Biomedical subjects

V Krchnak

Publications and source records attributed to V Krchnak.

9 recordsLinked to original sources

PubMed2000

New approach for preparation of 2,3,7-trisubstituted 3,4-dihydroisoquinolinone libraries on solid phase.

In an attempt to prepare 7-substituted 3,4-dihydroisoquinolinone family of compounds, we observed an unexpected decarboxylation. The reaction of 4-nitrohomophthalic anhydride with a Schiff base formed on solid support leads to the formation of core structure. LC-MS and 1H NMR analysis confirmed the structure of unexpected intermediate. A library of 38,400 compounds was produced using this new synthetic approach.

Anhydrides↗

PubMed1999

Combinatorial chemistry reveals a new motif that binds the platelet fibrinogen receptor, gpIIbIIIa.

Among cell adhesion molecules, the classic Arg-Gly-Asp (RGD) motif is the best studied. We used combinatorial chemical and affinity immunochemical methods to find a novel motif of unnatural peptide ligands for the fibrinogen receptor of platelets, gpIIbIIIa (alphaIIbbeta3). The new d-amino acid motif, p(f/y)l, is unique among the ligands that bind the RGD pocket: It lacks the carboxylic acid group that is believed to coordinate with calcium in the MIDAS motif of the receptor. With an IC50 of 14 microM for the most potent compound, these linear p(f/y)l peptides had affinities similar to those of linear peptides containing RGD, and reversed sequences failed to compete with binding up to 1 mM. As the new motif was so different, molecular modeling was employed to suggest a model for molecular recognition. A reversed binding mechanism common for d-amino acid mimics of natural l-amino acid peptides offers an attractive hypothesis that suggests three points of contact similar to those made by the RGD-mimicking monoclonal antibody, OPG2. Interestingly, the model proposes that pi-electrons in the new motif may substitute for the carboxylate group present in all other RGD-types of ligands. Although modeling linear peptides is subjective, the pi-bonding model provides intriguing possibilities for medicinal chemistry after appropriate confirmatory studies.

Amino Acid Sequence↗

PubMed1996

A One-Bead One-Peptide Combinatorial Library Method for B-Cell Epitope Mapping

The one-bead one-peptide combinatorial library method represents a powerful approach to the discovery of binding peptides for various macromolecular targets. It involves the synthesis of millions of peptides on beads such that each bead displays only one peptide entity. The peptide-beads that interact with a specific macromolecular target are then isolated for structure determination. We have applied this method to discovering peptide ligands for several murine monoclonal antibodies: (i) anti-beta-endorphin (continuous epitope), (ii) anti-vmos peptide, (iii) anti-human insulin (discontinuous epitope), and (iv) surface immunoglobulins (μkappa) of two murine B-cell lymphoma cell lines (antigen unknown).

Journal Article↗

PubMed1995

Neutralizing antibodies against highly cytopathic Zairian human immunodeficiency type-1 virus (HIV-1) NDK are present in sera outside Africa.

The prototype virus HIV-1 LAV and highly cytopathic Zairian virus HIV-1 NDK belong to the genetic subtypes B and D and represent low and highly cytopathic phenotypes, respectively. Their neutralization pattern and serotype were studied with respect to differences in their genotypes and phenotypes. Sera from HIV-1-infected persons living in four geographically distant areas, Philadelphia (USA), Ribeirao Preto (Brazil), Marseille (France) and Kinshasa (Zaire), were tested for the presence of type-specific and group-specific cross-reacting neutralizing antibodies against HIV-1 LAV and HIV-1 NDK in a continuous cell line MT4. The majority of type-specific antibodies were directed against HIV-1 LAV in Philadelphia, Ribeirao Preto and Marseille, and against HIV-1 NDK in Kinshasa. However, some sera with an HIV-1 NDK type-specific neutralization pattern were also found in Philadelphia, Ribeirao Preto and Marseille. These results indicate that strains with an HIV-1 NDK-like serotype could be found outside Africa. The presence of type-specific neutralizing antibodies against HIV-1 NDK in sera from North and South America and Europe should be taken into account during attempts to serotype HIV as well as in the course of selection of HIV-1 candidate strains for an AIDS vaccine.

Amino Acid Sequence↗

PubMed1993

Discovery of D-amino-acid-containing ligands with selectide technology.

When all L-amino acid (aa) random peptide libraries synthesized on solid-phase particles were screened (Selectide Technology), we identified several peptide ligands (YG_F_) that interacted specifically with an anti-beta-endorphin monoclonal antibody (clone 3E7) (single-letter aa symbols; symbols '_' indicate variable aa). Here, we report on the screening of three different D-aa-containing pentapeptide libraries (XxXxX, xXxXx and xxxxx, wherein X = L-aa, and x = D-aa) with the same antibody, in which several D-aa-containing ligands were identified. The binding affinities of many of these D-aa-containing ligands were as least two orders of magnitude lower than that of YGGFL, for which the Ki is 17.5 nM.

Amino Acid Sequence↗

PubMed1993

Isolation and characterization of a 2.3-kilobase-pair cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor.

An immunoscreening strategy was used to isolate a cDNA clone encoding the binding domain for the external glycoprotein gp51 of the bovine leukemia virus (BLV). Three recombinant phages demonstrating BLV binding activity and containing 2.3-kbp cDNA inserts with identical nucleotide sequences were isolated from a lambda gt11 cDNA library of bovine kidney cells (MDBK). One clone, BLVRcp1, hybridized with a 4.8-kb mRNA from cells of bovine origin and was also found to be conserved as a single-copy gene in murine, bovine, ovine, primate, canine, feline, and porcine DNAs. The same gene is amplified in caprine DNA isolated from a BLV-induced tumor. The longest open reading frame of BLVRcp1 encodes a protein fragment of 729 amino acids with a putative receptor structure. BLVRcp1 cDNA was cloned in the eucaryotic expression vector pXT-1 and transfected into murine NIH 3T3 and human HEp-2 cells. Cells expressing BLVRcp1 mRNA became susceptible to BLV infection. BLVRcp1 has no known physiological function and has no significant homology with sequences registered in the GenBank and EMBL data libraries (31 July 1992). Expression of deleted constructs of BLVRcp1 indicates that the BLV binding region is encoded at the 5' side of the receptor clone.

Amino Acid Sequence↗

PubMed1993

Mapping of B-neutralizing and T-helper cell epitopes on the bovine leukemia virus external glycoprotein gp51.

A battery of 19 synthetic peptides was used to characterize efficient neutralizing and helper T-cell epitopes on the bovine leukemia virus (BLV) external envelope glycoprotein gp51. Four of the antipeptide antisera raised in rabbits inhibited the formation of BLV-induced syncytia; these antisera are directed against peptides 64-73, 98-117, and 177-192. Only antisera directed against the 177-192 region also neutralized vesicular stomatitis virus-BLV pseudotypes. This study clearly demonstrates that neutralizing properties can be observed with antibodies raised to regions undescribed so far and included in both the amino-terminal and central parts of the antigen. In addition, some helper T-cell determinants were defined from gp51-immunized mice and from BLV-infected cattle. Although none of the peptides tested behaved as a universal helper T-cell epitope, peptide 98-117 stimulated T-cell proliferation from BALB/c mice and from three infected cows, while peptide 169-188 strongly stimulated T-cell proliferation from one infected cow. Further experiments performed with three peptides overlapping the 169-188 region (177-192, 179-192, 181-192) demonstrated the particular relevance of residue(s) P-177 and/or D-178 in the helper T-cell epitope. These data should assist in the design of an efficient subunit vaccine against BLV infection that contains peptides possessing both B-neutralizing and helper T-cell determinants.

Amino Acid Sequence↗

PubMed1992

Mapping of sequential epitopes recognized by monoclonal antibodies on the bovine leukaemia virus external glycoproteins expressed in Escherichia coli by means of antipeptide antibodies.

A lambda gt11 cDNA library prepared from bovine leukaemia virus (BLV)-producing ovine cells was screened with a cocktail of anti-BLV gp51 monoclonal antibodies (MAbs). Four recombinant phages with inserts of about 2-5 kbp were isolated. One, lambda BLV-gp51-1, was sequenced and shown to encode the C-terminal part of gp51 and all of gp30. This insert was subcloned into pEV-vrf1 and expressed in Escherichia coli N-4830-1 cells. The BLV product and a series of antipeptide antibodies were used to localize the sequential epitopes defined on BLV envelope glycoprotein gp51 by their reactivity with MAbs. Epitope B was localized to amino acids 180 to 205, B' to residues 195 to 205, D and D' to residues 218 to 237, and A to amino acids 249 to 260. All the mapped sequential epitopes were localized in the C-terminal half of BLV gp51. The results of epitope mapping with bacterially produced gp51 confirm the map obtained using native viral glycoprotein.

Amino Acid Sequence↗

PubMed1991

Mapping of two immunodominant structures on human interferon alpha 2c and their role in binding to cells.

Structure-function studies of human recombinant interferon (IFN) alpha 2c were performed using a panel of specific monoclonal antibodies in the binding and neutralizing assays. Two immunodominant structures, designated sites I and II, were detected and localized within two conserved hydrophilic regions of IFN-alpha molecule. Using the NK2 antibody as a marker, site I was mapped into a carboxy-terminal domain around residues 112-148. This site was shown to be, most probably, responsible for inducing the antiviral and antiproliferative activities of the receptor-bound IFN-alpha 2c in the cell. Site II that mapped into the amino-terminal domain of IFN-alpha 2c was, at least partially, formed by the amino acid residues 36-41. This region was shown to be most probably involved in the binding of IFN to its cellular receptor. These findings fit with Sternberg and Cohen's model (Int. J. Biol. Macromol. 4, 137-144, 1982) for the tertiary structure of human IFN-alpha.

Animals↗