[Show abstract][Hide abstract] ABSTRACT: Abstract The αIIb cytoplasmic domain of platelet integrin αIIbβ3 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 αIIbβ3 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 αIIbβ3.
[Show abstract][Hide abstract] ABSTRACT: Peptide mimics of epitopes for pathogen-specific antibodies present in patient sera can be selected based on the phage display technology. Such mimotopes potentially represent vaccine candidates in case they are able to induce neutralizing antibodies upon vaccination. Here we analyze the immunogenicity of different conjugates of epitope EC26-2A4 localizing to the membrane proximal external region (MPER) of the HIV-1 transmembrane protein gp41. The EC26-2A4 epitope, which overlaps with that of the broadly neutralizing monoclonal antibody (mAb) 2F5, was coupled to sequential oligopeptide carriers (SOC) or to palmitoyl acid for better immunogenicity. Upon prime-boost immunizations of mice, the peptide conjugates induced EC26-2A4 specific antibodies in all settings and mice sera neutralized HIV-1SF162.LS in standardized neutralization assays. Thus, the EC26-2A4 MPER epitope represents a promising vaccine candidate for further analysis in larger animals with respect to the breadth of the neutralizing antibodies induced.
[Show abstract][Hide abstract] ABSTRACT: The terminal parts of the influenza hemagglutinin (HA) receptors α2,6- and α2,3-sialyllactoses were conjugated to an artificial carrier, named sequential oligopeptide carrier (SOC(4) ), to formulate human and avian receptor mimics, respectively. SOC(4) , formed by the tripeptide unit Lys-Aib-Gly, adopts a rigid helicoids-type conformation, which enables the conjugation of biomolecules to the Lys-N(ε) H(2) groups. By doing so, it preserves their initial conformations and functionalities of the epitopes. We report that SOC(4) -glyco-conjugate bearing two copies of the α2,6-sialyllactose is specifically recognized by the biotinylated Sambucus nigra (elderberry) bark lectin, which binds preferentially to sialic acid in an α2,6-linkage. SOC(4) -glyco-conjugate bearing two copies of the α2,3-sialyllactose was not recognized by the biotinylated Maackia amurensis lectin, despite its well-known α2,3-sialyl bond specificity. However, preliminary immune blot assays showed that H1N1 virus binds to both the SOC(4) -glyco-conjugates immobilized onto nitrocellulose membrane. It is concluded that Ac-SOC(4) [(Ac)(2) ,(3'SL-Aoa)(2) ]-NH(2) 5 and Ac-SOC(4) [(Ac)(2) ,(6'SL-Aoa)(2) ]-NH(2) 6 mimic the HA receptors. These findings could be useful for easy screening of binding and inhibition assays of virus-receptor interactions.
Journal of Peptide Science 11/2011; 18(1):52-8. · 2.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apolipoprotein A-I (apoA-I), which constitutes the principal protein component of high-density lipoprotein, is responsible for its major antiatherogenic functions. Aiming at contributing to the development of potent inhibitors of low-density lipoprotein (LDL) peptide models of helices 4,6 and 9,10 of apoA-I were designed and synthesized. Specific amino acid substitutions, resulting in transformation of the original helix class A and Y to G according to the Schiffer and Edmundson helical wheel representation, were introduced in order to validate the contribution of these modifications in the inhibitory activity of the synthesized peptide models against the LDL oxidation. The role of Met at positions 112 (helix 4) and 148 (helix 6) as oxidant scavenger was also investigated. The helical characteristics of all the peptide models were studied by CD in membrane-mimicking microenvironments and compared with the original helices.
Journal of Peptide Science 09/2011; 17(11):720-5. · 2.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The influenza virus, major surface glycoprotein hemagglutinin (HA) is one of the principal targets for the development of protective immunity. Aiming at contributing to the development of a vaccine that remains the first choice for prophylactic intervention, a reconstituted model of HA, mimicking its antigenic properties was designed, synthesized and tested in mice for the induction of protective immunity. Four helper T lymphocyte [HTL (T(1) , T(3) , T(7) and T(8) )] and four cytotoxic lymphocyte [CTL (T(2) , T(4) , T(5) and T(6) )] epitopes were coupled in two copies each to an artificial carrier, SOC(4) , which was formed by the repeating tripeptide Lys-Aib-Gly. The helical conformation of the SOC(4) -conjugates preserves the initial topology of the attached epitopes, which is critical for their immunogenic properties. Survival of immunized animals, ranged from 30 to 50%, points out the induction of protective immunity by using the SOC(4) -conjugates.
Journal of Peptide Science 03/2011; 17(3):226-32. · 2.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is convincing evidence from numerous clinical and epidemiological studies that physical activity can reduce the risk for breast and prostate cancer. The biological mechanisms underlying this phenomenon remain elusive. Herein we suggest a role for naturally produced antibodies reactive with the vasoactive intestinal peptide (VIP) in the suppression of breast and prostate cancer, which we believe could offer a possible molecular mechanism underlying control of these cancers by physical exercise.
We found that sera from individuals having breast and prostate cancers have decreased titers of VIP natural antibodies as demonstrated by a lower reactivity against peptide NTM1, having similar informational and structural properties as VIP. In contrast, sera collected from elite athletes, exhibited titers of natural NTM1-reactive antibodies that are significantly increased, suggesting that physical activity boosts production of these antibodies.
Presented results suggest that physical exercise stimulates production of natural anti-VIP antibodies and likely results in suppression of VIP. This, in turn, may play a protective role against breast and prostate cancers. Physical exercise should be further investigated as a potential tool in the treatment of these diseases.
PLoS ONE 01/2011; 6(11):e28304. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ability of N-acetyl-cysteine and glutathione to inhibit the oxidation of corn oil stripped of tocopherols and refined olive oil was examined. The oxidative stability of corn oil stripped of tocopherols at 50 o C, 120 o C and 180 o C was evaluated. The absorbances at 234 nm and 270 nm, and p-anisidine value were monitored. N-acetyl-cysteine and glutathione inhibited the oxidation of oil, N-acetyl-cysteine at a higher degree. In comparison to BHA at 200 mg/L, both thiols at 20-40 mg/L exhibited lower (50 o C and 120 o C) or similar (180 o C) antioxidant activities. Compounds similar to the two thiols but not containing –SH group, N-acetyl-serine and oxidized glutathione, exhibited very low or no antioxidant activities. The oxidative stability of refined olive oil at 20 o C was determined by monitoring the peroxide value. N-acetyl-cysteine and glutathione at 20 mg/L exhibited antioxidant activities comparable to that of BHA at 200 mg/L. Present results show the ability of N-acetyl-cysteine and glutathione to inhibit the oxidation of corn oil stripped of toco-pherols and refined olive oil indicating that thiols can inhibit the oxidation of any oil.
The Open Food Science Journal 01/2010; 3(1):103-107.
[Show abstract][Hide abstract] ABSTRACT: Activation of the platelet integrin-receptor αIIbβ3 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 β3-endonexin that bind to N744PLY747 and N756ITY759 motif of the β3 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 β3 cytoplasmic tail and specifically the N743NPLYKEA750 and T755NITYRGT762 sequence that contains an NXXY motif, in platelet aggregation, secretion, αIIbβ3 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-β3743–750 conjugate exhibited similar inhibitory activity in PRP and in washed platelets whereas the Tat-β3755–762 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-β3755–762 conjugate being more potent than Tat-β3743–750. The Tat(48–60) peptide and the peptides β3743–750 and β3755–762, 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 β3 subunit β3743–750 and β3755–762 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 αIIbβ3, 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 αIIbβ3.
[Show abstract][Hide abstract] ABSTRACT: 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).
[Show abstract][Hide abstract] ABSTRACT: Despite the effectiveness of HAART in controlling HIV-1 replication, the emergence of drug-resistant viruses in infected patients and the severe side effects caused by the currently used drug regimens and the lack of an effective vaccine necessitate the continued search for new therapeutic strategies for prevention and therapy of HIV disease. Previously we reported that natural autoantibodies, recognizing peptide FTDNAKTI (peptide NTM1) derived from the C2 domain of HIV-1 gp120, contribute to the control of HIV disease. Here we demonstrated that sera from well-trained athletic (HIV-negative) subjects showed high reactivity with peptide NTM1. This result confirms that aerobic exercise training stimulates production of natural autoantibodies, which recognize peptide NTM1. Bioinformatics analysis indicates that these natural autoantibodies could slow down disease progression by blocking the superantigenic site on HIV-1 gp120. The results suggest that aerobic exercise training may be a promising non-toxic and inexpensive adjunctive anti-HIV therapy.
Scandinavian Journal of Medicine and Science in Sports 07/2009; 20(3):469-74. · 3.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Modulation of protein-protein interactions involved in the immune system by using small molecular mimics of the contact interfaces may lead to the blockage of the autoimmune response and the development of drugs for immunotherapy. The nonpolymorphic beta-regions, exposed to the microenvironment, of the modeled HLA-DQ7, which is genetically linked to autoimmune diseases, were determined. Peptides 132-141 and 58-67, located at the beta(1) and beta(2) domains of HLA-DQ7, respectively, were tested for their involvement in the interactions with CD4(+) T lymphocytes. Linear, cyclic, and dimeric analogs that mimic the exposed surfaces of HLA-DQ7 were designed and synthesized. Their immunosuppressory activities, found in the secondary, humoral immune response to sheep erythrocytes (SRBC) in mice in vitro, ranged from 11% to 53%. The significance of the total charge of the peptides, the pattern of the hydrogen bonding, and the presence of secondary structure were investigated in relation to the immunomodulatory effect of the peptides. Two dimeric analogs of the HLA-DQ7 58-67 fragment, consisting of the two monomers covalently linked by a polyethylene glycol (PEG) spacer, able to mimic the superdimers, were also synthesized and studied. As the 58-67 segment is located at the beta(1) region of HLA-DQ7, close to the major histocompatibility complex (MHC) groove, one may assume that the 58-67 peptide could accommodate the association between T-cell receptor (TCR) and human leukocyte antigen (HLA) by activating a co-stimulatory molecule of the TCR/HLA interaction. This hypothesis is supported by the confocal laser image of the fluorescein-labeled 58-67 peptide and by the fact that it is an immunostimulator at low concentration.
Journal of Peptide Science 03/2009; 15(4):296-304. · 2.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ability of glutathione, oxidized glutathione, N-acetyl-cysteine, and N-acetyl-serine to protect wine aroma volatiles was examined. Muscat-white and Xinomavro-red wine were stored in open bottles at 20 o C and aroma volatiles were determined using solid phase microextraction along with GC-MS. Glutathione and N-acetyl-cysteine inhibited the decrease of several ace-tate esters, ethyl esters and terpenols, while oxidized glutathione and N-acetyl-serine did not. Present results show that the free –SH is responsible for the ability of glutathione and N-acetyl-cysteine to protect white and red wine aroma volatiles. Consequently, the protective action of any thiol in any wine may be taken into account.
[Show abstract][Hide abstract] ABSTRACT: A series of (L-Arg-X-Gly)n sequential polypeptides, where X represents the amino acid residues L-Ala, Val, Leu, Ile, Nva, and Nle, have been prepared and their conformational properties as well as their association with nucleic acids in various water fluoroalcohol mixtures have already been reported. In order to gain further insight into the conformational behavior of these polypeptides in amphiphilic environments, we now report their secondary structure, in the presence of increasing amounts of sodium dodecylsulfate and dodecylphosphorylcholine using CD spectroscopy. The CD spectral pattern of all the polypeptides studied is indicative of an equilibrium between α-helix, β-turn, and random coil. They are analyzed in relation to the nature of the X side-chain (length, branching, linearity). The results are explained on the basis of Lundahl's model for polypeptide-micelle interactions.
International journal of peptide and protein research 01/2009; 33(3):195-201.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Complementary peptide epitopes, derived from complementary RNA sequences, have been used for suppressing the autoimmune response in experimental autoimmune diseases as myasthenia gravis, allergic neuritis and allergic encephalomyelitis. Aiming at contributing to the development of a tool that could regulate the autoantibody production against La/SSB, which is the main target of autoantibodies in Sjogren's syndrome (SS) and systemic lupus erythematosus (SLE), the complementary epitope, cpep349-364, of the minor T/major B cell epitope of La/SSB, pep349-364, was utilized for the induction of neutralizing anti-cpep349-364 antibodies in rabbit immunizations. Complementary peptides were coupled to an artificial carrier, developed in our laboratory, in order to enhance the complementary potency of cpep349-364 and its counterpart. This carrier, named Sequential Oligopeptide Carrier, SOC(n), formed by the repeating tripeptide Lys-Aib-Gly, adopts helical conformation, which allows the anchored peptide epitopes to preserve their initial reactivity such as molecular recognition, antigenicity/immunogenicity. Our study provides proof of evidence of specific interactions between idiotypic (Id)/anti-idiotypic (anti-Id) antibodies generated in immunized animals by the sense epitope (conjugate I) of La/SSB and its complementary counterpart (conjugate II). It was also demonstrated that the Id/anti-Id association is specifically disrupted by adding either the sense epitope (conjugate I) or its complementary counterpart (conjugate II). A mutual neutralization of Id/anti-Id antibodies was observed in vivo, which implies that generation of anti-Id antibodies by immunization with the complementary La/SSB epitope could scavenge the anti-La/SSB response.
Journal of Peptide Science 07/2008; 14(9):1069-76. · 2.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The C-terminus of the second conserved region of HIV-1 gp120 represents a functionally important domain, as it encompasses amino acids directly involved in the binding to the CD4 receptor and in post-receptor binding events. Previous studies have suggested that antibodies with specific affinity to a 23 amino acids-long NTM polypeptide, derived from this HIV-1 gp120 domain, may be involved in the control of HIV disease progression. In the current work, we searched for NTM-recognizing antibodies in specific cohorts of HIV-1 infected individuals, including long-term nonprogressors (LTNP) and progressors. For this purpose, we employed a previously defined bioinformatics criterion for design of an NTM peptide mimetic to select an octapeptide, NTMs (FTDNAKTI), which is more suitable for use in a solid-state enzyme-linked immunosorbent assay (ELISA). Our results show that NTMs-reactive antibodies are significantly more prevalent (p < 0.01) in LTNP as compared to progressors and healthy control subjects, indicating their association with non-progressive infection. The presence of antibodies recognizing the second conserved region of the HIV-1 gp120 derived peptide, NTMs, in LTNP sera suggest that these antibodies could be of considerable interest for development of anti-HIV immune-based therapies and vaccines.
Current HIV research 10/2007; 5(5):443-8. · 1.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The main guideline in designing effective immunogens as vaccine candidates capable of eliciting potent and specific immune responses is to combine B/T cell epitopes and adjuvants as immunostimulators on the same carrier that links the major histocompatibility complex with T cell receptors. Aiming at contributing to the development of carriers for human usage a helicoid type sequential oligopeptide carrier, SOC(n)-II, formed by the repeating tetrapeptide unit (Aib-Lys-Aib-Gly)(n), n=2-7, elongated from the amino-terminus by the palmitoyl group, known for its adjuvanticity, is now presented. The main B cell epitope, PPGMRPP, of the Sm autoantigen against which the majority of antibodies in patients with systemic lupus erythematosus is directed, was coupled to the Lys-N(epsilon)H(2) groups of the carrier in four copies and the resulting conjugate Palm-SOC(4)-II-Sm(4) was subjected to animal immunizations without utilizing any adjuvant. The induced immune response was comparable with that produced when Ac-SOC(4)-II-Sm(4) was administered in animals following the conventional immunization protocol of complete/incomplete Freund's adjuvant. High titers of anti-Palm-SOC(4)-II-Sm(4) antibodies were generated, which recognize the priming immunogenic conjugate, as well as reconstituted Sm mimics but not the carrier alone. It is concluded that Palm-SOC(n)-II carrier is a valuable tool for engineering immunogens eliciting enhanced and specific humoral immune responses.
[Show abstract][Hide abstract] ABSTRACT: Development of antimicrobial peptides has attracted considerable attention in recent years due to the excessive use of antibiotics, which has led to multiresistant bacteria. Cationic amphiphilic Aib-containing peptide models Ac-(Aib-Arg-Aib-Leu)(n)-NH2, n = 1-4, and sequential cationic polypeptides (Arg-X-Gly)(n), X = Ala, Val, Leu, were prepared and studied for their antimicrobial and hemolytic activity, as well as for their proteolytic stability. Ac-(Aib-Arg-Aib-Leu)(n)-NH2, n = 2, 3 and the polypeptide (Arg-Leu-Gly)(n) exhibited significant antimicrobial activity, and they were nontoxic at their MIC values and resistant, in particular the Aib-peptide models, to enzymatic degradation. The conformational characteristics of the peptide models were studied by circular dichroism (CD). Structure-activity relationship studies revealed the importance of the amphipathic alpha-helical conformation of the reported peptides in inducing antimicrobial effects. It is concluded that peptide models comprising cationic amino acids (Arg), helicogenic and noncoding residues (Aib) and/or hydrophobic and helix-promoting components (Leu) may lead to the development of antimicrobial therapeutics.
Journal of Peptide Science 08/2007; 13(7):481-6. · 2.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proteinaceous substances such as collagen, casein and albumin have been widely used as binding media in a variety of works of art. Damages of these 'sensitive' materials, mainly caused of the influence of the environment, are responsible for the overall decay of works of art, and their identification is essential to understand ancient technologies, determine the extent of deterioration and help in future restoration and preservation processes. The most commonly used techniques for the identification of proteinaceous binding media are staining techniques, chromatography, spectrometry and immunological methods, although for the latter no systematic studies have been carried out until now. Aiming at contributing to the development of a reliable and reproducible immunoassay for the evaluation of the collagen-based decay of works of art, sequential polypeptides (Pro-X-Gly)n where X represents amino acid residues Val, Lys, Glu and (Hyp-Val-Gly)n were prepared as models of collagen fragments derived from artificially and naturally aged animal collagens. Conformational studies of the polypeptides by CD revealed the occurrence of polyproline II-like structures comparable with those of collagen. Polypeptides and collagen I were administered to animals, and the induced antibodies were used for the immunochemical detection and differentiation of collagen and collagen fragments. The combined application of (i) anti-collagen antibodies, which strongly interact with native collagen, but poorly recognized by artificially aged collagen and (ii) anti-polypeptide antibodies, which do not associate with native collagen, but are strongly recognized by collagen fragments in naturally or artificially aged collagen, is a valuable tool in determining the extent of decay in works of art.
Journal of Peptide Science 03/2007; 13(2):121-7. · 2.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aiming at contributing to the development of potential atheroprotective agents, we report on the concept and design of two peptide models, which mimic the amphipathic helices of apoA-I and incorporate Met into their sequences to validate its role as oxidant scavenger: Ac-ESK(Palm)KELSKSW(10)SEM(13)LKEK(Palm)SKS-NH(2) (model 1 [W(10), M(13)]) and Ac-ESK(Palm)KELSKSM(10)SEW(13)LKEK(Palm)SKS-NH(2) (model 2 [M(10), W(13)]). Hydrophobic residues of both models cover about the half of the surface, while the positively and negatively charged residues constitute two separate clusters on the hydrophilic face. Palmitoyl groups were introduced into the Lys-N(epsilon)H(2) groups at positions 3 and 17 to contribute to the amphipathic character of the peptides and stabilize the nonpolar face of the helix. Conformational study by the combined application of 2D-NMR and molecular dynamics simulations, CD, FTIR, and fluorescence spectroscopy revealed that model 1 adopts helical conformation and Met is well exposed to the microenvironment. Model 2 that derives from model 1 by exchanging W(10) (model 1) with M(10) and M(13) (model 1) with W(13) also displays helical characteristics, while Met is rather shielded. Oxidation experiments indicated that model 1 exhibits a 2-fold more potent antioxidant activity towards LDL oxidation, compared to model 2, confirming the role of Met, when is devoid of steric hindrances, as oxidant scavenger for the protection of LDL.