Search PubMed⌕ Search

Biomedical subjects

E Giralt

Publications and source records attributed to E Giralt.

At least 55 records · Page 3Linked to original sources
PubMed1994

Kaliotoxin (1-37) shows structural differences with related potassium channel blockers.

The three-dimensional structure of kaliotoxin (1-37), KTX(1-37), a toxin from the scorpion Androctonus mauretanicus mauretanicus that blocks calcium-dependent potassium channels, has been determined by NMR. This toxin is homologous with other scorpion toxins such as charybdotoxin (ChTX) or iberiotoxin (IbTX) for which the structures are already known, but the presence of prolines in the expected alpha-helical region suggested that there may be some major difference in the structure of KTX that could be related to its different selectivity. Proline residues are also found in the homologous region of other scorpion toxins such as noxiustoxin or margatoxin. Our results indicate that KTX(1-37) contains the same sequence of secondary structure elements as ChTX but that the helical region is shorter and distorted due to the presence of two prolines. The distortion consists of a bending in the alpha-helix and in the presence of a 3(10) helix turn in the last three residues. Furthermore, the increased length of the extended structure preceding the helix favors a different packing of this part of the molecule with respect to the secondary structure elements. This change in folding modifies the accessibility of the conserved 27Lys which is known, from mutation studies, to be involved in channel blocking by ChTX.

Amino Acid Sequence↗
PubMed1994

Crystallization and preliminary X-ray diffraction studies of a monoclonal antibody Fab fragment against foot-and-mouth disease virus and of its complex with the main antigenic site peptide.

The Fab fragment of the neutralizing monoclonal antibody SD6 elicited against foot-and-mouth disease virus (FMDV) C-S8c1 and its complex with a peptide, corresponding to the major antigenic site of FMDV (VP1 residues 136-150, YTASARGDLAHLTTT), have been crystallized using the hanging drop vapor diffusion techniques. For the isolated Fab, crystals diffracting to 2.5 A resolution were obtained at room temperature using ammonium sulfate as precipitant. These crystals are monoclinic, space group C2, and unit cell parameters a = 109.53 A, b = 89.12 A, c = 64.04 A, and beta = 112.9 degrees and contain one Fab molecule per asymmetric unit. Crystals from the complex diffract, at least, to 2.8 A resolution and were obtained, at room temperature, using PEG as precipitant. These crystals are monoclinic, space group P2, and unit cell parameters a = 56.11 A, b = 60.67 A, c = 143.45 A, and beta = 95.4 degrees. Density packing considerations indicate that there are two Fab molecules in the asymmetric unit.

Amino Acid Sequence↗
PubMed1994

Association between double mutation in gyrA gene of ciprofloxacin-resistant clinical isolates of Escherichia coli and MICs.

The mutations in the quinolone resistance-determining region of the gyrA and gyrB genes from 27 clinical isolates of Escherichia coli with a range of MICs of ciprofloxacin from 0.007 to 128 micrograms/ml and of nalidixic acid from 2 to > 2,000 micrograms/ml were determined by DNA sequencing. All 15 isolates with ciprofloxacin MICs of > or = 1 micrograms/ml showed a change in Ser-83 to Leu of GyrA protein, whereas in clinical isolates with a MIC of > or = 8 micrograms/ml (11 strains), a double change in Ser-83 and Asp-87 was found. All isolates with a MIC of nalidixic acid of > or = 128 micrograms/ml showed a mutation at amino acid codon Ser-83. Only 1 of the 27 clinical isolates of E. coli analyzed showed a change in Lys-447 of the B subunit of DNA gyrase. A change in Ser-83 is sufficient to generate a high level of resistance to nalidixic acid, whereas a second mutation at Asp-87 in the A subunit of DNA gyrase may play a complementary role in developing the strain's high levels of ciprofloxacin resistance.

Amino Acid Sequence↗
PubMed1994

Antigenic heterogeneity of a foot-and-mouth disease virus serotype in the field is mediated by very limited sequence variation at several antigenic sites.

Antigenic variation in a major discontinuous site (site D) of foot-and-mouth disease virus (FMDV) of serotype C has been evaluated with neutralizing monoclonal antibodies. Isolates representing the major evolutionary sublines previously defined for serotype C were compared. Extensive variation, comparable to that of continuous epitopes within the hypervariable immunodominant site A (the VP1 G-H loop), was found. The amino acid sequences of the complete capsids of three antigenically highly divergent FMDVs (C1 Haute Loire-Fr/69, C5 Argentina/69, and C3 Argentina/85) have been determined and compared with the corresponding sequences previously determined for seven additional type C viruses. Differences in antigenicity are due to a very limited number of substitutions of surface amino acids accessible to antibodies and located within antigenic sites previously identified on FMDV. A significant number of residues at these positions were also replaced in monoclonal antibody escape mutants. Depending on the variants compared, replacements within site A or at site D, or at both sites, contributed significantly to their antigenic differences. Examples of divergence mediated by a few amino acid replacements were found among FMDVs of Europe and South America. The results suggest that within a serotype of FMDV, antigenically highly divergent viruses can arise in the field by very limited sequence variation at exposed key residues of each of several antigenic sites.

Amino Acid Sequence↗
PubMed1993

Use of substituted and tandem-repeated peptides to probe the relevance of the highly conserved RGD tripeptide in the immune response against foot-and-mouth disease virus.

Antigenic site A of foot-and-mouth disease virus (FMDV) is an exposed, mobile loop which includes a central, highly conserved Arg-Gly-Asp tripeptide (RGD, VP1 residues 141-143 in serotype C) thought to be part of the cell attachment site. We have analyzed the contribution of RGD to the interaction of site A with antibodies by incorporating selected amino acid replacements at RGD into synthetic peptides representing site A, and analyzing the reactivity of substituted peptides with site A-specific monoclonal antibodies (MAbs). Replacement of Arg-141, Gly-142 or Asp-143 by alanine resulted in the loss of one, three and five epitopes, respectively, out of seven epitopes probed. Other replacements resulted in the loss of even larger numbers of epitopes, suggesting that the amino acids of the RGD region are either directly involved in interaction with antibodies or that they exert an important influence on the interaction of surrounding residues with antibodies. Thus, we explored the ability of tandem repeats of the RGDL sequence (corresponding to FMDV C-S8c1) to evoke neutralizing antibodies in rabbits and guinea pigs. Neutralizing activity was generally low but with a broad specificity for different FMDV serotypes and variants. Significant decreases in neutralizing titers were observed with boosting, suggesting a possible suppression of those anti-peptide antibodies which may also be directed to cellular RGD sequences. The results point to an involvement of RGD in the antigenic structure of site A, and open the possibility that broadly neutralizing antibodies might be induced by tandem repeats of the critical, conserved domain.

Amino Acid Sequence↗
PubMed1993

Cyclic disulfide model of the major antigenic site of serotype-C foot-and-mouth disease virus. Synthetic, conformational and immunochemical studies.

A cyclic disulfide peptide representing antigenic site A of foot-and-mouth disease virus (FMDV) strain C-S8c1 (residues 134 to 155 of viral protein 1 (VP1) with Tyr136 and Arg153 replaced by cystine; TTCTASARGDLAHLTTTHACHL) was synthesized by solid phase methods. Formation of the cyclic disulfide was carried out by air oxidation of the fully deprotected and reduced bis-cysteine precursor, under high dilution conditions. The identity of the cyclic peptide was confirmed by both physical and enzymatic methods. A conformational study of the cyclic peptide and of its linear parent structure (YTASARGDLAHLTTTHARHLP, residues 136-156 of VP1 of FMDV C-S8c1) by circular dichroism in the presence of a structure-inducing solvent showed the cyclic disulfide analog to adopt lower levels of alpha-helix than its linear counterpart. In competitive ELISA assays both peptides reacted with similar affinity against a representative panel of neutralizing monoclonal antibodies directed towards antigenic site A. Thus, a high inherent flexibility of this loop may preclude a conformational restriction strong enough to alter recognition by anti-virus antibodies.

Amino Acid Sequence↗
PubMed1993

CD of proline-rich polypeptides: application to the study of the repetitive domain of maize glutelin-2.

An overview of CD of proline-rich peptides is reported. First, structural characteristics, theoretical CD studies, and the biological relevance of polyproline II structure in such peptides are discussed. Second, a CD study of peptides belonging to the repetitive domain of maize glutelin-2, H-(Val-His-Leu-Pro-Pro-Pro)n-OH (n = 3, 5, 8), is described. This series of peptides displayed the CD features of polyproline II structure in water (5 degrees C, pH 5). Moreover, it was shown that the addition of increasing amounts of the polyanionic molecule heparin forced a displacement of the conformational equilibrium of those peptides toward higher proportions of the polyproline II structure. In contrast, when the temperature is raised such a structure gradually disappears, leading to more disordered conformations.

Amino Acid Sequence↗
PubMed1993

Distinct repertoire of antigenic variants of foot-and-mouth disease virus in the presence or absence of immune selection.

Antigenic variants of foot-and-mouth disease virus (FMDV) were generated and frequently became dominant in clonal populations of FMDV (clone C-S8c1) grown in the absence of anti-FMDV antibodies. We have now passaged eight samples of the same FMDV clone in the presence of a limited amount of neutralizing polyclonal antibodies directed to the major antigenic site A of capsid protein VP1. Complex populations of variants showing increased resistance to polyclonal sera and to site A-specific monoclonal antibodies were selected. Some populations exhibited marked decreases in viral fitness. Multiple amino acid replacements within site A--and also elsewhere in VP1--accumulated upon passage of the virus in either the absence or the presence of neutralizing antibodies. However, antigenically critical replacements at one position in site A occurred repeatedly in FMDV passaged under antibody selection, but they were never observed in many passages carried out either in the absence of antiviral antibodies or in the presence of an irrelevant antiviral serum. Thus, even though antigenic variation of FMDV can occur in the absence or presence of immune selection, critical replacements which lead to important changes in antigenic specificity were observed only as a result of selection by neutralizing antibodies.

Amino Acid Sequence↗
PubMed1992

Non-additive effects of multiple amino acid substitutions on antigen-antibody recognition.

Synthetic peptides have been used to mimic the main antigenic site of foot-and-mouth disease virus (FMDV) of serotype C and of several variant isolates. This region includes multiple continuous B cell epitopes. The effect of single amino acid replacements, individually or in combination, on antigen specificity has been evaluated using monoclonal antibodies. Quantitative enzyme immunodot assays have shown that both additive and non-additive effects of multiple replacements occur in continuous B cell epitopes, with regard to antibody recognition. Antigenically critical single replacements may be compensated by other, non-critical replacements. Thus, the role of a single amino acid on antibody recognition depends on the sequence context in the antigenic domain. The non-additive effects of multiple replacements may modulate the extent of antigenic diversification of highly variable RNA viruses, and keep viruses confined within antigenic groups by precluding linear antigenic divergence.

Amino Acid Sequence↗
PubMed1992

Conformational study of a nine residue fragment of the antigenic loop of foot-and-mouth disease virus.

The nine-residue peptide Ac-TASARGDLA-NHMe was selected as model peptide in order to understand the conformational features of the antigenic loop of foot-and-mouth disease virus (FMDV). A throughout exploration of the conformational space has been carried out by means of molecular dynamics (MD) and energy minimization. The calculations have been carried out using the AMBER force field. Solvent effects have been included by an effective dielectric constant of epsilon = 4r. The lowest energy conformation presents a secondary structure constituted by an alpha-helix at the N-terminal end followed by two gamma-turns in the central region. The rest of the accessible minima found present also a high tendency to form gamma-turns. Finally, a 100 ps MD trajectory calculation at 298 K suggest a stability of the secondary structure elements of the lowest energy conformation.

Amino Acid Sequence↗
PubMed1992

Studies on antigenic variability of C strains of foot-and-mouth disease virus by means of synthetic peptides and monoclonal antibodies.

Peptides representing the sequence of the immunodominant loop of foot-and-mouth disease virus strain C-S8 (YTASARGDLAHLTTTHARHLP, residues 136-156 of VP1) and of several variant viruses have been prepared by solid phase methods. In addition, five peptides with single-residue replacements at Leu147 (Ile, Nle, Val, Ala, Gly) have been synthesized. Tosyl and dinitrophenyl protections for histidine have been compared, the latter being found to give better synthetic products. The peptides have been tested in an immunodot assay against a panel of monoclonal antibodies directed towards the VP1 loop. Immunochemical results are discussed on the basis of the mobility of the region reproduced by the peptides and the nature of the side chain of residue 147.

Amino Acid Sequence↗
PubMed1992

RNA binding characteristics of a 16 kDa glycine-rich protein from maize.

We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MA16-encoded protein binds preferentially to uridine- and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.

Amino Acid Sequence↗
PubMed1991

Diurnal rhythm of rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA synthase.

Rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase exhibits a diurnal rhythm of enzyme activity which coincides with the diurnal rhythm of HMG-CoA synthase protein. The peaks of activity and protein (determined by SDS/PAGE and immunoblotting) both occur at D10 (the tenth hour of the daily 12 h dark cycle). The peak of mRNA levels (measured by slot-blot hybridization of liver RNA) is slightly advanced with respect to that of protein, by about 4 h, and shows a maximum at D6. Cytosolic HMG-CoA synthase activity and protein in rats fed on a normal diet were approx. 2-fold higher during the peak at D10 than in the nadir at D2. HMG-CoA synthase mRNA levels were approx. 4-fold higher during the peak at D6 than in the nadir at D2. These results point to a transcriptional and translational regulation of the cytosolic HMG-CoA synthase.

Amino Acid Sequence↗
PubMed1991

Determination of the enantiomeric purity of synthetic peptides by gas chromatography-mass spectrometry.

Hydrolysis using 2H-labelled HCl and H2O, derivatization of free amino acids as N.O.S-trifluoroacetyl isobutyl esters, separation by gas chromatography on a chiral stationary phase and detection by mass spectrometry in selected-ion monitoring mode have been used in order to determine the enantiomeric purity of several synthetic peptides. Chromatographic separation has been optimized for proline, whose two enantiomers are difficult to resolve under standard conditions. Electron impact and methane chemical ionization mass spectra and chromatographic resolution of unnatural amino acids, such as 3-(1-naphthyl)-alanine and p-chlorophenylalanine, are reported. For both natural and unnatural amino acids selected-ion monitoring of the different fragmentation peaks has been carried out. The results are interpreted from the point of view of whether or not the fragments contain a hydrogen atom on the alpha-carbon, and a comparison between electron impact and methane chemical ionization has been carried out. The main advantage of the latter method is that a quasimolecular ion can be observed for all the amino acids studied.

Acetates↗
PubMed1991

Conformational analysis of bacitracin A, a naturally occurring lariat.

The proton nmr spectra of bacitracin A in H2O and DMSO-d6 have been assigned and the conformational behavior of the peptide in the two solvents has been compared. Although bacitracin A shows a conformational equilibrium between at least two conformations differing in the relative position of the cyclic and linear domains of the molecule, the spectra in water can be interpreted in terms of a preferred conformation in which the linear part is folded over the cyclic moiety and a turn is present around Ile(8)-DPhe(9).

Amino Acid Sequence↗
PubMed1991

Conformational analysis of two cyclic disulfide peptides.

Complete nmr and CD studies of two cyclic tetrapeptides with disulfide bonds, Ac-L-Pen-L-Pro-D-Val-L-Cys-NH2 (1) and Ac-L-Cys-L-Pro-D-Val-L-Cys-NH2 (2) bonds have been carried out in different solvents to investigate the formation and stabilization of beta-turn structures and to determine the stereochemistry of the disulfide linkage. Both peptides have three-dimensional structures with a type II beta-turn, as derived from quantitative nuclear Overhauser effect data. The combined use of CD and nmr indicates that the dihedral angle of the disulfide bridge is different in the two peptides, although the chirality is maintained.

Amino Acid Sequence↗
PubMed1991

A new ionizable chromophore of 1,4-bis(alkylamino)benzo[g]phthalazine which interacts with DNA by intercalation.

The tricyclic heteroaromatic nucleus of 1,4-bis(alkylamino)benzo[g]phthalazine can be protonated at physiological pH, depending on the nature of the side chains. The interaction of the 3-methoxypropyl derivative with calf thymus and closed, circular DNA has been studied with UV-vis spectroscopy and NMR. The effect of drug binding on the topology of closed, circular DNA was determined by topoisomerase-I catalyzed relaxation of the complex followed by gel electrophoresis. The results strongly support intercalative binding and suggest that this series of compounds are promising targets for anticancer activity evaluation.

DNA↗