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J Sire

Publications and source records attributed to J Sire.

At least 37 records · Page 2Linked to original sources
PubMed1990

The env gene variability is not directly related to the high cytopathogenicity of an HIV1 variant.

HIV1-NDK is a Zairian HIV1 isolate which is unique because of its high cytopathic effect on T lymphoblastoid cell lines. Its sequence analysis has indicated 30% divergence with HIV1-LAV/BRU HTLVIII B prototype in the env gene encoding for the envelope glycoprotein gp120. In order to correlate the highly cytopathic properties with the env genetic variability, recombinants between the HIV1 prototype and HIV1-NDK have been constructed, including HIV1-NDK env gene, and their cytopathic phenotypes were analyzed. The viral hybrid containing all HIV1 prototype sequences except a large fragment including the total HIV1-NDK env gene remained at a low cytopathic phenotype. Our results suggest that sequences other than the env gene namely p18 gag, vif, and vpr are required for the high virulence of HIV1-NDK.

Animals↗
PubMed1990

Differences in replication and cytopathogenicity of human immunodeficiency virus type 1 (HIV-1) are not determined by long terminal repeats (LTR).

The growth properties of molecular clones of a highly cytopathic Zairian HIV1-NDK and prototype viruses were compared to correlate genetic variations with biological changes. The cloned HIV1-NDK retained the highly replicating cytopathic phenotype and formed larger syncytia than the prototype. One of the major differences in the alignment of the nucleotide sequence of the HIV1-NDK and HIV1-BRU prototypes was localized in the negative regulatory element (NRE) of the long terminal repeat (LTR). In a chloramphenicol acetyl transferase (CAT) assay, we failed to detect a significant difference between LTR promoter activity of the prototype and HIV1-NDK, suggesting that the LTR of both phenotypes had a similar function. The complete recombinant provirus DNA molecules bearing HIV1 LTR derived from one phenotype and the rest of the genomes from the other phenotype were constructed and transfected. The high cytopathogenicity of both the original and the chimeric viruses was correlated with the high speed of virus replication. Cytopathogenicity, morphology of syncytia, and replication kinetics of the recombinant viruses were determined by the functions coded within an internal part of HIV1 genome, covering the gag to env region, which were, however, not within LTR.

Animals↗
PubMed1989

Nucleotide sequence of HIV1-NDK: a highly cytopathic strain of the human immunodeficiency virus.

A highly cytopathic strain of HIV1, named HIV1-NDK, has been isolated from a Zaïrian patient affected with AIDS. This isolate is 10(4) times more cytopathic and infectious than the prototype. To correlate the high cytopathic properties of this strain with genetic variations, we have cloned and sequenced the genome of this isolate. The principal feature which could be drawn from the fine analysis of the HIV1-NDK sequence is that the variability is not clustered in one particular region but rather spread out all along the genome. Only minor differences seem to be responsible for the acute biological effect of HIV1-NDK.

Amino Acid Sequence↗
PubMed1989

Recognition of oligonucleotide-encoded T cell epitopes introduced into a gene unrelated to the original antigen.

We have previously demonstrated that H-2Kd-restricted CTL specific for HLA-CW3 or HLA-A24 can recognize synthetic peptides corresponding to residues 170-182 of the HLA molecules. Synthetic oligonucleotides encoding region 170-182 of CW3 or A24 were inserted into the influenza nucleoprotein (NP) gene. We demonstrate herein that P815 (H-2d) cells transfected with the NP-oligo recombinant genes are specifically lysed by HLA-specific Kd-restricted CTL clones. Our results imply that there must be a high degree of flexibility for the expression of T cell epitopes in different molecular contexts.

Animals↗
PubMed1988

A study of functionally active amino acids involved in the interaction of HLA-A2 or HLA-A3 molecules with cytolytic T lymphocytes.

A large series of HLA-A2/HLA-A3 recombinant genes were generated by using the in vivo recombination technique. These genes have each been modified in the last two-thirds of the third exon such that one or several HLA-A2-specific substitutions have been made in the HLA-A3 gene and vice versa. The recombinant genes were transfected into the murine cell line P815 and the transfectants were used as targets for a series of 20 human CTL lines or clones specific for HLA-A2 or HLA-A3, or restricted by HLA-A2 and specific for influenza A. Several patterns of anti-HLA-A2, anti-HLA-A3, and HLA-A2-restricted anti-influenza CTL activity were observed and when uncloned cell lines were studied, a progressive selection of some clones with a similar pattern of activity was regularly found. From the comparison of these different patterns the following conclusions can be drawn: 1) In most but not all cases both domains of the class I molecule were essential for CTL recognition, but residue 152 was critically important for the majority of CTL tested; 2) amino acids 114/116 were also critical in most cases, and their position close to amino acid 152 in the tertiary structure of the molecule may have some functional significance; and 3) amino acid 161, although highly conserved, plays an unexpected but very important role in CTL function.

Amino Acids↗
PubMed1988

Epitope mapping of HLA-B27 and HLA-B7 antigens by using intradomain recombinants.

To study the HLA-B7 and HLA-B27 antigenic determinants, hybrid genes between these two alleles were constructed by in vivo recombination in Escherichia coli. After transfection of these genes into P815 (high transfection efficiency recipient) murine cells, the bindings of Bw6, HLA-B7, and HLA-B27 allele-specific mAb were studied, as well as that of human anti-HLA-B7 and anti-HLA-B27 monospecific alloantisera. Most of the HLA-B7 antigenic determinants were assigned to the first external domain of the molecule. Four different epitopic areas could be defined: the Bw6 epitope was associated with residues 82 and 83; the BB7.1 epitope to amino acids 63, 67, and 70; the MB40.2 and MB40.3 epitope to amino acid sequence 177-180, and human alloantisera identified as an epitope associated with residue 9. HLA-B27 antigenicity studied by TM-1 mAb was found to involve residues 77 and 80 in the alpha-1 domain. Results obtained with human monospecific alloantisera allowed the definition of an additional allospecific site associated with the NH2 terminal part on the alpha-1 domain of HLA-B27. Epitope mapping fits with data obtained by sequence comparisons and is discussed with reference to the crystallographic three-dimensional structure of the HLA-A2 molecule.

Amino Acid Sequence↗
PubMed1988

Hybrid genes between HLA-A2 and HLA-A3 constructed by in vivo recombination allow mapping of HLA-A2 and HLA-A3 polymorphic antigenic determinants.

HLA-A2 and -A3 genes have been modified in their third exon (second domain) by using in vivo recombination. In this method Escherichia coli are transfected with a plasmid which contains two highly homologous sequences (e.g., the third exons of HLA-A2 and -A3) and has been linearized by cleavage between these two sequences. Circularization takes place in the bacteria by homologous recombination leading to hybrid A2-A3 sequences. The analysis by DNA sequencing of a number of such recombinants shows that they indeed occur by homologous recombination (no insertions or deletions) and that the probability of crossing over decreases as the distance from the free end of DNA in the homologous region increases. No double recombinants were observed. These hybrid exons were reinserted into either HLA-A2 or HLA-A3 genes, thus generating a panel of functional hybrid genes containing one or several HLA-A2 specific substitutions in an HLA-A3 background or vice versa. These genes were expressed by transfection into murine P815-high transfection efficiency recipient cells. Serologic analysis leads to the conclusion that expression of polymorphic antigenic determinants specific for HLA-A2 (detected with M58, A2A28M1, and CR11.351 mAb) is linked to the presence of threonine residue (amino acid (AA) 142) and/or histidine residue (AA 145) and valine residue (AA 152). The expression of specific HLA-A3 polymorphic determinants (recognized by GAP-A3 mAb) is correlated with the existence of a asparagine residue (AA 127) and a aspartic residue (AA 161). But aspartic residue 161 contributes with glutamic acid residue 152 in the formation of the A3 epitope recognized by the anti-A3 mAb X1.23.2.

Antibodies, Monoclonal↗
PubMed1987

Creation of an HLA-A2/HLA-Aw69 alloantigenic determinant on an HLA-A3 molecule by site-directed mutagenesis.

The HLA-A2 and HLA-Aw69 molecules share an antigenic determinant not expressed by HLA-Aw68 and HLA-A3. Comparison of the amino acid (aa) sequences of these molecules and previous studies of the antigenic determinant expressed by different HLA-A2 X HLA-A3 hybrid molecules had established that three aa at positions 95, 97, and 107 were possibly involved in the formation of this determinant. The HLA-A3 gene was therefore mutagenized to replace successively at these positions the HLA-A3-specific aa by the HLA-A2 residues. A single substitution at position 107 of a glycine by a tryptophan residue is sufficient for full expression by HLA-A3 molecules of the HLA-A2/Aw69 shared antigenic determinant without modification of the other serological reactivities characteristic of the HLA-A3 molecules. Previous studies of ethyl methanesulfonate mutants having shown the involvement of aa 161 in this determinant, we assume that the two aa residues 107 and 161 are close to each other.

Amino Acid Sequence↗
PubMed1986

Transfection of murine LMTK- cells with purified HLA class I genes. VII. Association of allele- and locus-specific serological reactivities with respectively the first and second domains of the HLA-B7 molecule.

The individual contributions of the first two external domains of the HLA-B7 heavy chain to the expression of allele-specific (B7) and locus-specific (B and C) antigenic determinants were investigated using hybrid class I genes. Hybrid genes were constructed in vitro by exon shuffling between the parent genes HLA-B7, HLA-Cw3, HLA-A3, and H-2Kd, and their expression was monitored following transfection into mouse L cells. The results show that most allele-specific antigenic determinants are associated with the first external domain of the B7 heavy chain, whereas all the locus-specific antigenic determinants tested map to the second external domain.

Animals↗
PubMed1982

Rat immunoglobulin delta heavy chain gene: nucleotide sequence derived from cloned cDNA.

Rat immunoglobulin delta heavy-chain mRNA has been isolated. RNA blot analysis revealed that this mRNA with a length of 1.8 kb encodes for the secreted form of IgD. The corresponding cDNA was cloned in plasmid pBR322 and its sequence was determined. The hybrid plasmid contains a 775-bp insert comprising a partial C delta 1 sequence and complete C delta H, C delta 3, C delta DC and 3' untranslated sequences. Rat and mouse IgD amino acid sequences show striking homology in C delta 3 and C delta DC regions.

Amino Acid Sequence↗
PubMed1981

Rat IgD myeloma protein: cell-free translation of the delta mRNA and biochemical analysis of intracellular and membrane delta chain.

The analysis of IgD molecules produced by the tumor-derived IR 731 cell line showed that these molecules are not secreted and are only present at the cell surface as covalent dimers of 48 000 dalton delta chains. The original IR 731 tumor secreted complete IgD; thus, the disppearance of secretion in the cell line was correlated with the absence of light chain synthesis. The mRNA coding for the cell line delta chain was isolated and translated. The analysis of the translated delta chain (mol. wt.: 40 000) confirmed the absence of a region having the size of a domain as previously shown by partial amino acid sequence. The observation that the absence in delta chain of this region, which comprises most of the CH2 domain, could be a general feature of murine species is discussed.

Animals↗
PubMed1980

Biochemical characterization of an Fc receptor of rabbit lymphocytes.

Isolation of Fc-binding molecules was performed by complexing IgG antibodies and the corresponding antigens in lysates of radiolabeled rabbit lymphoid cells. A single-chain molecule of 110 000 apparent mol. wt. (unreduced) or 120 000 (reduced) was observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis when the antibodies were in the IgG form but not as F(ab')2. This molecule was very susceptible to proteolysis, but the fragments thus produced remained associated by disulfide bridges, and the binding properties of the molecule were conserved. The molecule and its proteolytic fragments (mol. wts. 75 000, 45 000 and 20 000) were very similar to those obtained for the mouse Fc receptor under similar conditions, and therefore the molecule was designated as rabbit Fc receptor. Among several precipitating systems used, some Ig-anti-Ig complexes appeared to be the most efficient in coprecipitating the rabbit Fc receptor. Remarkably high titers of Fc receptor were found on lymphocyte membrane of infected rabbits, in agreement with a possible role of this molecule in immune regulation.

Animals↗
PubMed1980

Putative conformation of mouse Fc gamma-receptor.

Three cell surface molecules (m.w. = 115,000, 90,000, and 70,000) binding to the Fc portion of complexed IgG have been isolated from the murine mastocytoma line P815. Various results suggested that the 90,000 and 70,000 dalton components are generated from the 115,00 dalton molecule by spontaneous proteolytic clevages and release of 23,000 dalton fragments. It was demonstrated that these cleavages occur during cell culture and not when freshly harvested mouse spleen cells are used as an Fc gamma R cell source. The survey of the Fc gamma R molecular forms obtained from P815 and spleen cells together with their reduction products led us to conclude that the mouse Fc gamma-receptor for complexed IgG is a single chain molecule (115,000 daltons) folded into five globular subunits (m.w. eta 23,000) linked by loose connecting regions accessible to proteolytic enzymes. Three of these subunits that compose the 70,000-dalton fragment are linked by di-sulfide bonds. Furthermore, a 140,000-dalton Fc gamma-binding molecule, not identified after cell surface labeling, could be detected after internal labeling. This component could be a cytoplasmic precursor of the Fc gamma R molecule. The structural model we present here might in addition shed some light on the discrepancy that appears through the various biochemical studies performed so far on Fc gamma-receptors.

Animals↗
PubMed1979

Identification of an IgD-like surface immunoglobulin on rabbit lymphocytes.

Rabbit immunoglobulin antigen receptor molecules were analyzed by electrophoresis in sodium dodecyl sulfate polyacrylamide gels. By sequential precipitation, using first anti-mu and then anti-L chain antisera, two different immunogloublins of similar molecular weight could be demonstrated. After tryptic digestion, two fractions were identified: an IgMs and an Fab fragment, the latter appearing after a few minutes of incubation. Thus, as in man and in the mouse, two main immunoglobulin antigen receptors were found in the rabbit. One receptor on rabbit lymphocytes is an IgMs molecule, and the other is easily split in the hinge region like human and mouse IgD receptors. In addition, a molecule similar to the mouse Fc receptor was nonspecifically precipitated.

Animals↗