Induction of immunity in sheep to Fasciola hepatica with mimotopes of cathepsin L selected from a phage display library.

An M13 phage random 12-mers peptide library was used to screen cathepsin L mimotopes of Fasciola hepatica and to evaluate their immunogenicity in sheep. Seven clones showed positive reactivity to a rabbit anti-cathepsin L1/L2 antiserum in ELISA, and their amino acid sequences deduced by DNA sequencing were tentatively mapped on the protein. Twenty sheep were randomly allocated into 4 groups of 5 animals each, for immunization with 1x10(14) phage particles of clones 1, 20, a mixture of 7 clones and PBS, without adjuvant at the beginning, and 4 weeks later. All groups were challenged with 300 metacercariae at week 6 and slaughtered 16 weeks later. The mean worm burdens after challenge were reduced by 47.61% and 33.91% in sheep vaccinated with clones 1 and 20, respectively; no effect was observed in animals inoculated with the clone mixture. Also, a significant reduction in worm size and burden was observed for those sheep immunized with clone 1. Animals receiving clone 20, showed a significant reduction in egg output. Immunization induced a reduction of egg viability ranging from 58.92 to 82.11%. Furthermore, vaccinated animals produced clone-specific antibodies which were boosted after challenge with metacercariae of F. hepatica.

L-Homoserine: a novel excreted metabolic marker of hepatitis B abnormally produced in liver from methionine.

The hepatitis B infection leads to various profound pathological processes in liver metabolism. Some biochemical alterations detectable by blood analysis are currently used for a preliminary evaluation of the infection. Based on existing data we present here evidence that non-protein amino acid L-homoserine is a pathological, hepatitis B-induced metabolite that is formed and excreted into urine from methionine via splitting S-adenosylmethionine. The urine L-homoserine is proposed as a new marker in the pre-diagnosis examinations that is easier for the clinical analysis than currently used blood test, and is applicable to large-scale epidemiological surveys of the probability of hepatitis B.

Post-panning computer-aided analysis of phagotope collections selected with neurocysticercosis patient polyclonal antibodies: separation of disease-relevant and irrelevant peptide sequences.

The homology of peptide sequences selected from a 7mer phage display library with antibodies elicited by the multicelled parasite Taenia solium in cerebrospinal fluid and serum of neurocysticercosis (NCC) patients and by antibodies of uninfected control patients with similar neurological complications of other ethiology (non-NCC) were analyzed using a PILEUP-Tudos sequence alignments program. The analysis generated dendrograms bearing two types of sequence clusters, those containing (1) only NCC patients-derived peptides and (2) both NCC- and control non-CC -- patient derivatives. By using ELISA, peptides that were selected by the antibodies were identified predominantly in the NCC-derived clusters. In repeated analysis in which sequences were added or removed, the first type of clusters maintained their structure, while the second type of clusters were split into many separate homology units dispersed throughout the guide tree. These results are interpreted as the ability of the analysis to segregate NCC-specific peptide sequences from other sequences. Altogether, this study demonstrates the high potential of the PILEUP-Tudos computer program to analyze phagotope collections recovered through biopanning with polyclonal antibodies elicited in patients by complex and as yet unknown multiple pathogenic antigens and to separate all phagotopes that are disease-relevant on the basis of the sequence homology.

Epitope mapping on N-terminal region of taenia solium paramyosin.

Epitope mapping of the amino-terminal 20aa sequence from Taenia solium paramyosin (TPmy), an immunodominant protein involved in the complex host-parasite relationship in human and porcine cysticercosis is reported. A 12-mer random peptide phage display library was screened with antibodies raised against a synthetic peptide corresponding to the amino-terminal 20aa sequence of TPmy, its highly immunodominant region. In total, 57 clones isolated in two panning conditions were analyzed, of which a single group of 14 sequences found in 25 clones shared a consensus motif showing structural similarity with the antigen Arg10-Thr16 region.

Isolation and structure-functional characterization of phage display library-derived mimotopes of noxiustoxin, a neurotoxin of the scorpion Centruroides noxius Hoffmann.

Noxiustoxin (NTX) is a short-chain toxin from the venom of the scorpion Centruroides noxius Hoffmann, whose molecular structure and physiological effects have been characterized in detail, whereas the antigenic properties of this and other K(+) channel-blocking toxins are poorly studied. A monoclonal antibody against NTX, BNTX18, able to inhibit the binding of NTX to rat brain synaptosomes, was used in the present study for selecting immunoreactive peptides, mimotopes, from a 12mer and a 7mer phage library. The peptides were characterized immunologically and used for mapping the epitope on NTX. In total, 75 phage clones carrying 43 different peptides were analyzed of which 42 clones carrying 17 different peptides, twelve 12mer and five 7mer peptides, presented a single consensus motif: Leu(Ile, Val)-Tyr(Phe, Trp, Leu)-Gly-Met(Ala). All but three of the peptides containing this motif were reactive with selected mAb BNTX18 in a dot-blot assay of which eight were clearly positive in ELISA and exhibited in competition-inhibition assay the antibody binding specificity of the NTX epitope recognized by BNTX18. The two most reactive mimotopes injected into mice showed the ability to induce antibodies reacting with NTX, thus, to mimic the epitope of NTX antigenically. Sequence comparison and the analysis of the three-dimensional structure of NTX led to the proposal that residues Glu19-Leu20-Tyr21-Gly22 and the hydrophobic part of the side chain of Lys18 form the C-terminal part of the epitope. Due to the frequent presence of residues Pro, Leu, Thr, Arg, and Gln in the N-terminal part of the mimotopes, corresponding homologous residues in the N-terminal proximity of the partial epitope may be part of an additional more hydrophilic epitope element.

[Determination of homoserine kinase activity by chromatographic separation and measurement of reaction products]. / Opredelenie aktivnosti gomoserinkinazy putem khromatograficheskogo razdeleniia i izmereniia produktov reaktsii.

A highly specific procedure for quantitative assay of the homoserine kinase activity in an optimized enzymatic reaction using 14C-labelled homoserine or [gamma 33P]-ATP as substrates, and paper or thin-layer chromatography for separation of the formed o-phosphohomoserine, is described. The procedure is simple, sensitive and allows the assay for the activity of both purified and non-purified homoserine kinases.

[A short repeat in the genome of the DNA sequences flanking bovine growth hormone genes]. / Umerennoe povtorenie v genome posledovatel'nostei DNK, okruzhaiushchikh gen gormona rosta byka.

The phage clones containing a gene coding for bovine growth hormone were isolated from a bovine genomic library. Comparison of the 5' and 3' regions flanking the bovine growth hormone gene by Southern blot hybridization revealed that they share homology. Screening the bovine genomic library by nick-translated DNA fragment from 5' flanking region leads to conclusion that this sequence is present in 0.1% of clones. Each analysed clone carrying the sequence contains some copies of it.

[Immunochemical properties of erythrocyte-specific histone H5 in salmonid fishes]. / Immunokhimicheskie svoistva éritrotsitspetsificheskogo gistona H5 u lososevykh ryb.

Comparative studies have been made on the immunochemical properties of histone H5 in Salmonidae species. High degree of homology between these histones and H5 histone from Oncorhynchus tschawytscha was demonstrated by microcomplement fixation. Properties of H5 histones in fish and birds are discussed.