Biomedical subjects

D Landais

Publications and source records attributed to D Landais.

9 recordsLinked to original sources

PubMed1998

CTS1: a p53-derived chimeric tumor suppressor gene with enhanced in vitro apoptotic properties.

The clinical potential of the p53 tumor suppressor gene is being evaluated currently for gene therapy of cancer. We have built a variant of wild-type p53, chimeric tumor suppressor 1 (CTS1), in which we have replaced the domains that mediate its inactivation. CTS1 presents some very interesting properties: (a) enhanced transcriptional activity; (b) resistance to the inactivation by oncogenic forms of p53; (c) resistance to the inactivation by MDM2; (d) lower sensitivity to E6-induced degradation; (e) ability to suppress cell growth; and (f ) faster induction of apoptosis. Thus, CTS1 is an improved tumor suppressor and an alternative for the treatment of wild-type p53-resistant human tumors by gene therapy.

Amino Acid Sequence↗

PubMed1993

The Saccharomyces cerevisiae gene product SDC25 C-domain functions as an oncoprotein in NIH3T3 cells.

Ras proteins in mammalian cells cycle between a GTP-bound 'on' state and a GDP-bound 'off' state. Activation of Ras p21 results from the dissociation of tightly bound GDP and the exchange of bound GDP for GTP. A guanine nucleotide exchange factor is required for this activation. Activation promotes interaction with effector molecules and allows the signal to be transduced. In Saccharomyces cerevisiae, the function of guanine nucleotide exchange has been ascribed to the product of the CDC25 gene. The C-terminus domain of SDC25, a homologue of CDC25, can substitute for the CDC25 protein in yeast. We have demonstrated that the SDC25 C-terminus domain promotes GTP binding to Ras p21 in CHO cells. In the present study, we found that the stable expression of the SDC25 C-terminus domain induced transformation of NIH3T3 cells. Ras proteins in these tumorigenic cells were GTP bound. In addition, the coexpression of wild-type Ha-Ras protein with the SDC25 C-terminus was found to enhance the tumorigenic properties of the NIH3T3 cells. These results imply that, in subsets of human tumours, cellular Ras p21 might be found in its GTP-bound active form as a consequence of an oncogenic activation of a mammalian Ras guanine nucleotide exchange factor.

3T3 Cells↗

PubMed1992

Design of yeast-secreted albumin derivatives for human therapy: biological and antiviral properties of a serum albumin-CD4 genetic conjugate.

Due to its remarkably long half-life, together with its wide in vivo distribution and its lack of enzymatic or immunological functions, human serum albumin (HSA) represents an optimal carrier for therapeutic peptides/proteins aimed at interacting with cellular or molecular components of the vascular and interstitial compartments. As an example, we designed a genetically engineered HSA-CD4 hybrid aimed at specifically blocking the entry of the human immunodeficiency virus into CD4+ cells. In contrast with CD4, HSA-CD4 is correctly processed and efficiently secreted by Kluyveromyces yeasts. In addition, its CD4 moiety exhibits binding and antiviral in vitro properties similar to those of soluble CD4. Finally, the elimination half-life of HSA-CD4 in a rabbit experimental model is comparable to that of control HSA and 140-fold higher than that of soluble CD4. These results indicate that the genetic fusion of bioactive peptides to HSA is a plausible approach toward the design and recovery of secreted therapeutic HSA derivatives with appropriate pharmacokinetic properties.

Animals↗

PubMed1989

Functional sites on the A alpha-chain. Polymorphic residues involved in antigen presentation to insulin-specific, Ab alpha:Ak beta-restricted T cells.

The interaction between the clonally selected TCR, the processed Ag peptide and the Ia molecule is not fully understood in molecular terms. Our study intended to delineate the residues of Ab alpha molecules that function as contact sites for Ag and for the TCR of a panel of T cells specific for the A chain of insulin in combination with mixed haplotype Ab alpha:Ak beta molecules. Multiple L cell transfectants expressing alpha,beta-heterodimers composed of wild-type A beta- and chimeric or mutant A alpha-chains served as antigen presenting cells. The recombinant A alpha-chains had been generated by an exchange of allelically hypervariable regions (ahv) or amino acids. The results point out a broad spectrum of b sequence requirements for the bovine insulin-specific activation of the various T cell populations. Activation of some T cells seemed quite permissive, requiring b-haplotype amino acids in any one of the three ahv, while others had strict requirements, demanding b-haplotype sequence in all three ahv. Our data stress the role of ahvII and especially ahvIII in T cell activation. Interestingly, single amino-acid substitutions in ahvII or ahvIII of Ak alpha were sufficient to bring up full stimulation potential for two T cell hybridomas. We also found that some ahv permutations influenced the Ag preference (beef insulin versus pig insulin) of some T cells. These data suggest a critical role for the three-dimensional structure of the complex formed by Ia and the processed Ag peptide. The stability of the trimolecular complex essential for T cell activation is envisioned as being the sum of the interactions between Ag/I-A, TCR/Ag, and TCR/I-A, each variable in strength and compensated for by the others.

Animals↗

PubMed1989

I-Ak polymorphisms define a functionally dominant region for the presentation of hen egg lysozyme peptides.

The class II molecules of the MHC not only bind processed antigenic peptides but also interact with the TCR. This latter interaction is thought to be the basis for allele specific "restriction" of Ag presentation to T cells. The specificity of this interaction is likely due to amino acid differences in a small number of polymorphic or "hypervariable" regions located in the amino terminal domains of the alpha- and beta-chains. We have explored the functional significance of these polymorphic regions in an I-Ak-restricted, hen egg lysozyme specific Ag presentation system in which the measurement of IL-2 production by T cell hybridomas was used as the indicator of TCR recognition of the I-A/Ag complex. Chimeric I-A molecules, in which b allelic residues were substituted in one or more of the polymorphic regions of the A alpha k chain or in which d allelic residues were substituted in one or more of the polymorphic regions of the A beta k chain, were used to examine the contribution of each polymorphic region of the molecule to its function. The results obtained demonstrate that the regions between residues 69 to 76 of the A alpha k chain and the regions between residues 63 to 67 and 75 to 78 of the A beta k-chain exert a dominant effect on the presentation of lysozyme peptides by I-Ak to the T cell hybridomas in our panel. These observations were confirmed and extended by the analysis of Ag presentation by seven serologically selected mutants, all of which have amino acid interchanges in or around the dominant polymorphic regions. The results suggest that the serologically selected mutants fail to present Ag not because they fail to bind the peptide Ag but because the amino acid substitutions destabilize the interaction between the Ia/peptide complex and the TCR. Use of the recently published hypothetical model for class II structure to interpret the Ag presentation results suggests that the dominant polymorphic regions lie across from one another near one end of the alpha-helices that form the two walls of the proposed Ag-binding cleft located on the top surface of the class II molecule. Furthermore, the majority of the amino acids which have been changed in the serologically selected mutants have side chains which are postulated to point up toward the exterior of the molecule and would, therefore, be potential contact residues for the TCR.

Amino Acid Sequence↗

PubMed1989

I-A alpha polymorphic residues that determine alloreactive T cell recognition.

An individual's T lymphocytes are highly reactive to allogeneic MHC molecules. As a step in deciphering the mechanism of allorecognition by T lymphocytes, we have attempted to identify the TCR's target on MHC class II molecules, in particular the polymorphic residues that determine the specificity of recognition. We have generated a panel of Ak-reactive, Ab-nonreactive T cell hybridomas, and sets of L cell transfectants displaying A alpha A beta molecules with wild-type, chimeric or single site-mutated A alpha chains, with reciprocal interchanges between Ak and Ab. We then measured the stimulation of the T hybridomas in response to the transfectants. The results indicate that the hybridomas recognize diverse and complex determinants, with contributions from both A alpha and A beta chains, and from several regions or amino acids of the A alpha chain. The data are most consistent with a model in which alloreactivity results from the presentation of peptides to the T cell by an allogeneic MHC molecule, peptides that cannot be presented by the responder's own MHC complexes. The specificity of allorecognition seems to be imparted mainly by peptide/MHC molecule rather than TCR/MHC molecule contacts.

Animals↗

PubMed1988

Slot-machine mutagenesis of a polymorphic residue on the A kappa alpha-chain.

This study explores the limitations on variability at a polymorphic position of an MHC class II molecule. Using a convenient and rapid method termed "slot-machine mutagenesis," we have converted Glu75 on the A kappa-chain to 15 alternative amino acids. This residue is of interest because it is an immunodominant site on the A kappa alpha chain and because it participates in certain T cell epitopes. The wild-type and mutant A kappa alpha cDNA were transfected into L cells (together with the A kappa beta cDNA and a selection marker), and transfectants displaying high surface levels of the A kappa complex were selected and expanded. We sought to examine three questions: what is the effect of these mutations on the expression and overall conformation of the A alpha: A beta complex? How do these diverse mutations influence mAb epitopes for which Glu75 makes a direct contribution to specificity? Do such substitutions affect T cell recognition of the A kappa alpha:A kappa beta complex? The answers to these three questions are quite different. Position 75 of the A alpha chain can accommodate essentially all chemically divergent amino acids without major consequences for expression and overall A alpha:A beta structure. In contrast, mAb that recognize Glu75-dependent epitopes are extremely particular about the amino acid residing at this position. T cells are less fastidious: those that are affected by the mutations still recognize a number of substitutions. These data emphasize the tolerance of MHC molecules to evolutionary tampering.

Amino Acid Sequence↗

PubMed1986

Functional sites on Ia molecules: a molecular dissection of A alpha immunogenicity.

Ia antigens are polymorphic cell-surface molecules that control the immune response. We have begun to localize important functional sites on one of the Ia molecules, A alpha. Herein, we focus on the A alpha k and A alpha b alleles and ask what defines "b-ness" and "k-ness" for a panel of monoclonal antibodies. Two independent experimental strategies are employed: the ability of 12 monoclonal antibodies to recognize L cell transfectants bearing chimeric and mutant A alpha chains is assessed, and the amino acid sequences of A alpha chains expressed by immunoselected B lymphoma mutants are deduced. For each antibody, we identify a stretch of polymorphic residues critical for recognition; for several, we can pinpoint a single amino acid. Certain stretches of A alpha (depending on the allele) appear strikingly immunodominant.

Amino Acid Sequence↗

PubMed1985

A molecular basis for the Ia.2 and Ia.19 antigenic determinants.

The murine Ia antigens, heterodimeric glycoproteins on the surface of immunocompetent cells, restrict immune recognition by their influence on cell-cell interactions. Many serological specificities have been mapped to these molecules, and monoclonal antibodies directed against some of these determinants block antigen presentation to T lymphocytes. As a step toward a better understanding of Ia function, we sought to define the molecular basis of Ia.2 and Ia.19, specificities found on the A alpha chain of only the k or of both k and r haplotypes, respectively. We report nucleotide sequences for the A alpha chain cDNAs of the k, r, and s haplotypes, which, when compared to previously published A alpha sequences, demonstrate the existence of one k-specific amino acid residue and of another present only in the k and r haplotypes. These residues must thus play an important role in the generation of Ia.2 and Ia.19 specificities.

Alleles↗