Identification of Domains and Amino Acids Essential to the Collagen Galactosyltransferase Activity of GLT25D1

Collagen is modified by hydroxylation and glycosylation of hydroxylysine residues. This glycosylation is initiated by the β1,O galactosyltransferases GLT25D1 and GLT25D2. The structurally similar protein cerebral endothelial cell adhesion molecule CEECAM1 was previously reported to be inactive when assayed for collagen glycosyltransferase activity. To address the cause of the absent galactosyltransferase activity, we have generated several chimeric constructs between the active human GLT25D1 and inactive human CEECAM1 proteins. The assay of these chimeric constructs pointed to a short central region and a large C-terminal region of CEECAM1 leading to the loss of collagen galactosyltransferase activity. Examination of the three DXD motifs of the active GLT25D1 by site-directed mutagenesis confirmed the importance of the first (amino acids 166–168) and second motif (amino acids 461–463) for enzymatic activity, whereas the third one was dispensable. Since the second DXD motif is incomplete in CEECAM1, we have restored the motif by introducing the substitution S461D. This change did not restore the activity of the C-terminal region, thereby showing that additional amino acids were required in this C-terminal region to confer enzymatic activity. Finally, we have introduced the substitution Q471R-V472M-N473Q-P474V in the CEECAM1-C-terminal construct, which is found in most animal GLT25D1 and GLT25D2 isoforms but not in CEECAM1. This substitution was shown to partially restore collagen galactosyltransferase activity, underlining its importance for catalytic activity in the C-terminal domain. Because multiple mutations in different regions of CEECAM1 contribute to the lack of galactosyltransferase activity, we deduced that CEECAM1 is functionally different from the related GLT25D1 protein

Web Usage Mining in Tourism — A Query Term Analysis and Clustering Approach

According to current research, one of the most promising applications for web usage mining (WUM) is in identifying homogenous user subgroups (Liu, 2008). This paper presents a prototypical workflow and tools for analyzing user sessions to extract business intelligence hidden in web log data. By considering a leading Swedish destination gateway, we demonstrate how query term analysis in combination with session clustering can be utilized to effectively explore the information needs of website users. The system thus overcomes many of the limitations of typical web site analysis tools that only offer general statistics and ignore the opportunities offered by unsupervised learning techniques

Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae

Identification and characterization of collagen glycosyltransferases of human and viral origin

Collagens are a superfamily of glycoproteins mainly found in the extracellular matrix. They are the most abundant proteins in the human body. Collagens are characterized by a right handed triple helix formed out of three lefthanded α-chains representing repeats of the motif G-X-Y, where (hydroxy)proline and (hydroxy)lysine are often found at positions X and Y. To act as a functional collagen, selected hydroxylysine residues have to be further modified by the addition of either galactose or the disaccharide glucosylgalactose. This glycosylation of collagen takes place in the endoplasmic reticulum before the formation of the triple helix and is mediated by specific β(1-O) galactosyl- and α(1-2) glucosyltransferase enzymes. The molecular nature of these glycosyltransferases has remained unknown to date. The present study describes the identification of collagen galactosyltransferase enzymes using a strategy based on affinity chromatography and protein sequencing by mass spectrometry. Three structurally related candidate genes were cloned and expressed in Sf9 insect cells using the baculovirus system. Two of the three candidate glycosyltransferases (GLT25D1 and GLT25D2) were confirmed to be active collagen galactosyltransferases. The collagen galactosyltransferase genes are differentially expressed in human tissues, suggesting that these enzymes may show preference for different types of collagens or contribute to the varying extent of collagen glycosylation throughout tissues. This was supported by showing a selective preference of GLT25D1 and GLT25D2 for collagen type III and collagen type IV acceptors. GLT25D1 showed a higher enzymatic activity on deglycosylated collagen type I to type V than GLT25D2. Collagen glycosylation is conserved in animals and collagen is also found in several prokaryotic genomes. Proteins sharing structural similarity with the collagen galactosyltransferases have been found in prokaryotes and even in virus. The Acanthamoeba polyphaga mimivirus has been detected as an unique member of the nucleo-cytoplasmic large DNA virus family being clearly a virus but also showing features never seen before in viruses. Mimivirus is the largest known DNA virus with a 1.2 Mbp linear dsDNA genome. It was reported that mimivirus encodes eight proteins with a collagen triple helix motif. These collagens are most probably found in the fibrils of the virus capsid. These fibrils cover the whole icosahedral virus capsid, which are specific for mimivirus. As the Gram staining of mimivirus is positive, it is supposed that the viral fibrils are glycosylated. This suggests that the mimiviral collagens might be post-translationally modified by hydroxylation and subsequent glycosylation. In this study, the protein L230 was identified as a mimiviral collagen glucosyltransferase transferring glucose on the acceptor hydroxylysine on animal and on mimiviral collagens. This addition of glucose on the acceptor hydroxylysine in collagen has not been reported up to now. It seems that collagen glycosylation in mimivirus is different than in animal collagens. In conclusion, this work reports the identification and characterization of the two human collagen galactosyltransferases GLT25D1 and GLT25D2 and the identification of the mimiviral collagen glucosyltransferase L230

eFitness of Destination Websites – Still more to go

Website evaluation has been examined by researchers for many years resulting in various suggestions concerning which key success factors to consider and how to proceed. However, due to advances in technology there is a need to continuously reconsider dimensions and criteria. This study evaluates the performance of 40 DMO websites from Austria and Switzerland applying an updated catalogue of criteria called eFitness Check-Update. Results show that DMOs have room for improvements regarding Interactivity and Legal Aspects. Furthermore, Common State-, Competitive Advantage-, and Future Potential Dimensions are revealed, and Austrian and Swiss results are compared

Distribution Channels and Management in the Swiss Hotel Sector

This research investigates how the hospitality sector in Switzerland has embraced the new world of (online) distribution. It analyses the role of existing distribution channels, and gives insights into channel management methods used. Data is gathered through an online survey among Swiss hotels resulting in 196 usable questionnaires. Findings show that direct booking channels remain the dominant sales tools in Switzerland, although their proportion in the distribution mix has been decreasing steadily in the last years. The Internet Distribution System channel reaches 16.4% and thus is the channel with the highest growth rate in 2011. A cluster analysis on multi-channel distribution strategies results in four groups: multi-channel distributors, electronic distributors, real time distributors, and traditional distributors. Further, it is shown that more than half of the hotels manage rates and availabilities manually and only one out of four hotels has implemented a channel manager able to manage different channels

Mimivirus collagen is modified by bifunctional lysyl hydroxylase and glycosyltransferase enzyme

Collagens, the most abundant proteins in animals, are modified by hydroxylation of proline and lysine residues and by glycosylation of hydroxylysine. Dedicated prolyl hydroxylase, lysyl hydroxylase, and collagen glycosyltransferase enzymes localized in the endoplasmic reticulum mediate these modifications prior to the formation of the collagen triple helix. Whereas collagen-like proteins have been described in some fungi, bacteria, and viruses, the post-translational machinery modifying collagens has never been described outside of animals. We demonstrate that the L230 open reading frame of the giant virus Acanthamoeba polyphaga mimivirus encodes an enzyme that has distinct lysyl hydroxylase and collagen glycosyltransferase domains. We show that mimivirus L230 is capable of hydroxylating lysine and glycosylating the resulting hydroxylysine residues in a native mimivirus collagen acceptor substrate. Whereas in animals from sponges to humans the transfer of galactose to hydroxylysine in collagen is conserved, the mimivirus L230 enzyme transfers glucose to hydroxylysine, thereby defining a novel type of collagen glycosylation in nature. The presence of hydroxylysine in mimivirus proteins was confirmed by amino acid analysis of mimivirus recovered from A. polyphaga cultures. This work shows for the first time that collagen post-translational modifications are not confined to the domains of life. The utilization of glucose instead of the galactose found throughout animals as well as a bifunctional enzyme rather than two separate enzymes may represent a parallel evolutionary track in collagen biology. These results suggest that giant viruses may have contributed to the evolution of collagen biology

Evidence that anti-muscarinic antibodies in Sjögren's syndrome recognise both M3R and M1R

Inhibitory anti-muscarinic receptor type 3 (M3R) antibodies may contribute to the pathogenesis of Sjögren's syndrome (SS), and putative anti-M3R blocking antibodies in intravenous immunoglobulin (IVIg) have been suggested as a rationale for treatment with IVIg. We investigated the presence of subtype-specific anti-MR autoantibodies in healthy donor and SS sera using MR-transfected whole-cell binding assays as well as M1R and M3R peptide ELISAs. Control antibodies against the second extracellular loop of the M3R, a suggested target epitope, were induced in rabbits and found to be cross-reactive on the peptides M3R and M1R. The rabbit antibodies had neither an agonistic nor an antagonistic effect on M3R-dependent ERK1/2 signalling. Only one primary SS (out of 5 primary SS, 2 secondary SS and 5 control sera) reacted strongly with M3R transfected cells. The same SS serum also reacted strongly with M1R and M2R transfectants, as well as M1R and two different M3R peptides. Strong binding to M1R and low-level activities against M3R peptides were observed both in SS and control sera. IVIg showed a strong reactivity against all three peptides, especially M1R. Our results indicate that certain SS individuals may have antibodies against M1R, M2R and M3R. Our results also suggest that neither the linear M3R peptide nor M3R transfectants represent suitable tools for discrimination of pathogenic from natural autoantibodies in SS

A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability

Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-l-alanine amidase domain from the endolysin of the thermophilic bacteriophage phi GVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage phi CP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50 degrees C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production

The mechanism of action of the antidiuretic peptide Tenmo ADFa in Malpighian tubules of Aedes aegypti.

The mechanism of action of Tenebrio molitor antidiuretic factor \u27a\u27 (Tenmo ADFa) was explored in isolated Malpighian tubules of Aedes aegypti. In the Ramsay assay of fluid secretion, Tenmo ADFa (10(-9) mol l(-1)) significantly inhibited the rate of fluid secretion from 0.94 nl min(-1) to 0.44 nl min(-1) without significant effects on the concentrations of Na+, K+ and Cl- in secreted fluid. In isolated perfused tubules, Tenmo ADFa had no effect on the transepithelial voltage (Vt) and resistance (Rt). In principal cells of the tubule, Tenmo ADFa had no effect on the basolateral membrane voltage (Vbl) and the input resistance of principal cells (Rpc). Tenmo ADFa significantly increased the intracellular concentration of cyclic guanosine monophosphate (cGMP) from 2.9 micromol l(-1) (control) to 7.4 micromol l(-1). A peritubular [cGMP] of 20 micromol l(-1) duplicated the antidiuretic effects of Tenmo ADFa without inducing electrophysiological effects. In contrast, 500 micromol l(-1) cGMP significantly depolarized V(bl), hyperpolarized Vt, and reduced Rt and Rpc, without increasing antidiuretic potency beyond that of 20 micromol l(-1) cGMP. A plot of peritubular cGMP concentration vs Vbl revealed a steep dose-response between 300 micromol l(-1) and 700 micromol l(-1) with an EC50 of 468 micromol l(-1). These observations suggest a receptor- and cGMP-mediated mechanism of action of Tenmo ADFa. Tenmo ADFa and physiological concentrations of cGMP (\u3c 20 micromol l(-1)) reduce the rate of isosmotic fluid secretion by quenching electroneutral transport systems. The inhibition reveals that as much as 50% of the normal secretory solute and water flux can stem from electrically silent mechanisms in this highly electrogenic epithelium