Biomedical subjects

J C Taveau

Publications and source records attributed to J C Taveau.

28 records · Page 2Linked to original sources

PubMed1990

Three-dimensional reconstruction of native Androctonus australis hemocyanin.

A sample of native 4 x 6-meric hemocyanin of Androctonus australis was negatively stained with the double-layer technique, and was observed by transmission electron microscopy under low-dose conditions with a 50 degree and 0 degree tilt. The three-dimensional reconstruction method from "Single-exposure, random conical tilt series" was then applied. Independent three-dimensional reconstructions were obtained from the top, side and 45 degree views. Despite a pronounced flattening effect, presumably due to the specimen preparation technique, the positions of the 24 subunits composing the oligomer were unequivocally determined. This experiment definitely solves the problem of the architectural organization of the subunits in the cheliceratan 4 x 6-meric hemocyanins. Moreover, distinction between the flip and flop faces and an attenuated rocking effect were observed.

Animals↗

PubMed1990

Topological mapping of 13 epitopes on a subunit of Androctonus australis hemocyanin.

A topological localization of epitopes on the surface of the Aa6 subunit of Androctonus australis hemocyanin has been carried out. First, immunocomplex strings composed of native hemocyanin and monoclonal antibodies were examined in the electron microscope and submitted to an image processing by correspondence analysis. The average images were then compared to a three-dimensional model of the 24-mer suggesting that 11 of the 13 epitopes are located in three zones of the subunit surface. Second, the overlaps between the epitopes were then studied by polyacrylamide gel electrophoresis, competitive binding inhibition, and immunoelectron microscopy. Four groups of epitopes were identified. One group was capable of binding exclusively to the free subunit. The other three groups were identical to those found in immunoelectron microscopy. The data are consistent with the existence of a small number of immunodominant regions on the surface of the Aa6 subunit.

Animals↗

PubMed1989

Image processing of proteinase- and methylamine-transformed human alpha 2-macroglobulin. Localization of the proteinases.

Comparative x-ray scattering experiments and electron microscopic observations have been performed on native S-form, and on different F-forms of human plasma alpha 2-macroglobulin (alpha 2M), obtained by proteinase (chymotrypsin, plasmin, and thrombin) or methylamine treatment. Image processing of electron micrographs of the alpha 2M molecules transformed by chymotrypsin, plasmin, and methylamine displayed average images which could be compared. The proteinase-complex alpha 2M molecules exhibited the usual H-like structure, but the methylamine-inactivated ones showed a different organization, with almost no stain-excluding material in the central region of the molecule, which therefore presented a central cavity filled with stain. By subtracting average images of alpha 2M-methylamine from alpha 2M-chymotrypsin or alpha 2M-plasmin, a putative localization of the proteinases inside the alpha 2M molecule, very close to its center was revealed. The values of the radii of gyration for the S- and F-forms obtained by x-ray scattering were very different (78 and 67.7 A, respectively). All four scattering curves of the F-forms were comparable in shape and showed maxima and minima different from that of the S-form alpha 2M. Image processing of electron micrographs and x-ray scattering have provided independent results which indicate that a large cavity exists in the alpha 2M-methylamine molecule and that the proteinases might be located in a very central position inside the alpha 2M-proteinase molecules.

Chymotrypsin↗

PubMed1989

Concerning the axial rotational flexibility of the Fab regions of immunoglobulin G.

By electron microscopy, we have observed immunocomplexes with both negative stain and in amorphous ice using monoclonal antibodies directed against one of the 24 subunits of scorpion haemocyanin. A copy of this subunit occurs at each of the corners of the square-shaped haemocyanin molecule. Three distinct orientations of adjacent haemocyanin molecules may be observed in immunocomplex pairs or chains using both the above-mentioned methods. These observations, coupled with low-resolution computer simulations of immunocomplex formation, argue strongly in favour of the existence of a considerable degree of rotational flexibility within the IgG molecule and around the long axis of the Fab arms, as was suggested by previous observations with negative stain. We find that the arms can rotate by up to 180 degrees with respect to the Fc region.

Animals↗

PubMed1988

Mapping of six epitopes on haemocyanin subunit Aa 6 by immunoelectron microscopy.

Monoclonal antibodies directed against the haemocyanin of the scorpion Androctonus australis were raised in order to map antigenic determinants (epitopes). The method of mapping employed in this study is molecular immunoelectron microscopy. It consists of a direct electron microscopic observation of antigenic molecules labelled with monoclonal antibodies. The epitopes are then localized in a small region of the external surface of the antigenic molecule whose architecture and quaternary structure are well known. Six monoclonal antibodies have been selected and epitopes have been circumscribed within a small area of one subunit among the 24 subunits composing the whole antigenic molecule.

Animals↗

PubMed1988

Intramolecular localization of epitopes within an oligomeric protein by immunoelectron microscopy and image processing.

Three epitopes have been localized by immunoelectron microscopy on subunit Aa6 of the 4 x 6-meric hemocyanin of the scorpion Androctonus australis. Soluble immunocomplexes composed of monoclonal antibodies and of native hemocyanin were purified, negatively stained with uranyle acetate by the single-layer technique, and examined under the electron microscope (EM). The molecule images were digitized, aligned, and submitted to correspondence analysis according to the method of Van Heel and Frank (Ultramicroscopy 6:187-194, 1981). A high-precision localization of the attachment point of the Fab arm to the antigen was achieved through a careful analysis of the average images. This method easily allowed the discrimination of epitopes located in different domains (Mr 20 kDa) of the same subunit. Nonoverlapping epitopes located in the same structural domain of subunit Aa6 could be distinguished by the stain exclusion patterns of their Fab arms. The method is general and may be used for epitope mapping in any antigen producing definite EM views.

Antibodies, Monoclonal↗

PubMed1988

Image analysis and three-dimensional model of chymotrypsin-transformed human alpha 2-macroglobulin complexed with a monoclonal antibody specific for this conformation.

Alpha 2-macroglobulin (alpha 2M) is a plasma inhibitor of proteinases, the steric mechanism of which is based on a considerable conformational change. The typical and distinct H-like shape of alpha 2M-chymotrypsin (alpha 2M-chy) complexes seen by electron microscopy led us to an ultrastructural study of the binding of a monoclonal antibody (Mab) specific for this conformation of alpha 2M. The epitope of this Mab is located near the extremities of the 4 arms of the H-like alpha 2M-chy, at a site that is not accessible on the native molecule. The identical binding of the Mab on the 4 arms of the tetrameric molecule demonstrates that these arms are equivalent portions of the 4 monomers. Various types of immune complexes between alpha 2M and IgG are described, and images of individual immune complexes were processed by correspondence analysis. This extracts new information concerning the organization of chymotrypsin-transformed alpha 2M. The molecule appears asymmetrical, presents 2 conformational states (which we describe as relaxed and twisted), and has flexible arms. These intramolecular motions are supposed to be related to IgG binding. The results are discussed in comparison with previously published models of proteinase-transformed alpha 2M.

Antibodies, Monoclonal↗

PubMedDate not supplied

Three-dimensional immunoelectron microscopy of scorpion hemocyanin labeled with a monoclonal Fab fragment.

An immunocomplex of the 4 x 6-meric hemocyanin of the scorpion Androctonus australis with the monoclonal Fab fragment L104 was reconstructed from electron micrographs of a negatively stained specimen, using the double-carbon-layer technique. The resulting structure enables a clear visualization of the Fab fragments bound to the four copies of the Aa6 subunit and directly confirms a previous localization of the L104 epitope deduced from two-dimensional image processing. Despite a strong flattening effect produced by the negative-staining technique the orientations of the Fab fragments are well characterized. Moreover, the observation of a central hole within the elbow bends of the Fab fragments provides information about the disposition of the Fabs around their main axis.

Animals↗

PubMedDate not supplied

Three-dimensional reconstruction of Androctonus australis hemocyanin labeled with a monoclonal Fab fragment.

An immunocomplex formed by the 4 x 6 meric hemocyanin of the scorpion Androctonus australis with the monoclonal antibody L104 was studied by cryoelectron microscopy and subjected to three-dimensional (3D) reconstruction. The reconstructed particle reflects the structure of the immunocomplex in its hydrated state and is devoid of the flattening that was previously observed with a negatively stained preparation. A 3D fitting of the X-ray data of the Panulirus interruptus hemocyanin and of the Fab R19.9 to the reconstruction volume allowed the first quantitative measurement of the structural parameters of the antigen, and the localization of the epitope at the surface of subunit Aa6. The independent alignment of the Fabs and the hexamers provides a direct verification for the accuracy of the fit and allows the building of the most detailed model of a cheliceratan 4 x 6meric complex and its attached monoclonal antibodies.

Animals↗

PubMedDate not supplied

Presentation of the SIGMA software: Software of Imagery and Graphics for Molecular Architecture.

Sigma (Software of Imagery and Graphics for Molecular Architecture) is a software package designed for visualization and exploration of three-dimensional (3D) molecular electron microscopy data. It comprises a text and two graphic modes. In the text mode, a language allows the writing of scripts for repetitive tasks. The two graphic modes are based on the multiwindow environment with a mouse as pointer. The first graphic mode is dedicated to vectorial objects and the second to pixel/voxel objects. The data imported by SIGMA have various origins such as 3D reconstruction volumes, Protein Data Bank data, or geometrical objects like spheres, cubes, or cylinders. These 3D objects are interactively rotated, translated, and rendered with SIGMA. This software, written in C language, runs on Silicon Graphics workstations using the X-Window plus Motif environment. The specific 3D routines are based on the Silicon Graphics GL library.

Animals↗