PubMed Health⌕ Search

Biomedical subjects

J Rocca-Serra

Publications and source records attributed to J Rocca-Serra.

At least 19 recordsLinked to original sources
PubMed1997

Thy-1 immunolabeled thymocyte microdomains studied with the atomic force microscope and the electron microscope.

The atomic force microscope (AFM) and the transmission electron microscope (TEM) have been used to study the morphology of isolated mouse thymocyte microdomains and Thy-1 antigen distribution at the surface of these structures. AFM images were recorded in air in the contact mode on membrane vesicles deposited on previously heated tissue culture plastic sheets and indirectly immunolabeled for Thy-1 expression with colloidal gold-conjugated secondary antibodies. AFM images of untreated plastic plates showed a very characteristic network of streaks 20-200 nm wide. Heating the plastic removed the streaks and provided flat surfaces (r.m.s. 1 nm). This substrate allowed strong adsorption and homogeneous spreading of the vesicles and easy manipulations during immunolabeling experiments. Vesicles flattened on the substrate without losing their morphology. The 10-nm membrane-bound gold beads were reproducibly imaged without degradation by repeated tip scanning. The observed microdomains had a mean diameter of 184 +/- 76 nm, and 65% of them were specifically labeled. Images obtained with the TEM on the same vesicles, deposited on carbon-coated grids and negatively stained, confirmed the AFM observations. The size distribution of the microdomains was quite similar, but the number of beads per vesicle was significantly higher, and 76% of the vesicles were labeled. The difference may be explained 1) by removal of beads from the vesicles in the additional washing step with water, which was necessary for the AFM; 2) by tip-sample convolution; and 3) by statistical fluctuations.

Animals↗
PubMed1988

Combinatorial association of V genes: one VH gene codes for three non-cross-reactive monoclonal antibodies each specific for a different antigen (phoxazolone, NP or gat).

Two anti-phenyloxazolone (phOx3) and one anti-GAT MAbs from C57BL mice are shown to be coded by VH gene 186.2. This gene has been found earlier to code for several anti-NP (NNP) antibodies (Bothwell et al., 1981) and anti-GT antibodies (Rocca-Serra et al., 1983; Carmack and Pincus, 1986). The L chain partner of the VH 186.2 gene is different in anti-NP and anti-GAT antibodies (Bothwell et al., 1981; Rocca-Serra et al., 1983; Carmack and Pincus, 1986); in anti-phOx antibodies two new unrelated kappa chain V regions were found. Both of the new VK genes involved code frequently for anti-phOx antibodies in BALB/c mice but then with different VH genes. We tested five 186.2-coded antibodies for cross-reactions. Four antibodies were specific, one bound only to NNP, one only to phOx and two only to GT (GAT). The fifth antibody (anti-phOx) bound also to NNP, GAT and ABA-HOP though probably with a low affinity. This is the first demonstration that one V gene can code for three different antibody specificities. It emphasizes the role of the combinatorial element in antibody diversity.

Animals↗
PubMed1985

The idiotypic network and the internal image: possible regulation of a germ-line network by paucigene encoded Ab2 (anti-idiotypic) antibodies in the GAT system.

Heavy and light chain variable regions from eight monoclonal Ab2 (anti-idiotypic) antibodies of the GAT antigen, a (Glu60 Ala30 Tyr10)n co-polymer, have been analyzed by direct mRNA sequencing. Three mAb2s were directed against private idiotopes and used various VH-D-JH and Vk-Jk combinations. By contrast, the five 'anti-public' mAb2 antibodies used a very restricted repertoire, including all gene segments. Interestingly, within their D regions, Glu-Glu-Tyr or Tyr-Tyr-Glu sequences were reminiscent of the original (GAT) antigen and may act as possible internal images. A striking observation was that two mAb2 antibodies shared the same V-D-J sequence although derived from separate fusions. As this D sequence, 33 nucleotides long, has not been described so far, it is suggested that it may be encoded by a new germ-line D gene, acting as a crucial regulatory element in a GAT germ-line idiotypic network. An alternative model that may lead to the construction of this D segment by 'odd' rearrangements from pre-existing already reported sequences is also presented.

Animals↗
PubMed1985

A single VH-gene associated with a variety of D- and J-segments encodes for a large family of ABPC48-related antibodies induced by antiidiotypic immunization.

Two series of monoclonal antibodies have been obtained from BALB/c mice immunized against two antiABPC48 antiidiotypic antibodies. They are divided into two serologically different classes. Class I antibodies bind only the immunizing antibody; class II antibodies display a broad binding capacity to various antiidiotypic antibodies, and some bind levan, as does ABPC48. Northern blot analyses and partial mRNA sequencing show that all class II antibodies express the VH-gene coding for ABPC48 and UPC10 antilevan antibodies associated with a variety of D- and J-segments. The third hypervariable region of the sequenced antibody with antilevan activity is structurally related to that of ABPC48 and UPC10 antibodies but has a different genetic origin. This study indicates that the identification of idiotype-related antibodies arising from antiidiotypic immunization may be misleading, if based on their antigen-binding properties; and it stresses the importance of structural approaches for the analysis of regulatory mechanisms ruling immune responses.

Amino Acids↗
PubMed1985

Gene repertoire of the anti-poly(Glu60Ala30Tyr10) (GAT) immune response: comparison of VH, V kappa, and D regions used by anti-GAT antibodies and monoclonal antibodies produced after anti-idiotypic immunization.

Eight monoclonal antibodies were selected from BALB/c mice immunized with two different monoclonal anti-idiotypic antibodies recognizing two discrete idiotopes characteristic of the anti-poly(Glu60Ala30Tyr10) (GAT) antibody response. These monoclonal antibodies were previously classified as Ab1 (anti-GAT-like) and Ab3 (anti-anti-idiotype) on the basis of expression of the public idiotypic specificity (p.GAT) studied with a xenogeneic serum, anti-GAT activity, and expression of various public idiotopes. All the heavy chain variable region (VH) sequences from Ab1 are nearly identical to the VH sequences of Ab1 anti-GAT monoclonal antibodies. The same type of results has been found with the Ab1 kappa light chain variable region (V kappa) sequences. Confirming our classification, Ab3 VH and V kappa sequences were found to be completely different from Ab1 VH and V kappa sequences. The Ab1 diversity (D) regions are different from one another and different from the D regions found on monoclonal anti-GAT antibodies but function similarly. These D regions are not simply derived from already described D genes. Finally, our results suggest that in the anti-GAT response VH and V kappa sequence are mainly responsible for idiotype expression.

Amino Acid Sequence↗
PubMed1984

Phosphate uptake in the yeast Candida tropicalis: purification of phosphate-binding protein and investigations about its role in phosphate uptake.

The purification of a phosphate-binding protein (PiBP2) by immunoadsorption is described. The entire anti phosphate-binding protein 2 antibodies as well as the Fab fragments obtained from these antibodies inhibit Pi uptake by whole cells. The inhibition is a mixed type of inhibition (Vm and Km are affected). These results should be regarded as a possible involvement of phosphate-binding protein 2 in Pi uptake. The binding of 125I-labelled fragments prepared from anti phosphate-binding protein 2 antibodies to whole cells, to shocked cells and to protoplasts has been investigated. The results confirm the release of phosphate-binding protein by osmotic shock and during protoplast formation. From these findings, a cell-wall localisation, near the cell surface of the phosphate-binding protein should be proposed.

Antibodies↗
PubMed1983

V kappa gene family in (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies that express CGAT (or pGAT) public idiotypic specificities. Protein and mRNA sequencing of eight monoclonal V kappa chains.

A large proportion of (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies expresses public idiotypic specificities, termed CGAT (or pGAT), that require the presence of both the heavy and the light chains in order to be expressed. We report in this paper the complete sequence of eight V kappa regions pertaining to eight anti-GAT monoclonal antibodies derived from three strains of mice: BALB/c, DBA/2, and C57BL/6. The methodology used a combination of NH2-terminal amino acid and mRNA nucleotide sequencing. All eight sequences analyzed, although highly homologous and all pertaining to the same V kappa 1 subgroup, allowed definition of three germline genes that are likely to be present in all three strains of mice and also in NZB. It seems likely, however, that any given strain may not necessarily use all three genes for making anti-GAT antibodies. The search for structural correlates of idiotypes could not be framed in a simple picture, but our data suggest that similar idiotopes may result from different interacting primary structures, leading to structural homologies that should be visualized at three-dimensional level.

Amino Acid Sequence↗
PubMed1983

Analysis of antibody diversity: V-D-J mRNA nucleotide sequence of four anti-GAT monoclonal antibodies. A paucigene system using alternate D-J recombinations to generate functionally similar hypervariable regions.

The nucleotide sequence of four anti-(Glu60-Ala30-Tyr10)n (GAT) monoclonal gamma 1 heavy chain mRNAs was determined from codon 10 to 120. This sequence overlaps with the NH2-terminal amino acid sequence, allowing elucidation of the complete protein sequence encompassing regions VH, D and JH. These sequences, which are highly conserved, indicate that anti-GAT antibodies expressing the same public idiotypic specificities represent a paucigene system, which uses at least two D-J combinations leading to functionally similar hypervariable regions involved in the recognition of the dominant Glu-Tyr determinant. D regions are encoded by D genes which are closely related either to the D-SP2 or the D.FL16 germ line gene cores.

Amino Acid Sequence↗
PubMed1983

Structural bases for public idiotypic specificities of monoclonal antibodies directed against poly(Glu60Ala30Tyr10) and poly(Glu60Ala40) random copolymers.

NH2-terminal amino acid sequences of heavy and light chains of seven poly(Glu60Ala30Tyr10) (GAT) specific hybridoma products derived from DBA/2 and (DBA/2 X BALB/c)F1 hybrid mice and those of BALB/B polyclonal antibodies have been determined over the first 40 residues. Comparison of these sequences with those of nine other GAT or poly(Glu60Ala40) (GA) specific hybridoma products previously reported allowed the following conclusions. (i) Sequences of hybridoma H and L chains are present in the pool of polyclonal antibodies. (ii) The public CGAT (or pGAT) idiotypic specificities are strictly confined to antibodies exhibiting limited heterogeneity with regard to both the variable heavy (VH) and the variable kappa (V kappa) sequences that may be accounted for by one and two germ-line genes, respectively. (iii) The public idiotypic specificities GA-1, expressed by some anti-GAT and most anti-GA antibodies, make use of the same (or similar) VH germ-line genes as the CGAT or pGAT antibodies but possess a distinctive V kappa sequence. (iv) Antibodies expressing neither of the alternative public specificities mentioned above appear to be more heterogeneous and express VH and V kappa sequences that were found to differ from the basic structures defining the CGAT (pGAT) or GA-1 correlates. It is concluded that CGAT (or pGAT) and GA-1 public idiotypic specificities are germ-line markers of both VH and V kappa regions, an observation in agreement with previously reported serological data.

Animals↗
PubMed1982

Inorganic phosphate uptake by protoplasts and whole cells of yeast Candida tropicalis: absence of high affinity transport system in protoplasts.

Pi-uptake by protoplasts obtained from the yeast Candida tropicalis as well as in whole cells is an energy dependent process. However, the apparent affinity constant of Pi transport by protoplasts about 0.4-0.5 mM whereas Km of 5-6 microM, occurs in whole cells as previously shown. Therefore it can be concluded that protoplasts take up actively Pi but the high affinity capacities are lost. In addition, the Pi-binding proteins are released during protoplasts formation. The presence of Pi-binding proteins in the "cellulase fluid" is demonstrated by immunological tests and by their binding activities after partial purification. These results are in good agreement with the involvement of Pi-binding proteins in Pi uptake in the yeast Candida tropicalis.

Biological Transport, Active↗
PubMed1982

The limited diversity of the mouse gamma-chains anti-GAT repertoire does not seem to be noticeably amplified upon class switch.

NH2-terminal sections of H and L chains isolated from five monoclonal anti-GAT antibodies derived from BALB/c mice have been sequenced upon to residue 43. Four among these five antibodies, sharing similar public idiotypic determinants, possess extremely conserved sequences, both for the H, which is apparented to the VH II type, and the L chains, which belong to the V kappa I subgroup. VH sequences are identical up to residue 43 and contain the common sequences (residues 1 to 32) defined for the H chains derived from the DBA/2 IgM anti-GAT monoclonal antibodies. Light chains are also remarkably conserved, a rather unusual situation for kappa-chains. The fifth antibody that expresses only part of the public idiotypic determinants contains very distinctive H and L chains. Its heavy chains are close to the VH I subgroup, whereas its kappa-chains permit definition of a new V kappa subgroup. The repertoire appears to be highly conserved between BALB/c and DBA/2 mice, and does not seem larger in IgG than in IgM antibodies. This latter observation does not speak in favor of a switch-linked amplification of diversity.

Amino Acid Sequence↗
PubMed1975

Determination of the primary structure of a mouse IgG2a immunoglobulin. Amino-acid sequence of the H4 cyanogen-bromide fragment.

The complete amino acid sequence of CNBr fragment H4 of the murine immunoglobulin MOPC 173 (IgG2a,chi) has been determined, thus completing the sequence determination of the entire heavy chain. The H4 fragment contains 150 residues, and extends from residue 105 to residue 254 of the heavy chain, which appears thus to be composed of 447 amino acids residues. This fragment contains the end of the V region, the switch peptide, the CH1 domain, the hinge region and the beginning of CH2. Sequence comparisons suggest that the CH1 domain is highly conserved in evolution, and allows the definition of two additional isotypic-specific regions.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular analysis of the anti-GAT repertoire in three strains of mice.

GAT-specific antibodies which express the CGAT (or pGAT) public idiotypic specificities are encoded by a paucigene system. Antibodies expressing discrete specificities, i.e. anti-GAT and anti-NP, appeared to use the same VH germ-line genes, an observation which directly supports the importance of random H-L pairing to generate a large number of distinct antibody molecules.

Amino Acid Sequence↗
PubMedDate not supplied

"Dideoxy" RNA sequencing: a rapid method for studying genetic information.

The chain termination sequencing technique on RNA is a very rapid, direct method for the analysis of genetic information. It is particularly suitable for analysing systems on which microgram amounts of 50% pure RNA can be made available and for which numerous data are needed in order to derive genetic or structural information. The usefulness and limitations of the technique are discussed.

Animals↗
PubMedDate not supplied

The internal image and the structural idiotypic network (Ab1, Ab2, Ab3) in the GAT system.

The GAT repertoire expressed at the different levels of the classical idiotypic cascade Ag----Ab1----Ab2----Ab3 has been analysed by direct nucleotide sequencing of H- and L-enriched mRNA. Ab1 and Ab3 expressing the major public idiotypes used similar, if not identical, VH and VK genes. The VH Ab3 of the Ab1 type (Ab1') appeared highly conserved. Ab2 also use a small number of germ-line genes. The D region of Ab2 is of particular interest since it contains either a Tyr-Tyr-Glu or a Glu-Glu-Tyr sequence, characteristic of the GAT major determinants. It is therefore suggested that this D region contains the internal image of the antigen. A large number of VH germ-line genes have been isolated and sequenced. They all pertain to the VH-II family, which contains a large number of members, some of them being very close in sequence.

Amino Acid Sequence↗