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M Ferrand

Publications and source records attributed to M Ferrand.

34 records · Page 2Linked to original sources
PubMed1991

Stored mRNA in early embryos of a fern Marsilea vestita: a paternal and maternal origin.

In early embryos of the fern Marsilea vestita, we have shown that transcription did not begin until the 8-cell stage, even though protein synthesis was taking place. As in animal species, the pre-existence of stored maternal mRNA has been suspected in embryonic cells. To investigate the presence of such long-lived mRNA we have used in situ hybridization method using [3H]polyuridylic acid, [3H]poly(U), as a probe. Temporal and spatial changes in the distribution of poly(A)+ RNA were followed throughout all the different phases of embryogenesis. Poly(A)+ RNA was present in the cytoplasm of both unfertilized and fertilized eggs and early embryos, while the nucleus exhibited no, or a moderate, [3H]-poly(U) binding activity. New poly(A)+ RNA was detected in the nucleus only at the 16-cell stage, when cytological differentiation of the root apical cell became morphologically detectable. After in situ hydridization with the labeled probes, the numerous silver grains detected autoradiographically over the mature sperm, indicate the presence of poly(A)+ RNA molecules associated with the male genome. We discuss the possible role of poly(A)+ RNA of maternal origin in supporting early translation prior to synthesis of new mRNA. Additional studies are needed to elucidate the role of stored paternal mRNA.

Autoradiography↗
PubMed1989

Relationship between immunological structure and biochemical properties of human thyroid peroxidase.

Although the primary structure of human thyroid peroxidase (hTPO) has been recently deciphered, little is known about its spatial conformation. Such information is of crucial importance in any attempt to relate the structure with the function of hTPO. To probe the antigenic surface of hTPO and to correlate its immunological structure to its biochemical properties, we used 13 monoclonal antibodies (mAb) displaying various affinity for hTPO. Criss-cross experiments showed 7 clusters of reactivity which were interpreted as reflecting 7 epitopes on the surface of the hTPO molecule. Extending our analysis to partial and nonsymmetrical cross-reactivities, these epitopes were shown to be localized in 4 antigenic domains of the hTPO. We further investigated the nature of these 7 hTPO epitopes by testing mAb binding to peroxidases from various origins and chemically modified hTPO; 3 epitopes were shown to be evolutionary conserved, and 5 resistant to reduction and denaturation. We also analyzed the role of the hTPO epitopes in the enzymatic activity and autoimmune targeting of the molecule. Nine epitopes were shown to be localized at the vicinity of both catalytic sites as the binding of their respective mAb modulated the enzyme activity. Autoantibodies from patients presenting with autoimmune thyroid disorders were essentially directed to epitopes similar or adjacent to those recognized by 8 of the 13 mAb and present on only 2 antigenic domains of hTPO. Taken together these data allowed us to propose a tentative map of the surface of the hTPO molecule which associates its epitopic structure with its biochemical functions.

Antibodies, Monoclonal↗
PubMed1988

Novel routine assay of thyroperoxidase autoantibodies.

This radioimmunoassay was developed for specific and large-scale routine measurement of autoantibodies to thyroperoxidase (TPO), an enzyme recently identified as the thyroid microsomal antigen. Because of the scarcity of purified thyroperoxidase, we did not base the assay on the antigen-coated method but rather on autoantibody inhibition of the binding of labeled TPO to a solid-phase-bound monoclonal antibody to TPO. This assay design ensured highly specific measurements without interference from irrelevant thyroid antigens and autoantibodies. When we used affinity-purified autoantibodies to TPO as standards, the range of the curve extended over 10(3)-fold differences in the autoantibodies' concentrations, which allowed us to assay most sera without dilution. Within- and between-assay coefficients of variation (CVs) ranged from 6.1% to 11.5% and from 6.6% to 12.0%, respectively. The correlation between anti-TPO and antimicrosomal autoantibodies, as assessed by hemagglutination test, was highly significant (r = 0.90, P less than 0.0001). This assay is sensitive, easy to perform, and requires only trace amounts of purified TPO.

Antibodies, Monoclonal↗
PubMed1987

Thyroid peroxidase is the organ-specific 'microsomal' autoantigen involved in thyroid autoimmunity.

Autoantibodies (aAb) in serum of patients with autoimmune thyroid diseases (AITD) are directed to an antigen associated with thyroid microsomes. Although it has been investigated over almost three decades, the nature of this autoantigen remained unknown. Taking advantage of monoclonal antibodies (mAb) produced in our laboratory, we have demonstrated that thyroid peroxidase (TPO) is the 'microsomal' antigen. Sera of patients with AITD strongly inhibited the binding of only one of 19 mAb raised against human thyroid plasma membranes. This mAb did not react with thyroglobulin but achieved significant binding to preparations of human, bovine and porcine TPO, bovine lactoperoxidase and human myeloperoxidase without altering the enzyme activity. The mAb has been used to immunopurify the human TPO from solubilized thyroid microsomes. The procedure allowed high purification (approximately 3500-fold) of the native enzyme with a reasonable yield (approximately 10 mg TPO/kg thyroid tissue). Human TPO exhibited a specific activity of 350-400 guaiacol U/mg, a peak in the Soret region and a ratio of A411 nm to A280 nm of 0.20-0.25. Upon SDS-polyacrylamide gel electrophoresis, the purified enzyme gave two contiguous bands in the 100 kDa region. Performed in non-reducing conditions, electrophoresis of TPO showed one band in the same 100 kDa region. Sera with aAb to the microsomal antigen immunoprecipitated purified TPO to an extent ranging from 80 to 100% of the initial enzyme amount while sera from normal subjects or from patients with undectable level of anti-microsomal aAb elicit a decrease of less than 30% of the total TPO activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1986

Interaction of highly purified thyroid peroxidase with anti-microsomal antibodies in autoimmune thyroid diseases.

Among the several antigens involved in autoimmune thyroid diseases, the microsomal antigen has been recently identified as the human thyroid peroxidase (TPO). The availability of highly purified TPO and anti-TPO monoclonal antibody (mAb) allowed us to study in more details anti-TPO autoimmune antibodies (aAb) and their relationship with anti-microsomal aAb. Only sera with anti-microsomal aAb, as assayed by passive hemagglutination, highly immunoprecipitated purified TPO; anti-thyroglobulin aAb did not contribute to this effect. IgG binding to TPO and inhibition of mAb binding to TPO were also observed using anti-microsomal positive and anti-thyroglobulin negative sera. The correlation between anti-TPO and anti-microsomal aAb titers was found significant (p less than 0.02). These data suggest that anti-microsomal and anti-TPO immunoreactivity are presented by the same aAb. It could not be ruled out, however, that some of the anti-microsomal aAb detected by passive hemagglutination could be directed against antigens different from TPO. The use of highly purified TPO and anti-TPO mAb would allow large scale studies of anti-TPO aAb, necessary for investigating their pathological significance in thyroid disorders.

Antibodies, Monoclonal↗
PubMed1985

Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases.

Human thyroid peroxidase (TPO) has been purified from thyroid microsomes by immunoaffinity chromatography using a monoclonal antibody (mAb) to TPO. The eluted material had a specific activity of 381 U/mg and exhibited a peak in the Soret region. The ratio of A411 to A280 ranged from 0.20 to 0.25. Upon SDS-polyacrylamide gel electrophoresis, the purified enzyme gave two contiguous bands in the 100 kDa region. Further, it has been demonstrated that sera with anti-microsomal autoantibodies from patients presenting Graves' or Hashimoto's thyroiditis diseases were able to bind to purified TPO and to inhibit in a dose-dependent manner the mAb binding to purified TPO. This suggests that TPO is the thyroid antigen termed to date the microsomal antigen.

Antibodies, Monoclonal↗
PubMed1985

Cell membrane coating with glutaraldehyde: application to a versatile solid-phase assay for thyroid membrane proteins and molecules interacting with thyroid membranes.

In defined conditions, glutaraldehyde was shown to tightly bind cell membranes to flexible microtiter plates without significant alteration of the antigenic and functional properties of membrane proteins. In the presence of 0.06% glutaraldehyde, human thyroid membranes were bound to plastic firmly enough to resist numerous washing and flicking steps; the coated membranes remained almost unaltered with regard to monoclonal antibody and thyrotropin binding as well as adenylate cyclase and peroxidase activities. Based on the use of thyroid membrane-coated microtiter plates, a versatile solid-phase assay was developed which allowed screening of anti-membrane monoclonal antibodies, detection of thyrotropin-displacing activity in hormone and antibody preparations, and monitoring of fractionation experiments of solubilized membrane antigens and thyrotropin receptor. It was concluded that the use of glutaraldehyde for coating cell membranes to flexible microtiter plates enabled the establishment of simple, rapid, and reliable assays for detection and quantitation of membrane proteins and molecules interacting with membranes.

Adenylyl Cyclases↗
PubMed1985

[Antigenic relation between thyroid peroxidase and the microsomal antigen implicated in auto-immune diseases of the thyroid].

Pools of sera from patients with Graves' disease or Hashimoto's thyroiditis highly inhibit the binding to human thyroid membranes of one of 19 monoclonal antibodies raised against preparations of human thyroid membranes. This monoclonal antibody reacts with human and bovine thyroid peroxidase and bovine lactoperoxidase but not with human hemoglobin, cytochrome c and other related molecules. These results indicate that the thyroid peroxidase and the microsomal antigen are antigenically related. These data taken together with those from other groups, highly suggest that thyroid peroxidase is the microsomal antigen involved in autoimmune thyroid diseases.

Adult↗
PubMed1985

Fertilization of a colchicine-treated gamete of the fern Marsilea vestita.

In the fern, Marsilea vestita, the addition of colchicine to the culture medium during the final stages of archegogenesis gives rise to a new distribution of cellular organelles and lipid droplets within the egg cytoplasm. These changes affect primarily the usually free space through which the spermatozoid moves towards the female nucleus. Depending on the length of the treatment this fertilization cone may disappear. Completion of fertilization, i.e. karyogamy, is only observed in the presence of this cone. When it is missing, progress of the male nucleus, which is no longer directly channeled towards the female nucleus, is notably slowed down and karyogamy does not occur. Then, male chromatin decondensation takes place in the egg cytoplasm. This event enables us to describe the structure of the sperm nucleus: it is composed of two cylindrical parallel units which, subsequently, break up into short fragments which may be the "male chromosomes".

Cell Nucleus↗