LOXL2-mediated H3K4 oxidation reduces chromatin accessibility in triple-negative breast cancer cells.

Oxidation of H3 at lysine 4 (H3K4ox) by lysyl oxidase-like 2 (LOXL2) generates an H3 modification with an unknown physiological function. We find that LOXL2 and H3K4ox are higher in triple-negative breast cancer (TNBC) cell lines and patient-derived xenografts (PDXs) than those from other breast cancer subtypes. ChIP-seq revealed that H3K4ox is located primarily in heterochromatin, where it is involved in chromatin compaction. Knocking down LOXL2 reduces H3K4ox levels and causes chromatin decompaction, resulting in a sustained activation of the DNA damage response (DDR) and increased susceptibility to anticancer agents. This critical role that LOXL2 and oxidized H3 play in chromatin compaction and DDR suggests that functionally targeting LOXL2 could be a way to sensitize TNBC cells to conventional therapy.

The antiproliferative peptide Ctn[15-34] is active against multidrug-resistant yeasts Candida albicans and Cryptococcus neoformans.

AIMS: Crotalicidin (Ctn), a cathelicidin-related antimicrobial peptide from the South American rattlesnake venom gland, and its C-terminal Ctn[15-34] fragment, have exhibited important activities against micro-organisms, trypanosomatid protozoa and certain lines of tumour cells. Herein, the activity against clinical strains of fluconazole-resistant Candida albicans and of amphotericin B and fluconazole-resistant Cryptococcus neoformans was investigated. METHODS AND RESULTS: Microdilution and luminescent cell viability tests were used to evaluate and compare the susceptibility of pathogenic yeasts to these peptides. The time-kill curves of the most active Ctn[15-34] alone or in combination with fluconazole against drug-resistant yeasts were determined. Concomitantly, the fungicidal and/or fungistatic effects of Ctn[15-34] were visualized by the spotting test. The peptides were active against all strains, including those resistant to antifungal agents. The association of fluconazole with both Ctn and Ctn[15-34], although not synergic, was additive. In contrast, such pattern was not observed for C. neoformans. CONCLUSIONS: Overall, Ctn and Ctn[15-34] are potential antifungal leads displaying anti-yeast activities against clinical isolates endowed with drug resistance mechanisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The effective peptide activity against resistant strains of pathogenic yeasts demonstrates that crotalicidin-derived peptides are promising templates to develop new antifungal pharmaceuticals.

Immune Response and Partial Protection against Heterologous Foot-and-Mouth Disease Virus Induced by Dendrimer Peptides in Cattle.

Synthetic peptides mimicking protective B- and T-cell epitopes are good candidates for safer, more effective FMD vaccines. Nevertheless, previous studies of immunization with linear peptides showed that they failed to induce solid protection in cattle. Dendrimeric peptides displaying two or four copies of a peptide corresponding to the B-cell epitope VP1 [136-154] of type O FMDV (O/UKG/11/2001) linked through thioether bonds to a single copy of the T-cell epitope 3A [21-35] (termed B2T and B4T, resp.) afforded protection in vaccinated pigs. In this work, we show that dendrimeric peptides B2T and B4T can elicit specific humoral responses in cattle and confer partial protection against the challenge with a heterologous type O virus (O1/Campos/Bra/58). This protective response correlated with the induction of specific T-cells as well as with an anamnestic antibody response upon virus challenge, as shown by the detection of virus-specific antibody-secreting cells (ASC) in lymphoid tissues distal from the inoculation point.

VT Recurrence After Ablation: Incomplete Ablation or Disease Progression? A Multicentric European Study.

AIM: To determine whether ventricular tachycardia (VT) recurrences in arrhythmogenic RV cardiomyopathy (ARVC) and nonischemic cardiomyopathy (NICM) are related to incomplete ablation or disease progression. METHODS: ARVC and NICM patients with two substrate maps of the same diseased ventricle with an interprocedural delay of ≥12 months were included. Disease progression was defined as ≥1 factor: scar area progression (PROG, +5%), ventricular remodeling (dilatation [+25 mL] or decreased ejection fraction [-5%EF]). Incomplete ablation was defined as index VT recurrence or ablation in previously unablated regions inside index scar without PROG. RESULTS: Twenty patients from nine centers were included (80% male 55 ± 16 years, 7 ARVC and 13 NICM, LVEF 43 ± 14%). Mean delay was 28 ± 18 months. Disease progression occurred in 75% with ventricular remodeling in 70%: ventricular dilation in 45% (ARVC [71%]; NICM [38%]), decreased EF in 60% [RVEF in ARVC (71%); LVEF in NICM (54%)], and scar progression in 50% (in ARVC [57%] and NICM [46%]). Index VT recurrence was observed in 40%. Redo ablation sites were located in previously unablated regions inside the index scar in 70% of patients. VT recurrence following the second procedure was seen in 25%. Fifteen percent died during a follow-up of 17 ± 17 months. CONCLUSION: Disease progression is the rule in ARVC and NICM while scar progression occurs in half. However, even if disease progression is frequently observed, incomplete index ablation is the most common finding, strongly suggesting the need for more extensive ablation.

Vipericidins: a novel family of cathelicidin-related peptides from the venom gland of South American pit vipers.

Cathelicidins are phylogenetically ancient, pleiotropic host defense peptides-also called antimicrobial peptides (AMPs)-expressed in numerous life forms for innate immunity. Since even the jawless hagfish expresses cathelicidins, these genetically encoded host defense peptides are at least 400 million years old. More recently, cathelicidins with varying antipathogenic activities and cytotoxicities were discovered in the venoms of poisonous snakes; for these creatures, cathelicidins may also serve as weapons against prey and predators, as well as for innate immunity. We report herein the expression of orthologous cathelicidin genes in the venoms of four different South American pit vipers (Bothrops atrox, Bothrops lutzi, Crotalus durissus terrificus, and Lachesis muta rhombeata)-distant relatives of Asian cobras and kraits, previously shown to express cathelicidins-and an elapid, Pseudonaja textilis. We identified six novel, genetically encoded peptides: four from pit vipers, collectively named vipericidins, and two from the elapid. These new venom-derived cathelicidins exhibited potent killing activity against a number of bacterial strains (S. pyogenes, A. baumannii, E. faecalis, S. aureus, E. coli, K. pneumoniae, and P. aeruginosa), mostly with relatively less potent hemolysis, indicating their possible usefulness as lead structures for the development of new anti-infective agents. It is worth noting that these South American snake venom peptides are comparable in cytotoxicity (e.g., hemolysis) to human cathelicidin LL-37, and much lower than other membrane-active peptides such as mastoparan 7 and melittin from bee venom. Overall, the excellent bactericidal profile of vipericidins suggests they are a promising template for the development of broad-spectrum peptide antibiotics.

Influence of conjugation chemistry and B epitope orientation on the immune response of branched peptide antigens.

Multimeric presentation, a well-proven way of enhancing peptide immunogenicity, has found substantial application in synthetic vaccine design. We have reported that a combination of four copies of a B-cell epitope with one of a T-cell epitope in a single branched construct results in a peptide vaccine conferring total protection against foot-and-mouth disease virus in swine, a natural host (Cubillos et al. (2008) J. Virol. 82, 7223-7230). More recently, a downsized version of this prototype with only two copies of the B epitope has proven as effective as the tetravalent one in mice. Here we evaluate three approaches to bivalent platforms of this latter type, involving different chemistries for the conjugation of two B epitope peptides to a branching T epitope. Comparison of classical thioether, "reverse" thioether (Monsó et al. (2012) Org. Biomol. Chem. 10, 3116-3121) and thiol-ene conjugation chemistries in terms of synthetic efficiency clearly singles out the latter, maleimide-based strategy as most advantageous. We also examine how minor structural differences among the conjugates--including the N- or C-terminal attachment of the B epitope to the branching T epitope--bear on the immunogenicity of these vaccine candidates, with the maleimide-based conjugate again emerging as the most successful.

Usefulness of transoesophageal echocardiography before circumferential pulmonary vein ablation in patients with atrial fibrillation: is it really mandatory?

AIMS: Transoesophageal echocardiography (TEE) is recommended prior to circumferential pulmonary vein ablation (CPVA) in patients with atrial fibrillation (AF) to identify left atrial (LA) or left atrial appendage (LAA) wall thrombi. It is not clear whether all patients undergoing CPVA should receive pre-procedural TEE. We wanted to assess the incidence of LA thrombus in these patients and to identify factors associated with its presence. METHODS AND RESULTS: Consecutive patients referred for CPVA from 2004 to 2009 underwent TEE within 48 h prior to the procedure. Of 408 patients included in the study, 6 patients (1.47%) had LA thrombi, persistent AF, and LA dilation. Compared with patients without thrombus, these six patients had larger LA diameter (P = 0.0001) and more frequently were women (P = 0.002), had persistent AF (P = 0.04), and had underlying structural cardiac disease (P = 0.014). The likelihood of presenting LA thrombus increased with the number of these four risk factors present (P < 0.001). None of the patients with paroxysmal AF and without LA dilation had LA thrombus. A cut-off value of 48.5 mm LA diameter yielded 83% sensitivity, 92% specificity, and a 10.1 likelihood ratio to predict LA thrombus appearance. CONCLUSION: The incidence of LA thrombus prior to CPVA is low. Persistent AF, female sex, structural cardiopathy, and LA dilation were associated with the presence of LA thrombus. Our data suggest that the use of TEE prior to CPVA to detect LA thrombi might not be needed in patients with paroxysmal AF and no LA dilation or structural cardiopathy.

Effect of acylation on the interaction of the N-Terminal segment of pulmonary surfactant protein SP-C with phospholipid membranes.

SP-C, the smallest pulmonary surfactant protein, is required for the formation and stability of surface-active films at the air-liquid interface in the lung. The protein consists of a hydrophobic transmembrane alpha-helix and a cationic N-terminal segment containing palmitoylated cysteines. Recent evidence suggests that the N-terminal segment is of critical importance for SP-C function. In the present work, the role of palmitoylation in modulating the lipid-protein interactions of the N-terminal segment of SP-C has been studied by analyzing the effect of palmitoylated and non-palmitoylated synthetic peptides designed to mimic the N-terminal segment on the dynamic properties of phospholipid bilayers, recorded by spin-label electron spin resonance (ESR) spectroscopy. Both palmitoylated and non-palmitoylated peptides decrease the mobility of phosphatidylcholine (5-PCSL) and phosphatidylglycerol (5-PGSL) spin probes in dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) bilayers. In zwitterionic DPPC membranes, both peptides have a greater effect at temperatures below than above the main gel-to-liquid-crystalline phase transition, the palmitoylated peptide inducing greater immobilisation of the lipid than does the non-palmitoylated form. In anionic DPPG membranes, both palmitoylated and non-palmitoylated peptides have similar immobilizing effects, probably dominated by electrostatic interactions. Both palmitoylated and non-palmitoylated peptides have effects comparable to whole native SP-C, as regards improving the gel phase solubility of phospholipid spin probes and increasing the polarity of the bilayer surface monitored by pK shifts of fatty acid spin probes. This indicates that a significant part of the perturbing properties of SP-C in phospholipid bilayers is mediated by interactions of the N-terminal segment. The effect of SP-C N-terminal peptides on the chain flexibility gradient of DPPC and DPPG bilayers is consistent with the existence of a peptide-promoted interdigitated phase at temperatures below the main gel-to-liquid-crystalline phase transition. The palmitoylated peptide, but not the non-palmitoylated version, is able to stably segregate interdigitated and non-interdigitated populations of phospholipids in DPPC bilayers. This feature suggests that the palmitoylated N-terminal segment stabilizes ordered domains such as those containing interdigitated lipids. We propose that palmitoylation may be important to promote and facilitate association of SP-C and SP-C-containing membranes with ordered lipid structures such as those potentially existing in highly compressed states of the interfacial surfactant film.

Lack of oestrogen protection in amyloid-mediated endothelial damage due to protein nitrotyrosination.

Amyloid beta-peptide (Abeta) cytotoxicity, the hallmark of Alzheimer's disease, implicates oxidative stress in both neurons and vascular cells, particularly endothelial cells. Consequently, antioxidants have shown neuroprotective activities against Abeta-induced cytotoxicity. Among the different antioxidants used in both in vitro and in vivo studies, 17beta-oestradiol (E2) has garnered the most attention. Oestrogen attenuated Abeta(E22Q)-induced toxicity in neurons but failed to protect endothelial cells. Here we show that E2-mediated activation of endothelial nitric oxide synthase (eNOS) increases the production of nitric oxide (NO), which, under Abeta(E22Q)-induced oxidative damage, results in the formation of peroxynitrite and increased nitration of tyrosine residues. Inhibition of eNOS prevents nitrotyrosination and permits E2-mediated protection against Abeta(E22Q) on endothelial cells. The main nitrotyrosinated proteins in the presence of E2 and Abeta(E22Q) were identified by MALDI-TOF mass spectrometry. These proteins are key players in the regulation of energy production, cytoskeletal integrity, protein metabolism and protection against oxidative stress. Our data highlight the potential damaging consequences of E2 in vascular disorders dealing with oxidative stress conditions, such as cerebral amyloid angiopathy, stroke and ischaemia-reperfusion conditions.

Conformational study of linear and cyclic peptides corresponding to the 276-284 epitope region of HSV gD-1.

The results of conformational analysis of linear and cyclic peptides from the 276SALLEDPVG(284) sequence of glycoprotein D of Herpes simplex virus are presented. The epitope peptides were synthesized by SPPS and on resin cyclization was applied for preparation of cyclic compounds. Circular dichroism spectroscopy, Fourier-transform infrared spectroscopy and nuclear magnetic resonance (NMR) were used to determine of the solution structure of both linear and cyclic peptides. The results indicated that the cyclopeptides containing the core of the epitope (DPVG) as a part of the cycle have more stable beta-turn structure than the linear peptides or the cyclic analogues, where the core motif is not a part of the cycle. NMR study of H-SALLc(EDPVGK)-NH(2) confirm presence of a type I beta-turn structure which includes the DPVG epitope core.

Probing degeneracy in antigen-antibody recognition at the immunodominant site of foot-and-mouth disease virus.

Antigen-antibody binding is regarded as one of the most representative examples of specific molecular recognition in nature. The simplistic view of antigenic recognition in terms of a lock-and-key mechanism is obsolete, as it is evident that both antigens and antibodies are flexible and can undergo substantial mutual adaptation. This flexibility is the source of complexities such as degeneracy and nonadditivity in antigenic recognition. We have used surface plasmon resonance to study the effects of combining multiple amino acid replacements within the sequence of the antigenic GH loop of foot-and-mouth disease virus. Our aim was 2-fold: to explore the extent to which antigenic degeneracy can be extended in this particular case, and to search for potential nonadditive effects in introducing multiple amino acid replacements. Combined analysis of one such multiply substituted peptide by SPR, solution NMR and X-ray diffraction shows that antigenic degeneracy can be expected as long as residues directly interacting with the paratope are conserved and the peptide bioactive folding is unaltered.

In vitro activity of CA(1-8)M(1-18), a synthetic cecropin A-melittin hybrid peptide, against multiresistant Acinetobacter baumannii strains.

The in vitro antibiotic activity of CA(1-8)M(1-18), a synthetic cecropin A-melittin hybrid peptide, was determined by broth microdilution on 20 clinical Acinetobacter baumannii isolates with different resistance profiles. The MIC(50), MIC(90) and ranges were 4 mg/l, 4 mg/l and 2-8 mg/l, respectively, and were independent of resistance pattern. Different assay parameters such as microplate plastic (polystyrene or polypropylene), addition of supplements (5-10% fetal calf serum or 5% horse blood), inoculum size (10(5), 10(6), 10(7) and 10(8) CFU/ml) or incubation period (24 or 48 h) were studied. MIC was independent of the first two parameters, although the MIC values increased both with inoculum size or incubation period. Killing curves were obtained both for a standard strain and a multiresistant isolate over a 45.7-2.8 mg/l (16-1 mM) peptide range, using an initial inoculum of 10(5)-10(6) CFU/ml and 10(9)-10(10) CFU/ml. A concentration of 45.7 mg/l was required for complete killing. Accordingly, CA(1-8)M(1-18) showed good in vitro activity against the A. baumannii strains tested irrespective of the resistance to classical antibiotics, and could be a future candidate for multiresistant A. baumannii infections, although further cytotoxicity and pharmacological studies will be needed.

Screening of antifeedant activity in brain extracts led to the identification of sulfakinin as a satiety promoter in the German cockroach. Are arthropod sulfakinins homologous to vertebrate gastrins-cholecystokinins?

The feeding cycle of the adult female cockroach Blattella germanica parallels vitellogenesis. The study of the mechanisms that regulate this cycle led us to look for food-intake inhibitors in brain extracts. The antifeedant activity of brain extracts was tested in vivo by injecting the extract and measuring the carotenoids contained in the gut from carrot ingested after the treatment. By HPLC fractionation and tracking the biological activity with the carrot test, we isolated the sulfakinin EQFDDY(SO3H) GHMRFamide (Pea-SK). A synthetic version of the peptide inhibited food intake when injected at doses of 1 microg (50% inhibition) and 10 microg (60% inhibition). The sulfate group was required for food-intake inhibition. These biological and structural features are similar to those of the gastrin-cholecystokinin (gastrin-CCK) family of vertebrate peptides. However, heterologous feeding assays (human CCK-8 tested on B. germanica, and Pea-SK tested on the goldfish Carassius auratus) were negative. In spite of this, alignment and cluster analysis of these and other structurally similar peptide families suggest that sulfakinins and gastrin-CCKs are homologous, and that mechanisms of feeding regulation involving these regulatory peptides may have been conserved during evolution between insects and vertebrates.

Conformationally restricted PACAP27 analogues incorporating type II/II' IBTM beta-turn mimetics. Synthesis, NMR structure determination, and binding affinity.

To probe the importance of a proposed beta-turn within residues S9-R12 of PACAP for recognition by VIP/PACAP receptors, compounds 1 and 2, two conformationally restricted analogues of PACAP27 incorporating respectively (S)- or (R)-IBTM as type II or II' beta-turn dipeptide mimetic at the Y10-S11 position, were synthesized. According to 1H NMR conformational analyses in aqueous solution and 30% TFE, both PACAP27 and the [S-IBTM(10,11)]PACAP27 analogue 1 adopt similar ordered structures. PACAP27 shows an N-terminal disordered region (residues H1-F6) and an alpha-helical conformation within segment T7-L27. For residues S9-R12, our data seem more compatible with a segment of the alpha-helix than with the beta-turn previously proposed for this fragment. In compound 1 the alpha-helix, also spanning T7-L27 residues, appears slightly distorted at the N-terminus relative to the native peptide. Although this distortion could lead to the marked decrease in binding affinity of this compound at the VIP/PACAP receptors, the lack of the Y10 side chain in analogues 1 and 2 could also significantly affect the binding of these compounds.

Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase.

Determinants for the recognition of a mitochondrial presequence by the mitochondrial processing peptidase (MPP) have been investigated using mutagenesis and bioinformatics approaches. All plant mitochondrial presequences with a cleavage site that was confirmed by experimental studies can be grouped into three classes. Two major classes contain an arginine residue at position -2 or -3, and the third class does not have any conserved arginines. Sequence logos revealed loosely conserved cleavage motifs for the first two classes but no significant amino acid conservation for the third class. Investigation of processing determinants for a class III precursor, Nicotiana plumbaginifolia F1beta precursor of ATP synthase (pF1beta), was performed using a series of pF1beta presequence mutants and mutant presequence peptides derived from the C-terminal portion of the presequence. Replacement of -2 Gln by Arg inhibited processing, whereas replacement of either the most proximally located -5 Arg or -15 Arg by Leu had only a low inhibitory effect. The C-terminal portion of the pF1beta presequence forms a helix-turn-helix structure. Mutations disturbing or prolonging the helical element upstream of the cleavage site inhibited processing significantly. Structural models of potato MPP and the C-terminal pF1beta presequence peptide were built by homology modelling and empirical conformational energy search methods, respectively. Molecular docking of the pF1beta presequence peptide to the MPP model suggested binding of the peptide to the negatively charged binding cleft formed by the alpha-MPP and beta-MPP subunits in close proximity to the H111XXE114H115X(116-190)E191 proteolytic active site on beta-MPP. Our results show for the first time that the amino acid at the -2 position, even if not an arginine, as well as structural properties of the C-terminal portion of the presequence are important determinants for the processing of a class III precursor by MPP.

N-terminal fatty acid substitution increases the leishmanicidal activity of CA(1-7)M(2-9), a cecropin-melittin hybrid peptide.

In order to improve the leishmanicidal activity of the synthetic cecropin A-melittin hybrid peptide CA(1-7)M(2-9) (KWKLFKKIGAVLKVL-NH(2)), a systematic study of its acylation with saturated linear fatty acids was carried out. Acylation of the N(epsilon)-7 lysine residue led to a drastic decrease in leishmanicidal activity, whereas acylation at lysine 1, in either the alpha or the epsilon NH(2) group, increased up to 3 times the activity of the peptide against promastigotes and increased up to 15 times the activity of the peptide against amastigotes. Leishmanicidal activity increased with the length of the fatty acid chain, reaching a maximum for the lauroyl analogue (12 carbons). According to the fast kinetics, dissipation of membrane potential, and parasite membrane permeability to the nucleic acid binding probe SYTOX green, the lethal mechanism was directly related to plasma membrane permeabilization.

Identification of T-cell epitopes in nonstructural proteins of foot-and-mouth disease virus.

Porcine T-cell recognition of foot-and-mouth disease virus (FMDV) nonstructural proteins (NSP) was tested using in vitro lymphoproliferative responses. Lymphocytes were obtained from outbred pigs experimentally infected with FMDV. Of the different NSP, polypeptides 3A, 3B, and 3C gave the highest stimulations in the in vitro assays. The use of overlapping synthetic peptides allowed the identification of amino acid regions within these proteins that were efficiently recognized by the lymphocytes. The sequences of some of these antigenic peptides were highly conserved among different FMDV serotypes. They elicited major histocompatibility complex-restricted responses with lymphocytes from pigs infected with either a type C virus or reinfected with a heterologous FMDV. A tandem peptide containing the T-cell peptide 3A[21-35] and the B-cell antigenic site VP1[137-156] also efficiently stimulated lymphocytes from infected animals in vitro. Furthermore, this tandem peptide elicited significant levels of serotype-specific antiviral activity, a result consistent with the induction of anti-FMDV antibodies. Thus, inclusion in the peptide formulation of a T-cell epitope derived from the NSP 3A possessing the capacity to induce T helper activity can allow cooperative induction of anti-FMDV antibodies by B cells.

L and D presequence peptides derived from the precursor of F1beta subunit of the ATP synthase inhibit mitochondrial protein import by interaction with import machinery.

We investigated the effect of L and D enantiomers of a 25-residue peptide derived from the N-terminal region of the presequence of Nicotiana plumbaginifolia F1beta subunit of the ATP synthase, pF1beta(1, 25), on import into spinach leaf mitochondria. Three in vitro synthesized precursor proteins using different import pathways were used. Import of the precursor proteins of F1beta subunit of the ATP synthase, pre-F1beta, and the alternative oxidase, pre-AOX, required addition of external ATP. whereas the chimeric precursor containing the N-terminal 84 amino acids of the cytochrome b2 precursor protein linked to dihydrofolate reductase, pre-b2(1, 84)-DHFR was not dependent on ATP. Import of pre-F1beta, and pre-AOX was inhibited already at 1 microM and 3 microM concentration of the L and D enantiomers, whereas inhibition of import of pre-b2(1, 84)-DHFR, occurred at concentrations >10 microM of both enantiomers. Binding efficiency of the precursor proteins was not affected by addition of the L and D enantiomers. There was no correlation between inhibition of import of pre-F1beta and pre-AOX and dissipation of membrane potential measured as a decrease of Rhodamine 123 fluorescence quenching. The inhibitory effect of the L and D presequence enantiomers on import of pre-F1beta and pre-AOX was concluded to occur within the outer membrane translocase machinery beyond the initial precursor receptor interaction. Furthermore, the fact that the D enantiomer had the same effect as the natural peptide showed that interaction of the presequence with the import machinery was not dependent on chiral properties of the presequence.

Synthetic peptides as functional mimics of a viral discontinuous antigenic site.

Functional reproduction of discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate into a single molecule each of the three protein loops that define the antigenic site. The site D mimics are designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulphide-linked heterodimers that consist of a larger unit containing VP2(71-84), followed by a polyproline module and by VP3(52-62), and a smaller unit corresponding to VP1(188-194). Guinea pig antisera to the peptides recognize the viral particle and compete with site D-specific monoclonal antibodies, while inoculation with a simple (non-covalently bound) admixture of the three VP1-VP3 sequences yields no detectable virus-specific serum conversion. Similar results have been reproduced in two cattle. Antisera to the peptides are also moderately neutralizing of FMDV in cell culture and partially protective of guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent peptide-based vaccine against FMDV.

Conjugation of epitope peptides with SH group to branched chain polymeric polypeptides via Cys(Npys).

Since bioconjugates may play an important role as therapeutics in the future, the development of new and effective conjugation strategies is necessary. For the attachment of peptide-like molecules to carriers, there are two main coupling methods involving amide or disulfide bonds. Conjugation through an amide bond can be achieved in several well-defined ways known from peptide chemistry. However, the formation of disulfide bridges between cysteine-containing peptides and carrier molecules still has some problems. In this paper, we describe a novel approach in which the carrier polypeptide is modified by 3-nitro-2-pyridinesulfenyl (Npys)-protected cysteine and this derivative has been applied for conjugation of Cys-containing epitope peptides with poly(L-lysine)-based branched polypeptides. Considering the stability of Npys group in the presence of pentafluorophenol, Boc-Cys(Npys)-OPfp dervivative was selected for introduction to the N-terminal of branches of polypeptides backbone. The branches of the polymers were built up from oligo(DL-alanine) (poly[Lys(DL-Ala(m))], AK) and elongated by an optically active amino acid [poly[Lys(X(i)-DL-Ala(m))], XAK]. We found that the nature of X (Glu, Ser, Thr) has great influence on the incorporation of the protected cysteine residue. Herpes simplex virus and adenovirus epitope peptides were conjugated to Boc-Cys(Npys)-modified polypeptides. Results indicate that the incorporation of epitope peptides depends on the number of Npys group on the polymers as well as on the presence/absence of Boc-protecting group on the Cys residue. This new class of Cys(Npys)-derivatized branched polypeptides is stable for a couple of months and suitable for effective preparation of epitope peptide conjugates possessing increased water solubility.