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F Hudecz

Publications and source records attributed to F Hudecz.

At least 73 records · Page 4Linked to original sources
PubMed1988

Application of Marfey's reagent in racemization studies of amino acids and peptides.

Reversed-phase high-performance liquid chromatography and pre-column derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent) were used for monitoring racemization in peptides, amino acids and their derivatives by separation of optical isomers of amino acids. The technique was applied to the analysis of biologically active peptides, amino acids, their N- and C-protected derivatives, branched polypeptides based on polylysine, and endothiopeptides, and to the detection of stereochemical consequences of side reactions and hydrolysis. The chromatographic samples were mixtures of L- and D-amino acids obtained by hydrolysis of different peptides and derivatives. Baseline separations could be achieved on an ODS-Hypersil column with methanol-acetonitrile-acetate buffer (pH 4) mixtures as the eluents. The rates of racemization were calculated.

Alanine↗
PubMed1988

Influence of side-chain-terminating moieties on the conformation of branched polypeptides and their conjugates with 4-ethoxymethylene-2-phenyl-5(4H)-oxazolone.

Poly(Lys-(Xi-DL-Alam] polypeptides carrying hydrophilic (X = His, Glu, Lys) or hydrophobic (X = Nle, Ile, Phe) amino acid residues and their conjugates with 4-ethoxymethylene-2-phenyl-5(4H)-oxazolone were synthesized. The conformational properties of carrier polypeptides and conjugates were studied by circular dichroism (CD) spectroscopy in the wavelength regions 190-250 and 310-380 nm, with the emphasis on analysis under near physiological conditions. Based on CD studies, it could be demonstrated that the helix-forming capacity appears to be related to the hydrophobic nature of the branch-terminating amino acid of the branched polypeptides. With respect to carrier function, the presence of a coupled derivative of oxazolone at the side chain termini generally promotes the formation of helical secondary structure. The absolute configuration of the side-chain-terminating amino acids was found to be important for the local orientation of the hapten molecule in the conjugates.

Circular Dichroism↗
PubMed1986

Isotype distribution and fine specificity of the antibody response of inbred mouse strains to four compounds belonging to a new group of synthetic branched polypeptides.

A new group of synthetic branched polypeptides was developed to initiate a systematic study of the relationships between the chemical structure (charge, size, primary structure, configuration and conformation), the carrier potential and the antigenic properties of these biodegradable and biocompatible macromolecules. This model system has two main advantages over the previously used ones: (i) the side chains grafted to the poly(L-lysine) backbone are composed of about three DL-Ala and a single chain-terminating amino acid with different absolute configuration and/or identity, and (ii) the conformation of these polypeptides is characterized in solution. The size, charge and inside area of the four molecules selected for this study were identical; however, the identity, the absolute configuration of the chain-terminating amino acids (D-Leu, Leu, Phe or D-Phe) and, in consequence, the conformation of the macromolecules were different. The qualitative and quantitative features of the antibody response induced by the four polypeptides were characterized in inbred mouse strains by IgM and IgG type antibody levels, as well as by isotype distribution and fine specificity of antibodies produced during the primary and memory response. The intensity of the memory response and the characteristics of subclass distribution were dependent on the conformation of the branched polypeptides. These molecules carry at least two types of antigenic determinants. One is ordered to the tetrapeptide side chain, the expression of which proved to be inversely correlated with the backbone-originated helix content of the molecules. The other antigenic determinant corresponds to the common inside area of the polypeptides which is less conformation-dependent and therefore common to all four polypeptides.

Animals↗
PubMed1986

Immune responses to isolated human cytomegalovirus envelope proteins.

A group of envelope proteins of human cytomegalovirus, gA protein (L. Pereira, M. Hoffman, M. Tatsuno, and D. Dondero, Virology 139:73-86, 1984; L. Pereira, p. 383-404, in B. Roizman, ed., The herpesviruses, vol. 3, 1985), and two protein mixtures (58,000-molecular-weight [58K]-66K and 130K-66K), separated by serial columns prepared with anti-gA immunoglobulin G from sera of immunized guinea pigs, induced neutralizing antibodies and a cellular immune response in the animals. The gA is a disulfide-linked protein complex consisting of high-molecular-weight (greater than 200K), 130K-150K, and 55K-58K proteins.

Animals↗
PubMed1986

Immunomodulatory effect of synthetic branched polypeptides. II.

A comparative investigation of various polypeptides was carried out in order to elucidate structure-activity correlations. The immunoadjuvant properties of the chemically well-characterized branched polypeptides, poly[Lys-(DL-Alam)] (Lys:Ala = 1:2.95) (AK), poly[Lys-(D-Leui-DL-Alam)] (Lys:Ala:Leu = 1:3.0:0.95) (D-LAK), and poly[Lys-Hisi-DL-Alam)] (Lys:Ala:His = 1:2.95:0.85) (HAK), were investigated. D-LAK and HAK were able to augment the antibody response of BDF inbred mice to immunization with sheep red blood cells (SRBC), as assessed by the hemolytic plaque-forming cell assay, whereas AK had no similar effect. The stimulating effect of D-LAK and HAK was dependent on dose and timing of treatment relative to SRBC immunization. However, the optimal dose levels were lower and the effective dose interval more restricted as compared to the previously described poly[Lys-(Leui-DL-Alam)] (LAK). Like LAK, both HAK and D-LAK were able to compensate for the immunosuppressive effect of the cytotoxic drugs dianhydrogalactitol, vincristine, and 5-fluorouracil, which all have different mechanisms of action, provided that combined treatment by polypeptide and drug was applied repeatedly before the SRBC immunization.

Adjuvants, Immunologic↗
PubMed1985

Preparation of highly purified human cytomegalovirus envelope antigen.

A human cytomegalovirus (HCMV) envelope preparation was highly purified by immunoaffinity column chromatography using an anti-cellular-IgG column. The purified envelope induced high titre antibodies to HCMV in guinea-pigs. Analysis of the guinea-pig immune sera by RIA and immunofluorescence (IF) showed that this envelope preparation, unlike its unpurified counterpart, did not induce antibody to cellular contaminants. Dot-blot assay revealed viral proteins in the flow-through fraction and cellular proteins in the bound fractions. Results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of flow-through and bound fractions suggest that a number of proteins previously identified as virus-specific may, in fact, reflect cellular contamination of the envelope preparation, or crossreactivity of some virus-specific proteins with cellular proteins.

Animals↗
PubMed1985

The T cell response to the glycoprotein D of the herpes simplex virus: the significance of antigen conformation.

Synthetic peptides corresponding to the first 23 amino acids of the glycoprotein D molecule of herpes simplex virus have been used to immunize mice and examine the role of antigen conformation in T cell responses. The structure of the peptides was determined by circular dichroism studies and was shown to be consistent with theoretical structure predictions. T cell clones were found that could respond to peptides that were nonhelical, and this response, which was directed to the C-terminal determinant (residues 8-23), could be modified by a constrained N-terminal sequence (residues 1-7) of the peptide. Namely, substitution of Ala for Pro at position 7 induced an alpha-helix and inhibited the response. Furthermore, a response to the N-terminal part of the molecule seemed to be directed to the alpha-helix and correlated positively with calculated degree of helicity. This response was also modified, in this case, by the C-terminal part of the molecule. These results suggest that local secondary structure of an antigen may regulate T cell responses and that structural changes in the peptide antigen downstream from the determinant modify recognition of that determinant. Furthermore, antigen processing by the macrophage and antigen presentation of T cells appears to conserve antigen structural integrity.

Amino Acid Sequence↗
PubMed1984

Fine structure analysis of type-specific and type-common antigenic sites of herpes simplex virus glycoprotein D.

The fine structure of the antigenic determinants of herpes simplex virus type 1 and 2 glycoprotein D (gD) was analyzed to determine whether structural differences underlie the differential immunogenicity of these glycoproteins. A region common to herpes simplex virus type 1 and 2 gD (amino acid residues 11 to 19) and two sites specific for herpes simplex virus type 2 gD (one determined by proline at position 7, the other determined by asparagine at position 21) were localized within the N-terminal 23 amino acids of gD by synthesis of peptides and comparison of their cross-reactivity with antisera raised to herpes simplex virus type 1 and 2 gD. The secondary structure of these peptides, as predicted by computer analysis, is discussed in relation to their immunogenicity.

Amino Acid Sequence↗
PubMed1984

Immunomodulatory effect of synthetic branched polypeptides I.

The immunomodulatory potential of poly-(Lys-[Leu-poly-DL-Ala]) (LAK) (Lys:Ala:Leu = 1:3:0.7) as the first prototype of a series of new branched polypeptides was studied. Circular dichroism spectra indicated a highly ordered structure of LAK at physiological pH. The ability of the polypeptide to stimulate antibody response to immunization with sheep red blood cells (SRBC) when applied in aqueous medium was assessed in BDF1 inbred normal mice by the hemolytic plaque-forming cell and rosette-forming cell (RFC) assays. LAK, similarly to levamisole (LEV), a clinically applied immunomodulator, stimulated the host's humoral immune response in a wide dose interval (0.02-20 mg/kg; optimal dose, 1 mg/kg); however, the number of RFC was not influenced considerably. LAK was not toxic in doses of up to 25 mg/kg. The varied timing of LAK treatment (1 mg/kg i.p.) relative to SRBC immunization resulted in an oscillating potentiation of the immune response. The immunoadjuvant activity elicited by LAK--similarly to LEV--was sufficient to produce effective compensation of immunosuppression induced by the cytotoxic drugs vincristine and dianhydrogalactitol when combined treatments on multiple schedules were applied before SRBC immunization.

Adjuvants, Immunologic↗
PubMed1981

Interaction of the antitumour drug 4'-(9-acridinylamino)-methanesulfon-m-anisidine.HCl (m-AMSA) with nucleic acids.

The interaction of AMSA with nucleic acids was studied by several techniques. Melting temperature and CD studies equally suggest that AMSA-binding is interfering with the secondary structure of DNA. An overlap by two mechanism of binding seems to exist. Based on the CD measurements at low drug concentration intercalation is the most likely way of binding. At higher drug concentration stacking interaction predominates leading to cooperativity and formation of oriented sheets of aromatic ring-systems as reflected in the optical activity induced in the metachromatic band of the achiral drug. No base-pair specificity could be confirmed; however, a high affinity of AMSA to poly(A) chains was demonstrated. The CD measurements did not indicate any significant interaction with RNA. The selectivity of the AMSA-DNA interaction can be regarded as an important argument in favour of the role of this interaction in the anti-tumour effect of the drug.

Aminoacridines↗
PubMed1979

Antitumor drug-protein interactions. Binding of 1-beta-D-arabinofuranosyl cytosine to albumin and chemically modified albumin.

Equilibrium dialysis measurements were carried out to study the binding of 1-beta-D-arabinofuranosyl cytosine (ara-C) to human and bovine serum albumin (HSA, BSA) and to chemically modified albumin. The binding of 4-phenylbutyric acid to HSA was studied, too. Binding data were presented as Scatchard plots. There are two types of binding sites of different affinity for ara-C both on HSA and BSA. The relatively small value of affinity constant indicates that the pharmacological properties of ara-C might not be influenced very strongly by the HSA interaction or by competitive binding of other drugs. Selective chemical modifications of HSA with diethylpyrocarbonate (DEP) or o-nitrophenylsulfenyl chloride (NPS-Cl) reduce significantly the affinity of the strong binding area. On the other hand, the attachment of poly-alpha-L-glutamyl or poly-DL-alanyl side-chains to BSA increase the number of the strong and secondary binding sites and also the affinity at the first group of sites. Experimental results suggest a correlation between the binding affinity and therapeutic efficacy of various cytotoxic drug-protein complexes.

Animals↗
PubMedDate not supplied

The influence of synthetic conditions on the stability of methotrexate-monoclonal antibody conjugates determined by reversed phase high performance liquid chromatography.

Methotrexate (MTX) has been convalently attached to an IgG-type monoclonal antibody (791T/36) directed to tumour-associated antigen gp72. Conjugates were synthesized by the active ester method using MTX N-succinimidyl ester at various pH values (7.5-10.5). Following purification by gel filtration, high performance liquid chromatography was used to assess the free drug or its derivatives in samples of MTX-791T/36 conjugates previously treated (or not) with hydroxylamine. Quantitative analysis, performed on a reversed phase column (pore size 300 A) with isocratic acetonitrile-sodium acetate buffer (pH 4.8) as mobile phase, indicated no detectable amount of free methotrexate in hydroxylamine-treated conjugates even six months after their preparation. Similar observations were made with conjugates, whose synthesis were performed at pH greater than or equal to 10. In contrast, the presence of increasing amounts of drug/metabolite could be demonstrated in samples produced at lower pH values. Based on these findings, the pH-dependent kinetics of MTX release has been determined and used to design conditions under which stable MTX-791T/36 conjugates could be prepared without hydroxylamine reaction.

Antibodies, Monoclonal↗
PubMedDate not supplied

Manipulation of epitope function by modification of peptide structure: a minireview.

We have explored various approaches to modify the immunrecognition of linear peptides representing sequential or continuous topographic B-cell or T-cell epitopes. For these studies, epitopes from herpes simplex virus (HSV) glycoprotein D (gD) and from mucin 1 and mucin 2 glycoproteins or T-cell epitopes from 16 kDa and 38 kDa proteins of Mycobacterium tuberculosis were selected. To increase antigenicity and immunogenicity we have prepared cyclic and chimaeric peptide variants as well as epitope peptides with altered flanking regions and epitope-carrier conjugates containing multiple epitope copies.

Amino Acid Sequence↗
PubMedDate not supplied

Immunoconjugate design: a predictive approach for coupling of daunomycin to monoclonal antibodies.

There is increasing interest in the development of daunomycin-antibody immunoconjugates for the targeting of drug to specific cells or tissues. To this end, we have examined the factors influencing the synthesis of daunomycin-monoclonal antibody conjugates linked covalently by an acid-labile cis-aconitic spacer (which is considered to aid drug release from immunoconjugates in the lysosomes and thus enhance their cytotoxic potential). A rapid and efficient procedure for the purification of drug from contaminants and stabilizers was first developed; conditions for the optimal preparation of cis-aconityldaunomycin were established; products were analyzed and identified by TLC and HPLC. The coupling of cis-aconityldaunomycin to antibody was accomplished by activating the modified drug with a carbodiimide before addition to antibody. Several factors were identified which influenced the efficiency of the conjugation; in particular, the compositional features of the antibody which determine its electrophoretic charge characteristics were of profound effect. However, by appropriate choice and control of buffer pH during conjugation, it was possible to define conditions resulting in the controlled substitution of antibody with drug. The consequent effects upon the cell-binding activity of immunoconjugates were established and related to the extent of substitution. The procedures described enable appropriate reaction conditions to be selected for the linkage of daunomycin to antibody (at set drug/antibody molar ratios) and in good yield, based upon consideration of the compositional and charge properties of the antibody.

Antibodies, Monoclonal↗
PubMedDate not supplied

Carrier design: biodistribution of branched polypeptides with a poly(L-lysine) backbone.

The biodistribution has been examined in mice of a range of synthetic branched polypeptides which are based on a polylysine backbone but which differ in ionic charge, side-chain structure, and molecular size. Polycationic polypeptides, regardless of their size or primary structure at the branches, were cleared rapidly from the circulation, the liver being the major site of clearance. Polypeptides with glutamic acid in the side chain, which would be amphoteric under physiological conditions, showed a significantly prolonged blood survival, and this was seen with polypeptides in the range of molecular weights of 46,000 up to 213,000. Such polypeptides provide a useful system with which to investigate the effect of structural parameters on the pharmacokinetic properties of carrier molecules and would allow the selection of candidate carriers for a variety of uses.

Amino Acid Sequence↗
PubMedDate not supplied

Synthesis, conformation, biodistribution, and in vitro cytotoxicity of daunomycin-branched polypeptide conjugates.

Daunomycin has been attached to various structurally related synthetic branched polypeptides with a polylysine backbone, using its acid-labile cis-aconityl derivative (cAD). Due to the importance of the side-chain structure in alpha-helix formation and immunological and pharmacological properties of branched polypeptides, we have investigated the conformation, biodistribution, and in vitro cytotoxicity of cAD-carrier conjugates with polypeptides containing amino acid residues of different identity and/or configuration at the side-chain end (XAK type) or at the position next to the polylysine backbone (AXK type), where X = Leu, D-Leu, Pro, Glu, or D-Glu. According to CD studies, polycationic conjugates with hydrophobic Leu in the side chains could assume a highly ordered conformation, while amphoteric conjugates containing Glu proved to be unordered in PBS. The reduction of in vitro cytotoxic activity of cAD by conjugation to carriers and the biodistribution profile of the conjugates were found to be dependent predominantly on the charge properties and on the side-chain sequence of the carrier polypeptide. It was demonstrated that by proper combination of structural elements of the carrier molecule, it is feasible to construct a cAD-branched polypeptide conjugate with significantly prolonged blood survival and with no reduction in in vitro cytotoxicity of the drug.

Amino Acid Sequence↗
PubMedDate not supplied

Influence of carrier on biodistribution and in vitro cytotoxicity of methotrexate-branched polypeptide conjugates.

Methotrexate (MTX) has been conjugated to various structurally related, synthetic, branched polypeptides containing a poly(L-Lys) backbone by the aid of water-soluble carbodiimide. The average degree of MTX incorporated was found to be dependent on the size of the polymer and on the identity of the terminal amino acid residue of the side chains. Consequently the average molar substitution ratio was in the range of 4.9-72.0 MTX per carrier molecule. CD spectra of conjugates showed significant differences in solution conformation correlating with the identity of the side-chain-terminating amino acid. Polycationic conjugates XAK-MTX (X = Leu or D-Leu) assumed essentially ordered (helical) secondary structure, while the CD spectrum of the amphoteric conjugate (X = Glu) corresponded to only a partially ordered conformation in PBS. The covalent attachment of MTX to branched polypeptides results in a reduction of drug in vitro cytotoxicity influenced by the carrier structure. Conjugation to amphoteric polymers, depending on the configuration and position of glutamic acid (XAK-MTX vs AXK-MTX type conjugates) resulted in a decrease of anti-791T cell activity. However polycationic conjugates bearing L-Leu at the side chain terminal position (LAK-MTX) produced a compound with cytotoxicity only about 60 times less effective than free MTX. The biodistribution in mice has been characterized by blood clearance, whole-body retention, and tissue distribution 24 h after iv administration. Blood clearance of MTX-branched polypeptides could be significantly prolonged by incorporation of glutamic acid into the side chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗