Biomedical subjects

D Andreu

Publications and source records attributed to D Andreu.

At least 19 recordsLinked to original sources

PubMed2005

Lack of oestrogen protection in amyloid-mediated endothelial damage due to protein nitrotyrosination.

Amyloid beta-peptide (Abeta) cytotoxicity, the hallmark of Alzheimer's disease, implicates oxidative stress in both neurons and vascular cells, particularly endothelial cells. Consequently, antioxidants have shown neuroprotective activities against Abeta-induced cytotoxicity. Among the different antioxidants used in both in vitro and in vivo studies, 17beta-oestradiol (E2) has garnered the most attention. Oestrogen attenuated Abeta(E22Q)-induced toxicity in neurons but failed to protect endothelial cells. Here we show that E2-mediated activation of endothelial nitric oxide synthase (eNOS) increases the production of nitric oxide (NO), which, under Abeta(E22Q)-induced oxidative damage, results in the formation of peroxynitrite and increased nitration of tyrosine residues. Inhibition of eNOS prevents nitrotyrosination and permits E2-mediated protection against Abeta(E22Q) on endothelial cells. The main nitrotyrosinated proteins in the presence of E2 and Abeta(E22Q) were identified by MALDI-TOF mass spectrometry. These proteins are key players in the regulation of energy production, cytoskeletal integrity, protein metabolism and protection against oxidative stress. Our data highlight the potential damaging consequences of E2 in vascular disorders dealing with oxidative stress conditions, such as cerebral amyloid angiopathy, stroke and ischaemia-reperfusion conditions.

Aged↗

PubMed2004

Safety and efficacy of antimicrobial peptides against naturally acquired leishmaniasis.

Leishmaniases, which are important causes of morbidity and mortality in humans and dogs, are extremely difficult to treat. Antimicrobial peptides are rarely used as alternative treatments for naturally acquired parasitic diseases. Here we report that the acylated synthetic antimicrobial peptide Oct-CA(1-7)M(2-9) is safe and effective for treating naturally acquired canine leishmaniasis.

Animals↗

PubMed2003

Conformational study of linear and cyclic peptides corresponding to the 276-284 epitope region of HSV gD-1.

The results of conformational analysis of linear and cyclic peptides from the 276SALLEDPVG(284) sequence of glycoprotein D of Herpes simplex virus are presented. The epitope peptides were synthesized by SPPS and on resin cyclization was applied for preparation of cyclic compounds. Circular dichroism spectroscopy, Fourier-transform infrared spectroscopy and nuclear magnetic resonance (NMR) were used to determine of the solution structure of both linear and cyclic peptides. The results indicated that the cyclopeptides containing the core of the epitope (DPVG) as a part of the cycle have more stable beta-turn structure than the linear peptides or the cyclic analogues, where the core motif is not a part of the cycle. NMR study of H-SALLc(EDPVGK)-NH(2) confirm presence of a type I beta-turn structure which includes the DPVG epitope core.

Circular Dichroism↗

PubMed2002

Probing degeneracy in antigen-antibody recognition at the immunodominant site of foot-and-mouth disease virus.

Antigen-antibody binding is regarded as one of the most representative examples of specific molecular recognition in nature. The simplistic view of antigenic recognition in terms of a lock-and-key mechanism is obsolete, as it is evident that both antigens and antibodies are flexible and can undergo substantial mutual adaptation. This flexibility is the source of complexities such as degeneracy and nonadditivity in antigenic recognition. We have used surface plasmon resonance to study the effects of combining multiple amino acid replacements within the sequence of the antigenic GH loop of foot-and-mouth disease virus. Our aim was 2-fold: to explore the extent to which antigenic degeneracy can be extended in this particular case, and to search for potential nonadditive effects in introducing multiple amino acid replacements. Combined analysis of one such multiply substituted peptide by SPR, solution NMR and X-ray diffraction shows that antigenic degeneracy can be expected as long as residues directly interacting with the paratope are conserved and the peptide bioactive folding is unaltered.

Antibodies, Monoclonal↗

PubMed2001

Antigenicity modulation upon peptide cyclization: application to the GH loop of foot-and-mouth disease virus strain C1-Barcelona.

Foot-and-mouth disease virus (FMDV) isolate C(1)-Barcelona (or C-S30) includes four replacements within its immunodominant site (GH loop, residues 136-150 of capsid protein VP1, YTTSTRGDLAHVTAT), relative to reference strain C-S8c1 (YTASARGDLAHLTTT). Although one of the mutations in C-S30 (147Leu-->Val) is known to be detrimental for antibody recognition, reactivity of this isolate with the neutralizing monoclonal antibody (mAb) 4C4, raised against FMDV C1-Brescia (GH loop: YTASTRGDLAHLTAT), was indistinguishable from those of strains C-S8c1 or C1-Brescia. A structural interpretation for these somewhat striking findings is available, based on the observation that 15-residue peptides reproducing the C-S30 and C-S8c1 GH loops adopt very similar, quasi-circular, conformations in crystal complexes with 4C4. Nevertheless, surface plasmon resonance (SPR) kinetic analyses of the interactions between these peptides and three anti-GH loop mAbs have now revealed that the linear C-S30 peptides were less antigenic in solution than their C-S8c1 and C1-Brescia counterparts. We have, therefore, tried to modulate peptide antigenicity in solution by cyclization. Functional SPR and structural two dimensional proton nuclear magnetic resonance (2D-1H NMR) studies of both linear and cyclic peptide antigens are discussed here. Conformation seems to have an important role in peptide antigenicity, even when continuous (i.e. linear) antigenic sites are involved.

Amino Acid Sequence↗

PubMed2001

Conformationally restricted PACAP27 analogues incorporating type II/II' IBTM beta-turn mimetics. Synthesis, NMR structure determination, and binding affinity.

To probe the importance of a proposed beta-turn within residues S9-R12 of PACAP for recognition by VIP/PACAP receptors, compounds 1 and 2, two conformationally restricted analogues of PACAP27 incorporating respectively (S)- or (R)-IBTM as type II or II' beta-turn dipeptide mimetic at the Y10-S11 position, were synthesized. According to 1H NMR conformational analyses in aqueous solution and 30% TFE, both PACAP27 and the [S-IBTM(10,11)]PACAP27 analogue 1 adopt similar ordered structures. PACAP27 shows an N-terminal disordered region (residues H1-F6) and an alpha-helical conformation within segment T7-L27. For residues S9-R12, our data seem more compatible with a segment of the alpha-helix than with the beta-turn previously proposed for this fragment. In compound 1 the alpha-helix, also spanning T7-L27 residues, appears slightly distorted at the N-terminus relative to the native peptide. Although this distortion could lead to the marked decrease in binding affinity of this compound at the VIP/PACAP receptors, the lack of the Y10 side chain in analogues 1 and 2 could also significantly affect the binding of these compounds.

Amino Acid Sequence↗

PubMed2001

L and D presequence peptides derived from the precursor of F1beta subunit of the ATP synthase inhibit mitochondrial protein import by interaction with import machinery.

We investigated the effect of L and D enantiomers of a 25-residue peptide derived from the N-terminal region of the presequence of Nicotiana plumbaginifolia F1beta subunit of the ATP synthase, pF1beta(1, 25), on import into spinach leaf mitochondria. Three in vitro synthesized precursor proteins using different import pathways were used. Import of the precursor proteins of F1beta subunit of the ATP synthase, pre-F1beta, and the alternative oxidase, pre-AOX, required addition of external ATP. whereas the chimeric precursor containing the N-terminal 84 amino acids of the cytochrome b2 precursor protein linked to dihydrofolate reductase, pre-b2(1, 84)-DHFR was not dependent on ATP. Import of pre-F1beta, and pre-AOX was inhibited already at 1 microM and 3 microM concentration of the L and D enantiomers, whereas inhibition of import of pre-b2(1, 84)-DHFR, occurred at concentrations >10 microM of both enantiomers. Binding efficiency of the precursor proteins was not affected by addition of the L and D enantiomers. There was no correlation between inhibition of import of pre-F1beta and pre-AOX and dissipation of membrane potential measured as a decrease of Rhodamine 123 fluorescence quenching. The inhibitory effect of the L and D presequence enantiomers on import of pre-F1beta and pre-AOX was concluded to occur within the outer membrane translocase machinery beyond the initial precursor receptor interaction. Furthermore, the fact that the D enantiomer had the same effect as the natural peptide showed that interaction of the presequence with the import machinery was not dependent on chiral properties of the presequence.

Amino Acids↗

PubMed2001

Screening of antifeedant activity in brain extracts led to the identification of sulfakinin as a satiety promoter in the German cockroach. Are arthropod sulfakinins homologous to vertebrate gastrins-cholecystokinins?

The feeding cycle of the adult female cockroach Blattella germanica parallels vitellogenesis. The study of the mechanisms that regulate this cycle led us to look for food-intake inhibitors in brain extracts. The antifeedant activity of brain extracts was tested in vivo by injecting the extract and measuring the carotenoids contained in the gut from carrot ingested after the treatment. By HPLC fractionation and tracking the biological activity with the carrot test, we isolated the sulfakinin EQFDDY(SO3H) GHMRFamide (Pea-SK). A synthetic version of the peptide inhibited food intake when injected at doses of 1 microg (50% inhibition) and 10 microg (60% inhibition). The sulfate group was required for food-intake inhibition. These biological and structural features are similar to those of the gastrin-cholecystokinin (gastrin-CCK) family of vertebrate peptides. However, heterologous feeding assays (human CCK-8 tested on B. germanica, and Pea-SK tested on the goldfish Carassius auratus) were negative. In spite of this, alignment and cluster analysis of these and other structurally similar peptide families suggest that sulfakinins and gastrin-CCKs are homologous, and that mechanisms of feeding regulation involving these regulatory peptides may have been conserved during evolution between insects and vertebrates.

Amino Acid Sequence↗

PubMed2001

Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase.

Determinants for the recognition of a mitochondrial presequence by the mitochondrial processing peptidase (MPP) have been investigated using mutagenesis and bioinformatics approaches. All plant mitochondrial presequences with a cleavage site that was confirmed by experimental studies can be grouped into three classes. Two major classes contain an arginine residue at position -2 or -3, and the third class does not have any conserved arginines. Sequence logos revealed loosely conserved cleavage motifs for the first two classes but no significant amino acid conservation for the third class. Investigation of processing determinants for a class III precursor, Nicotiana plumbaginifolia F1beta precursor of ATP synthase (pF1beta), was performed using a series of pF1beta presequence mutants and mutant presequence peptides derived from the C-terminal portion of the presequence. Replacement of -2 Gln by Arg inhibited processing, whereas replacement of either the most proximally located -5 Arg or -15 Arg by Leu had only a low inhibitory effect. The C-terminal portion of the pF1beta presequence forms a helix-turn-helix structure. Mutations disturbing or prolonging the helical element upstream of the cleavage site inhibited processing significantly. Structural models of potato MPP and the C-terminal pF1beta presequence peptide were built by homology modelling and empirical conformational energy search methods, respectively. Molecular docking of the pF1beta presequence peptide to the MPP model suggested binding of the peptide to the negatively charged binding cleft formed by the alpha-MPP and beta-MPP subunits in close proximity to the H111XXE114H115X(116-190)E191 proteolytic active site on beta-MPP. Our results show for the first time that the amino acid at the -2 position, even if not an arginine, as well as structural properties of the C-terminal portion of the presequence are important determinants for the processing of a class III precursor by MPP.

Amino Acid Sequence↗

PubMed2001

N-terminal fatty acid substitution increases the leishmanicidal activity of CA(1-7)M(2-9), a cecropin-melittin hybrid peptide.

In order to improve the leishmanicidal activity of the synthetic cecropin A-melittin hybrid peptide CA(1-7)M(2-9) (KWKLFKKIGAVLKVL-NH(2)), a systematic study of its acylation with saturated linear fatty acids was carried out. Acylation of the N(epsilon)-7 lysine residue led to a drastic decrease in leishmanicidal activity, whereas acylation at lysine 1, in either the alpha or the epsilon NH(2) group, increased up to 3 times the activity of the peptide against promastigotes and increased up to 15 times the activity of the peptide against amastigotes. Leishmanicidal activity increased with the length of the fatty acid chain, reaching a maximum for the lauroyl analogue (12 carbons). According to the fast kinetics, dissipation of membrane potential, and parasite membrane permeability to the nucleic acid binding probe SYTOX green, the lethal mechanism was directly related to plasma membrane permeabilization.

Acylation↗

PubMed2001

Identification of T-cell epitopes in nonstructural proteins of foot-and-mouth disease virus.

Porcine T-cell recognition of foot-and-mouth disease virus (FMDV) nonstructural proteins (NSP) was tested using in vitro lymphoproliferative responses. Lymphocytes were obtained from outbred pigs experimentally infected with FMDV. Of the different NSP, polypeptides 3A, 3B, and 3C gave the highest stimulations in the in vitro assays. The use of overlapping synthetic peptides allowed the identification of amino acid regions within these proteins that were efficiently recognized by the lymphocytes. The sequences of some of these antigenic peptides were highly conserved among different FMDV serotypes. They elicited major histocompatibility complex-restricted responses with lymphocytes from pigs infected with either a type C virus or reinfected with a heterologous FMDV. A tandem peptide containing the T-cell peptide 3A[21-35] and the B-cell antigenic site VP1[137-156] also efficiently stimulated lymphocytes from infected animals in vitro. Furthermore, this tandem peptide elicited significant levels of serotype-specific antiviral activity, a result consistent with the induction of anti-FMDV antibodies. Thus, inclusion in the peptide formulation of a T-cell epitope derived from the NSP 3A possessing the capacity to induce T helper activity can allow cooperative induction of anti-FMDV antibodies by B cells.

Amino Acid Sequence↗

PubMed2001

In vitro activity of CA(1-8)M(1-18), a synthetic cecropin A-melittin hybrid peptide, against multiresistant Acinetobacter baumannii strains.

The in vitro antibiotic activity of CA(1-8)M(1-18), a synthetic cecropin A-melittin hybrid peptide, was determined by broth microdilution on 20 clinical Acinetobacter baumannii isolates with different resistance profiles. The MIC(50), MIC(90) and ranges were 4 mg/l, 4 mg/l and 2-8 mg/l, respectively, and were independent of resistance pattern. Different assay parameters such as microplate plastic (polystyrene or polypropylene), addition of supplements (5-10% fetal calf serum or 5% horse blood), inoculum size (10(5), 10(6), 10(7) and 10(8) CFU/ml) or incubation period (24 or 48 h) were studied. MIC was independent of the first two parameters, although the MIC values increased both with inoculum size or incubation period. Killing curves were obtained both for a standard strain and a multiresistant isolate over a 45.7-2.8 mg/l (16-1 mM) peptide range, using an initial inoculum of 10(5)-10(6) CFU/ml and 10(9)-10(10) CFU/ml. A concentration of 45.7 mg/l was required for complete killing. Accordingly, CA(1-8)M(1-18) showed good in vitro activity against the A. baumannii strains tested irrespective of the resistance to classical antibiotics, and could be a future candidate for multiresistant A. baumannii infections, although further cytotoxicity and pharmacological studies will be needed.

Acinetobacter↗

PubMed2000

Direct single-step surface plasmon resonance analysis of interactions between small peptides and immobilized monoclonal antibodies.

Surface plasmon resonance (SPR) methods have been optimized to permit direct kinetic analysis of the antigenic peptide analytes interacting with immobilized monoclonal antibodies (mAbs). High reproducibility and a significant correlation between SPR and previous ELISA data on the same set of antibodies and peptides were observed. The kinetic data obtained provide further insight into the structure of the main antigenic site of foot-and-mouth disease virus (FMDV).

Amino Acid Sequence↗

PubMed2000

Molecular analysis of peptides from the GH loop of foot-and-mouth disease virus C-S30 using surface plasmon resonance: a role for kinetic rate constants.

A foot-and-mouth disease virus (FMDV) field variant, isolate C-S30 (also named C(1)-Barcelona), is known to contain four changes within the main antigenic site A (GH loop of capsid protein VP1, residues 136-150), at least one of which (Leu147-->Val) involves a highly conserved position, critical for antibody recognition in the reference strain C-S8c1. However, immunoenzymatic analysis of FMDV C-S30 showed it was recognised by 4C4, a monoclonal antibody that specifically targets site A. This remarkable behaviour has led us to analyse the individual and combined contributions of the four mutations to the antigenicity of C-S30, by surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA) studies of pentadecapeptides displaying all possible combinations of the four replacements. Analysis of this family of C-S30-derived analogues shows a certain level of antibody recognition by SPR. In addition, SPR data suggest that kinetic rate constants provide an indirect measure, on the one hand, of paratope accessibility (association rate constant) and, on the other hand, of peptide fitness to the same paratope (dissociation rate constant).

Amino Acid Sequence↗

PubMed2000

Nsc and Fmoc Nalpha-amino protection for solid-phase peptide synthesis: a parallel study.

The 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is an alternative to Fmoc for Nalpha-protection in solid-phase peptide synthesis. Nsc-amino acids may be particularly suitable for automatic synthesizers, in which the amino acids are stored in solution, and the incorporation of residues prone to racemization such as Cys and His. Owing to the hydrophilicity of the Nsc group, these derivatives are useful for the preparation of protected peptides in convergent solid-phase peptide synthesis strategies.

Amino Acid Sequence↗

PubMed2000

A multiply substituted G-H loop from foot-and-mouth disease virus in complex with a neutralizing antibody: a role for water molecules.

The crystal structure of a 15 amino acid synthetic peptide, corresponding to the sequence of the major antigenic site A (G-H loop of VP1) from a multiple variant of foot-and-mouth disease virus (FMDV), has been determined at 2.3 A resolution. The variant peptide includes four amino acid substitutions in the loop relative to the previously studied peptide representing FMDV C-S8c1 and corresponds to the loop of a natural FMDV isolate of subtype C(1). The peptide was complexed with the Fab fragment of the neutralizing monoclonal antibody 4C4. The peptide adopts a compact fold with a nearly cyclic conformation and a disposition of the receptor-recognition motif Arg-Gly-Asp that is closely related to the previously determined structure for the viral loop, as part of the virion, and for unsubstituted synthetic peptide antigen bound to neutralizing antibodies. New structural findings include the observation that well-defined solvent molecules appear to play a major role in stabilizing the conformation of the peptide and its interactions with the antibody. Structural results are supported by molecular-dynamic simulations. The multiply substituted peptide developed compensatory mechanisms to bind the antibody with a conformation very similar to that of its unsubstituted counterpart. One water molecule, which for steric reasons could not occupy the same position in the unsubstituted antigen, establishes hydrogen bonds with three peptide amino acids. The constancy of the structure of an antigenic domain despite multiple amino acid substitutions has implications for vaccine design.

Amino Acid Substitution↗

PubMed2000

Interspecies major histocompatibility complex-restricted Th cell epitope on foot-and-mouth disease virus capsid protein VP4.

T-cell epitopes within viral polypeptide VP4 of the capsid protein of foot-and-mouth disease virus were analyzed using 15-mer peptides and peripheral blood mononuclear cells (PBMC) from vaccinated outbred pigs. An immunodominant region between VP4 residues 16 and 35 was identified, with peptide residues 20 to 34 (VP4-0) and 21 to 35 (VP4-5) particularly immunostimulatory for PBMC from all of the vaccinated pigs. CD25 upregulation on peptide-stimulated CD4(+) CD8(+) cells-dominated by Th memory cells in the pig-and inhibition using anti-major histocompatibility complex class II monoclonal antibodies indicated recognition by Th lymphocytes. VP4-0 immunogenicity was retained in a tandem peptide with the VP1 residue 137 to 156 sequential B-cell epitope. This B-cell site also retained immunogenicity, but evidence is presented that specific antibody induction in vitro required both this and the T-cell site. Heterotypic recognition of the residue 20 to 35 region was also noted. Consequently, the VP4 residue 20 to 35 region is a promiscuous, immunodominant and heterotypic T-cell antigenic site for pigs that is capable of providing help for a B-cell epitope when in tandem, thus extending the possible immunogenic repertoire of peptide vaccines.

Amino Acid Sequence↗

PubMed2000

Cell recognition by foot-and-mouth disease virus that lacks the RGD integrin-binding motif: flexibility in aphthovirus receptor usage.

Cell surface molecules that can act as virus receptors may exert an important selective pressure on RNA viral quasispecies. Large population passages of foot-and-mouth disease virus (FMDV) in cell culture select for mutant viruses that render dispensable a highly conserved Arg-Gly-Asp (RGD) motif responsible for integrin receptor recognition. Here, we provide evidence that viability of recombinant FMDVs including a Asp-143-->Gly change at the RGD motif was conditioned by a number of capsid substitutions selected upon FMDV evolution in cell culture. Multiply passaged FMDVs acquired the ability to infect human K-562 cells, which do not express integrin alpha(v)beta(3). In contrast to previously described cell culture-adapted FMDVs, the RGD-independent infection did not require binding to the surface glycosaminoglycan heparan sulfate (HS). Viruses which do not bind HS and lack the RGD integrin-binding motif replicate efficiently in BHK-21 cells. Interestingly, FMDV mutants selected from the quasispecies for the inability to bind heparin regained sensitivity to inhibition by a synthetic peptide that represents the G-H loop of VP1. Thus, a single amino acid replacement leading to loss of HS recognition can shift preferential receptor usage of FMDV from HS to integrin. These results indicate at least three different mechanisms for cell recognition by FMDV and suggest a potential for this virus to use multiple, alternative receptors for entry even into the same cell type.

Amino Acid Sequence↗