Jiří Velek

Differential pulse voltammetric study of the complexation of Cd(II) by the phytochelatin (γ-GluCys)2Gly assisted by multivariate curve resolution

Abstract A multivariate curve resolution method by alternating least-squares (MCR-ALS) is applied to differential pulse voltammograms measured on the Cd(II)+(γ-GluCys) 2 Gly system as a model of metal–phytochelatin interactions at concentrations of both components in the range 10 −7 –10 −5 mol l −1 . The course of complexation is different when peptide is titrated with metal from that when metal is titrated with peptide. The combined analysis of both matrices from titrations of peptide with metal and of metal with peptide allowed the resolution of the system. The analysis of the resulting pure voltammograms and concentration profiles of the resolved components suggested the presence of four different types of bound Cd(II) and made possible the formulation of a complexation model.

Differences in immunogenicity and vaccine potential of peptides from Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase.

Six peptides, A, B1, B, C, D and E derived from the primary sequence of Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase (SG3PDH) were selected based on lowest homology to human G3PDH and used for immunization of BALB/c mice. Peptides B1 and D induced immunoglobulin (Ig) G1, IgG2a and IgG2b antibodies that reacted with native and denatured SG3PDH, and were associated with significant (P<0.05) increase in fecundity and burden of challenge worms, respectively. Peptides A, B, C and E elicited a modest cellular immune response, IgG2a and/or IgG2b antibodies, and no effect on challenge worm burden. In contrast, tetrameric multiple antigen peptide (MAP) constructs A, B, C or E elicited strong cellular immune responses and production of IgG1 and/or IgG2a and IgG2b antibodies against the homologous MAP and peptide and SG3PDH. The immune responses were associated with significant (P<0.05) decrease in challenge worm burden for MAP B, and significant (P<0.05) reduction in egg count per worm couple for MAP C or E. The data together indicated the nature and effect of immune responses vary for each SG3PDH-derived peptide.

Human and Murine Humoral Immune Recognition of Multiple Peptides from Schistosoma mansoni Glyceraldehyde 3-P Dehydrogenase is Associated with Resistance to Schistosomiasis

Schistosoma mansoni glyceraldehyde 3‐phosphate dehydrogenase (SG3PDH) is a target of cellular and humoral immune responses of Brazilian and Egyptian subjects putatively resistant to reinfection with S. mansoni. In an aim to develop a safe, stable and effective vaccine based on this promising molecule, six peptides derived from its primary sequence were selected based on the lowest homology to human G3PDH. The synthetic peptides were tested by ELISA against plasma of humans putatively susceptible or resistant to reinfection with S. mansoni or S. haematobium following chemotherapeutic cure of previous infection. Repeat experiments indicated that the six peptides bear human B‐cell epitopes that bind immunoglobulin (Ig)M, IgG1 and IgG3 antibodies. Resistance to reinfection appeared to be significantly associated with humoral immune responses to multiple peptides. This contention was supported by studies in the murine model, whereby we examined the B cell immune responses of Swiss and inbred BALB/c and C57BL/6 mice immunized with recombinant SG3PDH (rSG3PDH) to the six SG3PDH‐derived peptides. The serum antibodies of rSG3PDH‐immunized Swiss mice were directed to only one of the six peptides tested by ELISA. Antibodies from rSG3PDH‐immunized C57BL/6 and BALB/c mice bound, respectively, to four and six out of six peptides. In contrast to Swiss mice, immunization of C57BL/6 and BALB/c mice with rSG3PDH induced protection against challenge cercariae which reached the level of significance (P < 0.05) for BALB/c mice. The data together indicate that host recognition of multiple peptides of a candidate vaccine antigen is necessary for the expression of its ability to contribute to protective immunity against Schistosomiasis.

Solid phase synthesis of glycopeptide dendrimers with Tn antigenic structure and their biological activities. Part I

Multiple antigenic peptides containing dimeric Tn antigen [Ac‐(Tn)2‐γ‐Abu]4‐(Lys‐X)2‐Lys‐β‐Ala (V: X=0; VIII: X=γ‐Abu) and [Ac‐(Tn)2‐γ‐Abu]8‐(Lys‐X)4‐(Lys‐X)2‐Lys‐β‐Ala (XI: X=0; XIV: X=γ‐Abu), immobilized on biocompatible Tenta Gel S NH2 support were prepared by SPPS. Rosetting tests of V, VIII, XI and XIV showed positive reactions with anti‐Tn (DAKO) and Tn+ erythrocytes, with anti‐Tn/A (BRIC 66) and Tn+ and A erythrocytes, other combinations were negative. In all the animals immunized with XIV, we found a remarkable increase in the level of anti‐Tn (titre 2000–64 000, score 105–167) and no change of anti‐A levels (titre 8, score 13–17). Neither non‐immune nor immune sera showed any reactivity with T+, Cad+ and blood group O erythrocytes. Immunized mice did not exhibit any sign of adverse reaction to the administered conjugates. Biological activities were correlated with molecular modelling and molecular dynamic calculations. The biological activities of these synthetic Tn antigen conjugates (good availability for the immunological interactions, highly specific immunogenicity, good biological tolerance) together with their precise chemical characterization seem to be a promising approach to preparation of anti‐tumour vaccine and affinity purification of anti‐Tn antibodies. Copyright

A new epigenetic marker: the replication-coupled, cell cycle-dependent, dual modification of the histone H4 tail.

Recently, we described the cold-dependent detection of an epitope, epiC, that was selectively recognized by a monoclonal anti-actin antibody at 4 degrees C, but not at RT, in the early replicating chromatin domains of human fibroblast cell nuclei and chromosomes. EpiC was present in a distinct cell cycle window extending from S-phase throughout mitosis until early G1-phase of the next cell generation, indicating its possible involvement in the transfer/maintenance of epigenetic information on transcriptionally competent parts of the genome. However, the molecular nature of epiC remained unresolved. Here we identified epiC as a dual post-translational modification on the same histone H4 tail, which was immunodetected for the first time. We show that the antibody selectively recognized a synthetic peptide of the histone H4 region K12-L22 containing acetylated K16 and dimethylated K20 (H4K16ac-K20me2) at 4 degrees C, but not at RT. Moreover, we show that the peptide containing acetylated K16 and either unmodified or monomethylated K20 was recognized by this antibody at both temperatures. The present and previous results together indicate that, by acetylation of histone H4 K16 during S-phase, the early replicating chromatin domains acquire the H4K16ac-K20me2 epigenetic label that persists on the chromatin throughout mitosis and become deacetylated during early G1-phase of the next cell cycle.

Insect oostatic activity of GnRH and its fragments

Mammal,125I-mammal, salmon, chicken I and II GnRHs and three fragments of mammal GnRH were synthesized and their effect on oogenesis in the flesh flyNeobellieria (formerlySarcophaga) bullata (Diptera) was investigated. The peptides were prepared by the Merrifield solid phase synthesis on polystyrene/divinylbenzene polymer using the Nα-Boc strategy in DMF and were purified by preparative RP-HPLC in a gradient of water-MeOH. From the peptides assayed, only mammal GnRH and two of its carboxy-terminus truncated analogs remarkably affected the processes of egg development in ovarioles, causing changes in the follicular epithelium, proliferation of its nuclei and cell division towards the inner part of the egg chamber. The process led to the occurrence of multinuclear follicular epithelium which finally filled up almost the whole egg chamber and then it degenerated. The inability of GnRH of other animal species to evoke the changes in the egg development establishes the question of primary structures of GnRH responsible for these biological effects. The identity of sequences of GnRHs from position 1 up to 6 (with the exception of chicken GnRH II) points to functionality of amino acids located in positions 7 and 8 of the peptide chain. The radioactivity of the125I-labelled mammal GnRH with maintained oostatic activity and its receptor competition with the non-labelled mammal GnRH were measured in slected insect organs and exhibited different residual values according to the organ and the time after application of the peptide. A transfer of the radioactivity into the next (F1) generation was also observed.