Dyrithiopsis lakefuxianensis gen. et sp. nov. from Fuxian Lake, Yunnan, China, and notes on the taxonomic confusion surrounding Dyrithium

A new taxon with Dyrithium-like characteristics was collected from Lake Fuxian in China. The taxon is typical of the Amphisphaeriaceae in that it has relatively large, ostiolate, immersed ascomata, unitunicate asci with a J+ subapical ring, and brown ascospores. It is similar to Dyrithium in that it has muriform ascospores, but considerable confusion surrounds this genus. In Dyrithium asci are bitunicate and lack a J+ subapical ring, while this was not true of our species. A new genus, Dyrithiopsis, therefore is established to accommodate this new taxon. Details of its anamorph also are provided, based on cultural studies. Parsimony analyses of part of the large-subunit rDNA provide further evidence to support the familial placement of this new genus in the Amphisphaeriaceae. The taxonomic position of Dyrithium also is discussed.     

Microfungi on the Pandanaceae: Linocarpon lammiae sp. nov., L. siamensis sp. nov. and L. suthepensis sp. nov. are described with a key to Linocarpon species from the Pandanaceae

Linocarpon species are reported from Pandanaceae in Australia, Brunei, Hong Kong, Nepal, New Zealand, Philippines, Seychelles, Thailand and Vanuatu. Linocarpon lammiae sp. nov. were collected on decaying leaves of Pandanus tectorius in Hong Kong. Linocarpon siamensis sp. nov. and L. suthepensis sp. nov. were collected from decaying leaves of P. penetrans in Thailand. These taxa are described, illustrated and compared with Linocarpon species with similar ascospore morphology and dimensions. Included are a synoptic table, which compares the new species to similar known species, and a dichotomous key to species of Linocarpon known from members of the Pandanaceae.     

NMR and CD spectroscopy show that imino acid restriction of the unfolded state leads to efficient folding

Protein folding is determined by molecular features in the unfolded state, as well as the native folded structure. In the unfolded state, imino acids both restrict conformational space and present cis-trans isomerization barriers to folding. Because of its high proline and hydroxyproline content, the collagen triple-helix offers an opportunity to characterize the impact of imino acids on the unfolded state and folding kinetics. Here, NMR and CD spectroscopy are used to characterize the role of imino acids in a triple-helical peptide, T1-892, which contains an 18-residue sequence from type I collagen and a C-terminal (Gly-Pro-Hyp)(4) domain. The replacement of Pro or Hyp by an Ala in the (Gly-Pro-Hyp)(4) region significantly decreases the folding rate at low but not high concentrations, consistent with less efficient nucleation. To understand the molecular basis of the decreased folding rate, changes in the unfolded as well as the folded states of the peptides were characterized. While the trimer states of the peptides are all similar, NMR dynamics studies show monomers with all trans (Gly-Pro-Hyp)(4) are less flexible than monomers containing Pro --> Ala or Hyp --> Ala substitutions. Nucleation requires all trans bonds in the (Gly-Pro-Hyp)(4) domain and the constrained monomer state of the all trans nucleation domain in T1-892 increases its competency to initiate triple-helix formation and illustrates the impact of the unfolded state on folding kinetics.     

Discovery and characterization of cooperative ligand binding in the adaptive region of interleukin-2

The cytokine hormone interleukin-2 (IL-2) contains a highly adaptive region that binds small, druglike molecules. The binding properties of this adaptive region have been explored using a "tethering" method that relies on the formation of a disulfide bond between the protein and small-molecule ligands. Using tethering, surface plasmon resonance (SPR), and X-ray crystallography, we have discovered that the IL-2 adaptive region contains at least two cooperative binding sites where the binding of a first ligand to one site promotes or antagonizes the binding of a second ligand to the second site. Cooperative energies of interaction of -2 kcal/mol are observed. The observation that the adaptive region contains two adjacent sites may lead to the development of tight-binding antagonists of a protein-protein interaction. Cooperative ligand binding in the adaptive region of IL-2 underscores the importance of protein dynamics in molecular recognition. The tethering approach provides a novel and general strategy for discovering such cooperative binding interactions in specific, flexible regions of protein structure.     

Two new species of Spadicoides from Brunei and Hong Kong

Spadicoides hodgkissa sp. nov. and Spadicoides arengae sp. nov., recorded from submerged decaying wood in Hong Kong and from decaying palm fronds in Brunei, are described and illustrated. Spadicoides hodgkissa is characterized by versicolored, obovoid conidia with up to 2 septa, including a distal distoseptum and a proximal euseptum, while Spadicoides arengae is characterized by unicellular, ellipsoidal conidia with verruculose walls that are relatively large. Eight genera, including Dendrographium, Helminthosporium, Luzfridiella, Paliphora, Polyschema, Polytretophora, Porosubramaniania, and Weufia, have the same conidiogenesis as Spadicoides. A key to these genera is provided.     

Detection of fenspiride and identification of in vivo metabolites in horse body fluids by capillary gas chromatography-mass spectrometry: administration, biotransformation and urinary excretion after a single oral dose

Studies related to the in vivo biotransforrmation and urinary excretion of fenspiride hydrochloride in the horse are described. After oral administration, the drug is metabolised by both phase I functionalisation and phase II conjugation pathways. Following enzymatic deconjugation, fenspiride and its phase I metabolites were isolated from post-administration biofluids using bonded co-polymeric mixed mode solid-phase extraction cartridges to isolate the basic compounds. Following trimethylsilylation (TMS), the parent drug and metabolites were identified by capillary gas chromatography-mass spectrometry (GC-MS). Fenspiride (A) and seven metabolites (B-->G) arising from oxidation on both the aromatic and heterocyclic substructures were detected in urine. The positive ion electron ionisation mass spectra of the TMS derivatives of fenspiride and its metabolites provided useful information on its metabolism. Positive ion methane chemical ionisation-GC-MS of the derivatives provided both derivatised molecular mass and structural information. Unchanged fenspiride can be detected in post-administration plasma and urine samples for up to 24 h. Maximum urinary levels of 100-200 ng ml(-1) were observed between 3 and 5 h after administration. After enzymatic deconjugation, the major phenolic metabolite (G) can be detected in urine for up to 72 h. This metabolite is the analyte of choice in the GC-MS screening of post-race equine urine samples for detection of fenspiride use. However, a distinct difference was observed in the urinary excretion of this metabolite between the thoroughbred horses used in UK study and the quarterbred and standardbred horses used for the USA administrations.     

Regulation of light-dependent Gqalpha translocation and morphological changes in fly photoreceptors

Heterotrimeric G-proteins relay signals between membrane-bound receptors and downstream effectors. Little is known, however, about the regulation of Galpha subunit localization within the natural endogenous environment of a specialized signaling cell. Here we show, using live Drosophila flies, that light causes massive and reversible translocation of the visual Gqalpha to the cytosol, associated with marked architectural changes in the signaling compartment. Molecular genetic dissection together with detailed kinetic analysis enabled us to characterize the translocation cycle and to unravel how signaling molecules that interact with Gqalpha affect these processes. Epistatic analysis showed that Gqalpha is necessary but not sufficient to bring about the morphological changes in the signaling organelle. Furthermore, mutant analysis indicated that Gqbeta is essential for targeting of Gqalpha to the membrane and suggested that Gqbeta is also needed for efficient activation of Gqalpha by rhodopsin. Our results support the 'two-signal model' hypothesis for membrane targeting in a living organism and characterize the regulation of both the activity-dependent Gq localization and the cellular architectural changes in Drosophila photoreceptors.     

Genotyping of cagA and vacA, Lewis antigen status, and analysis of the poly-(C) tract in the alpha(1,3)-fucosyltransferase gene of Irish Helicobacter pylori isolates

Much work has focused on trying to identify markers in Helicobacter pylori that might allow the eventual disease outcome of an infection to be predicted. In this study we examined the cagA and vacA genotype, and Lewis status in a panel of 43 Irish H. pylori clinical isolates, and investigated a possible correlation with disease pathology. In addition, differences in the poly-(C) tract of the alpha(1,3)-fucosyltransferase gene were examined to identify a possible correlation with gene expression. Only three of 43 isolates were cagA-negative, whereas the remaining 40 isolates, independent of pathology, were cagA-positive. In all the strains we examined, the vacA signal-sequence was type s1a. For the vacA mid-region 12/43 isolates were type m1 and 31/43 isolates were type m2. These data, and examination of isolates from different pathology groups, suggests that there is no correlation between virulence and vacA genotype in the Irish population of H. pylori isolates. Western blotting of whole cell lysates from 32 H. pylori isolates showed 3/32 displayed only the Le(x) epitope, 12/32 only the Le(y), 13/32 both epitopes and 4/32 neither epitope. No apparent association between Lewis phenotype and disease pathology was evident. A range of lengths of poly-(C) tract were observed in the alpha(1, 3)-fucosyltransferase gene, however the length of the tract in an isolate did not correlate with the Lewis structures present. We conclude that future studies on H. pylori pathogenesis should not alone focus on the importance of molecular markers, but also on the host response, including genetic background and immune responsiveness.