Levels of Circulating IgM and IgY Natural Antibodies in Broiler Chicks: Association with Genotype and Farming Systems.

Naturally occurring antibodies (NAbs), which are major components of innate immunity, exist in circulation under healthy conditions without prior antigenic stimulation and are able to recognize both self- and non-self-constituents. The present study aimed at identifying potential immunological differences between commercial fast- and slow-growth broilers (n = 555) raised in conventional and free-range systems, respectively, through the use of the specificity, isotypes and levels of circulating NAbs. The possible beneficial effect of oregano-based dietary supplementation was also evaluated. To this end, serum IgM and IgY NAbs against self- (actin and DNA) and non-self- antigens (trinitrophenol and lipopolysaccharide) were measured by ELISA and further correlated with genotype, season and performance. Significantly higher levels of IgM NAbs against all antigens were found in slow-growth compared to fast-growth broilers. IgM NAb levels were also significantly increased in dietarily supplemented slow-growth broilers versus those consuming standard feed. Moreover, significantly elevated levels of anti-DNA IgY NAbs were found in fast-growth compared to slow-growth broilers, whereas the opposite was observed for anti-LPS IgY NAbs. Multivariate linear regression analysis confirmed multiple interactions between NAb levels, genotype, season and performance. Overall, serum NAbs have proven to be valuable innovative immunotools in the poultry industry, efficiently differentiating fast-growing versus slow-growing broilers, and dietary supplementation of plant extracts can enhance natural immunity.

Control of Tyrosyl Radical Stabilization by {SiO2@Oligopeptide} Hybrid Biomimetic Materials.

Tyrosine radicals are notoriously short-lived/unstable in solution, while they present an impressive degree of stability and versatility in bioenzymes. Herein, we have developed a library of hybrid biomimetic materials (HBMs), which consists of tyrosine-containing oligopeptides covalently grafted on SiO2 nanoparticles, and studied the formation, lifetime, and redox properties of tyrosyl radicals. Using electron paramagnetic resonance spectroscopy, we have studied the radical-spin distribution as a probe of the local microenvironment of the tyrosyl radicals in the HBMs. We find that the lifetime of the tyrosyl radical can be enhanced by up to 6 times, by adjusting three factors, namely, a proximal histidine, the length of the oligopeptide, and the interface with the SiO2 nanomatrix. This is shown to be correlated to a significant lowering of E1/2 from +736 mV, in free tyrosine, to +548 mV in the {12-peptide}@SiO2 material. Moreover, we show that grafting on SiO2 lowers the E1/2 of tyrosine radicals by ∼50 mV in all oligopeptides. Analysis of the spin-distribution by EPR reveals that the positioning of a histidine at a H-bonding distance from the tyrosine further favors tyrosine radical stabilization.

Development of novel GnRH and Tat48-60 based luminescent probes with enhanced cellular uptake and bioimaging profile.

There is a clear need to develop photostable chromophores for bioimaging with respect to the classically utilized green fluorescent dye fluorescein. Along these lines, we utilized a phosphorescent carboxy-substituted ruthenium(ii) polypyridyl [Ru(bipy)2(mcb)]2+ (bipy = 2,2'-bipyridyl and mcb = 4-carboxy-4'-methyl-2,2'-bipyridyl) complex. We developed two luminescent peptide conjugates of the cell-penetrating peptide Tat48-60 consisting of either [Ru(bipy)2(mcb)]2+ or 5(6)-carboxyfluorescein (5(6)-FAM) tethered on the Lys50 of the peptide through amide bond. We confirmed the efficient cellular uptake of both bioconjugates in HeLa cells by confocal microscopy and flow cytometry and proved that the ruthenium-based chromophore possesses enhanced photostability compared to a 5(6)-FAM-based peptide, after continuous laser scanning. Furthermore, we designed and developed a luminescent agent with high photostability, based on the ruthenium core, that could be selectively localized in cancer cells overexpressing the GnRH receptor (GnRH-R). To achieve this, we took advantage of the tumor-homing character of d-Lys6-GnRH which selectively recognizes the GnRH-R. The [Ru(bipy)2(mcb)]2+-d-Lys6-GnRH peptide conjugate was synthesized, and its cellular uptake was evaluated through flow cytometric analysis and live-cell imaging in HeLa and T24 bladder cancer cells as negative and positive controls of GnRH-R, respectively. Besides the selective targeting that the specific conjugate could offer, we also recorded high internalization levels in T24 bladder cancer cells. The ruthenium(ii) polypyridyl peptide-based conjugates we developed is an intriguing approach that offers targeted cell imaging in the Near Infrared region, and simultaneously paves the way for further advancements in the dynamic studies on cellular imaging.

Intracellular targets: A multiple cargo transporting molecule.

The generation of cell-penetrating peptides as cargo-delivery systems has produced an immense number of studies owing to the importance of these systems as tools to deliver molecules into the cells, as well as due to the interest to shed light into a yet unclear mechanism of the entrance of these peptides into the cells. However, many cell-penetrating peptides might present drawbacks due to causing cellular toxicity, or due to being entrapped in endosomes, or as a result of their degradation before they meet their target. In this work, a cargo transporting molecule, the Cell Penetrating Sequential Oligopeptide Carrier (CPSOC), formed by the repetitive -Lys-Aib-Cys- moiety, was tested for its ability to penetrate the cell membrane and transport the conjugated peptides into the cells. The cysteine residue anchors bioactive molecules through a stable thioether bond. The lysine supplies the positive charge to the construct, whereas the α-amino isobutyric acid is well known to induce helicoid conformation to the peptide backbone and protects from enzymatic degradation. The present study demonstrates that CPSOC penetrates the membrane transporting the conjugated cargo into the cell. When we tested CPSOC-conjugated peptides carrying critical domains of Cdc42, a small GTPase implicated in exocytosis, the internalized peptides were found to be functional because they inhibited exocytosis of von Willebrand factor from endothelial Weibel-Palade bodies a trafficking event depending on the Cdc42 protein. The data suggest that the carrier can deliver efficiently functional peptides into the cells, and thus, it can be used as a multiple-cargo transporting molecule.

Multidrug Resistance Protein 4 (MRP4/ABCC4): A Suspected Efflux Transporter for Human's Platelet Activation.

The main role of platelets is to contribute to hemostasis. However, under pathophysiological conditions, platelet activation may lead to thrombotic events of cardiovascular diseases. Thus, anti-thrombotic treatment is important in patients with cardiovascular disease. This review focuses on a platelet receptor, a transmembrane protein, the Multidrug Resistance Protein 4, MRP4, which contributes to platelet activation, by extruding endogenous molecules responsible for their activation and accumulation. The regulation of the intracellular concentration levels of these molecules by MRP4 turned to make the protein suspicious and at the same time an interesting regulatory factor of platelet normal function. Especially, the possible role of MRP4 in the excretion of xenobiotic and antiplatelet drugs such as aspirin is discussed, thus imparting platelet aspirin tolerance and correlating the protein with the ineffectiveness of aspirin antiplatelet therapy. Based on the above, this review finally underlines that the development of a highly selective and targeted strategy for platelet MRP4 inhibition will also lead to inhibition of platelet activation and accumulation.

Synergistic effect of peptide inhibitors derived from the extracellular and intracellular domain of αIIb subunit of integrin αIIbß3 on platelet activation and aggregation.

αIIbß3, the major platelet integrin, plays a central role in hemostasis and thrombosis. Upon platelet activation, conformation of αIIbß3 changes and allows fibrinogen binding and, subsequently, platelet aggregation. It was previously shown that a lipid-modified platelet permeable peptide, which corresponds to the intracellular acidic membrane distal sequence 1000LEEDDEEGE1008 of αIIb (pal-K-LEEDDEEGE or pal-K-1000-1008), inhibits thrombin-induced human platelet aggregation, by inhibiting talin association with the integrin. YMESRADR, a peptide corresponding to the extracellular sequence 313-320 of αIIb, is also a potent platelet aggregation inhibitor by mimicking the effect of a clasp between the head domains of αIIb and ß3. The aim of the present study was to investigate the synergistic effect of the intra- and extracellular- peptide inhibitors on platelet aggregation, as well as on the phosphorylation of two signaling proteins, focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Platelet preincubation with Pal-K-LEEDDEGE followed by YMESRADR showed a synergistic inhibitory activity on platelet aggregation. Platelet incubation with threshold inhibitory concentrations of both peptides provoked almost the total inhibition of aggregation, PAC-1 binding, and fibrinogen binding, but not P-selectin exposure on activated platelets' surface. Like RGDS peptide, this mixture inhibits FAK phosphorylation whose phosphorylation is well known to be consecutive to the aggregation (postoccupancy events). However, in contrast to RGDS peptide that enhances ERK phosphorylation and activation, the mixture of threshold inhibitory concentrations of Pal-K-LEEDDEEGE and YMESRADR inhibits ERK phosphorylation. We suggest that the use of the intracellular in combination with the extracellular peptide inhibitor, acting with a non-RGD-like mechanism, may provide an alternative way to antagonize integrin αIIbß3 activation.

Inhibition of αIIbß3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the ßI Domain of ß3.

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbß3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, p1YMESRADR8, corresponding to amino acids 313-320 of the ß-ribbon extending from the ß-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbß3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the ß-ribbon by forming a clasp restraining the ß3 hybrid and ßI domains in a closed conformation. The involvement of species-specific residues of the ß3 hybrid domain (E356 and K384) and the ß1 domain (E297) as well as an intrapeptide bond (pE315-pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbß3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb ß-ribbon in preventing integrin αIIbß3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.

A rapid and efficient method for the synthesis of selectively S-Trt or S-Mmt protected Cys-containing peptides.

Selective removal of protecting groups under different cleavage mechanisms could be an asset in peptide synthesis, since it provides the feasibility to incorporate different functional groups in similar reactive centres. However, selective protection/deprotection of orthogonal protecting groups in peptides is still challenging, especially for Cys-containing peptides, where protection of the cysteine side-chain is mandatory since the nucleophilic thiol can be otherwise alkylated, acylated or oxidized. Herein, we established a protocol for the synthesis of Cys-selective S-Trt or S-Mmt protected Cys-containing peptides, in a rapid way. This was achieved by, simply fine-tuning the carbocation scavenger in the final acidolytic release of the peptide from the solid support in the classic SPPS.

Palmitoylated peptide, being derived from the carboxyl-terminal sequence of the integrin αIIb cytoplasmic domain, inhibits talin binding to αIIbß3.

The αIIb cytoplasmic domain of platelet integrin αIIbß3 contains an unorganized acidic membrane-distal (1000)LEEDDEEGE(1008) region. We have shown that a platelet permeable peptide corresponding to the above region the palmitoyl-K-LEEDDEEGE (pal-K-1000-1008) inhibits platelet aggregation induced by thrombin or by pal-K-989-995, a palmitoylated peptide corresponding to the membrane-proximal αIIb cytoplasmic domain (989)KVGFFKR(995). We now tested the anti-aggregatory activity of (i) a lipid-modified scrambled acidic peptide (pal-K-GDDEELEEE), (ii) two smaller peptides derived from the acidic amino sequence: palmitoyl-K-(1000)LEEDDE(1005) (pal-K-1000-1005) and palmitoyl-K-(1005)EEGE(1008) (pal-K-1005-1008) and (iii) lipid-modified palmitoyl-acidic peptides with alanine (Ala) substitution at residues 1001, 1003, 1004 and 1005 and one peptide with a double Ala substitution at residues 1001 and 1004 of the 1000-1008 sequence. All the peptides tested showed an inhibitory activity, however, the palmitoylated peptide with the natural and the whole acidic sequence, being the most active. Our results suggest that the whole acidic sequence, rather than some specific amino acids, contributes to the aggregation inhibitory activity. The inhibitory peptide, pal-K-1000-1008, inhibited the association of talin with αIIbß3 in thrombin-activated platelets, as demonstrated by co-immunoprecipitation experiments, while the scrambled peptide was inefficient. We suggest that, by interacting with αIIb cytoplasmic domain, pal-K-1000-1008 has an anti-aggregatory inhibitory activity due to a specific inhibition of talin binding to αIIbß3.

Side reactions in the SPPS of Cys-containing peptides.

Alkylation of sensitive amino acids during synthesis of biologically important peptides is a common and well-documented problem in Fmoc-based strategy. Herein, we probed for the first time an unexpected S-alkylation of Cys-containing peptides that occur during the final TFA cleavage of peptides from the Wang solid support. Through a battery of approaches (NMR, UV and LC-MS) the formed by-product was assigned as the alkylation of the cysteine sulfydryl group by the p-hydroxyl benzyl group derived from the acidic Wang linker decomposition. Factors affecting this side reaction were monitored and a protocol that minimizes the presence of the by-product is reported.

A highly constrained cyclic (S,S)-CDC- peptide is a potent inhibitor of carotid artery thrombosis in rabbits.

Inhibition of platelet aggregation is indispensable for the treatment of acute arterial thrombotic episodes. We have previously reported the synthesis of a highly constrained cyclic peptide, that incorporates the -CDC- sequence, (S,S) PSRCDCR-NH(2), which potently inhibits aggregation and fibrinogen binding to human platelets in vitro. We have tested the safety and efficacy of the peptide on the electrically induced carotid artery thrombosis experimental rabbit model. The peptide's effects on carotid blood flow, thrombus weight, in vitro and ex vivo platelet aggregation, and bleeding and hemostatic parameters were evaluated. The peptide was administered via the femoral vein. Carotid blood flow was continuously monitored for 90 min after electrical thrombus formation. The peptide, at 12 mg/kg, prevented total artery occlusion and significantly preserved carotid artery's patency compared with placebo and eptifibatide. Furthermore, (S,S) PSRCDCR-NH(2) administration at 12 mg/kg reduced thrombus weight, whereas it inhibited ex vivo ADP, arachidonic acid (AA) and collagen-induced platelet aggregation. Moreover (S,S) PSRCDCR-NH(2) at 12 mg/kg presented significantly higher inhibitory effects on AA and collagen-induced ex vivo platelet aggregation compared to eptifibatide. The peptide at any dose did not affect the coagulation cascade, the bleeding times or the hemostatic response of the animals. Thus highly constrained cyclic peptides like (S,S) PSRCDCR-NH(2) that incorporate the -CDC- motif and fulfil certain conformational criteria represent novel compounds that potently inhibit thrombus formation, ex vivo platelet aggregation and carotid artery occlusion superiorly to other non-RGD peptides, such as YMESRADR, without causing hemorrhagic complications in a rabbit model of arterial thrombosis.

Inhibition of platelet activation by peptide analogs of the beta(3)-intracellular domain of platelet integrin alpha(IIb)beta(3) conjugated to the cell-penetrating peptide Tat(48-60).

Activation of the platelet integrin-receptor alpha(IIb)beta(3) is the final pathway of platelet aggregation, regardless of the initiating stimulus. Many studies suggest that there are several cytoplasmic proteins such as talin and beta(3)-endonexin that bind to N(744)PLY(747) and N(756)ITY(759) motif of the beta(3) cytoplasmic tail and play the major role in the receptor activation. In this study, we investigated the role of the membrane distal region of human beta(3) cytoplasmic tail and specifically the N(743)NPLYKEA(750) and T(755)NITYRGT(762) sequence that contains an NXXY motif, in platelet aggregation, secretion, alpha(IIb)beta(3) activation (PAC-1 binding) and fibrinogen binding. We synthesized two peptides corresponding to the above sequences as well as their conjugates with the Tat(48-60) cell-penetrating peptide. The capability of conjugates to penetrate the platelet membrane was investigated with confocal laser scanning microscopy using carboxyfluorescein (CF)-labeled peptides. Our results showed that the conjugated with the Tat(48-60) sequence peptides penetrate the platelet membrane and inhibit platelet aggregation in both PRP and washed platelets in a dose-dependent manner. The Tat-beta(3)743-750 conjugate exhibited similar inhibitory activity in PRP and in washed platelets whereas the Tat-beta(3)755-762 conjugate was more potent inhibitor of aggregation in washed platelets than in PRP. Both conjugated peptides were also able to inhibit P-selectin membrane expression as well as PAC-1 and fibrinogen binding to the platelets, the Tat-beta(3)755-762 conjugate being more potent than Tat-beta(3)743-750. The Tat(48-60) peptide and the peptides beta(3)743-750 and beta(3)755-762, which were not conjugated to the Tat(48-60) sequence, did not exhibit any inhibitory effect on the above parameters. In conclusion, the present study shows for the first time that the peptide analogs of the intracellular domain of the beta(3) subunit beta(3)743-750 and beta(3)755-762 conjugated to the cell-penetrating peptide Tat(48-60) are capable of penetrating the platelet membrane and expressing biological activity by inhibiting the activation of alpha(IIb)beta(3), the fibrinogen binding to the activated receptor as well as platelet aggregation. Further studies are necessary to support whether such conjugated peptides may be useful tools for the development of potent antiplatelet agents acting intracellularly through the platelet integrin alpha(IIb)beta(3).

Effect of a synthetic peptide corresponding to residues 313 to 320 of the alphaIIb subunit of the human platelet integrin alphaIIbbeta3 on carotid artery thrombosis in rabbits.

The platelet integrin receptor alpha(IIb)beta(3) plays a critical role in thrombosis. We have shown previously that the octapeptide YMESRADR, corresponding to sequences 313 to 320 of the human alpha(IIb) subunit, inhibits human platelet activation and fibrinogen binding to alpha(IIb)beta(3), possibly interacting with the ligand. We investigated the effect of YMESRADR on electrically induced carotid artery thrombosis in New Zealand white rabbits. Peptide was administered via the femoral vein, starting 60 min before and continuing for 90 min after the electrical stimulation. Carotid blood flow was monitored for 90 min after the electrical stimulation. The peptide effects on platelet aggregation, in vitro and ex vivo, and on various coagulation, bleeding, and hemostatic parameters were evaluated. YMESRADR significantly inhibited rabbit platelet aggregation in vitro in a dose-dependent manner. It is important that peptide administration in vivo, at doses ranging from 3 to 15 mg/kg, prolonged the duration of the patency of the carotid artery, and no artery occlusion was observed until the end of the study (90 min after electrical stimulation). Furthermore, YMESRADR administration reduced platelet aggregation ex vivo and thrombus weight; however, these reductions reached statistical significance, compared with the control group, at the peptide doses of 12 and 15 mg/kg. YMESRADR did not affect any coagulation parameter studied and the hemostatic response observed in control animals. Thus, YMESRADR represents a novel antiplatelet agent that can inhibit thrombus formation effectively and carotid artery occlusion without causing hemorrhagic complications in a rabbit model of arterial thrombosis.

A palmitoylated peptide, derived from the acidic carboxyl-terminal segment of the integrin alphaIIb cytoplasmic domain, inhibits platelet activation.

Platelet integrin alpha(IIb)beta(3) contains an acidic membrane distal motif, 1000LEEDDEEGE1008, in the cytoplasmic domain of the alpha(IIb) subunit. We showed that a lipid-modified peptide corresponding to the above region, palmitoyl-K-LEEDDEEGE (pal-K-1000-1008), is platelet permeable and has inhibited platelet aggregation induced by 0.4 U/ml of thrombin (IC50 = 164 microM). Moreover the peptide inhibited both Fibrinogen and PAC-1, binding to activated platelets. The non palmitoylated analog was inactive. A modified, scrambled acidic peptide (palmitoyl-K-GDDEELEEE), showed significant lower inhibitory activity than pal-K-1000-1008. A palmitoylated peptide corresponding to the membrane proximal cytoplasmic domain of alpha(IIb), 989KGVFFKR995 (pal-989-995), is known to specifically induce platelet aggregation. Pal-K-1000-1008 was an inhibitor of human washed platelet aggregation induced by pal-K-989-995 (IC50 = 15 microM). Moreover, pal-K-1000-1008 inhibited phosphorylation of ERK and FAK, two protein kinases involved in platelet activation and aggregation. Our results favour the assumption that the interaction of the membrane proximal sequence 989KGVFFKR995 of the cytoplasmic domain of alpha(IIb) with the acidic terminal 1000LEEDDEEGE1008 motif may be an important structural factor in platelet signaling, leading to platelet activation and aggregation.

Computational studies on the backbone-dependent side-chain orientation induced by the (S,S)-CXC motif.

Disulfide cyclization is a well-known procedure to impose conformational restriction to peptides undergoing backbone flexibility. Rigid conformations are induced only for small rings with a specific combination of amino acids. In this work, we present a computational search of the backbone and backbone-dependent side-chain orientation of two series of linear and cyclic peptide analogs. The -C[XY]C- scaffold (where X,Y is arginine, aspartic acid or alanine residue) in its open and (S,S) cyclic form was used for the design of the studied analogs. Thirty-six compounds, resulting from the extension with one residue at either the N- or the C-terminus were studied with classical MD. The local backbone conformation and the relative orientation of the X and Y side chains induced by either cyclization and/or the presence of the charged residues are discussed. From the present study it is concluded that cyclization has a great impact on the synplanar orientation of the X and Y side chains in the (S,S)Ac-XCYC-NH2 series of compounds while charge-charge interaction has only a weak synergic effect. On the contrary, the antiplanar orientation is favored in the case of (S,S)Ac-CXCY-NH2.

Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis.

Sheep fasciolosis is a devastating burden for the livestock industry. We herein report on immunodiagnosis of fasciolosis, and significant protection of sheep against challenge infection with Fasciola gigantica following immunization with a peptide based on the H-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH (Fas14p) sequence of F. gigantica cathepsin L-cysteine proteinase. This sequence was synthesized in three different forms: as N(alpha) acetylated (Ac-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAc14p), bearing at the amino-terminus an N(alpha) acetylated cystein (Ac-Cys-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAcCys14p), and conjugated to sequential oligopeptide carrier Ac-[Lys-Aib-Gly](4)-OH (Ac-SOC(4)) through an amide bond formed between Val(123) carboxylic group of the epitope and the lysine N(epsilon) groups of the carrier (Ac-[Lys(Fas14p)-Aib-Gly](4)-OH). Ac-[Lys(Fas14p)-Aib-Gly](4)-OH failed to readily discriminate between naïve and infected sheep. In contrast, the free peptides reproducibly differentiated between parasite-free sheep, sheep infected with parasites other than Fasciola, and experimentally Fasciola-infected sheep. The data together indicated that the peptides might be of considerable use for discriminating between early and late, and low and high burden, sheep infection with F. gigantica. FasAc14p was chosen to determine whether a peptide based on a critical enzymatic site of cathepsin L proteinase may induce protection against challenge infection. Sheep immunization with FasAc14p peptide induced significant expression of interleukin-4 mRNA, and humoral antibodies that bound to molecule(s) on the intact surface membrane of newly excysted juvenile worms, and mediated their attrition. The immune responses were associated with significant (P < 0.02) decrease of 23.1% in worm recovery, but with no decrease in the size or maturation of worms recovered.

Synthesis and antitumor activity of peptide-paclitaxel conjugates.

Paclitaxel (Pac) is the most important anticancer drug used mainly in treatment of breast, lung, and ovarian cancer and is being investigated for use as a single agent for treatment of lung cancer, advanced head and neck cancers, and adenocarcinomas of the upper gastrointestinal tract. In this work, we present the synthesis of five 2'-paclitaxel-substituted analogs in which paclitaxel was covalently bound to peptides or as multiple copies to synthetic carriers. Ac-Cys(CH(2)CO-2'-Pac)-Arg-Gly-Asp-Arg-NH(2), Folyl-Cys(CH(2)CO-2'-Pac)-Arg-Gly-Asp-Ser-NH(2), Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](2)-NH(2), Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](3)-NH(2) and Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](4)-NH(2) were synthesized using 2'-halogeno-acetylated paclitaxel derivatives. Paclitaxel conjugates showed greater solubility in water than paclitaxel and inhibited the proliferation of human breast, prostate, and cervical cancer cell lines. Although all synthesized compounds had an antiproliferative activity, the Ac-[Lys-Aib-Cys(CH(2)CO-2'-Pac)](4)-NH(2) derivative showed improved biological activity in comparison with paclitaxel in cervical and prostate human cancer cells.

Assessment of the interaction between a synthetic epitope of troponin C and its specific antibody using a label-free faradaic impedimetric immunosensor and alpha-Keggin silicotungstic heteropolyacid as a redox probe.

The development of an immunosensor for the direct probing of the interaction between a cysteine-modified synthetic peptide, which corresponds to the epitope cTnC-89-98 of troponin C, and its specific antibody is described. Following immobilization of the peptide onto gold electrodes through the formation of a self-assembled monolayer, the alteration of the interfacial properties of the electrodes upon peptide-antibody interaction was traced by faradaic electrochemical impedance spectroscopy (EIS) using a silicotungstic heteropolyacid, H(4)SiO(4).12WO(3), as a redox probe. The electrochemical behaviour of the redox probe was evaluated with cyclic voltammetry and EIS. The effect of milk protein or 4-mercaptophenol, which was used as post-blocking agents, on the performance of the immunosensor, was investigated. Treatment with 4-mercaptophenol resulted in immunoeffective electrodes that successfully tested in anti-serum samples. An optimum dilution ratio of the samples, where the effect of the matrix on the measuring signal is negligible, was also determined.

Investigation of the role of adjacent amino acids to the 313-320 sequence of the alphaIIb subunit on platelet activation and fibrinogen binding to alphaIIbbeta3.

The platelet integrin receptor alphaIIbbeta3 plays a critical role in thrombosis and haemostasis by mediating interactions between platelets and several ligands, primarily fibrinogen. We have previously shown that the synthetic peptide YMESRADRKLAEVGRVYLFL corresponding to residues 313-332 of alphaIIb, is a potent inhibitor of platelet aggregation and fibrinogen binding to alphaIIbbeta3, interacting with fibrinogen rather than the receptor. Furthermore, we have demonstrated that the biological activities of the above peptide are due to the sequence YMESRADR, which corresponds to residues 313-320. By using new synthetic peptide analogues we investigated the structural characteristics responsible for the biological activity of YMESRADR as well the possible influence of the adjacent amino acids on the peptide's biological potency. According to our results, the synthetic octapeptide YMESRADR, is a potent inhibitor of platelet aggregation and P-selectin expression. Furthermore, YMESRADR inhibits fibrinogen binding but it does not significantly influence the binding of PAC-1 to ADP-activated platelets. The inhibitory potency of YMESRADR was gradually diminished by deleting the YMES sequence from the amino terminus and prolonging the carboxyl terminus of this peptide with the KLAE sequence. Extension of YMESRADR towards the amino terminus with the GAPL sequence (GAPLYMESRADR) does not modify the biological activity of YMESRADR. Furthermore, extension of GAPLYMESRADR at its carboxy terminus with the KLAE sequence (GAPLYMESRADRKLAE) significantly diminished its biological potency. Substitution of E315 with D significantly enhances antiaggregatory potency and completely abolishes the inhibitory effect on P-selectin expression. Importantly, the D315-containing peptides inhibit to a similar extent both fibrinogen and PAC-1 binding to activated alphaIIbbeta3 in contrast to the E315-containing peptide which only inhibits fibrinogen binding. In conclusion, the present study suggests that the YMESRADR sequence 313-320 of alphaIIb, is an important functional region of the insert connecting the beta2 and beta3 antiparallel beta-strands of the W5 blade of the alphaIIb subunit. Structural changes significantly modify the biological properties of this region.

Influence of sequential oligopeptide carriers on the bioactive structure of conjugated epitopes: comparative study of the conformation of a Herpes simplex virus glycoprotein gD-1 epitope in the free and conjugated form, and protein "built-in" crystal structure.

Synthetic carriers play an important role in immunogen presentation, due to their ability of inducing improved and specific responses to conjugated epitopes. Their influence on the bioactive conformation of the epitope, though admittedly crucial for relevant in vitro and in vivo applications, is difficult to evaluate, given the usual lack of information on the complex conformational features determined by the nature of the carrier and the mode of ligation. Using the Herpes simplex virus glycoprotein D-1 epitope (Leu(9)-Lys-Nle-Ala-Asp-Pro-Asn-Arg-Phe-Arg-Gly-Lys-Asp-Leu(22)) as a model, we have performed a detailed conformational analysis on the free epitope peptide in solution and on three constructs in which the epitope was conjugated to sequential oligopeptide carriers {Ac-[Lys-Aib-Gly](4)-OH (SOC(4))} (through either a thioether or an amide bond; Ac: acetyl) and polytuftsin oligomers {H-[Thr-Lys-Pro-Lys-Gly](4)-NH(2) (T20)}, (through a thioether bond). The analysis of the epitope conformation in the parent protein, in carrier-conjugated and free form, suggests that the beta-turn structure of the -Asp(13)-Pro-Asn-Arg(16)- segment is highly conserved and independent of the epitope form. However, small conformational variations were observed at the C-terminal part of the epitope, depending on the nature of the carrier.