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J Vágner

Publications and source records attributed to J Vágner.

16 recordsLinked to original sources
PubMed1996

Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.

Amino Acid Sequence↗
PubMed1993

Aggregation of resin-bound peptides during solid-phase peptide synthesis. Prediction of difficult sequences.

Nonrandom incomplete aminoacylation of a pendent peptide chain on an insoluble polymeric support during solid-phase peptide synthesis is sequence-dependent and is caused by aggregation of peptide chains, manifested by a decreased swelling capacity. The volume of the swollen peptidyl-resin after each coupling during the syntheses of 87 sequence unrelated peptides was measured, and for each amino acid an aggregation parameter, , was derived that reflects the propensity of the swollen volume of peptidyl-resin to decrease during peptide synthesis. These aggregation parameters were used to predict potentially difficult sequences.

Amino Acid Sequence↗
PubMed1993

Identification of seroreactive epitopes of human papillomavirus type 18 E7 protein by synthetic peptides.

Nine everlapping peptides covering the entire sequence of early protein E7 of human papillomavirus type 18 (HPV-18) were synthesized and tested as antigens with pools of selected human sera in ELISA. Peptides denoted 18/E7-2, 18/E7-3, and 18/E7-5 (amino acid positions 11-33, 21-40, and 41-60, respectively) were reactive with pooled sera originating from HPV-18 DNA-positive cervical cancer patients but not with sera from HPV-16 DNA-positive cervical cancer patients or from condyloma acuminata patients. This suggested that the epitopes contained in these peptides were HPV-18 type-specific, relative to HPV types 16, 11, and 6. On the other hand, 18/E7-1 (aa 1-23) and 18/E7-6 (aa 51-70) peptides were cross-reactive. The prevalence of antibodies reactive with 18/E7-2, 18/E7-3, and 18/E7-5 peptides in cervical carcinoma patients was very low. Thus, the utilization of these peptides for monitoring HPV-18 infection seems to be rather limited.

Amino Acid Sequence↗
PubMed1993

Human breast milk contains procathepsin D--detection by specific antibodies.

The presence of the zymogen of cathepsin D in human milk was detected using antibodies specific for the proenzyme and by the proteolytic activity at low pH. The antibodies were raised against a synthetic propeptide of human cathepsin D and were tested using immunoprecipitations and western blots of samples from different breast cancer cell lines as well as cytosol fractions of human breast cancer tissues. In all experiments these antibodies recognized specifically procathepsin D. Procathepsin D from human milk was partially activated at low pH. The activity was monitored using hemoglobin 14C proteolytic assay, and it was abolished by pepstatin A--a specific inhibitor of aspartic proteinases. Western blots did not reveal presence of cathepsin B or cathepsin H. These data indicate specific secretion of cathepsin D in human breast milk.

Antibodies↗
PubMed1992

Epitope mapping of the human papillomavirus type 16 E4 protein by means of synthetic peptides.

Eight overlapping icosapeptides covering the entire sequence of the E4 protein of human papillomavirus type 16 (HPV-16), were prepared and tested for their reactivity with human sera in IgG-specific ELISA. The strongest reactivity of sera from HPV-16 DNA-positive invasive cervical carcinoma (INCA) patients was detected with the peptide denoted 16/E4-6, covering amino acids 51 to 70. Subsequently nearly 200 sera were tested for the presence of the 16/E4-6-specific antibody. Reactivity was more frequent in cervical intraepithelial neoplasia patients and INCA patients than in matched control subjects. Sera from INCA patients were also tested for antibody reactive with peptide 16/E7-2 covering the major type-specific reactive region of the HPV-16 E7 protein. Only four of 13 sera possessing the 16/E4-6-specific antibody were reactive with the 16/E7-2 peptide.

Adult↗
PubMed1991

Inhibition of aspartic proteinases by propart peptides of human procathepsin D and chicken pepsinogen.

Two propart peptides of aspartic proteinases, the propart peptide of chicken pepsin and human cathepsin D, respectively, were investigated from the point of view of their inhibitory activity for a set of aspartic proteinases. These peptides display a very broad inhibitory spectrum. The strongest inhibition was observed for pepsin A-like proteinases where propart peptides can be used as titrants of active enzymes.

Animals↗
PubMed1991

Comparison of ELISA and western blotting for human papillomavirus type 16 E7 antibody determination.

A total of 140 sera originating from healthy women and women with either cervical intraepithelial neoplasia or cervical cancer were tested for the presence of IgG antibody against E7 of human papillomavirus type 16 (HPV-16) by ELISA using a synthetic icosapeptide, denoted 16/E7-2, representing amino acids 11 to 30, and by Western blotting (WB) using a genetically engineered HPV-16 E7 fusion protein. Eighteen sera were found positive in either one or the other test. Positive reactions were more frequently detected in cervical carcinoma patients (12 of 34, 35.2%) than in the other individuals (six of 106, 5.7%). Ten children's (1 to 3 years of age) sera reacted in neither ELISA nor WB with HPV-16 E7. A high degree of concordance between the two tests was found suggesting that both tests detect the same or similar activity. To locate the reacting epitopes in the E7 protein, absorption tests were performed with peptides corresponding to various sections of the protein. Based on the results obtained, sera possessing antibody to HPV-16 E7 could be differentiated into those reactive with only the 16/E7-2 peptide and those reactive with other HPV-16 E7 epitopes.

Adult↗
PubMed1990

A general procedure for evaluation of immunological relevance of synthetic peptides: peptides synthesized on paper in enzyme-linked immunosorbent assay.

Multiple continuous-flow solid-phase peptide synthesis has been adapted for synthesis of peptides on a cellulose carrier (Whatman 3MM paper). Paper-bound synthetic peptides that represent antigenic determinants of particular proteins detected antibodies against the respective proteins in an enzyme-linked immunosorbent assay. The method is applied to the synthesis, and use in site-directed serology, of four peptides derived from the gp41 glycoprotein of HIV, the Epstein-Barr virus-determined nuclear antigen-1 and VCA proteins of the Epstein-Barr virus, and the early region of human papillomavirus type 11.

Amino Acid Sequence↗
PubMed1990

Synthetic peptides derived from E7 region of human papillomavirus type 16 used as antigens in ELISA.

Nine overlapping peptides (20 amino acid) covering the entire sequence of early antigen E7 of human papillomavirus type 16 (HPV-16) were synthesized and tested as antigens with human sera in ELISA. Five of these peptides (no. 1 to 5 counting from the N terminus of the E7 protein) reacted with a pool of sera from HPV-16-infected individuals (as determined by molecular hybridization with their biopsy specimens); one (no. 5) was also reactive with pools of HPV-18- and HPV-6- or 11-infected individuals. Sera from 24 patients with cervical intraepithelial neoplasia (CIN) and from 29 invasive cervical carcinoma (INCA) patients were tested for the presence of antibodies reactive with peptides, no. 1 to 4 covering amino acids 1 to 50 and with peptide no. 5 covering amino acids 41 to 60. Only one of the sera from CIN patients was reactive with peptides no. 1 (amino acids 1 to 20) and no. 4 (amino acids 31 to 50). However, the majority of these sera reacted with peptide no. 5. The occurrence of this antibody was only slightly less frequent in sera from healthy subjects compared to CIN patients. On the other hand, sera from the INCA patients were reactive with the peptides no. 1 to 3 more frequently than the sera from matched control subjects. Positive reactions of sera from INCA patients were most frequently seen with no. 2; 24% of these sera but only 7% of the controls were reactive with no. 2 peptide. The present data suggest that no. 1 to 3 are HPV-16-specific, whereas no. 5 is broadly cross-reactive.

Amino Acid Sequence↗
PubMed1990

Immunoregulatory effect of a synthetic peptide corresponding to a region of protein p24 of HIV.

The effect of a synthetic peptide, corresponding to a sequence of HIV-1 p24 protein (amino acids 218-237), on in vitro immune responses was studied. The peptide inhibited in a dose-dependent manner the induction of an anti-SRC antibody response and of a PPD-specific proliferative response of human PBL. On the other hand, PHA-induced proliferation of human PBL and PPD-induced proliferation of a PPD-specific human T-cell line were not modified by comparable amounts of the peptide. These results suggest that structures from a protein (p24), present in the serum throughout the course of HIV infection, are able to interfere with the inductive stages of specific immune responses. These findings may help to unravel some of the pathogenic mechanisms of AIDS and may contribute to the development of vaccine strategies.

Animals↗
PubMed1989

Multiple continuous-flow solid-phase peptide synthesis. Synthesis of an HIV antigenic peptide and its omission analogues.

Multiple continuous-flow solid-phase peptide synthesis was performed on a standard polystyrene-based resin under low-pressure conditions using a simple manually operated synthesizer. Stable-flow resin-packed columns were prepared in small polypropylene flow reactors, adjustable for volume. The concurrent synthesis of 10 peptides was carried out in flow reactors concatenated together; solvents and reactants were passed through this set of columns using moderate overpressure. One decapeptide, H-Val-Tyr-Tyr-Arg-Asp-Ser-Arg-Asn-Pro-Leu-NH2, containing an antigenic determinant of the p31 protein product of the pol gene of the human immunodeficiency virus, and its nine omission analogues were synthesized.

HIV Antigens↗
PubMed1989

Solid phase enzyme immunoassay of human interleukin 2 utilizing antibodies against synthetic IL-2 peptides.

In the present study, solid phase enzyme immunoassay utilizing antibodies against synthetic IL-2 peptides was used for quantitative measurements of human recombinant and lymphoid IL-2 preparations. The 27-peptide MCF-III-6 (Leu-Glu-His-Leu-Leu-Leu-Asp-Leu-Gln-Met-Ile-Leu-Asn-Gly-Ile-Asn-Asn-- Tyr-Lys-Asn-Pro-Lys-Leu-Thr-Arg-Met-Leu) that comprises the region 14-40 from the IL-2 amino acid sequence was synthesized and used for immunization of rabbits. Resulting anti-MCF-III-6 polyvalent rabbit antibodies reacted specifically in EIA up to dilution of 10(-7) with the MCF-III-6 peptide used for immunization as well as with 16-peptide I-16 (Cys-Nle-Gly-Ile-Asn-Asn-Tyr-Lys-Asn-Pro-Lys-Leu-Thr-Arg-Met-Leu) that comprises the region 27-40 from the IL-2 amino acid sequence. The anti-MCF-III-6 antibody reacted also with human recombinant IL-2 preparations obtained from three producers (Cetus, Riga and Amersham), to the concentration of 0.1 ng/ml, and with various human lymphoid IL-2 preparations. Direct correlation was observed between quantitative measurements of human lymphoid IL-2 by EIA and by CTLL bioassay. It can be concluded that utilization of synthetic IL-2 peptides provides a suitable and comparatively unexpensive immunogen for the production of IL-2 antibodies and that the solid phase EIA using such antibodies can be employed as a rapid, reproducible, and sensitive method for quantitative examination of both recombinant and lymphoid IL-2 preparations.

Amino Acid Sequence↗
PubMedDate not supplied

Continuous-flow synthesis of alpha-gliadin peptides in an ultrasonic field and assay of their inhibition of intestinal sucrase activity.

Three dodecapeptide amides derived from the amino acid sequence of alpha-gliadin and four of their analogues were synthesized by continuous-flow solid-phase multiple peptide synthesis. Ultrasonic field conditions accelerated the coupling reaction without affecting purity. Biological tests of the synthetic fragments showed the relevance of toxicity prediction for preselection of immunogenic alpha-gliadin fragments.

Adenovirus Early Proteins↗
PubMedDate not supplied

Color-monitored solid-phase multiple peptide synthesis under low-pressure continuous-flow conditions.

Exemplified on fifty peptides derived from proteins of the human immunodeficiency virus (HIV), we summarize our experiences with rapid solid-phase multiple peptide synthesis under low-pressure continuous-flow conditions on standard polystyrene-based resin in a series of concatenated flow reactors with adjustable volume. Alternatively, multiple batchwise synthesis in plastic syringes may be used as a still simpler but a more elaborate procedure. Completeness of acylation of resin-bound free amino groups was monitored by the acid-base indicator bromophenol blue. Efficiency of the coupling reaction was increased by exposure to elevated temperature in an ultrasonic bath. Up to 100 mg of crude peptides were purified on a mass-overloaded Vydac C18 reversed-phase semipreparative HPLC column in one run, this procedure ensuring high recovery of homogeneous peptides. The peptides synthesized were serologically relevant to the detection of anti-HIV-1 antibodies in positive sera.

Acylation↗