc finger motif Cys2/His2 of P2 protamines may play a role in stabilization of human sperm chromatin and in inhibition of transcription.

Amino Acid Sequence↗
PubMed1991

The immune response to synthetic peptides of human protamines HP1 and HP2.

Peptides representing the amino-terminal sequence of protamines HP1 (sequence 1-12) and HP2 (sequence 1-11), the two major nuclear proteins of human sperm, have been synthesized. Rabbits were immunized either with peptide conjugated with a carrier or with free peptide. The resulting antisera were examined for their capacities to bind the homologous peptide, other peptides from protamines HP1, HP2, from ram protamine, a protein resembling HP1, and finally with the whole protamine. Only free peptides were immunogenic. Antisera were found to react with the homologous peptide, but also with some other peptides. More especially, antibodies to peptide HP1 1-12 were found to recognize an epitope shared by the homologous peptide, peptide HP1 37-49 and peptide 1-12 of ram protamine. The common antigenic determinant seems to depend on the conformation of the peptides, rather than strictly related to common sequences. Anti-peptide antibodies react poorly and in a non-specific manner with the parent protein. The failure of reactivity with the protamines strongly suggest that these small basic proteins are folded and probably globular molecules in contrast with the totally random model postulated by several previous works.

Amino Acid Sequence↗
PubMed1990

Fractionation of basic nuclear proteins of human sperm by zinc chelate affinity chromatography.

Immobilized metal affinity chromatography was investigated for the fractionation of basic nuclear proteins of human sperm. Human sperm nuclei essentially contain two classes of protamines: a protamine of type P1 (HPl), rich in cysteine but with only one histidine, and three protamines of type P2 (HP2, HP3, HP4), rich in cysteine and histidine (nine in protamine HP2), potential ligands for transition metal ions. The critical conditions for metal affinity chromatography were defined: choice of metal, protein material and buffer, type of elution and sample loading. Chromatography of nuclear proteins, without histones and with cysteine residues alkylated by iodoacetamide, was optimum on zinc Chelating Sepharose in a Tris-acetate buffer and elution with an increasing concentration gradient of imidazole. Under these conditions, the two classes of protamines were completely separated. The intermediate basic proteins were further purified by reversed-phase high-performance liquid chromatography. Heterogeneity of binding to zinc of protamine HP1 was demonstrated. The proposed method is simple and reproducible and the recovery of proteins is high. It may be applied to study the expression and function of P1 and P2 protamines, e.g., in the case of infertile men.

Buffers↗
PubMed1989

Isolation and characterization of a VH domain fragment from monoclonal rat IgG of IgG1 and IgG2a subclasses.

Digestion with trypsin of monoclonal rat IgG of IgG1 and IgG2a subclasses produced two fragments, isolated only in dissociating media. The larger fragment (mol. wt. 120,000 Da) was comprised of the two light chains covalently bound to shortened gamma chains. Amino acid sequence of the shortened gamma chain indicated that the site of cleavage is located at the beginning of the C gamma 1 domain at a position homologous to residue 139 of mouse gamma heavy chain of IgG1 MOPC 21. The smaller fragment (mol. wt. 13,000 Da) was found to consist of the entire variable domain of the heavy chain and probably a short stretch of the C gamma 1 domain. The unique susceptibility of rat monoclonal IgG1 and IgG2a is likely to be the result of the presence of a lysine residue in a loop of C gamma 1 domain, which therefore is accessible to trypsin. Tryptic cleavage of rat monoclonal antibodies of IgG1 and IgG2a subclasses can be considered as a simple method to produce a fragment related to the VH domain.

Amino Acid Sequence↗
PubMed1989

Antibodies to sperm basic nuclear proteins detected in infertile patients by dot-immunobinding assay and by enzyme-linked immunosorbent assay.

The auto-antibody response in infertile men was investigated by means of immunoenzymatic methods, dot-immunobinding assay (DIBA), and ELISA, using, as antigens, human sperm basic nuclear proteins. Comparison was made, for the same patients, with antibody response to membrane antigens, detected by tray agglutination test (TAT), spermotoxic test (STT), and immunobead binding test (IBT). A very good agreement was observed between the two kinds of antibody responses. Thus, an ELISA or a dot-immunobinding test with sperm nuclear proteins may be considered as a simple and sensitive method for detection of auto-antibodies in infertile men. The reactivity in ELISA of various synthetic peptides corresponding to sequences of human protamines HP1 and HP2 was also studied: all the sera containing anti-nuclear antibodies do not react with synthetic peptides. This observation suggests that antibodies to sperm nuclear proteins recognize conformational epitopes that are not present on small synthetic peptides.

Autoantibodies↗
PubMed1987

Studies of the IgE binding sites to rat mast cell receptor with proteolytic fragments and with a monoclonal antibody directed against epsilon heavy chain: evidence that the combining sites are located in the C epsilon 3 domain.

The binding sites of rat IgE to mast cell receptor were investigated by the use of proteolytic fragments and a monoclonal antibody to epsilon chain (MARE-1). Three main fragments were characterized by short-time papain digestion of IgE: F(ab')2-E, a fragment related to the C, 4 domain, and an asymmetric fragment corresponding probably to an IgE molecule with one proteolyzed C, 3 domain. Neither F(ab')2-E nor C, 4 could interfere with the binding of IgE to rat mast cells. These two fragments did not show significant polymerization upon heating at 56 degrees C, while large amounts of polymers were produced from whole IgE, MARE-1 monoclonal antibody was found to react neither with F(ab')2 nor with C, 4, thereby suggesting its interaction with the C, 3 domain. MARE-1 was found to inhibit partially (about 55%) the binding of IgE to its receptor. Taken together the results indicate that the binding sites of IgE to rat mast cell receptor are located within the C, 3 domain. In addition, isolation of the C, 4 domain will be useful to evaluate its participation in the affinity of IgE to receptors of other cells such as lymphocytes or macrophages.

Animals↗
PubMed1984

Influence of vitamin A on the immune response of Schistosoma mansoni-infected rats.

Nutritional (vitamin A levels, weights), parasitological (adult worm burden, count of eggs in liver, stool examination) and immunological (IgE serum levels, anti-Schistosoma mansoni antibodies, lymphocyte stimulation by concanavalin A and S. mansoni antigenic extract) parameters were studied in three groups of rats, a non-infected and normally fed control group, a S. mansoni-infected but normally fed group, and a S. mansoni-infected group with experimentally induced vitamin A deficiency. The number of worms was found significantly higher in the third (53 +/- 19) than in the second group (2 +/- 2) (p less than 0.001). There were many eggs in the liver surrounded by granulomatous reactions in the third group (399 +/- 73 epg liver). All stool examinations were negative. IgE levels and anti-S. mansoni antibody titres were significantly lower (p less than 0.001) in the third than in the second group. The concanavalin A lymphocyte stimulation indexes did not differ significantly between groups 2 and 3; the S. mansoni lymphocyte stimulation index was only significantly positive in group 3 (p less than 0.001). These results indicate a decrease in the humoral immune response without alteration of cellular immune response in vitamin A-deficient rats infected with S. mansoni.

Animals↗
PubMed1982

Conformational studies of rat monoclonal immunoglobulin E.

Two monoclonal IgEs (IR 2 and IR 162) were studied in terms of their conformational features by circular dichroism and differential u.v. absorption. The studies were performed both on the native proteins and under different conditions known to affect the biological properties of IgE (heating at 56 degrees C, acid pH, and presence of reducing agents). Whereas IgE IR 162 appeared to undergo significant conformational changes upon heating and at acid pH, IgE IR 2 was found to be unaffected or less affected by these treatments. The two monoclonal IgEs were found to be equally effective in blocking passive sensitization by IgE antibodies. However, the blocking capacity of IgE IR 162 was affected much more by heating at 56 degrees C than that of IgE IR 2.

Animals↗
PubMed1981

Tryptic cleavage of rat IgG: a comparative study between subclasses.

Monoclonal rat IgG belonging to the 4 rat IgG subclasses, and some IgG subclasses isolated from normal rat serum were subjected to enzymatic degradation with trypsin. Differences in the products of tryptic digestion were observed according to the IgG subclass. IgG2b and IgG2c were degraded mainly into Fab and Fc fragments. IgG1 and IgG2a appeared resistant to such cleavage. However, tryptic digestion of the latter two produced two fragments separated only in dissociating media. Results of the studies suggest that one of the fragments (mol. wt. 13,000) probably consists of most of the variable domain of the gamma heavy chain, while the second (mol. wt. 120,000) consists of the IgG deleted of the VH regions.

Animals↗
PubMed1978

IgE in experimental schistosomiasis. II. Quantitative determination of specific IgE antibodies against S. mansoni: a follow-up study of two strains of infected rats. Correlation with protective immunity.

Parasite specific IgE antibodies in rats infected with Schistosoma manoni were measured by passive cutaneous anaphylaxis (PCA) reactions and by the technique of immuno-adsorption. Two strains, one a low IgE producer (Fischer rats) and the other a high IgE producer (Hooded-Lister rats) were studied. In Fischer rats, a time course study of the occurrence of IgE antibodies and resistance to reinfection was made. Parasite specific IgE levels measured by immuno-adsorpiton were much lower than total IgE levels and a similar percentage of specific IgE (about 8%) was in the two strains.IgE antibodies were maximum at day 30 and day 60 after infection; however, a third peak at day 90 was observed only in Fischer rats. Some discrepancies between results obtained by PCA and immunosorbent techniques have been observed, which could be explained by differences in the affinity of IgE antibodies during infection or by the presence of total IgE in the PCA assay. There was a close parallelism between specific IgE antibodies levels and the course of immunity in Fischer rats. This parallelism supports the view that IgE could play a pre-eminent role in protective immunity in rat schistosomiasis.

Animals↗
PubMed1978

Circulating antigens and immune complexes in Schistosoma mansoni-infected rats. Characterization by radioimmunoprecipitation-PEG assay (RIPEGA).

Circulating schistosome antigens (CSA) and circulating immune complexes (CLC) were investigated in rats infected with Schistosoma mansoni. The radioimmunoprecipitation-polyethylene glycol (PEG) assay (RIPEGA), with 125I-labelled anti-S. mansoni anti-serum, detected CSA during two distinct periods of the infection; the first between the 11th and the 14th week of infection and the second between the 11th and 14th week after infection. The CH50 deviation test revealed the presence of CIC in sera from infected rats, approximately at the two periods when CSA were detected. At 6 weeks of infection, the levels of CIC in infected rats were not different from those in control rats. However, a more sensitive method characterized IgG2a in C1q-binding C1C from infected rats. At weeks 5 and 6, IgE immune complexes were also detected in the serum from infected rats. In fact, the use of RIPEGA on the material eluted from infected rat serum after passage through an anti-IgE immunosorbent showed the presence of schistosome antigen at week 4, and at higher levels at week 6. Levels of 50% haemolytic complement in infected rat serum were lowered between the 2nd and the 4th week, the 5th and the 8th week and after the 12th week of infection. The possible role played by CIC in the protective mechanisms to a S. mansoni challenge infection in rats is discussed.

Animals↗
PubMed1977

IgE in experiemental schistosomiasis. I. Serum IgE levels after infection by Schistosoma mansoni in various strains of rats.

Serum IgE levels were measured by radioimmunoassay in four strains of rats, before and after infection by Schistosoma mansoni. Differences were observed in basal IgE levels, depending on the strain considered. Serum IgE rose 10--20 days after infection and the maximum level, between days 30 and 50, ranged from 14 to 60 times the basal level. One of the most characteristic features of IgE production in rat schistosomiasis was the persistence of high IgE levels for a long time after rejection of the worms.

Animals↗