Recognition of nonstructural protein-peptides by cytotoxic t-lymphocytes raised against japanese encephalitis-virus

We have previously reported that Lyt(2+) cytotoxic T lymphocytes (CTL) can be raised against Japanese encephalitis virus (JEV) in BALB/c mice. In order to confirm the presence of H-2K(d)-restricted CTL and to examine their cross-recognition of West Wile virus (WNV), we tested the capacity of anti-JEV CTL to lyse uninfected syngeneic target cells that were pulsed with synthetic peptides. The sequence of the synthetic peptides was predicted based upon the H-2K(d) binding consensus motif. We show here that preincubation of uninfected syngeneic targets (P388D1) with JEV NS1- and NS3-derived peptides [NS1 (891-899) and NS3 (1804-1812)], but not with JEV NS5-derived peptide [NS5 (3370-3378)], partially sensitized them for lysis by polyclonal anti-JEV CTL. These results indicate the CTL recognition of NS1- and NS3-derived peptides of JEV.

A CD8(+) T cell clone specific for antigen also recognizes peptidomimics present in anti-idiotypic antibody: Implications for T cell memory

The relay hypothesis [R. Nayak, S. Mitra-Kaushik, M.S. Shaila, Perpetuation of immunological memory: a relay hypothesis, Immunology 102 (2001) 387-395] was earlier proposed to explain perpetuation of immunological memory without requiring long lived memory cells or persisting antigen. This hypothesis envisaged cycles of interaction and proliferation of complementary idiotypic B cells (Burnet cells) and anti-idiotypic B cells (Jerne cells) as the primary reason for perpetuation of immunological memory. The presence of pepti-domimics of antigen in anti-idiotypic antibody and their presentation to antigen specific T cells was postulated to be primary reason for perpetuation of T cell memory. Using a viral hemagglutinin as a model, in this work, we demonstrate the presence of peptidomimics in the variable region of ail anti-idiotypic antibody capable of functionally mimicking the antigen derived peptides. A CD8(+) CTL clone was generated against the hemagglutinin protein which specifically responds to either peptidomimic synthesizing cells or peptidomimic pulsed antigen presenting cells. Thus, it appears reasonable that a population of activated antigen specific T cells is maintained in the body by presentation of peptidomimic through Jerne cells and other antigen presenting cells long after immunization. (C) 2007 Elsevier Inc. All rights reserved.

Immune responses to hemagglutinin-neuraminidase protein of peste des petits ruminants virus expressed in transgenic peanut plants in sheep

Peste des petits ruminants (PPR) is an acute, highly contagious disease of small ruminants caused by a morbillivirus, Peste des petits ruminants virus (PPRV). The disease is prevalent in equatorial Africa, the Middle East, and the Indian subcontinent. A live attenuated vaccine is in use in some of the countries and has been shown to provide protection for at least three years against PPR. However, the live attenuated vaccine is not robust in terms of thermotolerance. As a step towards development of a heat stable subunit vaccine, we have expressed a hemagglutinin-neuraminidase (HN) protein of PPRV in peanut plants (Arachis hypogea) in a biologically active form, possessing neuraminidase activity. Importantly. HN protein expressed in peanut plants retained its immunodominant epitopes in their natural conformation. The immunogenicity of the plant derived HN protein was analyzed in sheep upon oral immunization. Virus neutralizing antibody responses were elicited upon oral immunization of sheep in the absence of any mucosal adjuvant. In addition, anti-PPRV-HN specific cell-mediated immune responses were also detected in mucosally immunized sheep. (C) 2010 Elsevier B.V. All rights reserved.

A natural compound, methyl angolensate, induces mitochondrial pathway of apoptosis in Daudi cells

Natural products discovered from medicinal plants have played an important role in the treatment of cancer. In an effort to identify novel small molecules which can affect the proliferation of lymphoma cells, we tested methyl angolensate (MA), a plant derived tetranortriterpenoid, purified from the crude extract of the root callus of Soymida febrifuga commonly known as Indian red wood tree. We have tested MA for its cytotoxic properties on Burkitt's lymphoma cell lines, using various cellular assays. We observed that MA induces cytotoxicity in Daudi cells in a dose-dependent manner using trypan blue, MTT and LDH assays. We find that the treatment with MA led to activation of DNA double-strand break repair proteins including KU70 and KU80, suggesting the activation of nonhomologous DNA end joining pathway in surviving cells. Further, we find that methyl angolensate could induce apoptosis by cell cycle analysis, annexin V-FITC staining, DNA fragmentation and PARP cleavage. Besides, MA treatment led to reactive oxygen species generation and loss of mitochondrial transmembrane potential. These results suggest the activation of mitochondrial pathway of apoptosis. Hence, we identify MA as a potential chemotherapeutic agent against Daudi cells.

Hydrolysis of aromatic beta-glucosides by non-pathogenic bacteria confers a chemical weapon against predators

Bacteria present in natural environments such as soil have evolved multiple strategies to escape predation. We report that natural isolates of Enterobacteriaceae that actively hydrolyze plant-derived aromatic beta-glucosides such as salicin, arbutin and esculin, are able to avoid predation by the bacteriovorous amoeba Dictyostelium discoideum and nematodes of multiple genera belonging to the family Rhabditidae. This advantage can be observed under laboratory culture conditions as well as in the soil environment. The aglycone moiety released by the hydrolysis of beta-glucosides is toxic to predators and acts via the dopaminergic receptor Dop-1 in the case of Caenorhabditis elegans. While soil isolates of nematodes belonging to the family Rhabditidae are repelled by the aglycone, laboratory strains and natural isolates of Caenorhabditis sp. are attracted to the compound, mediated by receptors that are independent of Dop-1, leading to their death. The b-glucosides-positive (Bgl(+)) bacteria that are otherwise non-pathogenic can obtain additional nutrients from the dead predators, thereby switching their role from prey to predator. This study also offers an evolutionary explanation for the retention by bacteria of `cryptic' or `silent' genetic systems such as the bgl operon.

Inhibition of cancer cell proliferation and apoptosis-inducing activity of fungal taxol and its precursor baccatin III purified from endophytic Fusarium solani

Background: Taxol (generic name paclitaxel), a plant-derived antineoplastic agent, used widely against breast, ovarian and lung cancer, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. The limited supply of the drug has prompted efforts to find alternative sources, such as chemical synthesis, tissue and cell cultures of the Taxus species both of which are expensive and yield low levels. Fermentation processes with microorganisms would be the methods of choice to lower the costs and increase yields. Previously we have reported that F. solani isolated from T. celebica produced taxol and its precursor baccatin III in liquid grown cultures J Biosci 33: 259-67, 2008. This study was performed to evaluate the inhibition of proliferation and induction of apoptosis of cancer cell lines by the fungal taxol and fungal baccatin III of F. solani isolated from T. celebica. Methods: Cell lines such as HeLa, HepG2, Jurkat, Ovcar3 and T47D were cultured individually and treated with fungal taxol, baccatin III with or without caspase inhibitors according to experimental requirements. Their efficacy on apoptotic induction was examined. Results: Both fungal taxol and baccatin III inhibited cell proliferation of a number of cancer cell lines with IC50 ranging from 0.005 to 0.2 mu M for fungal taxol and 2 to 5 mu M for fungal baccatin III. They also induced apoptosis in JR4-Jurkat cells with a possible involvement of anti-apoptotic Bcl2 and loss in mitochondrial membrane potential, and was unaffected by inhibitors of caspase-9,-2 or -3 but was prevented in presence of caspase-10 inhibitor. DNA fragmentation was also observed in cells treated with fungal taxol and baccatin III. Conclusions: The cytotoxic activity exhibited by fungal taxol and baccatin III involves the same mechanism, dependent on caspase-10 and membrane potential loss of mitochondria, with taxol having far greater cytotoxic potential.

Evolution of Aromatic beta-Glucoside Utilization by Successive Mutational Steps in Escherichia coli

The bglA gene of Escherichia coli encodes phospho-beta-glucosidase A capable of hydrolyzing the plant-derived aromatic beta-glucoside arbutin. We report that the sequential accumulation of mutations in bglA can confer the ability to hydrolyze the related aromatic beta-glucosides esculin and salicin in two steps. In the first step, esculin hydrolysis is achieved through the acquisition of a four-nucleotide insertion within the promoter of the bglA gene, resulting in enhanced steady-state levels of the bglA transcript. In the second step, hydrolysis of salicin is achieved through the acquisition of a point mutation within the bglA structural gene close to the active site without the loss of the original catabolic activity against arbutin. These studies underscore the ability of microorganisms to evolve additional metabolic capabilities by mutational modification of preexisting genetic systems under selection pressure, thereby expanding their repertoire of utilizable substrates.

Analysis of the amino acid sequences of plant Bowman-Birk inhibitors

Plant seeds contain a large number of protease inhibitors of animal, fungal, and bacterial origin. One of the well-studied families of these inhibitors is the Bowman-Birk family(BBI). The BBIs from dicotyledonous seeds are 8K, double-headed proteins. In contrast, the 8K inhibitors from monocotyledonous seeds are single headed. Monocots also have a 16K, double-headed inhibitor. We have determined the primary structure of a Bowman-Birk inhibitor from a dicot, horsegram, by sequential edman analysis of the intact protein and peptides derived from enzymatic and chemical cleavage. The 76-residue-long inhibitor is very similar to that ofMacrotyloma axillare. An analysis of this inhibitor along with 26 other Bowman-Birk inhibitor domains (MW 8K) available in the SWISSPROT databank revealed that the proteins from monocots and dicots belong to related but distinct families. Inhibitors from monocots show larger variation in sequence. Sequence comparison shows that a crucial disulphide which connects the amino and carboxy termini of the active site loop is lost in monocots. The loss of a reactive site in monocots seems to be correlated to this. However, it appears that this disulphide is not absolutely essential for retention of inhibitory function. Our analysis suggests that gene duplication leading to a 16K inhibitor in monocots has occurred, probably after the divergence of monocots and dicots, and also after the loss of second reactive site in monocots.

PeptideMine - A webserver for the design of peptides for protein-peptide binding studies derived from protein-protein interactomes

Background: Signal transduction events often involve transient, yet specific, interactions between structurally conserved protein domains and polypeptide sequences in target proteins. The identification and validation of these associating domains is crucial to understand signal transduction pathways that modulate different cellular or developmental processes. Bioinformatics strategies to extract and integrate information from diverse sources have been shown to facilitate the experimental design to understand complex biological events. These methods, primarily based on information from high-throughput experiments, have also led to the identification of new connections thus providing hypothetical models for cellular events. Such models, in turn, provide a framework for directing experimental efforts for validating the predicted molecular rationale for complex cellular processes. In this context, it is envisaged that the rational design of peptides for protein-peptide binding studies could substantially facilitate the experimental strategies to evaluate a predicted interaction. This rational design procedure involves the integration of protein-protein interaction data, gene ontology, physico-chemical calculations, domain-domain interaction data and information on functional sites or critical residues. Results: Here we describe an integrated approach called ``PeptideMine'' for the identification of peptides based on specific functional patterns present in the sequence of an interacting protein. This approach based on sequence searches in the interacting sequence space has been developed into a webserver, which can be used for the identification and analysis of peptides, peptide homologues or functional patterns from the interacting sequence space of a protein. To further facilitate experimental validation, the PeptideMine webserver also provides a list of physico-chemical parameters corresponding to the peptide to determine the feasibility of using the peptide for in vitro biochemical or biophysical studies. Conclusions: The strategy described here involves the integration of data and tools to identify potential interacting partners for a protein and design criteria for peptides based on desired biochemical properties. Alongside the search for interacting protein sequences using three different search programs, the server also provides the biochemical characteristics of candidate peptides to prune peptide sequences based on features that are most suited for a given experiment. The PeptideMine server is available at the URL: http://caps.ncbs.res.in/peptidemine

Click Chemistry Inspired Synthesis of Novel Ferrocenyl-Substituted Amino Acids or Peptides

This work reports on the synthesis of a wide range of ferrocenyl-substituted amino acids and peptides in excellent yield. Conjugation is established via copper-catalyzed 1,3-dipolar cycloaddition. Two complementary strategies were employed for conjugation, one involving cycloaddition of amino acid derived azides with ethynyl ferrocene 1 and the other involves cycloaddition between amino acid derived alkynes with ferrocene-derived azides 2 and 3. Labeling of amino acids at multiple sites with ferrocene is discussed. A new route to 1,1'-unsymmetrically substituted ferrocene conjugates is reported. A novel ferrocenophane 19 is accessed via bimolecular condensation of amino acid derived bis-alkyne 9b with the azide 2. The electrochemical behavior of some selected ferrocene conjugates has been studied by cyclic voltammetry.

A Minimax Filter for Systems with Large Plant Uncertainties Using Measurements Corrupted by Colored Noise

A minimax filter is derived to estimate the state of a system, using observations corrupted by colored noise, when large uncertainties in the plant dynamics and process noise are presen.

Facile Synthesis of beta-Amino Disulfides, Cystines, and Their Direct Incorporation into Peptides

Herein, we report a simple and efficient methodology for the synthesis of beta-amino disulfides by regioselective ring opening of sulfamidates with benzyltriethylammonium tetrathiomolybdate [BnNEt3](2)MoS4. Stability and reactivity of different protecting groups under the reaction conditions have been discussed. This methodology has also been extended to serine and threonine derived sulfamidates to furnish cystine and 3,3'-dimethyl cystine derivatives.

Stereochemistry of peptides and polypeptides containing omega amino acids

The omega amino acids have a larger degree of conformational variability than the alpha amino acids, leading to a greater diversity of backbone structures in peptides and polypeptides. The synthetic accessibility of chiral beta-amino acids and the recent observation of novel helical folds in oligomers of cyclic beta-amino acids has led to renewed interest in the stereochemistry of omega-amino acid containing peptides. This review focuses on the conformational characteristics of the polymethylene chain in omega-amino acid segments and surveys structural features in peptides established by X-ray diffraction. The literature on polymers of achiral omega-amino acids (nylon derivatives) and chiral, substituted derivatives derived from trifunctional alpha-amino acids, reveals that while sheet-like, intermolecular hydrogen bonded structures are formed by the former, folded helices appear favoured by the latter. omega-Amino acids promise to expand the repertoire of peptide folds.

Cloning, overexpression, folding and purification of a biosynthetically derived three disulfide scorpion toxin (BTK-2) from Mesobuthus tamulus

BTK-2, a 32 residue scorpion toxin initially identified in the venom of red Indian scorpion Mesobuthus tamulus was cloned, overexpressed and purified using Cytochrome 155 fusion protein system developed in our laboratory. The synthetic gene coding for the peptide was designed taking into account optimal codon usage by Escherichia coli. High expression levels of the fusion protein enabled facile purification of this peptide. The presence of disulfide bonded isomers, occurring as distinctly populated states even in the fusion protein, were separated by gel filtration chromatography. The target peptide was liberated from the host protein by Tev protease cleavage and subsequent purification was achieved using RP-HPLC methods. Reverse phase HPLC clearly showed the presence of at least two isomeric forms of the peptide that were significantly populated. The oxidative folding of BTK-2 was achieved under ambient conditions during the course of purification. Structural characterization of the two forms, by solution homonuclear and heteronuclear NMR methods, has shown that these two forms exhibit significantly different structural properties, and represent the natively folded and a "misfolded" form of the peptide. The formation of properly folded BTK-2 as a major fraction without the use of in vitro oxidative refolding methods clearly indicate the versatility of the Cytochrome b(5) fusion protein system for the efficient production of peptides for high resolution NMR studies.

Perpetuation of immunological memory through common MHC-I binding modes of peptidomimic and antigenic peptides

Understanding the molecular mechanisms of immunological memory assumes importance in vaccine design. We had earlier hypothesized a mechanism for the maintenance of immunological memory through the operation of a network of idiotypic and anti-idiotypic antibodies (Ab2). Peptides derived from an internal image carrying anti-idiotypic antibody are hypothesized to facilitate the perpetuation of antigen specific T cell memory through similarity in peptide-MHC binding as that of the antigenic peptide. In the present work, the existence of such peptidomimics of the antigen in the Ab2 variable region and their similarity of MHC-I binding was examined by bioinformatics approaches. The analysis employing three known viral antigens and one tumor-associated antigen shows that peptidomimics from Ab2 variable regions have structurally similar MHC-I binding patterns as compared to antigenic peptides, indicating a structural basis for memory perpetuation. (C)) 2007 Elsevier Inc. All rights reserved.