An endoglucanase, GsCelA, from Geobacilus sp. undergoes an intriguing self- truncation process for enhancing activity and thermostability

An endoglucanase, GsCelA, was isolated and cloned from a thermophilic Geobacillus sp. 70PC53 grown in a rice straw compost in southern Taiwan. It was observed that highly purified GsCelA was able to self-truncate, removing a segment of 53 amino acid residues from its C-terminus. The purified GsCelA does not possess any protease activity and this self-truncation process is insensitive to standard protease inhibitors except EDTA and EGTA. This unique self-truncation process takes place at a temperature higher than 10C with an optimal pH between 6-7, and can be further enhanced with certain divalent ions such as Ca+2 and Mg+2. Crystal structure of GsCelA has a typical TIM-barrel configuration with 8 alpha-helices and 8 beta-strands, but with the presence of a divalent ion. Mutations of amino acids residues surrounding this metal ion do not affect the self-truncation process, but some of these mutants have enhanced enzymatic activities. Mutation of the cleavage site between K315 and G316 does not affect the self-truncation process. However, a deletion of ten amino acids near the cleavage site, i.e. from amino acid 310 to 320, slows down the truncation process but does not block it, and a truncated form around 315 amino acids in length eventually appears. This intriguing observation indicates that the self-truncation process is not site specific, but capable of measuring 315 amino acids from the N-terminus as the cleavage site. This self-truncation process also occurs in the native host of this enzyme, Geobacillus sp. 70PC53, with almost all secreted form of this enzyme being self-truncated. The 53 amino-acid-long C-terminal segment removed by this self-truncation process has binding affinity toward both crystal and amorphous cellulose as well as the s cell walls, yet its sequence bears no apparent homology to any known carbohydrate binding motifs. Various other mutation analyses and the structure-based recombination process, SCHEMA, have been carried out, and both the activity and thermostabilty of this enzyme are further improved. The truncated and improved GsCelA has almost twice the activity as the un-truncated form, and its thermostability is also further enhanced with T50 reaching 86C and TA50 higher than 100C, making this enzyme extremely useful in industrial processes carried out at high temperatures, such as the pre-treatment of cellulosic animal feeds during the final drying step. This research was supported by grants from Taiwan Ministry of Science and Technology and from Academia Sinica

Analysis of two pheromone-responsive conjugative multiresistance plasmids carrying the novel mobile optrA locus from Enterococcus faecalis

Background: The acquired optrA gene, which encodes a ribosomal protection protein of the ABC-F family, can confer cross-resistance to linezolid and florfenicol, posing a serious therapeutic challenge to both human and veterinary medicine. Purpose: The objective of this study was to investigate the two Enterococcus faecalis (E. faecalis) plasmids for their fine structure, their transferability and the presence of mobile antimicrobial resistance loci. Methods: To elucidate their fine structure, the two plasmids were completely sequenced and the sequences analysed. Besides conjugation experiments, inverse PCR assays were conducted to see whether minicircles are produced from the mobile antimicrobial resistance loci. Results: Two pheromone-responsive conjugative optrA-carrying plasmids from E. faecalis, pE211 and pE508 were identified, which can transfer with frequencies of 2.6 ×10−2 and 3.7 ×10−2 (transconjugant per donor), respectively. In both plasmids, optrA was located on the novel mobile optrA locus with different sizes (12,834 bp in pE211 and 7,561 bp in pE508, respectively), flanked by two copies of IS1216 genes in the same orientation. Inverse PCR revealed that circular forms can be generated, consisting of optrA and one copy of IS1216, indicating they are all active. The 77,562 bp plasmid pE211 also carried Tn558 and a mobile bcrABDR locus, and the 84,468 bp plasmid pE508 also harbored the genes fexA, tet(L), tet(O/W/32/O) and a mobile aac(A)-aph(D) locus. Conclusion: The presence of mobile genetic elements in these plasmids renders them flexible and these elements will aid to the persistence and dissemination of these plasmids among enterococci and potentially also other gram-positive bacteria

I worship, so I download? Idol worship, music purchase and piracy by young consumers in Taiwan

Design/methodology/approach – A stratified, two-stage, cluster sampling procedure was applied to a list of high schools obtained from the Ministry of Education in Taiwan. A return rate of 80 per cent yielded 723 usable questionnaires, the data from which were analysed by the LISREL structural equation modelling software. Findings – The results suggest that both social worship and personal worship have a significant and positive impact on the intention to purchase music. However, personal worship has a negative impact on the intention to pirate music while social worship appears to strengthen it. Research limitations/implications – The findings suggest that idol worship is more complex than previously understood. The constructs chosen in this research should be seen only as a snapshot but other variables such as vanity trait, autonomy, romanticism or involvement are not taken into account. Future studies would benefit from inclusion of these variables and a wider geographical scope. Practical implications – The findings contain many implications to help marketing executives and planners better revise their existing marketing and communication strategies to increase their revenue. Originality/value – Existing research has tended to examine the impact of idol worship as a whole on the reduction of music piracy, but overlook the two-dimensional aspects of idol worship, hence ignoring the fact that many music firms have not properly utilised idol worship to deal with the challenges associated with music piracy. The findings broaden existing understanding about the causes of two different dimensions of idol worship and their different impacts on the intention to music piracy

The First Example of Tb-3-Containing Metallopolymer-Type Hybrid Materials with Efficient and High Color-Purity Green Luminescence

In the series of homo-leptic trinuclear complexes {[Ln(3)(L)(4)Cl-4(MeOH)(H2O)]center dot Cl} (Ln = La, 1; Ln = Eu, 2; Ln = Tb, 3 or Ln = Gd, 4) self-assembled from the allyl-modified benzimidazole-type ligand HL (4-allyl-2-(1H-benzo[d]imidazol-2-yl)-6-methoxyphenol) and LnCl(3)center dot 6H(2)O, a suitable energy level match endows efficient green luminescence (Phi(overall) = 72%) of Tb-3-arrayed complex 3. The copolymerization between each of these complex monomers 1-4 and C=C-containing MMA (methyl methacrylate) or NBE (norbornene) shows that degradative chain transfer of the terminal four flexible allyl groups within restrains their radical polymerization with MMA while it does not hinder their effective ring-opening metathesis polymerization (ROMP) with NBE. Thus, two kinds of PMMA-supported doping hybrid materials 1@PMMA, 2@PMMA, 3@PMMA and 4@PMMA and PNBE-supported metallopolymer-type hybrid materials Poly( NBE-1), Poly(NBE-2), Poly(NBE-3) and Poly(NBE-4) are obtained, respectively. Especially for both 3@PMMA and Poly(NBE-3) with high color-purity characteristic green emission of Tb3+ ions, improved physical properties including significantly enhanced luminescence (Phi(overall) = 76% or 83%) are observed, and covalent-bonding endows a higher-concentration self-quenching as compared to physical doping.National Natural Science Foundation 21373160, 91222201, 21173165Program for New Century Excellent Talents in University from the Ministry of Education of China NCET-10-0936Doctoral Program of Higher Education 20116101110003Science and Technology and Innovation Project of Shaanxi Province 2012KTCQ01-37Graduate Innovation and Creativity Fund (Visiting Learner) of Northwest University in P. R. ChinaChemistr

Enhancement of activity and thermostability of a Geobacillus endoglucanase via a unique self-truncation process

The complete utilization of lignocellulosic biomass requires the hydrolysis of cellulose fibers via the synergistic action of three enzymes: exoglucanase, endoglucanase and beta-glucosidase. GsCelA is a 368-amino-acid endoglucanase secreted from a thermophilic Geobacillus sp. 70PC53 that was isolated form a rice straw compost in south Taiwan. GsCelA belongs to the glycosyl hydrolase family 5 and has a typical TIM barrel structure. This enzyme has excellent lignocellulolytic activity and high thermostability, with optimal temperature at 60℃ and pH at 5.0. The purified GsCelA is capable of carrying out a unique self-truncation process at temperature higher than 10 ℃ with optimal pH at 6-7. This self-truncation process is not due to the action of contaminating proteases and it can be suppressed by EDTA and EGTA, and enhanced by divalent metal ions. This self-truncation process also takes place in vivo in Geobacillus sp. 70PC53. The spontaneous or engineered C-terminal truncation up to 60 amino acids from the C-terminus improves GsCelA specific activity and renders the enzyme more thermostable. To investigate the importance of specific amino acids on the enzymatic activity of GsCelA, site-directed mutagenesis and protein engineering approach were employed to alter amino acid residues unique to this enzyme. It was demonstrated that point mutations Y195T , D55S, G288T and D289L replacements increase the activity of this enzyme by 30%

Expressions of Neuregulin 1β and ErbB4 in Prefrontal Cortex and Hippocampus of a Rat Schizophrenia Model Induced by Chronic MK-801 Administration

Recent human genetic studies and postmortem brain examinations of schizophrenia patients strongly indicate that dysregulation of NRG1 and ErbB4 may be important pathogenic factors of schizophrenia. However, this hypothesis has not been validated and fully investigated in animal models of schizophrenia. In this study we quantitatively examined NRG1 and ErbB4 protein expressions by immunohistochemistry and Western blot in the brain of a rat schizophrenia model induced by chronic administration of MK-801 (a noncompetitive NMDA receptor antagonist). Our data showed that NRG1β and ErbB4 expressions were significantly increased in the rat prefrontal cortex and hippocampus but in different subregions. These findings suggest that altered expressions of NRG1 and ErbB4 might be attributed to the schizophrenia. Further study in the role and mechanism of NRG1 and ErbB4 may lead to better understanding of the pathophysiology for this disorder

Long Non Coding RNA MALAT1 Promotes Tumor Growth and Metastasis by Inducing Epithelial-Mesenchymal Transition in Oral Squamous Cell Carcinoma

The prognosis of advanced oral squamous cell carcinoma (OSCC) patients remains dismal, and a better understanding of the underlying mechanisms is critical for identifying effective targets with therapeutic potential to improve the survival of patients with OSCC. This study aims to clarify the clinical and biological significance of metastasis-associated long non-coding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in OSCC. We found that MALAT1 is overexpressed in OSCC tissues compared to normal oral mucosa by real-time PCR. MALAT1 served as a new prognostic factor in OSCC patients. When knockdown by small interfering RNA (siRNA) in OSCC cell lines TSCCA and Tca8113, MALAT1 was shown to be required for maintaining epithelial-mesenchymal transition (EMT) mediated cell migration and invasion. Western blot and immunofluorescence staining showed that MALAT1 knockdown significantly suppressed N-cadherin and Vimentin expression but induced E-cadherin expression in vitro. Meanwhile, both nucleus and cytoplasm levels of β-catenin and NF-κB were attenuated, while elevated MALAT1 level triggered the expression of β-catenin and NF-κB. More importantly, targeting MALAT1 inhibited TSCCA cell-induced xenograft tumor growth in vivo. Therefore, these findings provide mechanistic insight into the role of MALAT1 in regulating OSCC metastasis, suggesting that MALAT1 is an important prognostic factor and therapeutic target for OSCC