In silico and in vitro studies to elucidate the role of Cu2+ and galanthamine as the limiting step in the amyloid beta (1–42) fibrillation process

The formation of fibrils and oligomers of amyloid beta (Aβ) with 42 amino acid residues (Aβ_1–42) is the most important pathophysiological event associated with Alzheimer''s disease (AD). The formation of Aβ fibrils and oligomers requires a conformational change from an α-helix to a β-sheet conformation, which is encouraged by the formation of a salt bridge between Asp 23 or Glu 22 and Lys 28. Recently, Cu²⁺ and various drugs used for AD treatment, such as galanthamine (Reminyl®), have been reported to inhibit the formation of Aβ fibrils. However, the mechanism of this inhibition remains unclear. Therefore, the aim of this work was to explore how Cu²⁺ and galanthamine prevent the formation of Aβ_1–42 fibrils using molecular dynamics (MD) simulations (20 ns) and in vitro studies using fluorescence and circular dichroism (CD) spectroscopies. The MD simulations revealed that Aβ_1–42 acquires a characteristic U-shape before the α-helix to β-sheet conformational change. The formation of a salt bridge between Asp 23 and Lys 28 was also observed beginning at 5 ns. However, the MD simulations of Aβ₁₋₄₂ in the presence of Cu²⁺ or galanthamine demonstrated that both ligands prevent the formation of the salt bridge by either binding to Glu 22 and Asp 23 (Cu²⁺) or to Lys 28 (galanthamine), which prevents Aβ₁₋₄₂ from adopting the U-characteristic conformation that allows the amino acids to transition to a β-sheet conformation. The docking results revealed that the conformation obtained by the MD simulation of a monomer from the 1Z0Q structure can form similar interactions to those obtained from the 2BGE structure in the oligomers. The in vitro studies demonstrated that Aβ remains in an unfolded conformation when Cu²⁺ and galanthamine are used. Then, ligands that bind Asp 23 or Glu 22 and Lys 28 could therefore be used to prevent β turn formation and, consequently, the formation of Aβ fibrils.     

Length–weight and length–length relationships of 139 Indo‐Pacific fish species (Teleostei) from the Davao Gulf,Philippines

Length–weight relationships (LWRs) of 139 coral reef and pelagic fish species (representing 34 fish families) were calculated based on 3806 individuals measured at local fish markets near the Davao Gulf in the southern Philippines during weekly visits between March 2009 and July 2011, as well as in June 2012. Fishes were caught with a variety of fishing methods, corroborated by abrasions and injuries. Forty‐seven of 139 LWRs were firstly reported and new to science. The mean slope b of the LWRs was 3.035, indicating that the majority of studied species followed isometric growth. Standard length – total length relationships were calculated for all measured fish species. Additionally, standard length – fork length relationships are presented for 108 species. Moreover, fifteen new records of maximum fish length and weight are reported.     

Kinase activity and genetic characterization of a growth related antigen of Drosophila

The Drosophila developmental antigen recognized by the monoclonal antibody F7D6 is expressed in dividing embryonic and imaginal cells but is lost from all differentiating tissues except electrogenic cells of the nervous system and spontaneously contracting muscles. The 63 kDa antigen is associated with the inner surface of plasma membranes and is expressed in several classes of tumorous mutants of Drosophila. The monoclonal antibody was used for immunoprecipitating the antigen for biochemical characterization and for screening expression vector cDNA libraries. Here we report that this oncodevelopmental antigen is a phosphoprotein and a serine-threonine specific protein kinase. A 1.6 kb cDNA isolated by immunological screening of an ovarian library hybridized to a single band on polytene chromosomes, localizing the gene to 72F on the left arm of the third chromosome. Immunofluorescence assays of deficiency stocks in the region confirmed the location of the gene and identity of the cDNA clone, and mapped the gene between the left breakpoints of Df(3L) st100.62 and Df(3L) stj7, i.e., between 72F3-7 and 73A1-2. The biochemical and genetic properties indicate that this is a novel growth-related kinase of Drosophila.     

Targeted inactivation of the mecB gene, encoding cystathionine-gamma-lyase, shows that the reverse transsulfuration pathway is required for high-level cephalosporin biosynthesis in Acremonium chrysogenum C10 but not for methionine induction of the cephalosporin genes

Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of the mecB gene by the double-marker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive mecB gene by Southern blot analysis and no cystathionine-gamma-lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain A. chrysogenum C10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in A. chrysogenum derives from the autotrophic (SH(2)) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB mutants, indicating that the induction effect is not mediated by cystathionine-gamma-lyase.     

Generation,annotation, and analysis of ESTs from four different <Emphasis Type="Italic">Trichoderma</Emphasis> strains grown under conditions related to biocontrol

The functional genomics project “TrichoEST” was developed focused on different taxonomic groups of Trichoderma with biocontrol potential. Four cDNA libraries were constructed, using similar growth conditions, from four different Trichoderma strains: Trichoderma longibrachiatum T52, Trichoderma asperellum T53, Trichoderma virens T59, and Trichoderma sp. T78. In this study, we present the analysis of the 8,160 expressed sequence tags (ESTs) generated. Each EST library was independently assembled and 1,000–1,300 unique sequences were identified in each strain. First, we queried our collection of ESTs against the NCBI nonredundant database using the BLASTX algorithm. Moreover, using the Gene Ontology hierarchy, we performed the annotation of 40.9% of the unique sequences. Later, based on the EST abundance, we examined the highly expressed genes in the four strains. A hydrophobin was found as the gene expressed at the highest level in two of the strains, but we also found that other unique sequences similar to the HEX1, QID3, and NMT1 proteins were highly represented in at least two of the Trichoderma strains. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Overexpression of the trichodiene synthase gene tri5 increases trichodermin production and antimicrobial activity in Trichoderma brevicompactum

Trichoderma brevicompactum produces trichodermin, a simple trichothecene-type toxin that shares the first steps of the sesquiterpene biosynthetic pathway with other phytotoxic trichothecenes from Fusarium spp. Trichodiene synthase catalyses the conversion of farnesyl pyrophosphate to trichodiene and it is encoded by the tri5 gene that was cloned and analysed functionally by homologous overexpression in T. brevicompactum. tri5 expression was up-regulated in media with glucose, H(2)O(2) or glycerol. tri5 repression was observed in cultures supplemented with the antioxidants ferulic acid and tyrosol. Acetone extracts of tri5-overexpressing transformants displayed higher antifungal activity than those from the wild-type. Chromatographic and spectroscopic analyses revealed that tri5 overexpression led to an increased production of trichodermin and tyrosol. Agar diffusion assays with these two purified metabolites from the tri5-overexpressing transformant T. brevicompactum Tb41tri5 showed that only trichodermin had antifungal activity against Saccharomyces cerevisiae, Kluyveromyces marxianus, Candida albicans, Candida glabrata, Candida tropicalis and Aspergillus fumigatus, in most cases such activity being higher than that observed for amphotericin B and hygromycin. Our results point to the significant role of tri5 in the production of trichodermin and in the antifungal activity of T. brevicompactum.     

Permanent genetic resources added to Molecular Ecology Resources Database 1 February 2011-31 March 2011

This article documents the addition of 111 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acipenser oxyrinchus desotoi, Anopheles nuneztovari sensu lato, Asellus aquaticus, Calopteryx splendens, Calopteryx virgo, Centaurea aspera, Centaurea seridis, Chilina dombeyana, Proctoeces cf. lintoni and Pyrenophora teres f. teres.     

A comparison of the phenotypic and genetic stability of recombinant Trichoderma spp. generated by protoplast- and Agrobacterium-mediated transformation

Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 transformants/microg DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 transformants/107 spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transformants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.     

Involvement of Trichoderma trichothecenes in the biocontrol activity and induction of plant defense-related genes

Trichoderma species produce trichothecenes, most notably trichodermin and harzianum A (HA), by a biosynthetic pathway in which several of the involved proteins have significant differences in functionality compared to their Fusarium orthologues. In addition, the genes encoding these proteins show a genomic organization differing from that of the Fusarium tri clusters. Here we describe the isolation of Trichoderma arundinaceum IBT 40837 transformants which have a disrupted or silenced tri4 gene, a gene encoding a cytochrome P450 monooxygenase that oxygenates trichodiene to give rise to isotrichodiol, and the effect of tri4 gene disruption and silencing on the expression of other tri genes. Our results indicate that the tri4 gene disruption resulted in a reduced antifungal activity against Botrytis cinerea and Rhizoctonia solani and also in a reduced ability to induce the expression of tomato plant defense-related genes belonging to the salicylic acid (SA) and jasmonate (JA) pathways against B. cinerea, in comparison to the wild-type strain, indicating that HA plays an important function in the sensitization of Trichoderma-pretreated plants against this fungal pathogen. Additionally, the effect of the interaction of T. arundinaceum with B. cinerea or R. solani and with tomato seedlings on the expressions of the tri genes was studied.     

Mimicking a p53‐MDM2 interaction based on a stable immunoglobulin‐like domain scaffold

Antibodies recognize protein targets with great affinity and specificity. However, posttranslational modifications and the presence of intrinsic disulfide‐bonds pose difficulties for their industrial use. The immunoglobulin fold is one of the most ubiquitous folds in nature and it is found in many proteins besides antibodies. An example of a protein family with an immunoglobulin‐like fold is the Cysteine Protease Inhibitors (ICP) family I42 of the MEROPs database for protease and protease inhibitors. Members of this protein family are thermostable and do not present internal disulfide bonds. Crystal structures of several ICPs indicate that they resemble the Ig‐like domain of the human T cell co‐receptor CD8α As ICPs present 2 flexible recognition loops that vary accordingly to their targeted protease, we hypothesize that members of this protein family would be ideal to design peptide aptamers that mimic protein‐protein interactions. Herein, we use an ICP variant from Entamoeba histolytica (EhICP1) to mimic the interaction between p53 and MDM2. We found that a 13 amino‐acid peptide derived from p53 can be introduced in 2 variable loops (DE, FG) but not the third (BC). Chimeric EhICP1‐p53 form a stable complex with MDM2 at a micromolar range. Crystal structure of the EhICP1‐p53(FG)‐loop variant in complex with MDM2 reveals a swapping subdomain between 2 chimeric molecules, however, the p53 peptide interacts with MDM2 as in previous crystal structures. The structural details of the EhICP1‐p53(FG) interaction with MDM2 resemble the interaction between an antibody and MDM2.