In vitro toxicity testing of alcohol-soluble proteins from diploid wheat triticum monococcum in celiac disease

Peptic-tryptic digests of alcohol-soluble proteins from flours of 10 accessions of Triticum monococcum with contrasting storage protein compositions and bread-making characteristics were found unable to agglutinate K562(S) cells even at a peptide concentration as high as 14 g/L, agglutination being strongly correlated with toxicity in celiac disease. When fractionated by affinity chromatography on Sepharose-6B coupled with mannan, peptic-tryptic digests separated into three fractions. Fraction C peptides were shown to agglutinate K562(S) cells, whereas peptides in fractions A and B and in the mixed fraction B + C were inactive, suggesting that fraction B contains “protective” peptides that interfere with toxic peptides in fraction C in their agglutinating activity. These results offer an opportunity to study the biochemical and genetic bases of wheat toxicity at the diploid level. Moreover, the reduced toxicity, if any, of Triticum monococcum in the celiac disease, along with the good grain characteristics of some “monococcum” accessions, greatly increases the economical prospects of this wheat species. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 11: 313–318, 1997.     

The actin nucleator Spir-1 is a virus restriction factor that promotes innate immune signalling

Cellular proteins often have multiple and diverse functions. This is illustrated with protein Spir-1 that is an actin nucleator, but, as shown here, also functions to enhance innate immune signalling downstream of RNA sensing by RIG-I/MDA-5. In human and mouse cells lacking Spir-1, IRF3 and NF-κB-dependent gene activation is impaired, whereas Spir-1 overexpression enhanced IRF3 activation. Furthermore, the infectious virus titres and sizes of plaques formed by two viruses that are sensed by RIG-I, vaccinia virus (VACV) and Zika virus, are increased in Spir-1 KO cells. These observations demonstrate the biological importance of Spir-1 in the response to virus infection. Like cellular proteins, viral proteins also have multiple and diverse functions. Here, we also show that VACV virulence factor K7 binds directly to Spir-1 and that a diphenylalanine motif of Spir-1 is needed for this interaction and for Spir-1-mediated enhancement of IRF3 activation. Thus, Spir-1 is a new virus restriction factor and is targeted directly by an immunomodulatory viral protein that enhances virus virulence and diminishes the host antiviral responses.     

Helix induction potential of N-terminal α-methyl, α-amino acids

Summary A series of longer analogues of the C-peptide of RNAse A has been synthesized with the aim of assessing the helix induction potential in water of α-methyl, α-amino acids at the N-terminus of the chain. The circular dichroism data indicate that one isovaline residue is effective in increasing the helix content of the 13-residue peptide by about 7%.     

The Complete Amino Acid Sequence of a Trypsin Inhibitor from Bauhinia variegata var. candida Seeds

Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with M _r of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show K _i values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)–Ile (64).     

Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat

The Bacillus subtilis endospore coat protein CotA shows laccase activity. By using comparative modeling techniques, we were able to derive a model for CotA based on the known x-ray structures of zucchini ascorbate oxidase and Cuprinus cereneus laccase. This model of CotA contains all the structural features of a laccase, including the reactive surface-exposed copper center (T1) and two buried copper centers (T2 and T3). Single amino acid substitutions in the CotA T1 copper center (H497A, or M502L) did not prevent assembly of the mutant proteins into the coat and did not alter the pattern of extractable coat polypeptides. However, in contrast to a wild type strain, both mutants produced unpigmented colonies and spores unable to oxidize syringaldazine (SGZ) and 2'2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The CotA protein was purified to homogeneity from an overproducing Escherichia coli strain. The purified CotA shows an absorbance and a EPR spectra typical of blue multicopper oxidases. Optimal enzymatic activity was found at < or =pH 3.0 and at pH 7.0 for ABTS or SGZ oxidation, respectively. The apparent K(m) values for ABTS and SGZ at 37 degrees C were of 106 +/- 11 and 26 +/- 2 microm, respectively, with corresponding k(cat) values of 16.8 +/- 0.8 and 3.7 +/- 0.1 s(-1). Maximal enzyme activity was observed at 75 degrees C with ABTS as substrate. Remarkably, the coat-associated or the purified enzyme showed a half-life of inactivation at 80 degrees C of about 4 and 2 h, respectively, indicating that CotA is intrinsically highly thermostable.     

Synthesis and preliminary conformational analysis of TOAC spin‐labeled analogues of the medium‐length peptaibiotic tylopeptin B

A set of analogues of the 14‐residue peptaibol tylopeptin B, containing the stable free‐radical 4‐amino‐1‐oxyl‐2,2,6,6,‐tetramethylpiperidine‐4‐carboxylic acid (TOAC) at one or two selected positions, was synthesized by the solid‐phase methodology. A solution conformational analysis performed by FTIR absorption and CD suggests that, in membrane‐mimicking solvents, the labeled tylopeptin B analogues preserve the helical propensity of the parent peptide, with a preference for the α‐helix or the 3₁₀‐helix type depending upon the nature of the solvent. In aqueous environment, the spin‐labeled analogues present a higher content of helical conformation as a consequence of the strong helix promoter effect of the conformationally constrained TOAC residue. We observed a progressive increase of the quenching effect of the nitroxyl radical on the fluorescence of the N‐terminal tryptophan as TOAC replaces the Aib residue at positions 13, 8, and 4, respectively. A membrane permeabilization assay performed on two selected analogues, TOAC⁸‐ and TOAC¹³‐tylopeptin B, showed that the labeled peptides exhibit membrane‐modifying properties comparable with those of the natural peptaibiotic. We conclude that our TOAC paramagnetic analogues of tylopeptin B are good models for a detailed ESR investigation of the mechanism of membrane permeabilization induced by medium‐length peptaibiotics. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Lake‐derived midges increase abundance of shoreline terrestrial arthropods via multiple trophic pathways

Aquatic insects link adjacent ecosystems by transporting nutrients, energy, and material as they move from bodies of water into terrestrial habitats. Insects emerging from streams and rivers are known to benefit arthropod predators such as spiders, but their influence may extend to other arthropod feeding groups as well. We conducted a terrestrial arthropod survey at a series of lakes spanning a strong gradient of midge (Chironomidae, Diptera) emergence. These small, short‐lived insects reach high densities in some areas such that their carcasses litter the ground, and serve as a potential resource for non‐predatory arthropods. Our study revealed that arthropod assemblages in areas of high midge density were significantly different from those with few midges, the result of an increase of all taxa rather than changes in taxonomic composition. Eight of nine terrestrial arthropod taxa sampled showed a strong positive response to the presence of midges including detritivores and herbivores in addition to predators. Taxa that could consume living or dead midges directly responded especially strongly to midge gradients. Our results strongly suggest that midges enter the terrestrial arthropod food web through multiple pathways, increasing numbers of a wide range of arthropods. Furthermore, they emphasize the importance of lakes as sources of aquatic insects that significantly alter processes in the neighboring terrestrial environment.     

Overexpression of cyclin E/CDK2 complexes overcomes FGF-induced cell cycle arrest in the presence of hypophosphorylated Rb proteins

FGF signaling inhibits chondrocyte proliferation and requires the function of the p107 and p130 members of the Rb protein family to execute growth arrest. p107 dephosphorylation plays a critical role in the chondrocyte response to FGF, as overexpression of cyclin D1/CDK4 complexes (the major p107 kinase) in rat chondrosarcoma (RCS) cells overcomes FGF-induced p107 dephosphorylation and growth arrest. In cells overexpressing cyclin D1/CDK4, FGF-induced downregulation of cyclin E/CDK2 activity was absent. To examine the role of cyclin E/CDK2 complexes in mediating FGF-induced growth arrest, this kinase was overexpressed in RCS cells. FGF-induced dephosphorylation of either p107 or p130 was not prevented by overexpressing cyclin E/CDK2 complexes. Unexpectedly, however, FGF-treated cells exhibited sustained proliferation even in the presence of hypophosphorylated p107 and p130. Both pocket proteins were able to form repressive complexes with E2F4 and E2F5 but these repressors were not translocated into the nucleus and therefore were unable to occupy their respective target DNA sites. Overexpressed cyclin E/CDK2 molecules were stably associated with p107 and p130 in FGF-treated cells in the context of E2F repressive complexes. Taken together, our data suggest a novel mechanism by which cyclin E/CDK2 complexes can promote cell cycle progression in the presence of dephosphorylated Rb proteins and provide a novel insight into the key Retinoblastoma/E2F/cyclin E pathway. Our data also highlight the importance of E2F4/p130 complexes for FGF-mediated growth arrest in chondrocytes.