Biomedical subjects

C Granier

Publications and source records attributed to C Granier.

At least 37 records · Page 2Linked to original sources

PubMed2000

Systematic mapping of regions of human cardiac troponin I involved in binding to cardiac troponin C: N- and C-terminal low affinity contributing regions.

The Spot method of multiple peptide synthesis was used to map in a systematic manner regions of the human cardiac troponin I sequence (hcTnI) involved in interactions with its physiological partner, troponin C (cTnC). Ninety-six 20-mer peptides describing the entire hcTnI sequence were chemically assembled; their reactivity with [125I]cTnC, in the presence of 3 mM Ca2+, enabled the assignment of six sites of interaction (residues 19-32, 45-54, 129-138, 145-164, 161-178 and 191-210). For several sites, a good correlation with literature data was obtained, thus validating this methodological approach. Synthetic peptides, each containing in their sequence an interaction site, were prepared. As assessed by BIACORE, all of them exhibited an affinity for cTnC in the range of 10(-6)-10(-7) M, except for hcTnI [39-58] which showed a nanomolar affinity. This peptide was also able to block the interaction between hcTnI and cTnC. We therefore postulate that despite the existence of multiple cTnC interaction sites on the hcTnI molecule, only that region of hcTnI allows a stabilization of the complex. Residues 19-32 from the N-terminal cardio-specific extension of hcTnI were also found to be involved in interaction with cTnC; residues 19-32 may correspond to the minimal sequence of the extension which could switch between the N- and C-terminal TnC domains, depending on its phosphorylation state. Finally, two Ca(2+)-dependent cTnC binding domains within the C-terminal part of hcTnI (residues 164-178 and 191-210) were also mapped. The latter site may be linked with the cardiac dysfunction observed in stunned myocardium.

Amino Acid Sequence↗

PubMed2000

Binding specificity of monoclonal antibody AD2: influence of the phosphorylation state of tau.

Using recombinant human tau protein phosphorylated by a brain extract and the glycogen synthase kinase-3beta in the absence or the presence of heparin, we showed that phosphorylation-dependent antibody AD2 recognition only requires phosphorylated Ser-396. By the Spot multiple peptide synthesis method, we showed that Tyr-394, Ser(P)-396 and Pro-397 are critical for AD2 binding. A decrease in the binding of AD2 was observed with increasing phosphorylation of residues in the vicinity of Ser(P)-396.

Alzheimer Disease↗

PubMed2000

Dissection of an antibody paratope into peptides discloses the idiotope recognized by the cognate anti-idiotypic antibody.

Using methods of parallel synthesis, the complete amino acid sequence of an Ab 1 antibody (Tg 10, an anti-human thyroglobulin monoclonal antibody) was made in the form of a set of 100 synthetic overlapping peptides. This set of immobilized peptides was allowed to react with the cognate Ab2 (AI 10, a highly purified rabbit anti-idiotypic polyclonal antibody to Tg 10). A dominant peptide idiotope, INTFSGVPTYA, was thus mapped, which corresponds mainly to the CDR2 region from the V(H) domain of the Tg 10 mAb. A synthetic peptide replica of this idiotope was found to bind to AI 10 with an affinity (K(D) in the 10(-8) M range, as measured using BIACORE technology) which represents a significant part of the affinity of the complete Tg 10 antibody (K(D) in the 10(-9) M range). The synthetic peptide also elicited anti-idiotypic antibodies in rabbits that recognized specifically the Ab1 antibody in an Ab1- and antigen-inhibitable manner. The peptide idiotope was further characterized chemically by the identification of residues important for binding to the Ab2 and by modelization of its structure. Our approach makes it readily possible to map and characterize functional, continuous-type idiotopes that could be further used to manipulate the immune response by peptide technologies.

Amino Acid Sequence↗

PubMed2000

Mapping the CD4 binding domain of gp17, a glycoprotein secreted from seminal vesicles and breast carcinomas.

gp17, a secretory CD4-binding factor isolated from the human seminal plasma, is identical to the gross cystic disease fluid protein-15, a specific marker for primary and metastatic breast tumors. We previously demonstrated that gp17 binds to CD4 with high affinity and strongly inhibits T lymphocyte apoptosis induced by sequential cross-linking of CD4 and T cell receptor (TCR). To further characterize the gp17/CD4 interaction and map the gp17 binding site, we produced a secreted form of recombinant gp17 fused to human IgG1 Fc, gp17-Ig. We showed that gp17-Ig exhibits a binding affinity for CD4 similar to that of native gp17. As no information about gp17 structure is presently available, 99 overlapping gp17 peptides were synthesized by the Spot method, which allowed the mapping of two CD4 binding regions. Alanine scanning of CD4-reactive peptides identified critical residues, selected for site-directed mutagenesis. Nine gp17-Ig mutants were generated and characterized. Three residues within the carboxy-terminal region were identified as the major binding domain to CD4. The Spot method combined with mutagenesis represents a refined approach to distinguish the contact residues from the ones contributing to the conformation of the CD4-binding domain.

Amino Acid Sequence↗

PubMed2000

Human anti-thyroid peroxidase single-chain fragment variable of Ig isolated from a combinatorial library assembled in-cell: insights into the in vivo situation.

In an attempt to explore the natural variable heavy and light chain (VH/VL) pairing of autoantibodies involved in Graves' disease, we constructed a phage-displayed Ab library obtained by in-cell PCR of thyroid-infiltrating cells. We report here the molecular cloning and characterization of human single-chain fragment variable regions (scFv) specific for thyroid peroxidase (TPO) generated from this library. On the basis of the nucleotide sequences, three different scFvs were obtained (ICA1, ICB7, and ICA5). All were encoded by genes derived from the VH1 and Vlambda1 gene families. Using BIACORE for epitope mapping and kinetic analysis, we showed that these scFvs exhibited high affinity (Kd = 1 nM) for TPO and recognized three different epitopes. The biological relevance of these scFvs as compared with serum anti-TPO autoantibodies was assessed by competition studies. Sera from all the 29 Graves' disease patients tested were able to strongly inhibit (60-100%) the binding of the 3 scFvs to TPO. These data demonstrate that the in-cell PCR library generated human anti-TPO scFvs that retained the VH/VL pairing found in vivo and that the different epitope specificities defined by these scFvs overlapped with those found in the sera of patients with autoimmune thyroid disease.

Adult↗

PubMed2000

Major T cell epitope-containing peptides can elicit strong antibody responses.

Peptides containing major T cell epitopes have the capacity to induce T cell anergy and have therefore been proposed for the treatment of allergic and autoimmune diseases. Such peptides should not be immunogenic, i. e. should not contain a B cell recognition site. We have evaluated in BALB/c mice the therapeutic potential of a 15-mer peptide (p21 - 35) derived from Der p2, a major allergen of the house dust mite Dermatophagoides pteronyssinus, which contains a dominant T cell epitope but is not recognized by antibodies to Der p2. Unexpectedly, p21 - 35 elicited strong immune responses, suggesting the presence of a cryptic B cell epitope. Similar results were obtained with mice of three additional MHC haplotypes. A core sequence of four amino acids (Ile-Ile-His-Arg) corresponding to residues 28 - 31 was shared by the B and T cell epitopes. Critical residues for B cell recognition were Arg31 and Lys33, while Ile28 was essential for T cell recognition. A Lys33Ala mutant of p21 - 35 still activated T cells but had much reduced immunogenic properties, making it a suitable alternative peptide for T cell anergy induction. Careful investigation of the immunogenic potential of peptides used to induce T cell anergy should be carried out prior to their clinical application.

Allergens↗

PubMed2000

Co-Ordination of Cell Division and Tissue Expansion in Sunflower, Tobacco, and Pea Leaves: Dependence or Independence of Both Processes?

Temporal analyses of cell division and tissue expansion in pea, tobacco, and sunflower leaves reveal that both processes follow similar patterns during leaf development. Relative cell division and relative tissue expansion rates are maximal and constant during early leaf development, but they decline later. In contrast, relative cell expansion rate follows a bell-shaped curve during leaf growth. Cell division and tissue expansion have common responses to temperature, intercepted radiation, and water deficit. As a consequence, final leaf area and cell number remain highly correlated throughout a large range of environmental conditions for these different plant species, indicating that cell division and tissue expansion are co-ordinated during leaf development. This co-ordination between processes has long been explained by dependence between both processes. Most studies on dicotyledonous leaf development indicate that leaf expansion rate depends on the number of cells in the leaf. We tested this hypothesis with a large range of environmental conditions and different plant species. Accordingly, we found a strong correlation between both absolute leaf expansion rate and leaf cell number. However, we showed that this relationship is not necessarily causal because it can be simulated by the hypothesis of independence between cell division and tissue expansion according to Green's theory of growth (1976).

Journal Article↗

PubMed2000

Modulation of TCR recognition of MHC class II/peptide by processed remote N- and C-terminal epitope extensions.

N- and C-terminal extensions of naturally processed MHC class II-bound peptides may affect TCR recognition. In fact, residues immediately flanking the minimal epitope on either side can contact the MHC groove and modify the interaction with a TCR. We report now that residues much farther away from the peptide core can also modulate TCR recognition in a functional antigen presentation system. To show this, we isolated from the same donor DR5-restricted T cell clones, specific for the HIV-1 RT(248-262) sequence and differing in their ability to respond to recombinant antigens obtained by insertion of the epitope in different positions of schistosomal, human, or murine glutathione-S-transferase (GST). We found that the reactivity profile of individual clones was related to their TCR fine specificity, suggesting that processing can generate determinants focused onto the same epitope, but antigenically distinct. In addition, we analyzed the response of this panel of T-helper cell clones against GST-derived recombinant antigens in which the epitope was flanked by stretches of polyalanine or polyserine on either side. These spacers had different effects on TCR recognition suggesting that secondary structures outside the core peptide may influence MHC/epitope complex recognition over a distance of 15-30 residues from the determinant.

Amino Acid Sequence↗

PubMed2000

Quantitative analysis of cell division in leaves: methods, developmental patterns and effects of environmental conditions.

In planta quantitative studies of cell cycle are necessary for examining the role of cell division in the response of plants to environmental conditions and to analyse the behaviour of transformed plants in this context. We present and discuss non-intrusive kinematic methods which allow estimating the duration of cell cycle with a high spatial resolution in the leaf. Different methods are proposed and discussed for monocotyledons and dicotyledons, and compared with methods involving the use of chemicals. In monocotyledon leaves, cell division is restricted to a limited zone near the leaf insertion point, twice as long in the mesophyll as in the epidermis. In dicotyledons, cell division occurs in the whole leaf with a uniform and constant cell cycle duration for a determinate number of cell cycles, representing about half of leaf development. Over several experiments, this number is well conserved in a given leaf zone in the absence of stresses, but larger near the leaf base than near the leaf tip. After that, cell cycle duration increases because cells are progressively blocked in G1 while the durations of S-G2-M phases do not change with time. Leaf temperature affects neither the distribution of nuclei in each phase of the cycle nor the number of cell cycles in a leaf. Water or light deficits both cause a partial blockage of nuclei in G1 during the stress only, thereby increasing cell cycle duration and decreasing final cell number. These results suggest that a strong developmental programme drives cell division in leaves, so a simple framework allows analysis of temporal patterns, of spatial gradients and of the effect of environmental conditions.

Biomechanical Phenomena↗

PubMed2000

Affinity for the cognate monoclonal antibody of synthetic peptides derived from selection by phage display. Role of sequences flanking thebinding motif.

Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are generally further used in the form of synthetic peptides; however, as such, their affinity for the selecting antibody is extremely variable and factors influencing this affinity have not been fully deciphered. We have used an f88.4 phage-displayed peptide library to identify ligands of mAb 11E12, an antibody reactive to human cardiac troponin I. A majority of the sequences thus selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y-TEPH motif identified by multiple peptide synthesis as the critical motif recognized by mAb 11E12 in the peptide epitope. A set of 15-mer synthetic peptides derived from the phage-selected sequences was used in BIACORE to characterize their interaction with mAb 11E12. Most peptides exhibited affinities in the 7-26 nM range. These affinities represented, however, only 1.9-7. 5% of the affinity of the 15-mer peptide epitope. In circular dichroism experiments, the peptide epitope showed a propensity to have some stabilized conformation, whereas a low-affinity peptide selected by phage-display did not. To try to decipher the molecular basis of this difference in affinity, new peptides were prepared by grafting the N- or the C-terminal sequence of the peptide epitope to the Y-TEPK motif of a low-affinity peptide selected by phage-display. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in competition ELISA), compared with the parent sequence. Thus, the sequences flanking the motif, although not containing critical residues, convey some determinants necessary for high affinity. The affinity of a given peptide strongly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether high- or low-affinity peptides will be obtained from phage display.

Amino Acid Sequence↗

PubMed2000

Spatial distributions of expansion rate, cell division rate and cell size in maize leaves: a synthesis of the effects of soil water status, evaporative demand and temperature.

The spatial distributions of leaf expansion rate, cell division rate and cell size was examined under contrasting soil water conditions, evaporative demands and temperatures in a series of experiments carried out in either constant or naturally fluctuating conditions. They were examined in the epidermis and all leaf tissues. (1) Meristem temperature affected relative elongation rate by a constant ratio at all positions in the leaf. If expressed per unit thermal time, the distribution of relative expansion rate was independent of temperature and was similar in all experiments with low evaporative demand and no water deficit. This provides a reference distribution, characteristic of the studied genotype, to which any distribution in stressed plants can be compared. (2) Evaporative demand and soil water deficit affected independently the distribution of relative elongation rate and had near-additive effects. For a given stress, a nearly constant difference was observed, at all positions of the leaf, between the relative elongation rates of stressed plants and those of control plants. This caused a reduction in the length of the zone with tissue elongation. (3) Methods for calculating cell division rate in the epidermis and in all leaf tissues are proposed and discussed. In control plants, the zone with cell division was 30 mm and 60 mm long in the epidermis and in whole tissues, respectively. Both this length and relative division rate were reduced by soil water deficit. The size of epidermal and of mesophyll cells was nearly unaffected in the leaf zone with both cell division and tissue expansion, suggesting that water deficit affects tissue expansion rate and cell division rate to the same extent. Conversely, cell size of epidermis and mesophyll were reduced by water deficit in mature parts of the leaf.

Cell Division↗

PubMed2000

Spatial distribution of cell division rate can be deduced from that of p34(cdc2) kinase activity in maize leaves grown at contrasting temperatures and soil water conditions.

We have investigated the spatial distributions of cell division rate, p34(cdc2) kinase activity, and amount of p34(cdc2a) in maize (Zea mays) leaves grown at contrasting temperatures and soil water conditions. An original method for calculating cell division rate in all leaf tissues is proposed. In all studied conditions, cell division rate was stable and maximum in the first 2 cm beyond the leaf insertion point, declined afterward, and reached zero at 7 cm from the insertion point. The spatial distribution of p34(cdc2) kinase activity, expressed on a per cell basis, followed the same pattern. In contrast, the amount of p34(cdc2a) was maximum in the first centimeter of the leaf, declined afterward, but remained at 20% of maximum in more distal zones with a near-zero cell division rate. A mild water deficit caused a reduction in cell division rate and p34(cdc2) kinase activity by approximately 45% in all leaf zones, but did not affect the amount of p34(cdc2a). Growth temperature affected to the same extent cell division rate and p34(cdc2) kinase activity, but only if p34(cdc2) kinase activity was assayed at growth temperature, and not if a standard temperature was used in all assays. A common linear relationship between cell division rate and p34(cdc2) kinase activity applied to all causes of changes in cell division rate, i.e. cell aging, water deficit, or changes in temperature. It is shown that temperature has two distinct and additive effects on p34(cdc2) kinase activity; first, an effect on the rate of the reaction, and second, an effect on the amount of p34(cdc2a).

CDC2 Protein Kinase↗

PubMed2000

alpha2-Macroglobulin, the main serum antiprotease, binds beta2-microglobulin, the light chain of the class I major histocompatibility complex, which is involved in human disease.

beta(2)-Microglobulin, a 12 kDa protein forming part of the class I HLA (histocompatibility locus antigen) major histocompatibility complex, has been used as a prognosis factor for multiple myeloma and as a marker of renal function, and has been shown to be involved in the pathogenesis of dialysis-related amyloidosis. alpha(2)-Macroglobulin has the ability to bind a wide range of physiologically important molecules, thereby influencing their metabolic impact. In this study we show by Western blotting analysis that beta(2)-microglobulin binds to alpha(2)-macroglobulin in vitro. This binding was confirmed by BIAcore analysis, and was shown by ELISA to be concentration-dependent. The sequences of the binding peptides in the mature beta(2)-microglobulin molecule were identified by Spot multiple peptide synthesis and alpha(2)-macroglobulin binding studies. In conclusion, beta(2)-microglobulin interacts specifically with the universal antiprotease a(2)-macroglobulin. The identification of this interaction brings into question some of the axioms on the metabolism of beta(2)-microglobulin, and may help to explain the clinical findings observed in b(2)-microglobulin-related diseases.

Amyloidosis↗

PubMed1999

Synthetic peptides derived from the variable regions of an anti-CD4 monoclonal antibody bind to CD4 and inhibit HIV-1 promoter activation in virus-infected cells.

The monoclonal antibody (mAb) ST40, specific for the immunoglobulin complementarity-determining region (CDR) 3-like loop in domain 1 of the CD4 molecule, inhibits human immunodeficiency virus type 1 (HIV-1) promoter activity and viral transcription in HIV-infected cells. To design synthetic peptides from the ST40 paratope that could mimic these biological properties, a set of 220 overlapping 12-mer peptides frameshifted by one residue, corresponding to the deduced ST40 amino acid sequence, was synthesized by the Spot method and tested for binding to recombinant soluble CD4 antigen. Several peptides that included in their sequences amino acids from the CDRs of the antibody and framework residues flanking the CDRs were found to bind soluble CD4. Eleven paratope-derived peptides (termed CM1-CM11) were synthesized in a cyclic and soluble form. All the synthetic peptides showed CD4 binding capacity with affinities ranging from 1.6 to 86.4 nM. Moreover, peptides CM2, CM6, CM7, CM9, and CM11 were able to bind a cyclic peptide corresponding to the CDR3-like loop in domain 1 of CD4 (amino acids 81-92 of CD4). Peptide CM9 from the light chain variable region of mAb ST40 and, to a lesser extent, peptides CM2 and CM11 were able to inhibit HIV-1 promoter long terminal repeat-driven beta-galactosidase gene expression in the HeLa P4 HIV-1 long terminal repeat beta-galactosidase indicator cell line infected with HIV-1. The binding of mAb ST40 to CD4 was also efficiently displaced by peptides CM2, CM9, and CM11. Our results indicate that the information gained from a systematic exploration of the antigen binding capacity of synthetic peptides from immunoglobulin variable sequences can lead to the identification of bioactive paratope-derived peptides of potential pharmacological interest.

Amino Acid Sequence↗

PubMed1999

T cell response pattern to glutamic acid decarboxylase 65 (GAD65) peptides of newly diagnosed type 1 diabetic patients sharing susceptible HLA haplotypes.

Autoantibodies and autoreactive T lymphocytes directed against several pancreatic beta cell proteins such as GAD65 have been identified in the circulation before and at the onset of clinical type 1 (insulin-dependent) diabetes. Using GAD65 synthetic peptides, we studied the proliferative response of peripheral blood mononuclear cells (PBMC) either from recently diagnosed type 1 diabetic patients, of whom the majority share the disease-associated HLA class II haplotype (DR4-DQB1*0201 or DR3-DQB1*0302), or from HLA-matched control subjects. We found that 67% (14/21) of the type 1 diabetic patients and 39% (9/23) of the control subjects exhibited a positive proliferative response. Compared with control subjects, however, PBMC from diabetic patients proliferated more frequently (P < 0.05) in the presence of peptide pools from the C-terminal region of GAD65 (amino acids 379-585). Diabetic patients with the same HLA-DQ or HLA-DR alleles showed partially identical T cell reactivity, but no clear correlation could be made between MHC class II specificity and T cell epitopes because of multiple combinations of class II alleles. In addition, by flow cytometry, we studied the direct binding of GAD65 peptides to MHC class II molecules of Epstein-Barr virus (EBV)-transformed B (EBV-B) cells obtained from a diabetic patient. We found that 11 GAD peptides were able to bind to the highly susceptible haplotype DRB1*0301/0401-DQA1*0301/0501-DQB1*0302/0201 on the surface of EBV-B cells in partial correlation with the results obtained in the proliferation assays.

Adolescent↗

PubMed1999

Water deficit and spatial pattern of leaf development. Variability In responses can Be simulated using a simple model of leaf development

We analyzed the effect of short-term water deficits at different periods of sunflower (Helianthus annuus L.) leaf development on the spatial and temporal patterns of tissue expansion and epidermal cell division. Six water-deficit periods were imposed with similar and constant values of soil water content, predawn leaf water potential and [ABA] in the xylem sap, and with negligible reduction of the rate of photosynthesis. Water deficit did not affect the duration of expansion and division. Regardless of their timing, deficits reduced relative expansion rate by 36% and relative cell division rate by 39% (cells blocked at the G0-G1 phase) in all positions within the leaf. However, reductions in final leaf area and cell number in a given zone of the leaf largely differed with the timing of deficit, with a maximum effect for earliest deficits. Individual cell area was only affected during the periods when division slowed down. These behaviors could be simulated in all leaf zones and for all timings by assuming that water deficit affects relative cell division rate and relative expansion rate independently, and that leaf development in each zone follows a stable three-phase pattern in which duration of each phase is stable if expressed in thermal time (C. Granier and F. Tardieu [1998b] Plant Cell Environ 21: 695-703).

Journal Article↗

PubMed1999

Determination of cardiac troponin I forms in the blood of patients with acute myocardial infarction and patients receiving crystalloid or cold blood cardioplegia.

To determine the forms of cardiac troponin I (cTnI) circulating in the bloodstream of patients with acute myocardial infarction (AMI) and patients receiving a cardioplegia during heart surgery, we developed three immunoenzymatic sandwich assays. The first assay involves the combination of two monoclonal antibodies (mAbs) specific for human cTnI. The second assay involves the combination of a mAb specific for troponin C (TnC) and an anti-cTnI mAb. The third assay was a combination of a mAb specific for human cardiac troponin T (cTnT) and an anti-cTnI mAb. Fifteen serum samples from patients with AMI, 10 serum samples from patients receiving crystalloid cardioplegia during heart surgery, and 10 serum samples from patients receiving cold blood cardioplegia during heart surgery were assayed by the three two-site immunoassays. We confirmed that cTnI circulates not only in free form but also complexed with the other troponin components (TnC and cTnT). We showed that the predominant form in blood is the cTnI-TnC binary complex (IC). Free cTnI, the cTnI-cTnT binary complex, and the cTnT-cTnI-TnC ternary complex were seldom present, and when present, were in small quantities compared with the binary complex IC. Similar results were obtained in both patient populations studied. These observations are essential for the development of new immunoassays with improved clinical sensitivity and for the selection of an appropriate cTnI primary calibrator.

Fetal Blood↗

PubMed1998

An agonist-like monoclonal antibody against the human beta2-adrenoceptor.

Monoclonal antibodies were produced against a peptide corresponding to the second extracellular loop of the human beta2-adrenoceptor. One of these monoclonals, inducing an agonist-like effect in neonatal rat cardiomyocytes, was used to define the structural and physiological basis of this activity. The epitope recognized by the antibody corresponds to the sequence Trp-Tyr-Arg-Ala-Thr-His-Gln-Glu as determined by peptide scanning. Analysis by alanine modification of the peptide epitope showed the importance of the Trp, and Glu residues in antibody recognition The apparent affinity of the antibody assessed either by surface plasmon resonance or by functional titration on its agonist-like activity showed a similar value (10(8) M(-1)). The antibody recognized the receptor in its native form as shown by immunofluorescence experiments on A431 cells but not in its denatured form as shown by its absence of staining in immunoblots. The positive chronotropic effect in vitro was specifically blocked by both the antigenic peptide and the specific beta2-antagonist (+/-)-1-[2,3-(Dihydro-7-methyl1H-inden-4-yl)oxy]-3-[(1-methy lethyl)amino]-2-butanol hydrochloride (ICI1118,551). This activity was mediated through activation of Ca2+ L-type channels as assessed in guinea pig cardiomyocytes. These results suggest that the epitope is located in an extracellular alpha-helix, whose recognition by the antibody could stabilize the receptor in its 'active' conformation.

Adrenergic beta-2 Receptor Agonists↗