The Diversity of Serum Anti-DSG3 IgG Subclasses Has a Major Impact on Pemphigus Activity and Is Predictive of Relapses After Treatment With Rituximab.

Introduction: We studied the distribution and pathogenicity of anti-DSG3 IgG subclasses during the course of pemphigus vulgaris (PV).

Methods: We longitudinally studied the distribution of anti-DSG3 IgG subclasses (before after treatment) in sera from PV patients, using an addressable-laser bead immunoassay (ALBIA). The pathogenicity of corresponding sera was tested using keratinocyte dissociation and immunofluorescence assays.

Longitudinal Pathogenic Properties and -Glycosylation Profile of Antibodies from Patients with Pemphigus after Corticosteroid Treatment.

Pemphigus vulgaris is an autoimmune disease that occurs due to pathogenic autoantibodies that recognize the following epidermal adhesion proteins: desmogleins. Systemic corticosteroids usually decrease the titers of anti-desmoglein autoantibodies and improve patients' conditions. Since modifications of IgG -glycosylation have been described in some autoimmune diseases, we hypothesized that changes in the pathogenic activity of pemphigus IgG could be related to changes in their -glycosylation profile.

Sequence, structure and evolution of the ecdysone-inducible Lsp-2 gene of Drosophila melanogaster.

The Lsp-2 gene encodes a major larval serum protein (hexamerin) of Drosophila melanogaster. Transcription of Lsp-2 is controlled by 20-hydroxyecdysone. Here we report the analysis of the structure of the Lsp-2 gene including the adjacent 5' and 3' sequences.

Evidence for the binding of a biologically active interleukin-2 to human alpha 2-macroglobulin.

Human alpha 2-macroglobulin (alpha 2M), which irreversibly entraps proteinases through a drastic conformational change, has also been reported to bind various cytokines. The meaning of cytokine binding to native and/or transformed alpha 2M molecules is, however, not understood. In an attempt to elucidate this question, we have studied the interaction of radioiodinated recombinant human interleukin-2 (125I-rhIL-2) with native and chymotrypsin (alpha 2M-C)- or methylamine-transformed (alpha 2M-MA) alpha 2M.

Changes of self-association, secondary structure, and biological activity properties of topoisomerase II under varying salt conditions.

Topoisomerase II overexpressed in yeast was purified to near homogeneity. The milligram amounts of active enzyme obtained allowed its study by joint UV-circular dichroism, ultracentrifugation, and biological assays at different protein and salt conditions. First, sedimentation equilibrium was preferred over the other analytical ultracentifuge methods as it is based on firm theoretical grounds and does not require assumptions about the shape of the molecule.

Observation of binding and polymerization of Fur repressor onto operator-containing DNA with electron and atomic force microscopes.

The Fur (ferric uptake regulation) protein is a global regulator that, in the presence of Fe2+, represses the expression of a number of iron-acquisition genes and virulence determinants such as toxins. Dark-field electron microscopy of positively stained Fur-DNA complexes in addition to atomic force microscopy allowed direct visualization of Fur interactions with the regulatory regions of aerobactin and hemolysin operons and provided complementary information about the structure of the complexes. According to the DNA used and the protein/DNA ratio, Fur binding to DNA results in partial or total covering of the fragments, indicating that the protein initiates polymerization along the DNA molecules at specific sites.

Electron microscopy of alpha 2-macroglobulin with a thiol ester bound ligand.

In order to covalently bind the hydrolyzed thiol ester groups of the human alpha 2-macroglobulin (alpha 2M) transformed by methylamine, the phospholipase A2 (PLA2), a small enzyme (M(r) = 13,000) from Naja nigricollis snake venom was activated by succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Average images determined from electron micrographs of the methylamine-transformed alpha 2M, with and without activated PLA2, were determined by image processing and compared. A localization of the PLA2 was achieved by subtracting the average image of alpha 2M transformed by methylamine from that containing PLA2.

Ultrastructure of alpha 2-macroglobulins.

New results concerning the ultrastructure of human alpha 2-macroglobulin (alpha 2M) molecules are presented in connection and comparison with the historical, the current and our own most recent, even unpublished results on the structure and function of alpha 2M and related proteins. The electron microscopic approach uses classical negative staining, combined with the new imaging mode "Electron Energy Loss Spectroscopy", which provides unusual contrast, resolution and readability of the electron micrographs. Immuno- and cryoelectron microscopy, as well as image processing has provided new data necessary to the building of tentative 3D models of the molecule.

Localization of the proteinases in the human alpha 2-macroglobulin-chymotrypsin complex by image processing of electron micrographs.

Human alpha 2-macroglobulin (alpha 2M), a large tetrameric plasma glycoprotein, inhibits a wide spectrum of proteinases by a particular "trapping" mechanism resulting from the proteolysis of peptide bonds at specific "bait" regions. This induces the hydrolysis of four thiol esters triggering both the possible covalent bonding of the proteinases and a considerable structural change in the alpha 2M molecule, also observed following direct cleavage of the thiol esters by methylamine. By subtracting average images of electron micrographs from two populations of alpha 2M molecules in the same biochemical state (with both the four cleaved bait regions and thiol esters), but containing either two or zero chymotrypsins, we are able to demonstrate the position of the two proteinases inside the tetrameric alpha 2M molecule.

Location of B- and Z-DNA in the chromosomes of a primitive eukaryote dinoflagellate.

The usual conformation of DNA is a right-handed double helix (B-DNA). DNA with stretches of alternating purine-pyrimidine (G-C or A-T) can form a left-handed helix (Z-DNA). The transition B----Z, facilitated by the presence of divalent cations, cytosine methylation, or constraints on DNA such as superhelicity may play a role in the regulation of gene expression and/or in DNA compaction (Zarling, D.

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.

Dissociation of alpha 2 M-macroglobulin into functional half-molecules by mild acid treatment.

A slight decrease in pH below neutrality causes the dissociation of alpha 2-macroglobulin (alpha 2M) into dimers formed of two disulfide-bonded subunits. Half-dissociation occurs at pH 6.30 (50 mM NaCl), as determined by gel filtration analysis.

The molecular organization of human alpha 2-macroglobulin. An immunoelectron microscopic study with monoclonal antibodies.

To study the three-dimensional organization of alpha 2-macroglobulin (alpha 2M) from human plasma, immunoelectron microscopy of negatively stained specimens was used. A panel of monoclonal antibodies (mAb) with specificities typical for the two major conformers of alpha 2M (native and protease-transformed) was explored. The mAb have been selected and were classified biochemically as specific for either native or transformed alpha 2M or as reactive with both conformers.

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.

Image processing of electron micrographs of human alpha 2-macroglobulin half-molecules induced by Cd2+.

Human alpha 2-macroglobulin is a tetrameric plasma inhibitor of proteinases. Its dissociation by Cd2+ gives functional dimers. Electron microscopy of negatively stained dimers shows their round-ended cylindrical shape with furrows delimiting 3 main stain-excluding domains.

Functional alpha 2-macroglobulin half-molecules induced by cadmium.

Human alpha 2-macroglobulin can be reversibly dissociated by Cd2+ at low ionic strength in half-molecules which retain their ability to bind tightly plasmin and chymotrypsin. The steady state kinetic parameters of these proteinases towards chromogenic substrates when bound to half-molecules are not greatly different from those determined for these enzymes linked to whole alpha 2M molecules. Cd2+ can also induce the dissociation of plasmin- and chymotrypsin - alpha 2M complexes into proteinase-alpha 2M half-molecule conjugates.

Ultrastructural and anti-proteinase activity modifications of human alpha 2-macroglobulin induced by zinc and other divalent cations.

Native tetrameric alpha 2-macroglobulin molecules (alpha 2M) can be converted into a population of dimers by incubation with various divalent cations such as Zn, Cd, Mg, Cu, Ni, Co. This dissociation is completed within 30 min at 37 degrees C. These dimers have a characteristic shape and a size of about 16 X 8 nm, and appear to be the half of the native alpha 2M molecule which has a clear tetrameric structure as seen in the electron microscope.