Purification and molecular cloning of antimicrobial peptides from Scots pine seedlings

A novel protocol for rapid and efficient purification of antimicrobial peptides from plant seedlings has been developed. Two peptides with antimicrobial activity, designated p1 and p2, were purified nearly to homogeneity from Scots pine seedlings by a combination of sulfuric acid extraction, ammonium sulfate precipitation, heat-inactivation and ion-exchange chromatography on phosphocellulose. Purified proteins had molecular masses of 11 kDa (p1) and 5.8 kDa (p2) and were identified by mass spectrometry as defensin and lipid-transfer protein, respectively. We demonstrated their growth inhibitory effects against a group of phytopathogenic fungi. Furthermore, we report for the first time molecular cloning and characterization of defensin I cDNA from Scots pine. A cDNA expression library from 7 days Scots pine seedlings was generated and used to isolate a cDNA clone corresponding to Scots pine defensin, termed PsDef1. The full-length coding sequence of PsDef1 is 252 bp in length and has an open reading frame capable to encode a protein of 83 amino residues. The deduced sequence has the typical features of plant defensins, including an endoplasmic reticulum signal sequence of 33 aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5601.6 Da, which correlates well with the results of SDS-PAGE analysis. Finally, the antimicrobial properties of PsDef1 against a panel of fungi and bacteria define it as a member of the morphogenic group of plant defensins. (C) 2009 Elsevier Inc. All rights reserved.

Genetic diversity at the Dhn3 locus in Turkish Hordeum spontaneum populations with comparative structural analyses

We analysed Hordeum spontaneum accessions from 21 different locations to understand the genetic diversity of HsDhn3 alleles and effects of single base mutations on the intrinsically disordered structure of the resulting polypeptide (HsDHN3). HsDHN3 was found to be YSK2-type with a low-frequency 6-aa deletion in the beginning of Exon 1. There is relatively high diversity in the intron region of HsDhn3 compared to the two exon regions. We have found subtle differences in K segments led to changes in amino acids chemical properties. Predictions for protein interaction profiles suggest the presence of a protein-binding site in HsDHN3 that coincides with the K1 segment. Comparison of DHN3 to closely related cereals showed that all of them contain a nuclear localization signal sequence flanking to the K1 segment and a novel conserved region located between the S and K1 segments [E(D/T)DGMGGR]. We found that H. vulgare, H. spontaneum, and Triticum urartu DHN3s have a greater number of phosphorylation sites for protein kinase C than other cereal species, which may be related to stress adaptation. Our results show that the nature and extent of mutations in the conserved segments of K1 and K2 are likely to be key factors in protection of cells.

Mapping the landscape of the lymphocytic choriomeningitis virus stable signal peptide reveals novel functional domains

The stable signal peptide (SSP) of the lymphocytic choriomeningitis virus surface glycoprotein precursor has several unique characteristics. The SSP is unusually long, at 58 amino acids, and contains two hydrophobic domains, and its sequence is highly conserved among both Old and New World arenaviruses. To better understand the functions of the SSP, a panel of point and deletion mutants was created by in vitro mutagenesis to target the highly conserved elements within the SSP. We were also able to confirm critical residues required for separate SSP functions by trans-complementation. Using these approaches, it was possible to resolve functional domains of the SSP. In characterizing our SSP mutants, we discovered that the SSP is involved in several distinct functions within the viral life cycle, beyond translocation of the viral surface glycoprotein precursor into the endoplasmic reticulum lumen. The SSP is required for efficient glycoprotein expression, posttranslational maturation cleavage of GP1 and GP2 by SKI-1/S1P protease, glycoprotein transport to the cell surface plasma membrane, formation of infectious virus particles, and acid pH-dependent glycoprotein-mediated cell fusion.

Delineation and modelling of a nucleolar retention signal in the coronavirus nucleocapsid protein

Unlike nuclear localization signals, there is no obvious consensus sequence for the targeting of proteins to the nucleolus. The nucleolus is a dynamic subnuclear structure which is crucial to the normal operation of the eukaryotic cell. Studying nucleolar trafficking signals is problematic as many nucleolar retention signals (NoRSs) are part of classical nuclear localization signals (NLSs). In addition, there is no known consensus signal with which to inform a study. The avian infectious bronchitis virus (IBV), coronavirus nucleocapsid (N) protein, localizes to the cytoplasm and the nucleolus. Mutagenesis was used to delineate a novel eight amino acid motif that was necessary and sufficient for nucleolar retention of N protein and colocalize with nucleolin and fibrillarin. Additionally, a classical nuclear export signal (NES) functioned to direct N protein to the cytoplasm. Comparison of the coronavirus NoRSs with known cellular and other viral NoRSs revealed that these motifs have conserved arginine residues. Molecular modelling, using the solution structure of severe acute respiratory (SARS) coronavirus N-protein, revealed that this motif is available for interaction with cellular factors which may mediate nucleolar localization. We hypothesise that the N-protein uses these signals to traffic to and from the nucleolus and the cytoplasm.

Decomposition of surface electromyographic signal using Hidden Markov Model

The detection of physiological signals from the motor system (electromyographic signals) is being utilized in the practice clinic to guide the therapist in a more precise and accurate diagnosis of motor disorders. In this context, the process of decomposition of EMG (electromyographic) signals that includes the identification and classification of MUAP (Motor Unit Action Potential) of a EMG signal, is very important to help the therapist in the evaluation of motor disorders. The EMG decomposition is a complex task due to EMG features depend on the electrode type (needle or surface), its placement related to the muscle, the contraction level and the health of the Neuromuscular System. To date, the majority of researches on EMG decomposition utilize EMG signals acquired by needle electrodes, due to their advantages in processing this type of signal. However, relatively few researches have been conducted using surface EMG signals. Thus, this article aims to contribute to the clinical practice by presenting a technique that permit the decomposition of surface EMG signal via the use of Hidden Markov Models. This process is supported by the use of differential evolution and spectral clustering techniques. The developed system presented coherent results in: (1) identification of the number of Motor Units actives in the EMG signal; (2) presentation of the morphological patterns of MUAPs in the EMG signal; (3) identification of the firing sequence of the Motor Units. The model proposed in this work is an advance in the research area of decomposition of surface EMG signals.