Isolation and Characterisation of Major and Minor Collagens from Hyaline Cartilage of Hoki (Macruronus novaezelandiae).

The composition and properties of collagen in teleost (bony fish) cartilage have never been studied. In this study, we aimed to identify and characterise all collagen species in the nasal cartilage of hoki (Macruronus novaezelandiae). Four native collagen species were extracted using two techniques, and isolated with differential salt precipitation. We were able to assign the identity of three of these collagen species on the basis of solubility, SDS-PAGE and amino acid analyses. We found that hoki cartilage contains the major collagen, type II, and the minor collagens, type IX and type XI, which are homologous to those found in mammal and chicken cartilage. Using these extraction protocols, we also isolated a full-length type IX collagen from cartilage for the first time. In addition, we detected a 90 kDa, highly glycosylated collagen that has not been identified in any other species. For each isolate, structural and biochemical characterisations were performed using circular dichroism and Fourier transform infrared spectroscopy analyses, and the thermal denaturation properties were determined. Our results showed that the properties of hoki cartilage-derived collagens are similar to those of collagens in mammalian cartilage, indicating that teleost cartilage could provide biological ingredients for the development of biomaterials to treat cartilage-related illnesses.

Selection and characterization of DNA aptamers for the rat major urinary protein 13 (MUP13) as selective biorecognition elements for sensitive detection of rat pests.

Among invasive mammalian predators, rats represent a major threat, endangering ecosystem functioning worldwide. After rat-control operations, detecting their continued presence or reinvasion requires more sensitive and lower cost detection technologies. Here, we develop a new sensing paradigm by using a specific rat urine biomarker (MUP13) to unambiguously signal the presence of rats. As the first step towards a new remote surveillance technology, aptamers were selected to MUP13 using the Flu-Mag SELEX method. Six aptamer candidates were initially screened by dot blot and two of them (Apt-2.5 and Apt-1.4) exhibited high affinity and specificity. Both aptamers were further characterized by bead-based assay to confirm affinity and selectivity. The lead aptamer candidates were then applied to fluorescence anisotropy (FA) and surface plasmon resonance (SPR)-based biosensor platforms, showing dissociation constants in the nanomolar range and high specificity towards their target. The SPR biosensor had limits of detection of 13.8 and 7.5 nM for Apt-2.5 and Apt-1.4, respectively, which are more than three orders of magnitude lower than the physiological concentrations found in rat urine. Selectivity of the aptamers, when comparing with other major urinary proteins, was excellent, indicating strong efficacy in specific detection of rats. In order to validate the aptamer Apt-2.5 for use with real world samples a FA-based assay was performed on a rat urine sample. The assay showed that the aptamer could detect recombinant MUP13 spiked in filtered urine and the natural MUP13 in unfiltered urine, as a first step into translation to real world application. These are the first known assays to detect and quantify a MUP biomarker of rats.

Expression, purification and characterisation of the recombinant possum lipocalin vulpeculin.

BACKGROUND: Lipocalins are a large family of proteins, which possess a highly conserved eight-stranded antiparallel beta-barrel structure as distinctive trait. This family includes Major Urinary Proteins (MUPs) from rats and mouse, studied for their role in urinary protein-mediated chemosignalling. Vulpeculin has been identified as the most abundant protein in the urine of the common brushtail possum, Trichosurus vulpecula. On the basis of high similarity with other MUPS, we hypothesised that vulpeculin might have a role in possum chemosignalling and investigated its stability and binding ability. METHODS: We expressed and purified vulpeculin using an E.coli-based system and confirmed correct folding by circular dichroism (CD) spectroscopy. Thermal stability was studied by CD and binding properties were investigated using two optical probes N-phenyl-naphthylamine (NPN) and 8-anilino-1-naphthalene sulphonic acid (ANS). RESULTS: CD revealed a secondary structure typical of a predominantly ß-sheet protein, consistent with the beta barrel structure of the lipocalin family. Vulpeculin showed a high level of thermostability, as assessed by CD, exhibiting a small shift in the secondary structure even at 95 °C. Binding assays indicated that vulpeculin cannot accommodate the NPN ligand but can bind ANS. CONCLUSION: The urinary secretion, high degree of sequence similarity with other lipocalins, its beta sheet structure assessed by CD and potential to bind hydrophobic ligands in the hydrophobic cavity or an external hydrophobic pocket, suggest vulpeculin may be involved in possum chemosignalling. GENERAL SIGNIFICANCE: This work represents a first step towards the further investigation of the newly discovered lipocalin and its role in possum chemosignalling.

Intra-fibrillar citric acid crosslinking of marine collagen electrospun nanofibres.

The physical strength of the collagen fibre network in the extracellular matrix is due to the covalent crosslinks between the molecules within the fibres (intra-fibrillar crosslinks). Citric acid was investigated as an agent to introduce crosslinks within marine collagen electrospun fibres. We used collagen films to understand the ideal conditions for citric acid crosslinking. This information was used to develop an optimised method for intra-fibrillar crosslinking in electrospun marine collagen fibres, which increased the stability of these fibres in aqueous environments. The optimised method included a spinning solution containing collagen and citric acid at pH3.5 at high concentrations (260:1 citric acid:collagen molar ratio) coupled with high temperature annealing (165°C), which resulted in the highest intra-fibrillar crosslinking density in electrospun fibres.

Complex formation between recombinant ATP sulfurylase and APS reductase of Allium cepa (L.).

Recombinant ATP sulfurylase (AcATPS1) and adenosine-5'-phosphosulfate reductase (AcAPR1) from Allium cepa have been used to determine if these enzymes form protein-protein complexes in vitro. Using a solid phase binding assay, AcAPR1 was shown to interact with AcATPS1. The AcAPR1 enzyme was also expressed in E. coli as the N-terminal reductase domain (AcAPR1-N) and the C-terminal glutaredoxin domain (AcAPR1-C), but neither of these truncated proteins interacted with AcATPS1. The solid-phase interactions were confirmed by immune-precipitation, where anti-AcATPS1 IgG precipitated the full-length AcAPR1 protein, but not AcAPR1-N and AcAPR1-C. Finally, using the ligand binding assay, full-length AcATPS1 has been shown to bind to membrane-localised full-length AcAPR1. The significance of an interaction between chloroplastidic ATPS and APR in A. cepa is evaluated with respect to the control of the reductive assimilation of sulfate.

Structures and kinetics for plant nucleoside triphosphate diphosphohydrolases support a domain motion catalytic mechanism.

Extracellular nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that hydrolyze extracellular nucleotides to the respective monophosphate nucleotides. In the past 20 years, NTPDases belonging to mammalian, parasitic and prokaryotic domains of life have been discovered, cloned and characterized. We reveal the first structures of NTPDases from the legume plant species Trifolium repens (7WC) and Vigna unguiculata subsp. cylindrica (DbLNP). Four crystal structures of 7WC and DbLNP were determined at resolutions between 1.9 and 2.6 Å. For 7WC, structures were determined for an -apo form (1.89 Å) and with the product AMP (2.15 Å) and adenine and phosphate (1.76 Å) bound. For DbLNP, a structure was solved with phosphate and manganese bound (2.60 Å). Thorough kinetic data and analysis is presented. The structure of 7WC and DbLNP reveals that these NTPDases can adopt two conformations depending on the molecule and co-factor bound in the active site. A central hinge region creates a "butterfly-like" motion of the domains that reduces the width of the inter-domain active site cleft upon molecule binding. This phenomenon has been previously described in Rattus norvegicus and Legionella pneumophila NTPDaseI and Toxoplasma gondii NTPDaseIII suggesting a common catalytic mechanism across the domains of life.

Birinapant, a smac-mimetic with improved tolerability for the treatment of solid tumors and hematological malignancies.

Birinapant (1) is a second-generation bivalent antagonist of IAP proteins that is currently undergoing clinical development for the treatment of cancer. Using a range of assays that evaluated cIAP1 stability and oligomeric state, we demonstrated that 1 stabilized the cIAP1-BUCR (BIR3-UBA-CARD-RING) dimer and promoted autoubiquitylation of cIAP1 in vitro. Smac-mimetic 1-induced loss of cIAPs correlated with inhibition of TNF-mediated NF-κB activation, caspase activation, and tumor cell killing. Many first-generation Smac-mimetics such as compound A (2) were poorly tolerated. Notably, animals that lack functional cIAP1, cIAP2, and XIAP are not viable, and 2 mimicked features of triple IAP knockout cells in vitro. The improved tolerability of 1 was associated with (i) decreased potency against cIAP2 and affinity for XIAP BIR3 and (ii) decreased ability to inhibit XIAP-dependent signaling pathways. The P2' position of 1 was critical to this differential activity, and this improved tolerability has allowed 1 to proceed into clinical studies.

Genotypic variation in sulphur assimilation and metabolism of onion (Allium cepa L.). II: Characterisation of ATP sulphurylase activity.

To investigate the regulation of sulphur (S)-assimilation in onion further at the biochemical level, the pungent cultivar W202A and the milder cultivar Texas Grano 438 PVP (TG) have been grown in S-sufficient (S(+); 4meqS(-1)) or S-deficient (S(-); 0.1meqS(-1)) growth conditions, and tissues excised at the seedling stage (pre-bulbing; ca. 10-weeks-old) and at the mature stage (bulbing; ca. 16-weeks-old). S-supply negatively influenced adenosine-5'-phosphosulphate (APS) reductase (APR) enzyme activity in both cultivars at bulbing only, and a higher abundance of APR was observed in both cultivars at bulbing in response to low S-supply. In contrast, S-supply significantly influenced ATP sulphurylase (ATPS) activity in leaf tissues of W202A only, and only at bulbing, while an increase in abundance in response to high S-supply was observed for both cultivars at bulbing. To investigate the regulation of the ATPS enzyme activity and accumulation further, activity was shown to decrease significantly in roots at bulbing in the S-deficient treatment in both cultivars, a difference that was only supported by western analyses in W202A. Phylogenetic analysis revealed that AcATPS1 groups in a broad monocot clade with the closest sequences identified in Sorghum bicolour, Zea mays and Oryza sativa, but with some support for a divergence of AcATPS1. Detection of ATPS in leaf extracts after two dimensional gel electrophoresis (2-DE) revealed that the protein may undergo post-translational modification with a differential pattern of ATPS accumulation detected in both cultivars over the developmental progression from the seedling to the bulbing stage. Treatment of leaf extracts of W202A to dephosphorylate proteins resulted in the loss of immuno-recognised ATPS spots after 2-DE separation, although enzyme activity was not influenced. These results are discussed in terms of the tiers of control that operate at the biochemical level in the reductive S-assimilation pathway in a S-accumulating species particularly during the high-S-demanding bulbing stage.

Genotypic variation in the sulfur assimilation and metabolism of onion (Allium cepa L.) I. Plant composition and transcript accumulation.

Organosulfur compounds are major sinks for assimilated sulfate in onion (Allium cepa L.) and accumulation varies widely due to plant genotype and sulfur nutrition. In order to better characterise sulfur metabolism phenotypes and identify potential control points we compared plant composition and transcript accumulation of the primary sulfur assimilation pathway in the high pungency genotype 'W202A' and the low pungency genotype 'Texas Grano 438' grown hydroponically under S deficient (S-) and S-sufficient (S+) conditions. Accumulation of total S and alk(en)yl cysteine sulfoxide flavour precursors was significantly higher under S+ conditions and in 'W202A' in agreement with previous studies. Leaf sulfate and cysteine levels were significantly higher in 'W202A' and under S+. Glutathione levels were reduced by S- treatment but were not affected by genotype, suggesting that thiol pool sizes are regulated differently in mild and pungent onions. The only significant treatment effect observed on transcript accumulation in leaves was an elevated accumulation of O-acetyl serine thiol-lyase under S-. By contrast, transcript accumulation of all genes in roots was influenced by one or more treatments. APS reductase transcript level was not affected by genotype but was strongly increased by S-. Significant genotype×S treatment effects were observed in a root high affinity-sulfur transporter and ferredoxin-sulfite reductase. ATP sulfurylase transcript levels were significantly higher under S+ and in 'W202A'.