Direct binding of phosphatidylglycerol at specific sites modulates desensitization of a ligand-gated ion channel

Pentameric ligand-gated ion channels (pLGICs) are essential determinants of synaptic transmission, and are modulated by specific lipids including anionic phospholipids. The exact modulatory effect of anionic phospholipids in pLGICs and the mechanism of this effect are not well understood. Using native mass spectrometry, coarse-grained molecular dynamics simulations and functional assays, we show that the anionic phospholipid, 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), preferentially binds to and stabilizes the pLGIC, Erwinia ligand-gated ion channel (ELIC), and decreases ELIC desensitization. Mutations of five arginines located in the interfacial regions of the transmembrane domain (TMD) reduce POPG binding, and a subset of these mutations increase ELIC desensitization. In contrast, a mutation that decreases ELIC desensitization, increases POPG binding. The results support a mechanism by which POPG stabilizes the open state of ELIC relative to the desensitized state by direct binding at specific sites

Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) for diagnosis of natural infection with canine distemper virus

<p>Abstract</p> <p>Background</p> <p>Canine distemper virus (CDV) is present worldwide and produces a lethal systemic infection of wild and domestic <it>Canidae</it>. Pre-existing antibodies acquired from vaccination or previous CDV infection might interfere the interpretation of a serologic diagnosis method. In addition, due to the high similarity of nucleic acid sequences between wild-type CDV and the new vaccine strain, current PCR derived methods cannot be applied for the definite confirmation of CD infection. Hence, it is worthy of developing a simple and rapid nucleotide-based assay for differentiation of wild-type CDV which is a cause of disease from attenuated CDVs after vaccination. High frequency variations have been found in the region spanning from the 3'-untranslated region (UTR) of the matrix (M) gene to the fusion (F) gene (designated M-F UTR) in a few CDV strains. To establish a differential diagnosis assay, an amplification refractory mutation analysis was established based on the highly variable region on M-F UTR and F regions.</p> <p>Results</p> <p>Sequences of frequent polymorphisms were found scattered throughout the M-F UTR region; the identity of nucleic acid between local strains and vaccine strains ranged from 82.5% to 93.8%. A track of AAA residue located 35 nucleotides downstream from F gene start codon highly conserved in three vaccine strains were replaced with TGC in the local strains; that severed as target sequences for deign of discrimination primers. The method established in the present study successfully differentiated seven Taiwanese CDV field isolates, all belonging to the Asia-1 lineage, from vaccine strains.</p> <p>Conclusions</p> <p>The method described herein would be useful for several clinical applications, such as confirmation of nature CDV infection, evaluation of vaccination status and verification of the circulating viral genotypes.</p

Generating oxidation-resistant variants of Bacillus kaustophilus leucine aminopeptidase by substitution of the critical methionine residues with leucine

Bacillus kaustophilus leucine aminopeptidase (bkLAP) was sensitive to oxidative damage by hydrogen peroxide. To improve its oxidative stability, the oxidation-sensitive methionine residues in the enzyme were replaced with leucine by site-directed mutagenesis. The variants, each with an apparent molecular mass of approximately 54 kDa, were overexpressed in recombinant Escherichia coli M15 cells and purified to homogeneity by nickel-chelate chromatography. The specific activity for M282L, M285L, M289L and M321L decreased by more than 43%, while M400L, M426L, M445L, and M485L showed 191, 79, 313, and 103%, respectively, higher activity than the wild-type enzyme. Although the mutations did not cause significant changes in the K-m value, more than 67.8% increase in the value of k(cat)/K-m was observed in the M400L, M426L, M445L and M485L. In the presence of 50 mM H2O2 most variants were more stable with respect to the wild-type enzyme, indicating that the oxidative stability of the enzyme can be improved by engineering the methionine residues

Canopy CO2 concentrations and Crassulacean acid metabolism in Hoya carnosa in a subtropical rain forest in Taiwan: consideration of CO2 avallability and the evolution of CAM in epiphytes

The potential importance of CO2 derived from host tree respiration at night as a substrate for night time CO2 uptake during CAM was investigated in the subtropical and tropical epiphytic vine Hoya carnosa in a subtropical rainforest in north-eastern Taiwan. Individuals were examined within the canopies of host trees in open, exposed situations, as well as in dense forests. Although night time CO2 concentrations were higher near the epiphytic vines at night, relative to those measured during the day, presumably the result Of CO2 added to the canopy air by the host tree, no evidence for substantial use of this CO2 was found. In particular, stable carbon isotope ratios of H. carnosa were not substantially lower than those of many other CAM plants, as would be expected if host-respired CO2 were an important source Of CO2 for these CAM epiphytes. Furthermore, laboratory measurements of diel CO2 exchange revealed a substantial contribution of daytime CO2 uptake in these vines, which should also result in lower carbon isotope values than those characteristic of a CAM plant lacking daytime CO2 uptake. Overall, we found that host-respired CO2 does not contribute substantially to the carbon budget of this epiphytic CAM plant. This finding does not support the hypothesis that CAM may have evolved in tropical epiphytes in response to diel changes in the CO2 concentrations within the host tree canopy

Specific characters of 16S rRNA gene and 16S-23S rRNA internal transcribed spacer sequences of Xylella fastidiosa pear leaf scorch strains

Pear leaf scorch, the only Xylella fastidiosa-induced disease reported from Taiwan, was found in area where the variety Hengshan (Pyrus pyrifolia) was grown. Strains of pear leaf scorch Xyl. fastidiosa (XF-PLS) shared similarities to strains of other host origins in the requirement of complex medium and the exhibition of rippled cell walls, however, recent serological and molecular biology studies showed difference among them. Five strains of XF-PLS were compared with 20 other strains originally isolated from almond, oleander, pecan, plum, peach, mulberry, grapes, citrus, coffee, and sycamore by sequence analyses of the 16S rRNA gene and 16S-23S rRNA internal transcribed spacer region (ITS). When sequences of 16S rRNA gene based on fragment size of 1,537-1,540 bp were compared, the similarity index among 5 XF-PLS strains was 99.3-99.8%, whereas it was 97.8-98.6% between XF-PLS strains and strains from other hosts. When sequences of 16S-23S rRNA ITS based on fragment size of 510-540 bp were compared, the similarity index among 5 XF-PLS strains was 99.0-100%, whereas it was 80.7-82% between XF-PLS strains and strains from other hosts. Multiple sequence alignments led to the identification of 5 polymorphic nucleotides in the 16S rRNA gene among the 25 Xyl. fastidiosa strains, and there were considerable variations in the nucleotide sequences of 16S-23S rRNA ITS between XF-PLS and the other 20 Xyl. fastidiosa strains. The phylogenetic trees revealed that XF-PLS strains were separated from strains of other hosts. Strains of other hosts were divided into four subgroups: strains from (1) oleander, (2) grape, almond M23 and mulberry, (3) citrus and coffee, and (4) pecan, peach, plum, sycamore and almond M12. Results indicate that XF-PLS strains were not closely related to the above-mentioned strains from other hosts and could possibly belong to a new subspecies of Xyl. fastidiosa

A single amino acid substitution in PthA of Xanthomonas axonopodis pv. citri altering canker formation on grapefruit leaves

The typical citrus canker lesions produced by Xanthomonas axonopodis pv. citri are erumpent, callus-like, with water-soaked margins. Three novel atypical symptom-producing variants of X. axonopodis pv. citri were described recently in Taiwan. Only the variant designated as A(f) type produces typical erumpent canker lesions on Mexican lime (Citrus aurantifolia) but induces flat necrotic with water-soaked margin lesions on grapefruit leaves (C. paradisi). Two homologous pthA were cloned and characterized from strains XW19 (a typical canker lesion producing strain) and XW47 (a strain of A(f) type). The pthA homolog from XW19 was transformed into XW47. The transformant of XW47 induced typical erumpent canker lesions on grapefruit leaves. Sequence analyses of transformants XW19 and XW47 revealed over 99% homology in nucleotide and deduced amino acid sequences compared with pthA homologs deposited in GenBank. The amino acid residues located at positions 49, 286, 742 and 767 of PthA were different between XW47 and XW19. The PthA mutants with a single amino acid substitution at each of these four positions were constructed by site-directed mutagenesis. Modified PthA (S286P) from XW47 in transformant 47SP induced erumpent canker lesions on grapefruit leaves, whereas another modified PthA (P286S) from XW19 in transformant 47PS only induced flat necrotic lesions. These results suggested that a single amino acid substitution from either serine to proline or proline to serine at position 286 of PthA can alter canker formation by X. axonopodis pv. citri on grapefruit leaves

Intestinal geotrichosis in a German shepherd

A 4-year-old sexually intact male German shepherd with a 3-month history of chronic watery diarrhea was referred to the Veterinary Medical Teaching Hospital. Dehydration and serum biochemistry revealed hypoalbuminemia, and fecal material contained a large quantity of round arthroconidia that were microscopically observed. A specimen was submitted for fungal culture and yielded a white to cream-colored subsurface colony. Microorganisms derived from the colony exhibited chains of smooth, septate hyaline hyphae that were producing 1-celled arthroconidia. Geotrichum candidum was thus identified. Colonoscopic features included erythema, edema, and loss of the usual fine vascular pattern, with granularity of the mucosa of the descending column. Treatment consisted of oral administration of ketoconazole and metronidazole for 3 weeks, while oral prednisolone was tapered after 1 week of therapy. The dog's feces gradually softened after the first treatment. Fecal smear examination revealed no trace of the yeast-like microbes 7 days after treatment was administered, and 2 weeks post-treatment the dog passed well-formed stools and had regained its normal body weight. The previously observed clinical signs did not reoccur, even after oral medication was withdrawn

Four Lessons in Versatility or How Query Languages Adapt to the Web

Exposing not only human-centered information, but machine-processable data on the Web is one of the commonalities of recent Web trends. It has enabled a new kind of applications and businesses where the data is used in ways not foreseen by the data providers. Yet this exposition has fractured the Web into islands of data, each in different Web formats: Some providers choose XML, others RDF, again others JSON or OWL, for their data, even in similar domains. This fracturing stifles innovation as application builders have to cope not only with one Web stack (e.g., XML technology) but with several ones, each of considerable complexity. With Xcerpt we have developed a rule- and pattern based query language that aims to give shield application builders from much of this complexity: In a single query language XML and RDF data can be accessed, processed, combined, and re-published. Though the need for combined access to XML and RDF data has been recognized in previous work (including the W3C’s GRDDL), our approach differs in four main aspects: (1) We provide a single language (rather than two separate or embedded languages), thus minimizing the conceptual overhead of dealing with disparate data formats. (2) Both the declarative (logic-based) and the operational semantics are unified in that they apply for querying XML and RDF in the same way. (3) We show that the resulting query language can be implemented reusing traditional database technology, if desirable. Nevertheless, we also give a unified evaluation approach based on interval labelings of graphs that is at least as fast as existing approaches for tree-shaped XML data, yet provides linear time and space querying also for many RDF graphs. We believe that Web query languages are the right tool for declarative data access in Web applications and that Xcerpt is a significant step towards a more convenient, yet highly efficient data access in a “Web of Data”

Functional expression of the recombinant ATPase of orf virus

Nucleotide sequence analysis has indicated that the A32L gene of orf virus can encode an ATPase (Chan et al. in Gene 432:44-53, 2009). In this work, we cloned the A32L gene into a prokaryotic expression vector, and the recombinant protein was expressed and purified. The antigenicity of recombinant ATPase was examined by immunoblotting, and its identity was confirmed by mass spectrometry. The ATP hydrolysis function of the purified recombinant protein was examined, and our results showed that it exhibited the ATPase activity. Similar to other viral ATPases, the ATPase of orf virus remained active in the presence of different divalent ions; nevertheless, unlike other viral ATPases, our recombinant ATPase exhibited similar enzymatic activity in reaction buffers of different pH

Identification and phylogenetic analysis of orf virus from goats in Taiwan

An outbreak of contagious ecthyma in goats in central Taiwan was investigated. The disease was diagnosed by physical and histopathologic examinations, and the etiology of the disease was identified as orf virus by electron microscopy and polymerase chain reaction (PCR) and sequence of major envelope protein (B2L) gene. The entire protein-coding region of B2L gene were cloned and sequenced. Phylogenetic analysis of B2L amino acid sequences showed that the orf virus identified in this outbreak was closer to the Indian ORFV-Mukteswar 59/05 isolate. This is the first report on the molecular characterization of orf virus in Taiwan