bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium Activation of Ryanodine Receptor Channels—Reconciling RyR Gating Models with Tetrameric Channel Structure
Authors: Zahradník Ivan , Györke Sándor , Zahradníková Alexandra
Published: November 2005

Despite its importance and abundance of experimental data, the molecular mechanism of RyR2 activation by calcium is poorly understood. Recent experimental studies involving coexpression of wild-type (WT) RyR2 together with a RyR2 mutant deficient in calcium-dependent activation (Li, P., and S.R. Chen. 2001. J. Gen. Physiol. 118:33–44) revealed large variations of calcium sensitivity of the RyR tetramers with their monomer composition. Together with previous results on kinetics of Ca activation (Zahradníková, A., I. Zahradník, I. Györke, and S. Györke. 1999. J. Gen. Physiol. 114:787–798), these data represent benchmarks for construction and testing of RyR models that would reproduce RyR behavior and be structurally realistic as well. Here we present a theoretical study of the effects of RyR monomer substitution by a calcium-insensitive mutant on the calcium dependence of RyR activation. Three published models of tetrameric RyR channels were used either directly or after adaptation to provide allosteric regulation. Additionally, two alternative RyR models with Ca binding sites created jointly by the monomers were developed. The models were modified for description of channels composed of WT and mutant monomers. The parameters of the models were optimized to provide the best approximation of published experimental data. For reproducing the observed calcium dependence of RyR tetramers containing mutant monomers (a) single, independent Ca binding sites on each monomer were preferable to shared binding sites; (b) allosteric models were preferable to linear models; (c) in the WT channel, probability of opening to states containing a Ca2+-free monomer had to be extremely low; and (d) models with fully Ca-bound closed states, additional to those of an Monod-Wyman-Changeaux model, were preferable to models without such states. These results provide support for the concept that RyR activation is possible (albeit vanishingly small in WT channels) in the absence of Ca2+ binding. They also suggest further avenues toward understanding RyR gating.
Generation of Functional Fluorescent BK Channels by Random Insertion of GFP Variants
Authors: Giraldez Teresa , Hughes Thomas E. , Sigworth Fred J.
Published: November 2005

The yellow and cyan variants of green fluorescent protein (GFP) constitute an excellent pair for fluorescence resonance energy transfer (FRET) and can be used to study conformational rearrangements of proteins. Our aim was to develop a library of fluorescent large conductance voltage- and Ca2+-gated channels (BK or slo channels) for future use in FRET studies. We report the results of a random insertion of YFP and CFP into multiple sites of the α subunit of the hslo channel using a Tn5 transposon-based technique. 55 unique fluorescent fusion proteins were obtained and tested for cell surface expression and channel function. 19 constructs are expressed at the plasma membrane and show voltage and Ca2+-dependent currents. In 16 of them the voltage and Ca2+ dependence is very similar to the wild-type channel. Two insertions in the Ca2+ bowl and one in the RCK2 domain showed a strong shift in the G-V curve. The remaining 36 constructs were retained intracellularly; a solubility assay suggests that these proteins are not forming intracellular aggregates. The “success rate” of 19 out of 55 hslo insertion constructs compares very favorably with other studies of random GFP fusions.
Coupled K+–Water Flux through the HERG Potassium Channel Measured by an Osmotic Pulse Method
Authors: Ando Hiroyuki , Kuno Miyuki , Shimizu Hirofumi , Muramatsu Ikunobu , Oiki Shigetoshi
Published: November 2005

The streaming potential (Vstream) is a signature feature of ion channels in which permeating ions and water molecules move in a single file. Vstream provides a quantitative measure of the ion and water flux (the water–ion coupling ratio), the knowledge of which is a prerequisite for elucidating the mechanisms of ion permeation. We have developed a method to measure Vstream with the whole-cell patch-clamp configuration. A HEK293 cell stably expressing the HERG potassium channel was voltage clamped and exposed to hyperosmotic solutions for short periods of time (<1 s) by an ultrafast solution switching system (the osmotic pulse [quick jump-and-away] method). The reversal potentials were monitored by a series of voltage ramps before, during, and after the osmotic pulse. The shifts of the reversal potentials immediately after the osmotic jump gave Vstream. In symmetrical K+ solutions (10 mM), the Vstreams measured at different osmolalities showed a linear relationship with a slope of −0.7 mV/ΔOsm, from which the water–ion coupling ratio (n, the ratio of the flux of water to the flux of cations; Levitt, D.G., S.R. Elias, and J.M. Hautman. 1978. Biochim. Biophys. Acta. 512:436–451) was calculated to be 1.4. In symmetrical 100 mM K+ solutions, the coupling ratio was decreased significantly (n = 0.9), indicating that the permeation process through states with increased ion occupancy became significant. We presented a diagrammatic representation linking the water–ion coupling ratio to the mode of ion permeation and suggested that the coupling ratio of one may represent the least hydrated ion flux in the single-file pore.
Pygmalion and the Metamorphosis of Meaning in Jean Molinet's
Le Roman de la Rose moralisé
Authors: Croft Claire M.
Published: November 2005

Jean Molinet's
Roman de la Rose moralisé
has aroused little scholarly interest. No modern edition of the work yet exists, and knowledge of it remains scant. Amounting to a moralized re-working of Guillaume de Lorris's and Jean de Meun's renowned thirteenth-century
Roman de la Rose
, this fifteenth-century work poses many interesting questions. What does Molinet attempt to do to the
Rose
, one of the most prominent texts of the Middle Ages? What are the implications of his use of the term
moralisé
in relation to the worldly subject matter of the
Rose
?

The figure of Pygmalion is a pertinent example through which to examine Molinet's strategy on the
Rose
for two reasons. First, a direct comparison can be drawn between Molinet's techniques of moralization on Pygmalion and those put into practice on the Pygmalion account in the fourteenth-century
Ovide moralisé.
Second, Jean de Meun's Pygmalion account in the
Rose
is not a free-standing narrative, it is part of an intra-textual debate on nature and art. This factor allows us to examine how Molinet reacts to the
Rose
as a large and complex whole, on addition to its individual episodes. An analysis of the practices of both exegesis and allegoresis sheds further light on Molinet's undertaking by providing a literary context in which to consider the
Roman de la Rose moralisé.

This article seeks to demonstrate the complexity of Molinet's undertaking in providing the
Rose
with moralized interpretations. His reactions to the subtleties, enigmas, and challenges inherent within the
Rose
provide a striking example of one reader's response to this most widely debated of medieval texts.

C'est le Romant de la Rose,

moralisé cler et net,

translaté de rime en prose

par vostre humble Molinet.

1
Actions et récit dans
La Vengeance d'une femme
de Barbey d'Aurevilly
Authors: Vassilev Kris
Published: November 2005

La sixième
Diabolique
offre une perspective privilégiée à qui s'interroge sur le rapport entre vengeance et récit. Le projet vengeur recoupe ici l'acte de narrer. Il s'agit de faire rayonner l'histoire d'une bassesse et d'une grandeur, dont témoignent les faits et gestes de la duchesse de Sierra-Leone, qui se prostitue afin de se venger de son époux, grand d'Espagne. La stratégie adoptée par l'aristocrate consiste à se faire consumer par l'acte même au moyen duquel elle se venge. Rendu public, le récit des circonstances de sa mort doit souiller le nom et le rang de son mari et sceller ainsi son déshonneur. Cependant, ce programme narratif relève, en dernière analyse, d'une ambiguïté, inhérente à l'exécution de l'acte vengeur. Celui-ci semble en effet osciller entre deux scénarios incompatibles. Tantôt il prétend résider dans la transmission même du récit par un intermédiaire, tantôt il rejette la délégation narrative et prétend s'immuniser contre toute diffusion discursive: il se révèle alors comme pure contemplation mentale. C'est que, paradoxalement, la duchesse/fille semble ne raconter son histoire que pour affirmer la gratuité de son récit. L'ambiguïté troublante qui sous-tend la réalisation de cette vengeance est-t-elle levée par la production même du texte de la nouvelle?
Life after Léa: War and Trauma in Colette's
La Fin de Chéri
Authors: Fell Alison S.
Published: November 2005

This article considers the effects of the First World War on gender relations as they are perceived and represented in Colette's 1926 novel
La Fin de Chéri.
It discusses two interconnected aspects of the novel's evocation of the upper echelons of post-war French society. First, it focuses on the ways in which the novel intersects with the 1920s debate on gender identity, which conflates with a more generalized sense of cultural despair that was discernible after 1918 in diverse political, social and literary discourses. Whereas some commentators have argued that the novel reproduces reactionary and anti-feminist post-war stereotypes, this article suggests that the ambiguities evident in the figures of Edmée and Chéri work to reveal the inconsistencies and limitations of the ideals of femininity and masculinity that were widely promoted during this period. Second, the article examines in more detail the depiction of a returning soldier suffering from war-induced neurasthenia. I argue that the eponymous hero's psychological trauma is contrasted with Léa's more positive adaptation to a post-war world. I conclude, however, that Colette's novel equally flags up the difficulties inherent for both characters as they attempt to negotiate a space for their identity within the competing gender discourses of 1920s France.
Genomic analysis of LexA binding reveals the permissive nature of the Escherichia coli genome and identifies unconventional target sites
Authors: Wade Joseph T. , Reppas Nikos B. , Church George M. , Struhl Kevin
Published: November 2005

Genomes of eukaryotic organisms are packaged into nucleosomes that restrict the binding of transcription factors to accessible regions. Bacteria do not contain histones, but they have nucleoid-associated proteins that have been proposed to function analogously. Here, we combine chromatin immunoprecipitation and high-density oligonucleotide microarrays to define the in vivo DNA targets of the LexA transcriptional repressor in Escherichia coli. We demonstrate a near-universal relationship between the presence of a LexA sequence motif, LexA binding in vitro, and LexA binding in vivo, suggesting that a suitable recognition site for LexA is sufficient for binding in vivo. Consistent with this observation, LexA binds comparably to ectopic target sites introduced at various positions in the genome. We also identify ∼20 novel LexA targets that lack a canonical LexA sequence motif, are not bound by LexA in vitro, and presumably require an additional factor for binding in vivo. Our results indicate that, unlike eukaryotic genomes, the E. coli genome is permissive to transcription factor binding. The permissive nature of the E. coli genome has important consequences for the nature of transcriptional regulatory proteins, biological specificity, and evolution.
Morphogen gradient interpretation by a regulated trafficking step during ligand–receptor transduction
Authors: Jullien Jerome , Gurdon John
Published: November 2005

Morphogen gradients are important in early development, but how cells recognize their position in such a gradient is not well understood. Cells need to correctly interpret a morphogen concentration when the morphogen is no longer present in the extracellular medium. This memory of morphogen exposure is necessary for correct cell fate decisions in the changing morphogen gradient concentration in an embryo. Our results demonstrate that a previously unrecognized step in gradient interpretation is a temporal stop that arrests the progression of a ligand–receptor complex between internalization and lysosomal destruction. Signaling continues during this arrested progression, which constitutes the basis of memory of morphogen concentration. We show that prolonged signaling requires Dynamin-dependent internalization of the complex. Rab5QL- and Rab7QL-mediated increases in the speed of the endo-lysosomal progression do not affect memory. In contrast, memory is abolished by increasing the targeting of receptors to the lysosome through expression of the Smad7/Smurf2 ubiquitin ligase. We conclude that the basis for memory is the long-lasting residence of a signaling complex in the endo-lysosomal pathway. The regulated duration of this step helps to determine the choice of gene expression resulting from gradient interpretation.
Endoplasmic Reticulum Glucosidase II Is Required for Pathogenicity ofUstilago maydis [W]
Authors: Schirawski Jan , Böhnert Heidi U. , Steinberg Gero , Snetselaar Karen , Adamikowa Lubica , Kahmann Regine
Published: November 2005

We identified a nonpathogenic strain of Ustilago maydis by tagging mutagenesis. The affected gene, glucosidase1 (gas1), displays similarity to catalytic α-subunits of endoplasmic reticulum (ER) glucosidase II. We have shown that Gas1 localizes to the ER and complements the temperature-sensitive phenotype of a Saccharomyces cerevisiae mutant lacking ER glucosidase II. gas1 deletion mutants were normal in growth and mating but were more sensitive to calcofluor and tunicamycin. Mutant infection hyphae displayed significant alterations in the distribution of cell wall material and were able to form appressoria and penetrate the plant surface but arrested growth in the epidermal cell layer. Electron microscopy analysis revealed that the plant–fungal interface between mutant hyphae and the plant plasma membrane was altered compared with the interface of penetrating wild-type hyphae. This may indicate that gas1 mutants provoke a plant response.
A novel approach to structural alignment using realistic structural and environmental information
Authors: Chen Yu , Crippen Gordon M.
Published: November 2005

In the era of structural genomics, it is necessary to generate accurate structural alignments in order to build good templates for homology modeling. Although a great number of structural alignment algorithms have been developed, most of them ignore intermolecular interactions during the alignment procedure. Therefore, structures in different oligomeric states are barely distinguishable, and it is very challenging to find correct alignment in coil regions. Here we present a novel approach to structural alignment using a clique finding algorithm and environmental information (SAUCE). In this approach, we build the alignment based on not only structural coordinate information but also realistic environmental information extracted from biological unit files provided by the Protein Data Bank (PDB). At first, we eliminate all environmentally unfavorable pairings of residues. Then we identify alignments in core regions via a maximal clique finding algorithm. Two extreme value distribution (EVD) form statistics have been developed to evaluate core region alignments. With an optional extension step, global alignment can be derived based on environment‐based dynamic programming linking. We show that our method is able to differentiate three‐dimensional structures in different oligomeric states, and is able to find flexible alignments between multidomain structures without predetermined hinge regions. The overall performance is also evaluated on a large scale by comparisons to current structural classification databases as well as to other alignment methods.
Pneumococcal phosphorylcholine esterase, Pce, contains a metal binuclear center that is essential for substrate binding and catalysis
Authors: Lagartera Laura , González Ana , Hermoso Juan A. , Saíz José L. , García Pedro , García José L. , Menéndez Margarita
Published: November 2005

The phosphorylcholine esterase from Streptococcus pneumoniae, Pce, catalyzes the hydrolysis of phosphorylcholine residues from teichoic and lipoteichoic acids attached to the bacterial envelope and comprises a globular N‐terminal catalytic module containing a zinc binuclear center and an elongated C‐terminal choline‐binding module. The dependence of Pce activity on the metal/enzyme stoichiometry shows that the two equivalents of zinc are essential for the catalysis, and stabilize the catalytic module through a complex metal‐ligand coordination network. The pH dependence of Pce activity toward the alternative substrate p‐nitrophenylphosphorylcholine (NPPC) shows that kcat and kcat/Km depend on the protonation state of two protein residues that can be tentatively assigned to the ionization of the metal‐bound water (hydrogen bonded to D89) and to H228. Maximum activity requires deprotonation of both groups, although the catalytic efficiency is optimum for the single deprotonated form. The drastic reduction of activity in the H90A mutant, which still binds two Zn2+ ions at neutral pH, indicates that Pce activity also depends on the geometry of the metallic cluster. The denaturation heat capacity profile of Pce exhibits two peaks with Tm values of 39.6°C (choline‐binding module) and 60.8°C (catalytic module). The H90A mutation reduces the high‐temperature peak by about 10°C. Pce is inhibited in the presence of 1 mM zinc, but this inhibition depends on pH, buffer, and substrate species. A reaction mechanism is proposed on the basis of kinetic data, the structural model of the Pce:NPPC complex, and the currently accepted mechanism for other Zn‐metallophosphoesterases.
Disruption of an intermonomer salt bridge in the p53 tetramerization domain results in an increased propensity to form amyloid fibrils
Authors: Galea Charles , Bowman Prentice , Kriwacki Richard W.
Published: November 2005

We describe in molecular detail how disruption of an intermonomer salt bridge (Arg337–Asp352) leads to partial destabilization of the p53 tetramerization domain and a dramatically increased propensity to form amyloid fibrils. At pH 4.0 and 37°C, a p53 tetramerization domain mutant (p53tet‐R337H), associated with adrenocortical carcinoma in children, readily formed amyloid fibrils, while the wild‐type (p53tet‐wt) did not. We characterized these proteins by equilibrium denaturation, 13Cα secondary chemical shifts, {1H}‐15N heteronuclear NOEs, and H/D exchange. Although p53tet‐R337H was thermodynamically less stable, NMR data indicated that the two proteins had similar secondary structure and molecular dynamics. NMR derived pKa values indicated that at low pH the R337H mutation partially disrupted an intermonomer salt bridge. Backbone H/D exchange results showed that for at least a small population of p53tet‐R337H molecules disruption of this salt bridge resulted in partial destabilization of the protein. It is proposed that this decrease in p53tet‐R337H stability resulted in an increased propensity to form amyloid fibrils.
Evolution of an acylase active on cephalosporin C
Authors: Pollegioni Loredano , Lorenzi Simona , Rosini Elena , Marcone Giorgia Letizia , Molla Gianluca , Verga Roberto , Cabri Walter , Pilone Mirella S.
Published: November 2005

Semisynthetic cephalosporins are synthesized from 7‐amino cephalosporanic acid, which is produced by chemical deacylation or by a two‐step enzymatic process of the natural antibiotic cephalosporin C. The known acylases take glutaryl‐7‐amino cephalosporanic acid as a primary substrate, and their specificity and activity are too low for cephalosporin C. Starting from a known glutaryl‐7‐amino cephalosporanic acid acylase as the protein scaffold, an acylase gene optimized for expression in Escherichia coli and for molecular biology manipulations was designed. Subsequently we used error‐prone PCR mutagenesis, a molecular modeling approach combined with site‐saturation mutagenesis, and site‐directed mutagenesis to produce enzymes with a cephalosporin C/glutaryl‐7‐amino cephalosporanic acid catalytic efficiency that was increased up to 100‐fold, and with a significant and higher maximal activity on cephalosporin C as compared to glutaryl‐7‐amino cephalosporanic acid (e.g., 3.8 vs. 2.7 U/mg protein, respectively, for the A215Y‐H296S‐H309S mutant). Our data in a bioreactor indicate an ∼90% conversion of cephalosporin C to 7‐amino‐cephalosporanic acid in a single deacylation step. The evolved acylase variants we produced are enzymes with a new substrate specificity, not found in nature, and represent a hallmark for industrial production of 7‐amino cephalosporanic acid.
Distinct interaction modes of an AKAP bound to two regulatory subunit isoforms of protein kinase A revealed by amide hydrogen/deuterium exchange
Authors: Burns‐Hamuro Lora L. , Hamuro Yoshitomo , Kim Jack S. , Sigala Paul , Fayos Rosa , Stranz David D. , Jennings Patricia A. , Taylor Susan S. , Woods Virgil L.
Published: November 2005

The structure of an AKAP docked to the dimerization/docking (D/D) domain of the type II (RIIα) isoform of protein kinase A (PKA) has been well characterized, but there currently is no detailed structural information of an AKAP docked to the type I (RIα) isoform. Dual‐specific AKAP2 (D‐AKAP2) binds in the nanomolar range to both isoforms and provided us with an opportunity to characterize the isoform‐selective nature of AKAP binding using a common docked ligand. Hydrogen/deuterium (H/D) exchange combined with mass spectrometry (DXMS) was used to probe backbone structural changes of an α‐helical A‐kinase binding (AKB) motif from D‐AKAP2 docked to both RIα and RIIα D/D domains. The region of protection upon complex formation and the magnitude of protection from H/D exchange were determined for both interacting partners in each complex. The backbone of the AKB ligand was more protected when bound to RIα compared to RIIα, suggesting an increased helical stabilization of the docked AKB ligand. This combined with a broader region of backbone protection induced by the AKAP on the docking surface of RIα indicated that there were more binding constraints for the AKB ligand when bound to RIα. This was in contrast to RIIα, which has a preformed, localized binding surface. These distinct modes of AKAP binding may contribute to the more discriminating nature of the RIα AKAP‐docking surface. DXMS provides valuable structural information for understanding binding specificity in the absence of a high‐resolution structure, and can readily be applied to other protein–ligand and protein–protein interactions.
Functional expression of eukaryotic membrane proteins in Lactococcus lactis
Authors: Monné Magnus , Chan Ka Wai , Slotboom Dirk‐Jan , Kunji Edmund R.S.
Published: November 2005

The overproduction of eukaryotic membrane proteins is a major impediment in their structural and functional characterization. Here we have used the nisin‐inducible expression system of Lactococcus lactis for the overproduction of 11 mitochondrial transport proteins from yeast. They were expressed at high levels in a functional state in the cytoplasmic membrane. The results also show that the level of expression is influenced by the N‐terminal regions of the transporters. Expression levels were improved >10‐fold either by replacing or truncating these regions or by adding lactococcal signal peptides. The observed expression levels are now compatible with a realistic exploration of crystallization conditions. The lactococcal expression system may be used for the high‐throughput functional characterization of eukaryotic membrane proteins and structural genomics.
Viral haemorrhagic septicaemia virus in marine fish and its implications for fish farming – a review
Authors: Skall H F , Olesen N J , Mellergaard S
Published: November 2005

Viral haemorrhagic septicaemia virus (VHSV) has, in recent decades, been isolated from an increasing number of free‐living marine fish species. So far, it has been isolated from at least 48 fish species from the northern hemisphere, including North America, Asia and Europe, and fifteen different species including herring, sprat, cod, Norway pout and flatfish from northern European waters. The high number of VHSV isolations from the Baltic Sea, Kattegat, Skagerrak, the North Sea and waters around Scotland indicate that the virus is endemic in these waters. The VHSV isolates originating from wild marine fish show no to low pathogenicity to rainbow trout and Atlantic salmon, although several are pathogenic for turbot. Marine VHSV isolates are so far serologically indistinguishable from freshwater isolates. Genotyping based on VHSV G‐ and N‐genes reveals four groups indicating the geographical origin of the isolates, with one group representing traditional European freshwater isolates and isolates of north European marine origin, a second group of marine isolates from the Baltic Sea, a third group of isolates from the North Sea, and a group representing North American isolates. Examples of possible transfer of virus from free‐living marine fish to farmed fish are discussed, as are measures to prevent introduction of VHSV from the marine environment to aquaculture.
Environmental and physiological conditions affecting Tetrahymena sp. infection in guppies, Poecilia reticulata Peters
Authors: Pimenta Leibowitz M , Ariav R , Zilberg D
Published: November 2005

Parasitic infections caused by Tetrahymena sp. constitute a serious problem in guppies, Poecilia reticulata. Tetrahymena was isolated from skin lesions of naturally infected guppies in a commercial aquaculture farm, cultured in vitro and used in subsequent experimental infections. In addition to guppies, angelfish, Pterophyllum scalare, platyfish, Xiphophorus maculates, and neontetra, Paracheirodon innesi, were susceptible, whereas tilapia (Oreochromis niloticus ×O. aureus) was resistant. The ciliate had a high affinity for dead fish. Skin abrasion did not affect the infection, but fish with gas bubble disease exhibited a significantly higher infection than non‐affected fish. Infection was significantly higher when fish were exposed to high levels of ammonia, high organic load and low water temperatures. Under shipment conditions, infection was significantly elevated. Full recovery was achieved at a low fish density. Results suggest that poor environmental and physiological conditions enhance infection with Tetrahymena sp.
Detection and identification of Flavobacterium psychrophilum from gill washings and benthic diatoms by PCR‐based sequencing analysis
Authors: Izumi S , Fujii H , Aranishi F
Published: November 2005

A nested polymerase chain reaction (PCR) amplification technique was used to detect Flavobacterium psychrophilum from washings of fish gill surfaces and benthic diatoms as environmental samples. Gill washing samples were prepared from kawamutsu, Zacco temminckii (Temminck & Schlegel) and oikawa, Z. platypus (Temminck & Schlegel). Benthic diatom samples were collected from stone surfaces. All samples were collected from rivers in Wakayama Prefecture, Japan from November 2003 to January 2004. Following simple DNA extraction using a chelating resin, nested PCR techniques targeting 16S‐rDNA and gyrB regions were performed, and PCR products were cloned and sequenced. With nested PCR amplification for the 16S‐rDNA gene, ambiguous PCR products were detected from two of six samples, and by cloning and sequencing analysis were found not to be DNA fragments amplified from F. psychrophilum. Using nested PCR for the gyrB gene, however, five of six samples were clearly positive for F. psychrophilum in agarose gel electrophoresis, and were found to be identical with nucleotide sequences of F. psychrophilumgyrB deposited in DNA databases by sequencing analysis. Results indicate that nested PCR for the gyrB region is a useful technique to detect low levels of F. psychrophilum from environmental samples contaminated with many other organisms.
Hypersensitivity to Forcipomyia taiwana (biting midge): clinical analysis and identification of major For t 1, For t 2 and For t 3 allergens
Authors: Chen Y.‐H. , Lee M.‐F. , Lan J.‐L. , Chen C.‐S. , Wang H.‐L. , Hwang G.‐Y. , Wu C.‐H.
Published: November 2005

Background:  Forcipomyia taiwana is a tiny, blood‐sucking midge that cause intense pruritis and swelling in sensitive individuals. It is distributed island‐wide in rural Taiwan and Southern China.

Objective:  This study aimed to study the allergic immune responses and identify F. taiwana allergens.

Methods:  Crude whole body F. taiwana extracts were prepared with phosphate‐buffered saline. The specific IgE antibody was determined by enzyme‐linked immunoassay and immunoblotting. Protein was analyzed by electrospray ionization tandem mass spectrometry.

Results:  Among the 372 subjects that were exposed to F. taiwana bites, 179 (48%) reported an immediate skin reaction with/without delay reaction and 41(11.1%) reported a solely delay reaction. The skin of 21 subjects was tested with F. taiwana extract. Of these 21 subjects, 12 (57.1%) produced immediate skin reactions and contained high levels of specific IgE antibody against F. taiwana. Immunoblotting revealed that 11 allergenic components are able to bind specific IgE. Allergens of 22, 24, 35, 36, and 64 kDa bound 50, 50, 75, 66.7, and 75% of IgE‐containing sera tested, respectively. Tryptic fragments of the 24, 35, 36, and 64 kDa allergens were analyzed by ESI‐MS/MS. Selected tryptic peptides of 24, 35, and 36, and 64 kDa allergens exhibited significant sequence identity with triosephosphate isomerase of Anopheles merus,Tenebrio molitor,Ochlerotatus togoi, and Chrysops vittatus, fructose 1,6‐bisphosphate aldolase of Antheraea yamamai and Homalodisca coagulata, and a slow muscle myosin S1 heavy chain of Homarusamericanus and a protein with unknown function from A. gambiae, respectively. The 35 and 36 kDa proteins may represent different isoforms of the fructose 1,6‐bisphosphate aldolase.

Conclusion:  We conclude that immediate reaction to F. taiwana bites is IgE mediated and the 24 (For t 1), 35 (For t 2), and 64 kDa (For t 3) proteins are candidates for major F. taiwana allergens. Further studies are needed to confirm these allergens.
Single dose topical corticosteroid inhibits IL‐5 and IL‐13 in nasal lavage following grass pollen challenge
Authors: Erin E. M. , Leaker B. R. , Zacharasiewicz A. S. , Higgins L. A. , Williams T. J. , Boyce M. J. , de Boer P. , Durham S. R. , Barnes P. J. , Hansel T. T.
Published: November 2005

Background:  Nasal lavage is a noninvasive method of obtaining inflammatory exudates following nasal allergen challenge (NAC), and permits cells and released mediators to be evaluated.

Objective:  To determine the effects of a single dose of topical steroid on eosinophils and levels of chemokines and cytokines in nasal lavage fluid following NAC in patients with allergic rhinitis.

Methods:  Patients with grass pollen seasonal allergic rhinitis (n = 32) out of the allergy season received either nasal budesonide (100 μg per nostril) or matched placebo before allergen challenge in a double blind two‐way crossover design. A semi‐automated mixed bead array system was employed to measure multiple chemokines and cytokines in small volumes (50 μl) of nasal lavage supernatants.

Results:  Following NAC there was a rapid onset of nasal symptoms together with nasal eosinophilia, and the appearance of IL‐5 and IL‐13 in lavages between 4 and 8 h. Elevated levels of eotaxin, RANTES, IL‐8 and MCP‐1 were also detected following allergen challenge. A single dose of nasal budesonide caused a decrease in symptoms (P < 0.05) and nasal eosinophils (P < 0.05) with selective abrogation of IL‐5 and IL‐13 responses (P < 0.05), but a lack of effect on levels of eotaxin, RANTES, IL‐8 and MCP‐1.

Conclusion:  This study suggests that a single dose of nasal steroid has the capacity to selectively abolish IL‐5 and IL‐13 responses following NAC. This model should be convenient for testing novel anti‐inflammatory and immunoregulatory agents intended for the treatment of allergic rhinitis.
Exhaled nitric oxide predicts airway hyper‐responsiveness to hypertonic saline in children that wheeze
Authors: de Meer G. , van Amsterdam J. G. C. , Janssen N. A. H. , Meijer E. , Steerenberg P. A. , Brunekreef B.
Published: November 2005

Background:  Exhaled nitric oxide (eNO) has shown good validity for the assessment of airway inflammation in asthmatic children. In large‐scale epidemiological studies, this method would be preferred above airway challenge tests, because it is a quick and easy applicable tool.

Objective:  In this study, we aimed to assess the discriminatory capacity of eNO, and prechallenge FEV1 for airway hyper‐responsiveness (AHR) in 8–13‐year old schoolchildren.

Materials and methods:  Parents completed the ISAAC questionnaire, and children were tested for atopy, AHR to hypertonic (4.5%) saline (HS), and eNO. Diagnostic value was assessed by the area under the receiver operating curves (ROC), and calculation of positive and negative predicted values at different cut‐off points for eNO and prechallenge FEV1.

Results:  Areas under the ROC‐curves of AHR were 0.65 for eNO and 0.62 for FEV1. Values increased to 0.71 and respectively 0.75 for a combined occurrence of AHR and current wheeze. Highest sensitivity and specificity were obtained at a cut‐off value of 43 ppb for eNO and 103% predicted for FEV1. At these cut‐off values, the positive predictive values for the presence of AHR in symptomatic children were respectively 83% (eNO) and 33% (FEV1), and negative predictive values in asymptomatic children were, respectively, 90 (eNO) and 80% (FEV1).

Conclusion:  Exhaled nitric oxide is a valid screening tool for AHR to HS in children that present with current wheeze, and it outperforms FEV1 as a predictor of AHR.
Fast foods – are they a risk factor for asthma?
Authors: Wickens K. , Barry D. , Friezema A. , Rhodius R. , Bone N. , Purdie G. , Crane J.
Published: November 2005

Background:  Lifestyle changes over the last 30 years are the most likely explanation for the increase in allergic disease over this period.

Aim:  This study tests the hypothesis that the consumption of fast food is related to the prevalence of asthma and allergy.

Methods:  As part of the International Study of Asthma and Allergies in Childhood (ISAAC) a cross‐sectional prevalence study of 1321 children (mean age = 11.4 years, range: 10.1–12.5) was conducted in Hastings, New Zealand. Using standard questions we collected data on the prevalence of asthma and asthma symptoms, as well as food frequency data. Skin prick tests were performed to common environmental allergens and exercise‐induced bronchial hyperresponsiveness (BHR) was assessed according to a standard protocol. Body mass index (BMI) was calculated as weight/height2 (kg/m2) and classified into overweight and obese according to a standard international definition.

Results:  After adjusting for lifestyle factors, including other diet and BMI variables, compared with children who never ate hamburgers, we found an independent risk of hamburger consumption on having a history of wheeze [consumption less than once a week (OR = 1.44, 95% CI: 1.06–1.96) and 1+ times a week (OR = 1.65, 95% CI: 1.07–2.52)] and on current wheeze [consumption less than once a week (OR = 1.17, 95% CI: 0.80–1.70) and 1+ times a week (OR = 1.81, 95% CI: 1.10–2.98)]. Takeaway consumption 1+ times a week was marginally significantly related to BHR (OR = 2.41, 95% CI: 0.99–5.91). There was no effect on atopy.

Conclusions:  Frequent consumption of hamburgers showed a dose‐dependent association with asthma symptoms, and frequent takeaway consumption showed a similar association with BHR.
Idiopathic rhinitis, the ongoing quest
Authors: Van Rijswijk J. B. , Blom H. M. , Fokkens W. J.
Published: November 2005

The term rhinitis in daily practice is used for nasal dysfunction causing symptoms‐like nasal itching, sneezing, rhinorrhea and or nasal blockage. Chronic rhinitis can roughly be classified into allergic, infectious or nonallergic/noninfectious. When allergy, mechanical obstruction and infections have been excluded as the cause of rhinitis, a number of poorly defined nasal conditions of partly unknown aetiology and pathophysiology remain. The differential diagnosis of nonallergic noninfectious rhinitis is extensive. Although the percentage of patients with nonallergic noninfectious rhinitis with a known cause has increased the last decades, still about 50% of the patients with nonallergic noninfectious rhinitis has to be classified as suffering from idiopathic rhinitis (IR), or rather e causa ignota. Specific immunological, clinical and sometimes radiological and functional tests are required to distinguish known causes. Research to the underlying pathophysiology of IR has moved from autonomic neural dysbalans to inflammatory disorders (local allergy), the nonadrenergic noncholinergic (NANC) sensory peptidergic neural system and central neural hyperaesthesia, still without solid ground or proof. This review summarizes the currently known causes for nonallergic noninfectious rhinitis and possible treatments. Also possible pathophysiological mechanisms of IR are discussed.
Attitudes toward advance directives and the impact of prognostic information on the preference for cardiopulmonary resuscitation in medical inpatients in Chiang Mai University Hospital, Thailand
Authors: Sittisombut Sudarat , Love Edgar J. , Sitthi‐amorn Chitr
Published: November 2005

Abstract  Our previous study revealed that cardiopulmonary resuscitation (CPR) was performed in 65.7% of 411 terminally ill patients who died in a tertiary‐care university hospital in northern Thailand. Advance directives (ADs) are needed to ensure that life‐sustaining therapies are used more appropriately. To investigate inpatients’ attitudes regarding ADs for CPR and the impact of providing prognostic information on treatment preferences for CPR, we interviewed a randomly selected group of 200 ambulatory medical inpatients in multiple sessions. The results showed that most subjects had a positive attitude towards ADs for CPR. The majority preferred to have CPR when no information was provided on the chance of survival. However, this proportion decreased depending on the prognostic scenarios. Our investigation suggested that the preference of patients for CPR should be assessed individually and gradually, with adequate information given on the chance of survival.
What characterizes the ‘usual’ preoperative education in clinical contexts?
Authors: Fitzpatrick Elizabeth , Hyde Abbey
Published: November 2005

Abstract  The literature on preoperative education is dominated by studies that employ experimental designs to measure the effects of structured programs on patient outcomes. These studies predominantly compare structured preoperative educational interventions with the usual care that patients receive. However, the notion of what the “usual” care comprises is largely elusive and unexplored. This study aimed to understand how the usual preoperative education is practiced in a number of surgical clinical units at one particular hospital in Ireland. Twelve experienced surgical nurses were interviewed in depth. A qualitative strategy resembling grounded theory was employed to analyze the data. The findings indicated that the content and quality of the preoperative education that patients received depended largely upon the individual nurse caring for the patient. Although there was a generic content of preoperative education that all participants identified as important, this related to physical and technical issues, and to the transmission of administrative or procedural information. Perspectives on the process of delivering preoperative education were diverse. The vast majority did not relate to the formal language of the discourses of teaching and learning; nonetheless, their descriptions of engaging in their work indicated the application of some elements of educational theory. The data suggested that the use of teaching tools was inconsistent, depending on their availability and the practices of individual nurses. On the whole, the usual care as described by participants in our study may be characterized as uneven, variable, and mutable. We conclude by raising some methodological issues relating to the use of the usual care in control groups in experimental studies on preoperative education.
Menstrual status and bone mineral density among female athletes
Authors: Punpilai Sriareporn , Sujitra Tiansawad , Ouyporn Tonmukayakul , Teraporn Vutyavanich , Sombut Boonyaprapa
Published: November 2005

Abstract  The present study investigated the relationship between menstrual status and bone mineral density (BMD). Sixty‐three elite female athletes competing at the regional level participated. Self‐reported menstrual status, stress during the past 6 months, dietary intake of calcium, blood samples for hormonal study, mid‐thigh skinfold thickness, triceps, iliac crest, spine and femoral neck BMD were determined. It was found that more than half of the athletes were eumenorrheic while almost half were menstrually dysfunctional. The bone mineral density at the lumbar spine and the femoral neck were within normal ranges. Menstrual dysfunction in female athletes was related to a low BMD at the lumbar spine but not at the femoral neck. Delayed menarche and menstrual dysfunction during the first 2 years after menarche were related to current menstrual dysfunction, but low percent body fat was not related to menstrual dysfunction. This study suggests that exercise in elite female athletes might be an underlying cause of menstrual dysfunction and that there is a relationship between lumbar spine BMD and menstrual dysfunction. The assessment of menstrual history and percent body fat could be used as a screening tool for menstrual dysfunction.
Critically appraising quantitative research
Authors: Duffy Joanne R.
Published: November 2005

Abstract  Critical appraisal of quantitative research is a skill that is necessary for adequate evidence‐based practice. This paper describes critical appraisal and offers a process for its implementation. Four categories of quantitative studies are reviewed and associated links to relevant critical appraisal frameworks are provided. The implications for students, nursing faculty members, and practicing nurses are discussed.
Educating nurses for the 21st century
Authors: Bartels Jean E.
Published: November 2005

Abstract  The current and projected demand for nurses and nursing services worldwide, coupled with dramatic changes in the delivery of health care, require nurses with more knowledge, more education, and more skills. Issues facing nursing require a reconceptualization of the approach and expectations for nursing practice and for the educational models and processes that lead to reformed nursing practice. New initiatives such as the Clinical Nurse Leader and the Doctor of Nursing Practice may lead the transformational process that is needed.
Tectal neurons signal impending collision of looming objects in the pigeon
Authors: Wu Le‐Qing , Niu Yu‐Qiong , Yang Jin , Wang Shu‐Rong
Published: November 2005

Although the optic tectum in non‐mammals and its mammalian homolog, the superior colliculus, are involved in avoidance behaviors, whether and how tectal neurons respond to an object approaching on a collision course towards the animal remain unclear. Here we show by single unit recording that there exist three classes of looming‐sensitive neurons in the pigeon tectal layer 13, which sends looming information to the nucleus rotundus or to the tectopontine system. The response onset time of tau cells is approximately constant whereas that for rho and eta cells depends on the square root of the diameter/velocity ratio of objects looming towards the animal, the cardioacceleration of which is also linearly related to the square root of this ratio. The receptive field of tectal cells is composed of an excitatory center and an inhibitory periphery, and this periphery does not inhibit responses to looming stimuli. These results suggest that three classes of tectal neurons are specified for detecting an object approaching on a collision course towards the animal, and that rho and eta cells may signal early warning of impending collision whereas tau cells initiate avoidance responses at a constant time before collision through the tectopontine system.
Oxygen and glucose deprivation induces major dysfunction in the somatosensory cortex of the newborn rat
Authors: Albrecht Juliane , Hanganu Ileana L. , Heck Nicolas , Luhmann Heiko J.
Published: November 2005

The mechanisms and functional consequences of ischemia‐induced injury during perinatal development are poorly understood. Subplate neurons (SPn) play a central role in early cortical development and a pathophysiological impairment of these neurons may have long‐term detrimental effects on cortical function. The acute and long‐term consequences of combined oxygen and glucose deprivation (OGD) were investigated in SPn and compared with OGD‐induced dysfunction of immature layer V pyramidal cortical neurons (PCn) in somatosensory cortical slices from postnatal day (P)0–4 rats. OGD for 50 min followed by a 10–24‐h period of normal oxygenation and glucose supply in vitro or in culture led to pronounced caspase‐3‐dependent apoptotic cell death in all cortical layers. Whole‐cell patch‐clamp recordings revealed that the majority of SPn and PCn responded to OGD with an initial long‐lasting ischemic hyperpolarization accompanied by a decrease in input resistance (Rin), followed by an ischemic depolarization (ID). Upon reoxygenation and glucose supply, the recovery of the membrane potential and Rin was followed by a Na+/K+‐ATPase‐dependent postischemic hyperpolarization, and in almost half of the investigated SPn and PCn by a postischemic depolarization. Whereas neither a moderate (2.5 mm) nor a high (4.8 mm) increase in extracellular magnesium concentration protected the SPn from OGD‐induced dysfunction, blockade of NMDA receptors with MK‐801 led to a significant delay and decrease of the ID. Our data demonstrate that OGD induces apoptosis and a profound dysfunction in SPn and PCn, and underline the critical role of NMDA receptors in early ischemia‐induced neuronal damage.