A screening method for endo-beta-1,4-xylanase substrate selectivity

Endoxylanase (EC 3.2.1.8) substrate selectivity, i.e., its relative activity toward water-unextractable arabinoxylan (WU-AX) and water-extractable arabinoxylan (WE-AX) substrates, is important for its functionality in biotechnological processes such as bread-making and gluten starch separation. A screening method for rapidly determining said substrate selectivity was developed. Endoxylanase activity toward WU-AX was estimated by incubation of insoluble chromogenic substrate with a range of enzyme concentrations in microtiter plates, followed by colorimetric measurement of the dye released in the supernatant. A similar approach using soluble substrate and ethanol precipitation of unhydrolysed AX fragments was used to estimate enzyme activity toward WE-AX. A substrate selectivity factor was defined as the ratio of enzyme activity toward insoluble substrate over enzyme activity toward soluble substrate. A Bacillus subtilis and an Aspergillus aculeatus endoxylanase, known to have widely varying relative rates of hydrolysis of WU-AX and WE-AX, varied most in their substrate selectivity, while the endoxylanases of Aspergillus niger, Trichoderma longibrachiatum, and Trichoderma viride displayed intermediate such relative activities.     

Trypanosome trans-sialidase targets TrkA tyrosine kinase receptor and induces receptor internalization and activation

Trypanosome trans-sialidase (TS) is a sialic acid-transferring enzyme that hydrolyzes alpha2,3-linked sialic acids and transfers them to acceptor molecules. Here we show that a highly purified recombinant TS derived from T. cruzi parasites targets TrkA receptors on TrkA-expressing PC12 cells and colocalizes with TrkA internalization and phosphorylation (pTrkA). Maackia amurensis lectin II (MAL-II) and Sambucus nigra lectin (SNA) block TS binding to TrkA-PC12 cells in a dose-dependent manner with subsequent inhibition of TS colocalization with pTrkA. Cells treated with lectins alone do not express pTrkA. The catalytically inactive mutant TSDeltaAsp98-Glu also binds to TrkA-expressing cells, but is unable to induce pTrkA. TrkA-PC12 cells treated with a purified recombinant alpha2,3-neuraminidase (Streptococcus pneumoniae) express pTrkA. Wild-type TS but not the mutant TSDeltaAsp98-Glu promotes neurite outgrowth in TrkA-expressing PC12 cells. In contrast, these effects are not observed in TrkA deficient PC12nnr5 cells but are reestablished in PC12nnr5 cells stably transfected with TrkA and are significantly blocked by inhibitors of tyrosine kinase (K-252a) and MAP/MEK protein kinase (PD98059). Together these observations suggest for the first time that hydrolysis of sialyl alpha2,3-linked beta-galactosyl residues of TrkA receptors plays an important role in TrkA receptor activation, sufficient to promote cell differentiation (neurite outgrowth) independent of nerve growth factor.     

Coordinated regulation and colocalization of αv integrin and its activating enzyme proprotein convertase PC5 in vivo

Integrin alphav is involved in intracellular-extracellular signaling important for cytoskeleton alterations and control of cell movement. In vitro experiments indicate that the integrin alphav-subunit undergoes post-translational endoproteolytic cleavage. This type of activation requires the presence of suitable kexin/subtilisin-like proprotein convertases. In vitro experiments have demonstrated that, among several proprotein convertases, PC5A, and to a threefold lesser extent furin, can activate alphav integrin. The biological significance of these in vitro data would be further supported by a coexpression and coordinated regulation of the gene expression of alphav integrin and its activating enzyme PC5 in vivo. In the present study we investigated the regulation of alphav integrin and PC5 following balloon injury in vivo. Comparative immunocytochemistry revealed a coordinated regulation of alphav integrin and PC5 during vascular remodeling in rodents. Integrin alphav was found to be upregulated in PCNA-positive, proliferating vascular smooth muscle cells. Northern blots revealed no significant regulation of furin mRNA, whereas PC5A mRNA increased during vascular remodeling, suggesting that PC5 is the major convertase during neointima formation in vivo. Incubation of vascular smooth muscle cells with the Golgi-disturbing agent brefeldin A inhibited alphav integrin maturation, indicating that endoproteolytic cleavage occurs in the trans-Golgi network, were PC5 is localized. Thus, the present study further supports the concept that activation of alphav integrin occurs in the trans-Golgi network in vascular smooth muscle cells and involves PC5.     

Molecular identification of wheat endoxylanase inhibitor TAXI-I1, member of a new class of plant proteins

Triticum aestivum endoxylanase inhibitors (TAXIs) are wheat proteins that inhibit family 11 endoxylanases commonly used in different (bio)technological processes. Here, we report on the identification of the TAXI-I gene which encodes a mature protein of 381 amino acids with a calculated molecular mass of 38.8 kDa. When expressed in Escherichia coli, the recombinant protein had the specificity and inhibitory activity of natural TAXI-I, providing conclusive evidence that the isolated gene encodes an endoxylanase inhibitor. Bioinformatical analysis indicated that no conserved domains nor motifs common to other known proteins are present. Sequence analysis revealed similarity with a glycoprotein of carrot and with gene families in Arabidopsis thaliana and rice, all with unknown functions. Our data indicate that TAXI-I belongs to a newly identified class of plant proteins for which a molecular function as glycoside hydrolase inhibitor can now be suggested.     

De novo production of (+)-aristolochene by sporulated surface cultures of Penicillium roqueforti

The de novo production of the fungal metabolite, (+)-aristolochene by sporulated surface cultures of Penicillium roqueforti is reported for the first time. The biosynthesis of fungal volatiles by various sporulated surface cultures was monitored by solid phase micro-extraction (SPME). When comparing malt extract agar with sabouraud dextrose agar, the highest yield of the fungal metabolite (0.04 mg/ml of culture) was obtained with the latter medium. The biosynthesis of (+)-aristolochene showed a maximum during the fourth day after inoculation.     

MARAN: normalizing micro-array data

SUMMARY: MARAN is a web-based application for normalizing microarray data. MARAN comprises a generic ANOVA model, an option for Loess fitting prior to ANOVA analysis, and a module for selecting genes with significantly changing expression. AVAILABILITY: http://www.esat.kuleuven.ac.be/maran/.     

Enhanced endogenous bone morphogenetic protein signaling protects against bleomycin induced pulmonary fibrosis

Background

Effective treatments for fibrotic diseases such as idiopathic pulmonary fibrosis are largely lacking. Transforming growth factor beta (TGFβ) plays a central role in the pathophysiology of fibrosis. We hypothesized that bone morphogenetic proteins (BMP), another family within the TGFβ superfamily of growth factors, modulate fibrogenesis driven by TGFβ. We therefore studied the role of endogenous BMP signaling in bleomycin induced lung fibrosis.

Methods

Lung fibrosis was induced in wild-type or noggin haploinsufficient (Nog^+/LacZ) mice by intratracheal instillation of bleomycin, or phosphate buffered saline as a control. Invasive pulmonary function tests were performed using the flexiVent® SCIREQ system. The mice were sacrificed and lung tissue was collected for analysis using histopathology, collagen quantification, immunohistochemistry and gene expression analysis.

Results

Nog^+/LacZ mice are a known model of increased BMP signaling and were partially protected from bleomycin-induced lung fibrosis with reduced Ashcroft score, reduced collagen content and preservation of pulmonary compliance. In bleomycin-induced lung fibrosis, TGFβ and BMP signaling followed an inverse course, with dynamic activation of TGFβ signaling and repression of BMP signaling activity.

Conclusions

Upon bleomycin exposure, active BMP signaling is decreased. Derepression of BMP signaling in Nog^+/LacZ mice protects against bleomycin-induced pulmonary fibrosis. Modulating the balance between BMP and TGFβ, in particular increasing endogenous BMP signals, may therefore be a therapeutic target in fibrotic lung disease.     

The OST‐complex as target for RNAi‐based pest control in Nilaparvata lugens

RNAi‐based pest control strategies are emerging as environment friendly and species‐specific alternatives for the use of conventional pesticides. Because N‐glycosylation is important for many biological processes, such as growth and development, the early steps of protein N‐glycosylation are promising targets for an RNAi‐based pest control strategy. Through injection of dsRNAs, the expression of the catalytic subunits of the oligosaccharyl transferase complex was efficiently silenced in nymphs of the notorious rice pest insect Nilaparvata lugens. Silencing of both STT3 isoforms resulted in a high mortality of the N. lugens nymphs. However, our data reveals the occurrence of a functional redundancy between the two isoforms when silencing only one of the isoforms. These observations confirm the potential to use the early genes in the N‐glycosylation pathway as targets for an RNAi‐based pest control strategy. In addition, the existence of a functional redundancy between the two STT3 isoforms presents a factor which one must take into account when designing RNAi‐based approaches.     

TLXI, a novel type of xylanase inhibitor from wheat (Triticum aestivum) belonging to the thaumatin family

Wheat (Triticum aestivum) contains a previously unknown type of xylanase (EC 3.2.1.8) inhibitor, which is described in the present paper for the first time. Based on its >60% similarity to TLPs (thaumatin-like proteins) and the fact that it contains the Prosite PS00316 thaumatin family signature, it is referred to as TLXI (thaumatin-like xylanase inhibitor). TLXI is a basic (pI> or =9.3 in isoelectric focusing) protein with a molecular mass of approx. 18-kDa (determined by SDS/PAGE) and it occurs in wheat with varying extents of glycosylation. The TLXI gene sequence encodes a 26-amino-acid signal sequence followed by a 151-amino-acid mature protein with a calculated molecular mass of 15.6-kDa and pI of 8.38. The mature TLXI protein was expressed successfully in Pichia pastoris, resulting in a 21-kDa (determined by SDS/PAGE) recombinant protein (rTLXI). Polyclonal antibodies raised against TLXI purified from wheat react with epitopes of rTLXI as well as with those of thaumatin, demonstrating high structural similarity between these three proteins. TLXI has a unique inhibition specificity. It is a non-competitive inhibitor of a number of glycoside hydrolase family 11 xylanases, but it is inactive towards glycoside hydrolase family 10 xylanases. Progress curves show that TLXI is a slow tight-binding inhibitor, with a K(i) of approx. 60-nM. Except for zeamatin, an alpha-amylase/trypsin inhibitor from maize (Zea mays), no other enzyme inhibitor is currently known among the TLPs. TLXI thus represents a novel type of inhibitor within this group of proteins.