Proteomics Identification of Azaspiracid Toxin Biomarkers in Blue Mussels, Mytilus edulis

Azaspiracids are a class of recently discovered algae-derived shellfish toxins. Their distribution globally is on the increase with mussels being most widely implicated in azaspiracid-related food poisoning events. Evidence that these toxins were bound to proteins in contaminated mussels has been shown recently. In the present study characterization of these proteins in blue mussels, Mytilus edulis, was achieved using a range of advanced proteomics tools. Four proteins present only in the hepatopancreas of toxin-contaminated mussels sharing identity or homology with cathepsin D, superoxide dismutase, glutathione S-transferase Pi, and a bacterial flagellar protein have been characterized. Several of the proteins are known to be involved in self-defense mechanisms against xenobiotics or up-regulated in the presence of carcinogenic agents. These findings would suggest that azaspiracids should now be considered and evaluated as potential tumorigenic compounds. The presence of a bacterial protein only in contaminated mussels was an unexpected finding and requires further investigation. The proteins identified in this study should assist with development of urgently required processes for the rapid depuration of azaspiracid-contaminated shellfish. Moreover they may serve as early warning indicators of shellfish exposed to this family of toxins. Molecular & Cellular Proteomics 8: 1811-1822, 2009.

Development of a novel immunobiosensor method for the rapid detection of okadaic acid contamination in shellfish extracts

The mouse bioassay is the methodology that is most widely used to detect okadaic acid (OA) in shellfish samples. This is one of the best-known toxins, and it belongs to the family of marine biotoxins referred to as the diarrhetic shellfish poisons (DSP). Due to animal welfare concerns, alternative methods of toxin detection are being sought. A rapid and specific biosensor immunoassay method was developed and validated for the detection of OA. An optical sensor instrument based on the surface plasmon resonance (SPR) phenomenon was utilised. A polyclonal antibody to OA was raised against OA-bovine thyroglobulin conjugate and OA-N-hydroxy succinimide ester was immobilised onto an amine sensor chip surface. The assay parameters selected for the analysis of the samples were: antibody dilution, 1/750; ratio of antibody to standard, 1:1; volume of sample injected, 25 mu l min(-1); flow rate, 25 mu l min(-1). An assay action limit of 126 ng g(-1) was established by analysing of 20 shellfish samples spiked with OA at the critical concentration of 160 ng g(-1), which is the action limit established by the European Union (EU). At this concentration of OA, the assay delivered coefficient of variations (CVs) of

The Ectocarpus genome and the independent evolution of multicellularity in brown algae

Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related(1). These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity (Fig. 1). We report the 214 million base pair (Mbp) genome sequence of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for brown algae(2-5), closely related to the kelps(6,7) (Fig. 1). Genome features such as the presence of an extended set of light-harvesting and pigment biosynthesis genes and new metabolic processes such as halide metabolism help explain the ability of this organism to cope with the highly variable tidal environment. The evolution of multicellularity in this lineage is correlated with the presence of a rich array of signal transduction genes. Of particular interest is the presence of a family of receptor kinases, as the independent evolution of related molecules has been linked with the emergence of multicellularity in both the animal and green plant lineages. The Ectocarpus genome sequence represents an important step towards developing this organism as a model species, providing the possibility to combine genomic and genetic(2) approaches to explore these and other(4,5) aspects of brown algal biology further.

Kassorins: Novel innate immune system peptides from skin secretions of the African hyperoliid frogs, Kassina maculata and Kassina senegalensis

From defensive skin secretions acquired from two species of African hyperoliid frogs, Kassina maculata and Kassina senegalensis, we have isolated two structurally related, C-terminally amidated tridecapeptides of novel primary structure that exhibit a broad spectrum of biological activity. In reflection of their structural novelty and species of origin, we named the peptides kassorin M (FLEGLLNTVTGLLamide; 1387.8 Da) and kassorin S (FLGGILNTITGLLamide; 1329.8 Da), respectively. The primary structure and organisation of the biosynthetic precursors of kassorins M and S were deduced from cloned skin secretion-derived cDNA. Both open-reading frames encoded a single copy of kassorin M and S, respectively, located at the C-terminus. Kassorins display limited structural similarities to vespid chemotactic peptides (7/13 residues), temporin A (5/13 residues), the N-terminus of Lv-ranaspumin, a foam nest surfactant protein of the frog, Leptodactylus vastus, and an N-terminal domain of the equine sweat surfactant protein, latherin. Both peptides elicit histamine release from rat peritoneal mast cells. However, while kassorin S was found to possess antibacterial activity against Staphylococcus aureus, kassorin M was devoid of such activity. In contrast, kassorin M was found to contract the smooth muscle of guinea pig urinary bladder (EC50 = 4.66 nM) and kassorin S was devoid of this activity. Kassorins thus represent the prototypes of a novel family of peptides from the amphibian innate immune system as occurring in defensive skin secretions.

Role of Delta Np63 gamma in Epithelial to Mesenchymal Transition

Although members of the p63 family of transcription factors are known for their role in the development and differentiation of epithelial surfaces, their function in cancer is less clear. Here, we show that depletion of the Delta Np63 alpha and beta isoforms, leaving only Delta Np63 gamma, results in epithelial to mesenchymal transition (EMT) in the normal breast cell line MCF10A. EMT can be rescued by the expression of the Delta Np63 alpha isoform. We also show that Delta Np63 gamma expressed in a background where all the other Delta Np63 are knocked down causes EMT with an increase in TGF beta-1, -2, and -3 and downstream effectors Smads2/3/4. In addition, a p63 binding site in intron 1 of TGF beta was identified. Inhibition of the TGF beta response with a specific inhibitor results in reversion of EMT in Delta Np63 alpha- and beta-depleted cells. In summary, we show that p63 is involved in inhibiting EMT and reduction of certain p63 isoforms may be important in the development of epithelial cancers.

Association and expression study of synapsin III and schizophrenia

The synapsin III gene, SYN3, which belongs to the family of synaptic vesicle-associated proteins, has been implicated in the modulation of neurotransmitter release and in synaptogenesis, suggesting a potential role in several neuropsychiatric diseases. The human SYN3 gene is located on chromosome 22q12-13, a candidate region implicated in previous linkage studies of schizophrenia. However, association studies of SYN3 and schizophrenia have produced inconsistent results. In this Study, four SYN3 SNPs (rs133945 (-631 C>G), rs133946(-196 G>A), rs9862 and rs1056484) were tested in three sets of totally 3759 samples that comprise 655 affected subjects and 626 controls in the Irish Case-Control Study of Schizophrenia (ICCSS). 1350 samples incorporating 273 pedigrees in the Irish Study of High Density Schizophrenia Families (ISHDSF), and 564 unrelated schizophrenia patients and 564 healthy individuals in a Chinese case-control sample. The expression levels of SYN3 in schizophrenic patients and unaffected controls were compared using postmortem brain cDNAs provided by the Stanley Medical Research Institute (SMRI). There was no significant association in either the Irish or Chinese case-control samples, nor in the combined samples. Consistent with this finding, we did not find any significant difference in allele or haplotype frequencies when we used the pedigree disequilibrium test to analyze the Irish family sample. In the expression Studies, no significant difference (p = 0.507) was observed between patients and controls. Both the association studies and expression studies didn't support a major role for SYN3 in the susceptibility of schizophrenia in Irish and Chinese populations. (C) 2009 Elsevier Ireland Ltd All rights reserved.

CCN3-A key regulator of the hematopoietic compartment

CCN3, a founding member of the CCN family of growth regulators, was linked with hematology in 2003(1) when it was detected in human serum. CCN3 is expressed and secreted by hematopoietic progenitor cells in normal bone marrow. CCN3 acts through the core stem cell signalling pathways including Notch and Bone Morphogenic Protein, connecting CCN3 with the modulation of self-renewal and maturation of a number of cell lineages including hematopoietic, osteogenic and chondrogenic. CCN3 expression is disrupted in Chronic Myeloid Leukemia as a consequence of the BCR-ABL oncogene and allows the leukemic clone to evade growth regulation. In contrast, naive cord blood progenitors undergo enhanced clonal expansion in response to CCN3. Altered CCN3 expression is associated with numerous solid tumors including glioblastoma, melanoma. adrenocortical tumours, prostate cancer and bone malignancies including osteosarcoma. Mature CCN3 protein has five distinct modules and truncated protein variants with altered function are found in many cancers. Regulation by CCN3 is therefore cell type and isoform specific. CCN3 has emerged as a key player in stem cell regulation, hematopoiesis and a crucial component within the bone marrow microenvironment. (c) 2008 Elsevier Ltd. All rights reserved.

Identification of PP2A as a novel interactor and regulator of TRIP-Br1

TRIP-Br proteins area novel family of transcriptional coregulators involved in E2F-mediated cell cycle progression. Three of the four mammalian members of TRIP-Br family, including TRIP-Br1, are known oncogenes. We now report the identification of the Bot regulatory subunit of serine/threonine protein phosphatase 2A (MA) as a novel TRIP-Br1 interactor, based on an affinity binding assay coupled with mass spectrometry. A GST-TRIP-Br1 fusion protein associates with catalytically active PP2A-AB alpha C holoenzyme in vitro. Coimmunoprecipitation confirms this association in vivo. Immunofluorescence staining with a monoclonal antibody against TRIP-Br1 reveals that endogenous TRIP-Br1 and PP2A-B alpha colocalize mainly in the cytoplasm. Consistently, immunoprecipitation followed by immunodetection with anti-phosphoserine antibody suggest that TRIP-Br1 exists in a serine-phosphorylated form. Inhibition of PP2A activity by okadaic acid or transcriptional silencing of the PP2A catalytic subunit by small interfering RNA results in downregulation of total TRIP-Br1 protein levels but upregulation of serine-phosphorylated TRIP-Br1. Overexpression of PP2A catalytic subunit increases TRIP-Br1 protein levels and TRIP-Br1 co-activated E2F1/DP1 transcription. Our data support a model in which association between PP2A-AB alpha C holoenzyme and TRIP-Br1 in vivo in mammalian cells represents a novel mechanism for regulating the level of TRIP-Br1 protooncoprotein. (C) 2008 Elsevier Inc. All rights reserved.

Structure-based design of selective high-affinity telomeric quadruplex-binding ligands.

A library of triazole-based telomeric quadruplex-selective ligands has been developed that mimic an established family of tri-substituted acridine-based ligands, using crystal structure data as a starting-point for computer-based design. Binding affinities, estimated by electrospray mass spectrometry, are in accord with the design concept.

A comparison of nonstaggered compact FDTD schemes for the 3D wave equation

This paper aims at providing a better insight into the 3D approximations of the wave equation using compact finite-difference time-domain (FDTD) schemes in the context of room acoustic simulations. A general family of 3D compact explicit and implicit schemes based on a nonstaggered rectilinear grid is analyzed in terms of stability, numerical error, and accuracy. Various special cases are compared and the most accurate explicit and implicit schemes are identified. Further considerations presented in the paper include the direct relationship with other numerical approaches found in the literature on room acoustic modeling such as the 3D digital waveguide mesh and Yee's staggered grid technique.

Measuring the blame of each formula for inconsistent prioritized knowledge bases.

It is increasingly recognized that identifying the degree of blame or responsibility of each formula for inconsistency of a knowledge base (i.e. a set of formulas) is useful for making rational decisions to resolve inconsistency in that knowledge base. Most current techniques for measuring the blame of each formula with regard to an inconsistent knowledge base focus on classical knowledge bases only. Proposals for measuring the blames of formulas with regard to an inconsistent prioritized knowledge base have not yet been given much consideration. However, the notion of priority is important in inconsistency-tolerant reasoning. This article investigates this issue and presents a family of measurements for the degree of blame of each formula in an inconsistent prioritized knowledge base by using the minimal inconsistent subsets of that knowledge base. First of all, we present a set of intuitive postulates as general criteria to characterize rational measurements for the blames of formulas of an inconsistent prioritized knowledge base. Then we present a family of measurements for the blame of each formula in an inconsistent prioritized knowledge base under the guidance of the principle of proportionality, one of the intuitive postulates. We also demonstrate that each of these measurements possesses the properties that it ought to have. Finally, we use a simple but explanatory example in requirements engineering to illustrate the application of these measurements. Compared to the related works, the postulates presented in this article consider the special characteristics of minimal inconsistent subsets as well as the priority levels of formulas. This makes them more appropriate to characterizing the inconsistency measures defined from minimal inconsistent subsets for prioritized knowledge bases as well as classical knowledge bases. Correspondingly, the measures guided by these postulates can intuitively capture the inconsistency for prioritized knowledge bases.

Biosynthetic Chlorination of the Piperazate Residue in Kutzneride Biosynthesis by KthP

Kutznerides 2 and 8 of the cyclic hexadepsipeptide family of antifungal natural products from the soil actinomycete Kutzneria sp. 744 contain two sets of chlorinated residues, a 6,7-dichlorohexahydropyrroloindole moiety derived from dichlorotryptophan and a 5-chloropiperazate moiety, as well as a methylcyclopropylglycine residue that may arise from isoleucine via a cryptic chlorination pathway. Previous studies identified KtzD, KtzQ and KtzR as three halogenases in the kutzneride pathway but left no candidate for installing the CS chlorine on piperazate. On the basis of analysis of the complete genome sequence of Kutzneria, we now identify a fourth halogenase in the pathway whose gene is separated from the defined kutzneride cluster by 12 open reading frames. KthP (kutzneride halogenase for piperazate) is a mononuclear nonheme iron halogenase that acts on the piperazyl ring tethered by a thioester linkage to the holo forms of thiolation domains. MS analysis of the protein-bound product confirmed chlorination of the piperazate framework from the (3S)- but not the (3R)-piperazyl-S-pantetheinyl thiolation proteins. After thioesterase-mediated release, nuclear magnetic resonance was used to assign the free imino acid as (3S,5S)-5-chloropiperazate, distinct from the 3S,5R stereoisomer reported in the mature kutznerides. These results demonstrate that a fourth halogenase, KthP, is active in the kutzneride biosynthetic pathway and suggest further processing of the (3S,5S)-5-chloropiperazate during subsequent incorporation into the kutzneride depsipeptide frameworks.

The role of the active site residues in human galactokinase: implications for the mechanisms of GHMP kinases.

Galactokinase catalyses the phosphorylation of galactose at the expense of ATP. Like other members of the GHMP family of kinases it is postulated to function through an active site base mechanism in which Asp-186 abstracts a proton from galactose. This asparate residue was altered to alanine and to asparagine by site-directed mutagenesis of the corresponding gene. This resulted in variant enzyme with no detectable galactokinase activity. Alteration of Arg-37, which lies adjacent to Asp-186 and is postulated to assist the catalytic base, to lysine resulted in an active enzyme. However, alteration of this residue to glutamate abolished activity. All the variant enzymes, except the arginine to lysine substitution, were structurally unstable (as judged by native gel electrophoresis in the presence of urea) compared to the wild type. This suggests that the lack of activity results from this structural instability, in addition to any direct effects on the catalytic mechanism. Computational estimations of the pK(a) values of the arginine and aspartate residues, suggest that Arg-37 remains protonated throughout the catalytic cycle whereas Asp-186 has an abnormally high pK(a) value (7.18). Quantum mechanics/molecular mechanics (QM/MM) calculations suggest that Asp-186 moves closer to the galactose molecule during catalysis. The experimental and theoretical studies presented here argue for a mechanism in which the C-1-OH bond in the sugar is weakened by the presence of Asp-186 thus facilitating nucleophilic attack by the oxygen atom on the gamma-phosphorus of ATP.

Collagenolytic Activities of the Major Secreted Cathepsin L Peptidases Involved in the Virulence of the Helminth Pathogen, Fasciola hepatica

The temporal expression and secretion of distinct members of a family of virulence-associated cathepsin L cysteine peptidases (FhCL) correlates with the entry and migration of the helminth pathogen Fasciola hepatica in the host. Thus, infective larvae traversing the gut wall secrete cathepsin L3 (FhCL3), liver migrating juvenile parasites secrete both FhCL1 and FhCL2 while the mature bile duct parasites, which are obligate blood feeders, secrete predominantly FhCL1 but also FhCL2.

Eosinophil peroxidase induces the expression and function of acid-sensing ion channel-3 in allergic rhinitis:in vitro evidence in cultured epithelial cells

BACKGROUND:
Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR.
OBJECTIVES:
The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects.
METHODS:
Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function.
RESULTS:
Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P < 0.02). Immunohistochemistry confirmed significantly higher ASIC-3 protein expression on nasal epithelial cells in AR patients than controls (P < 0.01). Immunoreactivity for EPO+ eosinophils in both nasal epithelium and submucosa was more prominent in AR compared with controls. A mechanism of induction of ASIC-3 expression relevant to AR was suggested by the finding that eosinophil peroxidase (EPO), acting via ERK1/2, induced the expression of ASIC-3 in epithelial cells. Furthermore, using a quantitative functional measure of epithelial cell secretory function in vitro, EPO increased the air-surface liquid depth via an ASIC-dependent chloride secretory pathway.
CONCLUSIONS:
This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.