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Biomedical subjects

E Giralt

Publications and source records attributed to E Giralt.

At least 73 records · Page 4Linked to original sources
PubMed1991

Comparative study of the kinetics of the renal tracers 99Tcm-MAG3 and 131I-hippuran.

Serial measurements of plasma activity, plasma protein binding and urine excretion were obtained in order to study 99Tcm-MAG3 (MAG) and 131I-Hippuran (OIH) kinetics after simultaneous injection of both tracers in 21 patients with various renal diseases. Results were compared on the basis of a compartmental model, calculating the rate constants as well as the clearance and volume of distribution. Protein binding was calculated in 10 patients (mean: MAG = 54.7%, OIH = 33.8%). The dependence of time, tracer and patient factors was shown by ANOVA. Time was independent, with tracer and patient factors and their interaction being significant. The mean value of the renal excretion constant was equal for the two tracers (k12 = 0.052 min-1). The clearance values were found to be highly correlated (r = 0.982) with a ratio of 0.57 between them. The volumes of distribution in litres were 4.1 (MAG) and 7.0 (OIH). One-hour urine excretion was nearly the same for both tracers (MAG: 64%, OIH: 63% of the injected dose).

Blood Proteins↗
PubMed1991

Cyclization of disulfide-containing peptides in solid-phase synthesis.

Disulfide-containing peptides may be obtained in good yields and purities when oxidations are carried out on peptide chains anchored to polymeric supports used for solid-phase synthesis. Such approaches take advantage of the pseudo-dilution phenomenon which favors intramolecular processes. A variety of procedures have been demonstrated using the related model peptides Ac-Cys-Pro-D Val-Cys-NH2 and Ac-Pen-Pro-D Val-Cys-NH2 (which both readily assume a type II beta-turn conformation that becomes stabilized by a 14-membered disulfide-containing intramolecular ring), and oxytocin (conformationally mobile 20-membered disulfide ring). Both Boc and Fmoc were used for N alpha-amino protection, the beta-thiols of cysteine or penicillamine were blocked by S-acetamidomethyl (Acm), S-9-fluorenylmethyl (Fm), or S-trityl (Trt), and compatible anchoring linkages included HF-labile 4-methylbenzhydrylamide (MBHA), TFA-labile tris (alkoxy)benzylamide (PAL), and photolabile o-nitrobenzylamide (Nonb). Assemblies of linear sequences proceeded smoothly, and polymer-supported oxidations were carried out in a variety of ways either directly or after deblocking to the resin-bound dithiol. Chains were released from the support without substantial damage to the disulfide bridges, and overall yields were as high as 60-90%.

Amino Acid Sequence↗
PubMed1991

Studies on the enzymatic coupling of peptide segments on the solid support.

Four different supports: polyacrylamide (I), polyethylene glycol-polystyrene MP-PEG (II), Kel-F-g-styrene (III) and controlled pore glass CPG (IV-VI) were tested in order to determine the most suitable carrier for both chemical and enzymatic methods. As a model of enzymatic reaction for comparative study of supports, the hydrolysis of peptide methyl esters bound to insoluble polymer was examined. Peptide segment couplings by means of papain were attempted on the CPG-support which was found to be the most suitable for performing enzymatic reactions. The racemization-free assemblies of partially protected peptides on this support were achieved in moderate yields (31-54%).

Acrylic Resins↗
PubMed1991

Convergent solid-phase peptide synthesis. VIII. Synthesis, using a photolabile resin, and purification of a methionine-containing protected peptide.

The solid-phase synthesis, photolytic detachment from the solid support and purification in solution, of a fully-protected octapeptide containing a methionine residue (protected as the sulphoxide) is described. Protection of methionine in this manner avoids problems associated with the oxidation of this residue during the photolysis. The peptide has been purified by medium pressure liquid chromatography using solvent mixtures containing a high proportion of dimethylformamide in order to avoid precipitation of the peptide on the column.

Amino Acid Sequence↗
PubMed1990

A single amino acid substitution affects multiple overlapping epitopes in the major antigenic site of foot-and-mouth disease virus of serotype C.

Neutralizing monoclonal antibodies (nMAbs) elicited against foot-and-mouth disease virus (FMDV) of serotype C were assayed with field isolates and variant FMDVs using several immunoassays. Of a total of 36 nMAbs tested, 23 recognized capsid protein VP1 and distinguished at least 13 virion conformation-independent epitopes involved in neutralization of FMDV C. Eleven epitopes of FMDV C-S8c1 have been located in segments 138-156 or 192-209 of VP1 by quantifying the reactivity of nMAbs with synthetic peptides and with nMAb-resistant mutants of FMDV C-S8c1 carrying defined amino acid substitutions. The main antigenic site of FMDV C-S8c1 (VP1 residues 138 to 150) consists of multiple (at least 10), distinguishable, overlapping epitopes. Some amino acid replacements abolished one of the epitopes, whereas other replacements affected several epitopes in this region. The conservative substitution His(146)----Arg, found in many nMAb-resistant mutants analysed, abolished the reactivity of the virus with all nMAbs that recognized epitopes in the main antigenic site of FMDV C-S8c1. This indicates that a minimum genetic change can result in a highly amplified phenotypic effect, as regards the antigenicity of FMDV.

Amino Acid Sequence↗
PubMed1990

Application of acetamidomethyl and 9-fluorenylmethyl groups for efficient side protection of penicillamine in solid-phase peptide synthesis.

Synthesis of S-acetamidomethyl and S-fluorenylmethyl derivatives of penicillamine is described. Both groups are completely stable to all the usual reagents in solid-phase peptide synthesis, including the HF cleavage step, and show an excellent degree of orthogonality to each other. Treatment of the protected peptides Ac-L-Pen(X)-L-Pro-D-Val-L-Cys(X)-NH2 with thallium (III) trifluoroacetate or iodine for X = Acm or piperidine/DMF (1:1) for X = Fm induced with good yield the formation of the intramolecular disulfide bridge. This cyclic peptide appears to assume a type II beta-turn conformation in d6-DMSO as evidenced by 1H-NMR spectra.

Amino Acid Sequence↗
PubMed1990

Unique amino acid substitutions in the capsid proteins of foot-and-mouth disease virus from a persistent infection in cell culture.

Maintenance of a persistent foot-and-mouth disease virus (FMDV) infection in BHK-21 cells involves a coevolution of cells and virus (J. C. de la Torre, E. Martínez-Salas, J. Díez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). The resident FMDV undergoes a number of phenotypic changes, including a gradual decrease in virion stability. Here we report the nucleotide sequence of the P1 genomic segment of the virus rescued after 100 passages of the carrier cells (R100). Only 5 of 15 mutations in P1 of R100 were silent. Nine amino acid substitutions were fixed on the viral capsid during persistence, and three of the variant amino acids are not represented in the corresponding position of any picornavirus sequenced to date. Cysteine at position 7 of VP3, that provides disulfide bridges at the FMDV fivefold axis, was substituted by valine, as determined by RNA, cDNA, and protein sequencing. The modified virus shows high buoyant density in cesium chloride and depicts the same sensitivity to photoinactivation by intercalating dyes as the parental FMDV C-S8c1. Amino acid substitutions fixed in VP1 resulted in altered antigenicity, as revealed by reactivity with monoclonal antibodies. In addition to defining at the molecular level the alterations the FMDV capsid underwent during persistence, the results show that positions which are highly invariant in an RNA genome may change when viral replication occurs in a modified environment.

Amino Acid Sequence↗
PubMed1989

Kinetic analysis of the carboxypeptidase A hydrolysis of oligopeptides by reversed-phase high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic (HPLC)-based method was developed to follow the time course of the hydrolytic action of pancreatic carboxypeptidase A on oligopeptide substrates of the general formula benzoyl(glycyl)n-L-Phe, n being in the range 1-5. The proposed procedure is sensitive at the nanomolar level of substrate, but also allows the kinetics of substrate hydrolysis to be investigated over a wide range of concentrations. The prior quenching of the carboxypeptidase activity and the use of isocratic conditions for the rapid separation of the substrates and their products (in less than 8 min for the slowest one), allow the automation of the procedure, which could become an easy and versatile alternative to the commonly used spectrophotometric methods. A comparative evaluation of the use of a spectrophotometric method to measure carboxypeptidase A activity on the same substrates indicated that the latter is not valid for long oligopeptides (n greater than or equal to 2) at concentrations higher than 5 mM owing to an interference of a physico-chemical nature. For benzoylglycyl-L-Phe (n = 1), the apparent kinetic parameters were calculated by means of the HPLC method and by a well established spectrophotometric procedure, and both yielded similar values.

Animals↗
PubMed1989

A synthetic strategy for simultaneous purification-conjugation of antigenic peptides.

A novel approach to the preparation of immunopeptide-carrier protein conjugates of improved chemical definition based on a solid-phase synthetic protocol combining incorporation of a Cys(Npys) N-terminal residue with systematic acetylation after every coupling, is described. The potential of the method is demonstrated in a synthesis of the tridecapeptide (Npys)-Cys-Val-Asn-Tyr-Ile-Arg-Lys-Arg-Ser-Leu-Gln-Thr-Val-OH in which the main product is purposely contaminated by a number of shorter truncated sequences resulting from intentionally defective couplings. From this peptide crude, and rather independently of its complexity, the target sequence can be selectively recovered and attached to a carrier molecule through a disulfide bond formed by reaction of the Npys-protected cysteine residue and a thiol function in the carrier. The process can be properly named purification-by-conjugation.

Amino Acid Sequence↗
PubMed1989

Implications of a quasispecies genome structure: effect of frequent, naturally occurring amino acid substitutions on the antigenicity of foot-and-mouth disease virus.

We provide evidence that the quasispecies nature (extreme genetic heterogeneity) of foot-and-mouth disease virus is relevant to the virus evading an immune response. A monoclonal antibody neutralizing the viral infectivity (clone SD6) recognizes an epitope located around a highly conserved sequence (amino acid sequence Arg-Gly-Asp-Leu-Ala at positions 141-145) in the capsid protein VP1 of foot-and-mouth disease virus of serotype C1. The amino acid substitutions Ala-138----Thr and Leu-147----Ile (or ----Val) reduced 100-fold the binding titer of monoclonal antibody SD6 to virions or to VP1. The effect of those substitutions was quantitatively reproduced with synthetic peptides representing the relevant sequences. This provides evidence that the two chemically conservative amino acids replacements--and not other substitutions present in the virus quasispecies--are responsible for the modified interaction with neutralizing monoclonal antibody SD6. The three substitutions were fixed in the viral capsid during one occurrence of foot-and-mouth disease and, furthermore, they are of a type found frequently among independent foot-and-mouth disease virus isolates. The results implicate the extreme heterogeneity of foot-and-mouth disease virus as an important element of viral pathogenesis.

Amino Acid Sequence↗
PubMed1989

Use of the Npys thiol protection in solid phase peptide synthesis. Application to direct peptide-protein conjugation through cysteine residues.

The protection of the thiol function of cysteine with the 3-nitro-2-pyridylsulfenyl (Npys) group has been successfully applied in the solid phase synthesis of nine peptides. A reexamination of the chemical stability of the protecting group has shown that, while Npys is essentially suitable for standard Boc/benzyl synthesis conditions, it is inadequate for the Fmoc strategy. Its proven stability to "high" HF acidolysis can not be extended to "low-high" conditions without significant thiol deprotection. On the other hand, the Npys group is quite compatible with standard photolytical cleavage conditions. The stability of Npys to HF and its thiol-activating character allow its application in peptide-carrier protein conjugation reactions by specific coupling through cysteine residues in the peptide.

Amino Acid Sequence↗
PubMed1987

Reversed-phase high-performance liquid chromatography of protected peptide segments.

There is little evidence that reversed-phase high-performance liquid chromatography can be successfully used in the analysis of protected peptide segments. The use of C18 and CN packings and mobile phases containing water-acetonitrile with or without propionic acid in the separation of complex mixtures of synthetic protected peptides is reported. CN packings show a lower efficiency and exhibit poorer resolution than C18 packings but provide different separations. The addition of propionic acid to the mobile phase increases the retention time of peptides but also provides dramatic and useful changes in selectivity. Retention is not related to the molecular mass of the protected peptides but mainly to their hydrophobicity.

Amino Acids↗
PubMed1987

Reactivity with monoclonal antibodies of viruses from an episode of foot-and-mouth disease.

A panel of 12 monoclonal antibodies (MAbs) raised against foot-and-mouth disease virus (FMDV) of serotype C1 (FMDV C-S8c1) and 11 MAbs raised against other FMDVs have been used to evaluate the reactivity of 14 isolates of FMDV of serotype C1 (series FMDV C-S), 12 of them from one disease episode (Spain 1979-1982). The assays used were immunoelectrotransfer blot, immunodot and neutralization of infectivity. None of the isolates could be clearly distinguished by its reactivity with 6 non-neutralizing and 2 neutralizing MAbs raised against FMDV C-S8c1. In contrast, the isolates were distinguished in two groups by a 10(2)-fold difference in their reactivity with 6 neutralizing MAbs. The reactivity of MAbs with synthetic peptides indicated that conserved and non-conserved epitopes recognised respectively by neutralizing MAbs 4G3 and SD6 are localized in the immunogenic region (amino acids 138-156) of VP1. Thus, epidemiologically related FMDVs differ in at least one epitope critical for virus neutralization.

Amino Acid Sequence↗
PubMed1984

[Metastatic breast cancer: a comparative study of the efficacy of tamoxifen and the sequential administration of tamoxifen and medroxyprogesterone acetate].

Seventy-nine patients with an histologically proven disseminated breast cancer, never treated before with additive hormonal therapy, entered into a randomized trial between june 1981 and december 1982. In the first group 44 patients were given continually a daily dose of tamoxifen (TAM) of 20 mg/m2. In the 2nd group 35 patients were given a daily dose of TAM of 20 mg/m2 for 15 days and then an oral daily dose of medroxyprogesterone acetate of 350 mg/m2 for the next 15 days. In both groups I and II, the treatment was stopped at the first manifestation of progression of the disease. The hormonal receptor status was determined in 30 patients of the group I and 23 patients of the group II. An objective response to treatment was observed in 48 per cent of the patients of the group I and 60 per cent of the group II. This difference is not significant (X2 = 1,05). However, the mean duration of therapeutic response is significantly higher in the group II (p = 0,01).

Adenocarcinoma↗
PubMed1984

[Phase-1 study of the tolerance for increasing doses of recombinant human alpha 2 interferon in patients with advanced cancer].

Thirteen patients with malignant tumors were entered into a phase I trial with recombinant DNA human alpha 2 interferon (IFN alpha 2). The patients were given I.M. escalating doses of IFN alpha 2 ranging from 1-10(6) to 200-10(6) IU with a 72 hours washout between injections. In the majority of the patients, subjective symptoms were noted: fever, headache, chills, nausea, myalgias. Asthenia, anorexia, drowsiness appeared after the highest doses and disappeared without any sequellae. Leucopenia and thrombopenia were seen in 11 out of 13 patients. Hepatocellular toxicity was observed in 9 cases. Cardiac and vascular functions were not impaired by IFN alpha 2. The pharmacokinetic studies showed a maximum serum concentration between 4 and 6 hours after injection and the peak value was directly proportional to the dose. No neutralizing INF alpha 2 serum factor was detected during the treatment. The peak value for serum beta 2 microglobulin occurred 48 hours after and the N.K. activity was variably modified by IFN alpha 2 injections. A major clinical response was observed in 1 case, a minor response in 3 cases and a stabilisation of the disease in 4 cases.

Adult↗
PubMed1983

Amino-acids condensations in the preparation of N alpha-9-fluorenylmethyloxycarbonylamino-acids with 9-fluorenylmethylchloroformate.

Synthesis of N alpha-9-fluorenylmethyloxycarbonyl (Fmoc) amino-acids by reaction of free amino-acids (glycine and alanine) with 9-fluorenylmethylchloroformate leads to formation of small amounts of Fmoc-dipeptide which are difficult to eliminate by crystallization. The alternative way to prepare Fmoc-amino-acids by reacting the Fmoc-chloride first with sodium azide and then with the free amino-acid eliminates this side reaction, at least for glycine and alanine.

Amino Acids↗