Anomalous reversed-phase high-performance liquid chromatographic behavior of synthetic peptides related to antigenic helper T cell sites.

Sets of overlapping synthetic peptides for three well characterized proteins (sperm whale myoglobin, hen egg lysozyme, and the circumsporozoite protein from Plasmodium falciparum) were prepared and examined by reversed-phase high-performance liquid chromatography (RP-HPLC). Using retention coefficients to predict the retention time of each peptide, several peptides in each protein set were found that exhibited anomalous behavior (i.e. eluted significantly later than predicted). Previous work with model peptides has shown that this anomalous behavior can be attributed to specific amphipathic arrangements induced by the lipid stationary phase during the RP-HPLC process. In the current study it was found that although not all of the peptides containing an antigenic T cell site displayed anomalously late behavior, all of the peptides which eluted anomalously late during RP-HPLC included the regions of these proteins known from earlier studies to be antigenic T cell sites.     

Large-scale differential display analysis of T helper cell differentiation

We have developed a novel large-scale multicapillary fluorescent differential display (FDD) platform amenable to further automation. The power of the method is demonstrated by the analysis of T helper cell differentiation. Eight RNA samples from wild type, Stat4 knockout and Stat6 knockout mice were analyzed with 16 anchoring primers and 24 arbitrary primers, resulting in 285 294 sample peaks. Visually selected patterns of differential expression suggest two major regulatory mechanisms: activation and Stat4 genotype. A subset of the findings is reproduced in the confirmatory differential display (DD) that included technical and biological replicates. In a small fragment identification pilot study, we identify Ifi27 and Cct8 to be up-regulated by T cell activation. We present a method for the analysis of electropherogram similarity across large datasets, based on correlation of low-resolution representations of electrophoretic data. We show how it can be applied to analyze experimental and technical variables. Using this method, we demonstrate the effect of activation and genotype. In addition, agreement of our real experimental data to the theoretical basis of DD, as well as issues in anchoring primer selectivity, are studied.     

Hydration, charge, size, and shape characteristics of peptides from their CZE analyses

A CZE model is presented for peptide characterization on the basis of well-established physicochemical equations. The effective mobility is used as basic data in the model to estimate relevant peptide properties such as, for instance, hydration, net and total electrical charge numbers, hydrodynamic size and shape, particle average orientation, and pH-microenvironment from the charge regulation phenomenon. Therefore 102 experimental effective mobilities of different peptides are studied and discussed in relation to previous work. An equation for the estimation of peptide hydration as a function of ionizing, polar, and non-polar amino acid residues is included in the model. It is also shown that the shape-orientation factor of peptides may be either lower or higher than one, and its value depends on a complex interplay among total charge number, molar mass, hydration, and amino acid sequence.     

The size and shape of silica particles

Negatively charged silica particles were investigated at pH 10.0. They were found to be rod-shaped (cylinder) with a diameter of 5–5.5 nm and a full length of 44–67 nm depending on the rod model used. Moreover, the particles were found to be stable against aggregation in the region 0.4–50 mM NaCl. Received: 2 December 1998 Accepted in revised form: 2 February 1999     

Nonribosomal cyclic peptides: specific markers of fungal infections

Some cyclic peptides and depsipeptides are synthesized in microorganisms by large multienzymes called nonribosomal peptide synthetases. The structures of peptide products originating in this way are complex and diverse and are microorganism-specific. This work proposes the use of fungal cyclic peptides and depsipeptides as extremely specific markers of fungal infections. Since a reliable molecular tool for diagnosing fungal infections at an early stage is still missing, we present mass spectrometry as a new, modern, broadband (with respect to fungal strain) and specific tool for clinical mycologists. More than 40 different fungal species can be rapidly characterized according to specific families of cyclic peptides, and in some cases, a particular fungal strain can be identified on the basis of its cyclopeptide profile. This paper is also aimed at initiating discussion on the biological role of these secondary metabolites, especially of those synthesized by medically important strains. Proven cytotoxic, anti-inflammatory or immunosuppressive activities of some cyclic peptides indicate that these molecules may contribute to the synergistic array of fungal virulence factors and support microbial invasion during fungal infection. In addition to an overview on recent mass spectrometric protocols for cyclic peptide sequencing, the structures of new peptides from Paecilomyces and Pseudallescheria are presented.     

Exploring the evaluation of net charge, hydrodynamic size and shape of peptides through experimental electrophoretic mobilities obtained from CZE

This work explores the validity of simple CZE models to analyze the electrophoretic mobilities of 102 peptides reported in literature. These models are based mainly on fundamental physicochemical theories providing analytical expressions amenable to relatively simple numerical analysis. Thus, the Linderstr?m-Lang capillary electrophoresis model (LLCEM) and its perturbed version (PLLCEM), proposed and applied previously to the CZE of globular proteins, are adapted and used here for peptides. Also the effects of pK-shifts on net charge, hydration and hydrodynamic size and shape of peptides are analyzed and discussed. Emphasis is placed on the fact that these parameters are physically coupled, and thus a variation in the net charge may produce an appreciable change in the hydrodynamic size of peptides. Within the framework of CZE, peptides may be assumed as having a hydrodynamic volume associated with either spherical or spheroidal particles. The effects on peptide net charge and hydrodynamic size, of electrostatic interaction between a pair of charged groups in the chain and electrical permitivitty around the peptide domain are studied. The predictions of the PLLCEM and LLCEM are in good agreement with results reported previously in the literature. Several limitations concerning these models and some needs for further research are also described.     

曼氏和日本血吸虫副肌球蛋白的抗原肽研究

副肌球蛋白是世界卫生组织选取的用来发展血吸虫疫苗的重要侯选蛋白之一。 在计算机辅助下，应用PC－Gene程序，根据曼氏和日本血吸虫副肌球蛋白的氨基酸 序列，通过对其亲水性、柔韧性、可接近性和二级结构分析预测出8个抗原肽，并 用固相法进行了合成。经Dot-ELISA法测定，其中的2个对抗日本血吸虫免疫球蛋白 多克隆抗体（抗－Sj-IgG-PcAb)显示出抗原性，可作为抗血吸虫病合成多肽疫苗的 候选肽段。     

Enzymatic coupling of specific peptides at nonspecific ligation sites: effect of Asp189Glu mutation in trypsin on substrate mimetic-mediated reactions

Two main drawbacks seriously restrict the synthetic value of proteases as reagents in peptide fragment coupling: (i) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be formed. While the latter can be overcome by the use of substrate mimetics achieving peptide bond formation at nonspecific ligation sites, the risk of proteolytic cleavage still remains and hinders the wide acceptance of this powerful strategy for peptide coupling. This paper reports on the effect of the trypsin point mutant Asp189Glu on substrate mimetic-mediated reactions. The effect of this mutation on the steady-state hydrolysis of substrate mimetics of the 4-guanidinophenyl ester type and on trypsin-specific Lys- and Arg-containing peptides was investigated. The results were confirmed by enzymatic coupling reactions using substrate mimetics as the acyl donor and specific amino acid-containing peptides as the acyl acceptor. The competition assay verifies the predicted shift in substrate preference from Lys and Arg to the substrate mimetics and, thus, from cleavage to synthesis of peptide bonds. The combination of results obtained qualifies the trypsin mutant D189E as the first substrate mimetic-specific peptide ligase.     

Bioactive peptides grafted silicone dressings: A simple and specific method

The need for bioactive dressings increases with the population aging and the prevalence of chronic diseases. In contrast, there are very few dressings on the market which are designed to display a chosen bioactivity. In this context, we investigated the surface-functionalization of silicone wound dressing with bioactive peptides. One of the challenges was to avoid multistep grafting reactions involving catalysts, solvents or toxic reagents, which are not suitable for the fabrication of medical devices at an industrial scale. In the other hand, a covalent bonding was necessary to avoid the loss of the biological effect by progressive removal of the peptide in biological fluids generated by the wound. To solve these limitations, we developed a strategy allowing an easy and direct functionalization of silicone. This strategy relies on hybrid silylated bioactive peptides, which chemoselectively react with plasma-activated silicone surfaces. We synthesized three hybrid peptides with wound healing properties, which were grafted on commercially available silicone dressings Cerederm^® and Mepitel^®. Grafted dressings were evaluated in vitro and enabled a quicker scare recovery and extracellular matrix deposition with human dermal fibroblasts. These results were confirmed by in vivo studies showing an enhanced wound-healing of the pig skin. By this simple method, we transformed inert dressing into bioactive dressing which showed properties of wound healing.     

T cell response in rheumatic fever: crossreactivity between streptococcal M protein peptides and heart tissue proteins

Molecular mimicry between streptococcal and human proteins has been proposed as the triggering factor leading to autoimmunity in rheumatic fever (RF) and rheumatic heart disease (RHD). In this review we focus on the studies on genetic susceptibility markers involved in the development of RF/RHD and molecular mimicry mediated by T cell responses of RHD patients against streptococcal antigens and human tissue proteins. We identified several M protein epitopes recognized by peripheral T cells of RF/RHD patients and by heart tissue infiltrating T cell clones of severe RHD patients. The regions of the M protein preferentially recognized by human T cells were also recognized by murine T cells. By analyzing the T cell receptor (TCR) we observed that some Vbeta families detected on the periphery were oligoclonal expanded in the heart lesions. These results allowed us to confirm the major role of T cells in the development of RHD lesions.     

TTAgP 1.0: A computational tool for the specific prediction of tumor T cell antigens

Nowadays, cancer is considered a global pandemic and millions of people die every year because this disease remains a challenge for the world scientific community. Even with the efforts made to combat it, there is a growing need to discover and design new drugs and vaccines. Among these alternatives, antitumor peptides are a promising therapeutic solution to reduce the incidence of deaths caused by cancer. In the present study, we developed TTAgP, an accurate bioinformatic tool that uses the random forest algorithm for antitumor peptide predictions, which are presented in the context of MHC class I. The predictive model of TTAgP was trained and validated based on several features of 922 peptides. During the model validation we achieved sensitivity = 0.89, specificity = 0.92, accuracy = 0.90 and the Matthews correlation coefficient = 0.79 performance measures, which are indicative of a robust model. TTAgP is a fast, accurate and intuitive software focused on the prediction of tumor T cell antigens.     

Tactoids of plate-like particles: size, shape, and director field

We studied, by means of polarized light microscopy, the shape and director field of nematic tactoids as a function of their size in dispersions of colloidal gibbsite platelets in polar and apolar solvents. Because of the homeotropic anchoring of the platelets to the interface, we found large tactoids to be spherical with a radial director field, whereas small tactoids turn out to have an oblate shape and a homogeneous director field, in accordance with theoretical predictions. The transition from a radial to a homogeneous director field seems to proceed via two different routes depending in our case on the solvent. In one route, the what presumably is a hedgehog point defect in the center of the tactoid transforms into a ring defect with a radius that presumably goes to infinity with decreasing drop size. In the other route, the hedgehog defect is displaced from the center to the edge of the tactoid, where it becomes virtual again going to infinity with decreasing drop size. Furthermore, quantitative analysis of the tactoid properties provides us with useful information on the ratio of the splay elastic constant and the anchoring strength and the ratio of the anchoring strength and the surface tension.     

Oligonuclear ferrocene amides: mixed-valent peptides and potential redox-switchable foldamers

Trinuclear ferrocene tris-amides were synthesized from an Fmoc- or Boc-protected ferrocene amino acid, and hydrogen-bonded zigzag conformations were determined by NMR spectroscopy, molecular modelling, and X-ray diffraction. In these ordered secondary structures orientation of the individual amide dipole moments approximately in the same direction results in a macrodipole moment similar to that of α-helices composed of α-amino acids. Unlike ordinary α-amino acids, the building blocks in these ferrocene amides with defined secondary structure can be sequentially oxidized to mono-, di-, and trications. Singly and doubly charged mixed-valent cations were probed experimentally by Vis/NIR, paramagnetic 1H NMR and M?ssbauer spectroscopy and investigated theoretically by DFT calculations. According to the appearance of intervalence charge transfer (IVCT) bands in solution, the ferrocene/ferrocenium amides are described as Robin-Day class II mixed-valent systems. M?ssbauer spectroscopy indicates trapped valences in the solid state. The secondary structure of trinuclear ferrocene tris-amides remains intact (coiled form) upon oxidation to mono- and dications according to DFT calculations, while oxidation to the trication should break the intramolecular hydrogen bonding and unfold the ferrocene peptide (uncoiled form).     

Proteins and peptides voltammetry: Trends,potential, and limitations

Numerous biological processes are connected with the efficient electron transfer reactions in proteins and peptides. In this review, we discuss briefly the relevance and current challenges associated with the voltammetric analysis of peptides and proteins with and without a metal redox center. Special attention is paid to the integration of electrochemical methods with new nanomaterials which offers amplification of multiplexing capabilities for simultaneous and very sensitive examination of various proteins. After critically discussing the most interesting approaches in the proteins/peptides voltammetric analysis reported so far, for the single or multiplexed examination of such biomolecules with demonstrated applicability in the real-sample analysis, existing challenges still need to be addressed and future directions in this field will be pointed out.     

Topological estimation of proton-ligand formation constants of potential antitumour agents: Salicylhydroxamic acids

Proton-ligand formation constants of salicylhydroxamic acids (SHA) and their nuclear substituted derivatives have been estimated topologically using the normalized Wiener index, referred to as mean square Wiener index (Wms). Regression analysis of the data indicates that Wms can be used successfully for estimating and monitoring proton-ligand formation constants.     

Hydrolytic reaction by zinc finger mutant peptides: successful redesign of structural zinc sites into catalytic zinc sites

To redesign a metal site originally required for the stabilization of a folded protein structure into a functional metal site, we constructed a series of zinc finger mutant peptides such as zf(CCHG) and zf(GCHH), in which one zinc-coordinating residue is substituted into a noncoordinating one. The mutant peptides having water bound to the zinc ion catalyzed the hydrolysis of 4-nitrophenyl acetate as well as the enantioselective hydrolysis of amino acid esters. All the zinc complexes of the mutant peptides showed hydrolytic activity, depending on their peptide sequences. In contrast, the zinc complex of the wild-type, zf(CCHH), and zinc ion alone exhibited no hydrolytic ability. These results clearly indicate that the catalytic abilities are predominantly attributed to the zinc center in the zinc complexes of the mutant peptides. Kinetic studies of the mutant peptides demonstrated that the catalytic hydrolysis is affected by the electron-donating ability of the protein ligands and the coordination environment. In addition, the pH dependence of the hydrolysis strongly suggests that the zinc-coordinated hydroxide ion participates the catalytic reaction. This report is the first successful study of catalytically active zinc finger peptides.     

The relevance of shape and size of Au55 clusters

This critical review deals with the history of Au(55)(PPh(3))(12)Cl(6) and its derivatives from the very beginning in 1981 to date. Au(55) clusters obtain their special interest from their ultimate size and their ideal cuboctahedral structure. They are part of the family of so-called full-shell clusters, particles with perfectly completed geometries, also represented by icosahedral Au(13) clusters. Bare as well as ligand protected Au(55) clusters not only exhibit special chemical and physical stability, but draw their attention particularly from their unique electronic properties. Single electron switching at room temperature becomes possible, giving rise for development of applications in future nanoelectronic devices. A predominantly size-determined property of the 1.4 nm particles becomes obvious with respect of biological response. Au(55) clusters indicate an unusual cytotoxicity which seems to be caused by the unusually strong interaction between the 1.4 nm particles and the major grooves of DNA. Only marginally smaller or larger particles show drastically reduced toxicity, whereas significantly larger gold nanoparticles are completely non-toxic. Both, the electronic perspectives as well as the relevance in toxicology are at very early stages of development (75 references).