Novel anoplin-based (lipo)-peptide models show potent antimicrobial activity.

The subject of this study is the synthesis and biological evaluation of anoplin-based (Gly-Leu-Leu -Lys-Arg -Ile-Lys-Thr -Leu-Leu-NH )-designed (lipo)-peptides, aiming at the development of new antibiotic substances. The design of synthetic compounds based on natural bioactive molecules is an optimistic strategy for the development of new pharmaceutics. Antimicrobial peptides (AMPs) and (lipo)-peptides are two classes of promising compounds, with characteristics that allow them to express their activity by differentiated mechanisms of action.

Cationic amphipathic peptide analogs of cathelicidin LL-37 as a probe in the development of antimicrobial/anticancer agents.

Cathelicidin LL-37 belongs to the class of human defense peptides and is overexpressed in many cancers. Segments of LL-37 derived through biochemical processes have a wide range of activities. In this study, novel analogs of the 13-amino acid cathelicidin 17-29 amide segment F KRIV QR IK DF LR-NH were prepared and examined for their antimicrobial and hemolytic activities, as well as for their cytotoxicity on cancer bronchial epithelial cells.

Synthesis and biological activity of lipophilic analogs of the cationic antimicrobial active peptide anoplin.

Anoplin is a short natural cationic antimicrobial peptide which is derived from the venom sac of the solitary wasp, Anoplius samariensis. Due to its short sequence G LLKR IKT LL-NH , it is ideal for research tests. In this study, novel analogs of anoplin were prepared and examined for their antimicrobial, hemolytic activity, and proteolytic stability.

Collagen and keratin polypeptide models for assessing the natural and artificial protein decay of organic materials.

Among the materials constituting the natural and cultural heritage, organic materials of proteinaceous origin as bone (collagen), parchment and woolen textiles (keratin) are the most susceptible to damage and decay because of their exposure to air pollution, inappropriate values of ambient temperature, humidity and light. Aiming at contributing to the development of a reliable and reproducible immunoassay for the evaluation of collagen and keratin decay, three polypeptide models of these proteins were designed, synthesized and studied. Polypeptide [Pro-Ser(OBzl)-Gly] incorporates the typical motif Pro-X-Gly of collagen; polypeptide [Pro-Cys(Acm)-Gly] is a model of the C-terminal domain of type I keratin, corresponding to the repeating unit Pro-Cys-X of keratin, while polypeptide Ac-YRSGGGFGYRSGGGFGYRS-βAla-NH encloses the characteristic repeating sequence GGGFGYRS of the N-terminal part of Type II keratin.

Palmitoylated peptide, being derived from the carboxyl-terminal sequence of the integrin αIIb cytoplasmic domain, inhibits talin binding to αIIbβ₃.

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.

Sialic acid and sialyl-lactose glyco-conjugates: design, synthesis and binding assays to lectins and swine influenza H1N1 virus.

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.

Influenza virus H5N1 hemagglutinin (HA) T-cell epitope conjugates: design, synthesis and immunogenicity.

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.

HLA-DQ7 beta(1) and beta(2) derived peptides as immunomodulators.

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.

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).

A complementary La/SSB epitope anchored to Sequential Oligopeptide Carrier regulates the anti-La/SSB response in immunized animals.

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.

Chirality and helix stability of polyglutamic acid enantiomers.

In this work the chirality and the relative thermal stability of ordered micellar aggregates of poly-L- and poly-D-glutamic acids with the cationic surfactant C14TAB is examined. The complexed mesophases poly-L-Glu/C14TAB and poly-d-Glu/C14TAB were characterized by circular dichroism (CD) in the temperature range 10-70 degrees C for their chirality and thermal stability as well as by X-ray diffraction (XRD) for the micellar ordered structure. Low angle XRD analysis showed that both micellar aggregates poly-L-Glu/C14TAB and poly-D-Glu/C14TAB are hexagonally packed in a MCM-41 fashion with an intermicellar distance identical and equal to 3.

A palmitoyl-tailed sequential oligopeptide carrier for engineering immunogenic conjugates.

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.

Collagen models as a probe in the decay of works of art: synthesis, conformation and immunological studies.

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.

Artificial carriers: a strategy for constructing antigenic/immunogenic conjugates.

The rational design of artificial carriers for anchoring multiple copies of B and/or T cell epitopes, built-in vaccine adjuvants and "promiscuous" T cell epitopes for the construction of conjugates as antigenic substrates or potent immunogens has been the stimulus of intensive efforts nowadays. The unambiguous composition, the reliability and the versatility of the production of reconstituted antigens or immunogens has found a great number of biochemical applications in developing immunoassays of high sensitivity, specificity and reproducibility and in generating site-specific antibodies for usage as human vaccine candidates. In this review are summarized different types of artificial carriers currently used as dendrimers bearing branching segments, multimeric core matrices and templates with built-in folding devices.

Phosphorylated and nonphosphorylated epitopes of the La/SSB autoantigen: comparison of their antigenic and conformational characteristics.

La/SSB phosphoprotein is the target antigen of autoantibodies in sera of patients with Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). Among other structural and function motifs, four phosphorylation sites are encompassed in the primary sequence of La/SSB. Two of them (Thr-362 and Ser-366) are located within GSGKGKVQFQGKKTKFASDD (346-368) and one (Thr-302) within VTWEVLEGEVEKEALKKI (301-318), which are main B-cell epitopes of La/SSB.

Innovative, multifunctional sequential oligopeptide carriers Soc(n)-I and SOC(n)-II: functions-technology-perspectives.

An innovative type of multifunctional helicoid artificial carriers, formed by the repetitive Lys-Aib-Gly (SOC(n)-I) or by the Aib-Lys-Aib-Gly (SOC(n)-II), with structural rigidity and regularity were successfully applied in our laboratory for anchoring antigenic/immunogenic peptides. The carriers, designed to display a predetermined 3D structure, adopt the 3(10) helical conformation, while the attached peptides preserve their original "active" conformation. The constructed conjugates were used as substrates in solid phase immunoassays, as well as for generating potent and specific immune responses.

A peptide carrier with a built-in vaccine adjuvant: construction of immunogenic conjugates.

A multifunctional carrier combining B/T cell epitopes (i), a built-in vaccine adjuvant (ii), and a universal T cell epitope (iii) for the construction of potent and specific immunogenic conjugates is presented. The IL-1beta(163-171) fragment known to reproduce the immunostimulatory and adjuvant effects of the whole IL-1beta without possessing any of the pro-inflammatory properties of IL-1beta was covalently anchored to the N-terminus of the Sequential Oligopeptide Carrier, SOC(n), formed by the repeating tripeptide unit Lys-Aib-Gly. A promiscuous T cell epitope derived from the tetanus toxin, TT(593-599), was also positioned in the carboxy terminus of SOC(n) as a universal immunogen to provide broad immunogenicity.

Zinc ion dependent B-cell epitope, associated with primary Sjogren's syndrome, resides within the putative zinc finger domain of Ro60kD autoantigen: physical and immunologic properties.

The Ro/La ribonucleoprotein (RNP) complex is composed of the proteins Ro60kD, Ro52kD, and La48kD that are in association with one small cytoplasmic RNA (YRNA). Specific protein-RNA and protein-protein interactions are thought to occur through the RNP and zinc-finger secondary structure elements of the Ro60kD protein. The aim of our study was to investigate the antigenic properties of the zinc finger domain of the Ro60KD autoantigen and its contribution to the formation of Ro/La RNP complex.