bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome
Authors: Saito Kuniaki , Nishida Kazumichi M. , Mori Tomoko , Kawamura Yoshinori , Miyoshi Keita , Nagami Tomoko , Siomi Haruhiko , Siomi Mikiko C.
Published: August 2006

In Drosophila, Piwi (P-element-induced wimpy testis), which encodes a protein of the Argonaute family, is essential for germ stem cell self-renewal. Piwi has recently been shown to be a nuclear protein involved in gene silencing of retrotransposons and controlling their mobilization in the male germline. However, little is known about the molecular mechanisms of Piwi-dependent gene silencing. Here we show that endogenous Piwi immunopurified from ovary specifically associates with small RNAs of 25–29 nucleotides in length. Piwi-associated small RNAs were identified by cloning and sequencing as repeat-associated small interfering RNAs (rasiRNAs) derived from repetitive regions, such as retrotransposon and heterochromatic regions, in the Drosophila genome. Northern blot analyses revealed that in vivo Piwi does not associate with microRNAs (miRNAs) and that guide siRNA was not loaded onto Piwi when siRNA duplex was added to ovary lysate. In vitro, recombinant Piwi exhibits target RNA cleavage activity. These data together imply that Piwi functions in nuclear RNA silencing as Slicer by associating specifically with rasiRNAs originating from repetitive targets.
Small RNAs just got bigger: Piwi-interacting RNAs (piRNAs) in mammalian testes
Authors: Kim V. Narry
Published: August 2006

Small RNAs constitute a large family of regulatory molecules with diverse functions in eukaryotes. Hallmarks of small RNAs are their dependence on double-stranded RNAs (dsRNA)-specific RNase III-type enzymes for biogenesis and their association with Argonaute family proteins for the silencing process. At least two classes of small RNAs have previously been described: microRNAs (miRNAs) derived from hairpin-shaped precursors and small interfering RNAs (siRNAs) generated from long dsRNAs. Recent articles reported a novel class of small RNAs that are expressed specifically and abundantly in the spermatogenic cells of mice. These RNAs are bigger (26–31 nucleotides [nt]) than most previously described small RNAs (21–23 nt) and are associated with Piwi-subclade members of the Argonaute protein family. Although the biogenesis and function of these RNAs are yet to be determined, these findings may add new dimensions in small RNA biology and germline cell biology.
Skin epidermis lacking the c-myc gene is resistant to Ras-driven tumorigenesis but can reacquire sensitivity upon additional loss of the p21Cip1 gene
Authors: Oskarsson Thordur , Essers Marieke Alida Gertruda , Dubois Nicole , Offner Sandra , Dubey Christelle , Roger Catherine , Metzger Daniel , Chambon Pierre , Hummler Edith , Beard Peter , Trumpp Andreas
Published: August 2006

The target gene(s) required for Myc-mediated tumorigenesis are still elusive. Here we show that while endogenous c-Myc is surprisingly dispensable for skin homeostasis and TPA-induced hyperplasia, c-Myc-deficient epidermis is resistant to Ras-mediated DMBA/TPAinduced tumorigenesis. This is mechanistically linked to p21Cip1, which is induced in tumors by the activated Ras–ERK pathway but repressed by c-Myc. Acute elimination of c-Myc in established tumors leads to the up-regulation of p21Cip1, and epidermis lacking both p21Cip1 and c-Myc reacquires normal sensitivity to DMBA/TPA-induced tumorigenesis. This identifies c-Myc-mediated repression of p21Cip1 as a key step for Ras-driven epidermal tumorigenesis.
CREB antisense oligodeoxynucleotide administration into the dorsal hippocampal CA3 region impairs long- but not short-term spatial memory in mice
Authors: Florian Cédrick , Mons Nicole , Roullet Pascal
Published: August 2006

The transcription factor cAMP response-element binding protein (CREB) has a pivotal role in hippocampal synaptic plasticity and hippocampus-dependent long-term memory. We recently demonstrated that the dorsal hippocampal CA3 region is involved in memory consolidation of spatial information tested on a Morris water maze in mice. To test whether activation of CREB in the CA3 region is required for memory consolidation of spatial information, bilaterally cannulated mice were infused 18 h before the beginning of the behavioral training with antisense or control sense CREB oligodeoxynucleotides (ODNs) or buffer. Mice were then subjected to massed training in a spatial version of the water maze and tested for retention 0 or 24 h after the last training session. We showed that CREB antisense ODN-infusion in the CA3 region impaired long-term memory when tested 24 h later but had no effect on spatial acquisition or short-term memory tested immediately after behavioral training. These findings provide evidence that the regionally restricted activation of CREB in the dorsal hippocampal CA3 region is critical for the long-term memory consolidation phase of spatial learning but not for short-term memory.
Effects of mutation at the D‐JH junction on affinity, specificity, and idiotypy of anti‐progesterone antibody DB3
Authors: He Mingyue , Hamon Maureen , Liu Hong , Corper Adam L. , Taussig Michael J.
Published: August 2006

The crystal structures of the Fab′ fragment of the anti‐progesterone monoclonal antibody DB3 and its complexes with steroid haptens have shown that the D‐JH junctional residue TrpH100 is a key contributor to binding site interactions with ligands. The indole group of TrpH100 also undergoes a significant conformational change between the bound and unliganded states, effectively opening and closing the combining site pocket. In order to explore the effect of substitutions at this position on steroid recognition, we have carried out mutagenesis on a construct encoding a three‐domain single‐chain fragment (VH/K) of DB3 expressed in Escherichia coli. TrpH100 was replaced by 13 different amino acids or deleted, and the functional and antigenic properties of the mutated fragments were analyzed. Most substitutions, including small, hydrophobic, hydrophilic, neutral, and negatively charged side chains, were reduced or abolished binding to free progesterone, although binding to progesterone‐BSA was partially retained. The reduction in antigen binding was paralleled by alteration of the idiotype associated with the DB3 combining site. In contrast, the replacement of TrpH100 by Arg produced a mutant that retained wild‐type antibody affinity and idiotype, but with altered specificity. Significant changes in this mutant included increased relative affinities of 104‐fold for progesterone‐3‐carboxymethyloxime and 10‐fold for aetiocholanolone. Our results demonstrate an essential role for the junctional residue H100 in determining steroid‐binding specificity and combining site idiotype and show that these properties can be changed by a single amino acid substitution at this position.
Fundamental processes of protein folding: Measuring the energetic balance between helix formation and hydrophobic interactions
Authors: Xian Wujing , Connolly Peter J. , Oslin Marcela , Hausrath Andrew C. , Osterhout John J.
Published: August 2006

Theories of protein folding often consider contributions from three fundamental elements: loops, hydrophobic interactions, and secondary structures. The pathway of protein folding, the rate of folding, and the final folded structure should be predictable if the energetic contributions to folding of these fundamental factors were properly understood. αtα is a helix‐turn‐helix peptide that was developed by de novo design to provide a model system for the study of these important elements of protein folding. Hydrogen exchange experiments were performed on selectively 15N‐labeled αtα and used to calculate the stability of hydrogen bonds within the peptide. The resulting pattern of hydrogen bond stability was analyzed using a version of Lifson‐Roig model that was extended to include a statistical parameter for tertiary interactions. This parameter, x, represents the additional statistical weight conferred upon a helical state by a tertiary contact. The hydrogen exchange data is most closely fit by the XHC model with an x parameter of 9.25. Thus the statistical weight of a hydrophobic tertiary contact is ∼5.8× the statistical weight for helix formation by alanine. The value for the x parameter derived from this study should provide a basis for the understanding of the relationship between hydrophobic cluster formation and secondary structure formation during the early stages of protein folding.
Changes in immune regulation in response to examination stress in atopic and healthy individuals
Authors: Höglund C. Olgart , Axén J. , Kemi C. , Jernelöv S. , Grunewald J. , Müller‐Suur C. , Smith Y. , Grönneberg R. , Eklund A. , Stierna P. , Lekander M.
Published: August 2006

Background
Stress can aggravate the allergic inflammation, but determinants of disturbed immune regulation are largely unknown.

Objective
To determine systemic immunological, local inflammatory and functional airway responses to stress in healthy and atopic individuals.

Methods
Forty‐one undergraduate students, 22 with allergy of whom 16 had asthma, and 19 healthy controls, were studied in a low‐stress period and in association with a large exam. Subjects completed questionnaires on stress and health behaviours, underwent lung function tests, bronchial methacholine challenge, measurements of exhaled nitric oxide and urine cortisol. Blood cells were phenotyped, and cytokines from mononuclear blood cells were analysed.

Results
Perceived stress and anxiety increased in both groups during the exam period while cortisol increased only in the atopy group. Cytokine production decreased broadly in response to stress in both groups, which was paralleled by an increase in the proportion of regulatory T cells (CD4+CD45RO+CD25bright). Interestingly, atopic individuals, but not controls, reacted with a decreased T‐helper type 1/T‐helper type 2 (Th1/Th2) ratio and a decrease in natural killer (NK) cell numbers in response to stress. In control subjects only, exhaled nitric oxide decreased and forced expiratory volume in one second increased during stress.

Conclusion
Atopic and non‐atopic subjects shared some immune changes in response to stress, such as a dramatic decline in cytokines and an increase in the number of regulatory T cells in peripheral blood. However, other stress‐induced immune changes were unique to atopic individuals, such as a skewed Th1/Th2 ratio and reduced NK cell numbers, indicating that some pathogenic mechanisms in atopics may be more strongly affected by stress than others.
Characterization of recombinant Mal d 4 and its application for component‐resolved diagnosis of apple allergy
Authors: Ma Y. , Zuidmeer L. , Bohle B. , Bolhaar S.T.H. , Gadermaier G. , Gonzalez‐Mancebo E. , Fernandez‐Rivas M. , Knulst A.C. , Himly M. , Asero R. , Ebner C. , Van Ree R. , Ferreira F. , Breiteneder H. , Hoffmann‐Sommergruber K.
Published: August 2006

Background
Profilins are ubiquitous panallergens that have been extensively characterized; yet, their clinical relevance is still unclear.

Objective
The aim of the present study was to produce recombinant apple profilin (rMal d 4) and to evaluate its allergenic activity and its potency for component‐resolved allergy diagnosis.

Methods
Complementary DNA‐derived Mal d 4 was cloned, expressed in Escherichia coli and subsequently purified via poly (l‐proline) sepharose. A total of 28 sera from apple‐allergic patients were used for IgE‐ELISA, immunoblot, RAST and basophil histamine release (BHR) test. In addition, skin prick tests (SPTs) were performed in five patients.

Results
Four different complementary DNA coding for apple profilin, Mal d 4, each with an open reading frame of 393 nucleotides, were identified. One isoform Mal d 4.0101 was expressed in Escherichia coli and subsequently purified. Mass spectroscopy revealed the expected mass of 13.826 for rMal d 4.0101, and circular dichroism analysis data were typical for a folded protein and small‐angle X‐ray scattering measurement identified the protein as a monomer. All the serum samples displayed IgE binding to rMal d 4.0101 in IgE ELISA, immunoblot and RAST. In immunoblotting, IgE binding to natural Mal d 4 was partially/completely inhibited by preincubation with rMal d 4.0101, and RAST values to apple extract were significantly reduced upon serum pretreatment with rMal d 4.0101. SPTs and BHR assays using purified rMal d 4.0101 were positive. Purified rMal d 4.0101 was destroyed within seconds when subjected to pepsin digestion.

Conclusions
Apple profilin complementary DNAs were identified. The physicochemical and allergenic properties of purified recombinant Mal d 4.0101 were evaluated showing that the recombinant protein was equal to the natural protein as shown by inhibition assays. Thus, Mal d 4 represents another example suitable for component‐resolved diagnosis of food allergy.
Presence of plasma complement regulatory proteins clusterin (Apo J) and vitronectin (S40) on circulating immune complexes (CIC)
Authors: Chauhan A K , Moore T L
Published: August 2006

The complement regulatory (CR) proteins clusterin and vitronectin bind to the membrane attack complex (MAC) and thus prevent cytolysis. In this report, we demonstrate the presence of both of these CR proteins on MAC bound to circulating immune complexes (CIC). We measured the amount of clusterin and vitronectin on MAC in plasma, also referred to as soluble MAC (SMAC), as well as on MAC bound to CIC (MAC–CIC), using antibody directed to polymerized C9 in systemic lupus erythematosus (SLE) patients. We observed a strong correlation among the quantities of SMAC and MAC–CIC. The amount of both clusterin and vitronectin associated with MAC–CIC was two- to threefold higher in comparison to the SMAC. Patients with high levels of clusterin and vitronectin demonstrated renal involvement. We hypothesize that these complement regulatory proteins besides regulating the insertion of MAC play other critical roles, in disease pathogenesis.
First experience with an oral combination therapy using bosentan and sildenafil for pulmonary arterial hypertension
Authors: Lunze K. , Gilbert N. , Mebus S. , Miera O. , Fehske W. , Uhlemann F. , Mühler E. G. , Ewert P. , Lange P. E. , Berger F. , Schulze‐Neick I.
Published: August 2006

Background  New oral substances such as beraprost, bosentan and sildenafil have proven effective in different forms of pulmonary arterial hypertension (PAH), both alone and in combination with standard treatment such as intravenous and inhaled prostacyclins. However, there are few reports so far on the effect of a combination of exclusively oral substances. In this paper, we present our initial findings of treatment using a combination of these oral substances in a heterogeneous group of patients with different forms of PAH.

Materials and methods  Eleven patients with a median age of 12·9 years (5·5–54·7 years) with both idiopathic PAH and forms associated with congenital cardiac defects (PAH‐CHD) with a mean pulmonary arterial pressure > 25 mmHg were enrolled in an observational, open‐label, prospective, single‐centre study. Either combination treatment with bosentan and sildenafil was started initially, or an existing bosentan treatment was complemented with sildenafil given as an add‐on therapy. Mean doses given were 2·3 ± 0·6 mg kg−1 for bosentan and 2·1 ± 0·9 mg kg−1 for sildenafil. Clinical status, exercise capacity, and haemodynamics were assessed at baseline and at the end of the observation period after a mean follow‐up time of 1·1 years (0·5–2·5 years).

Results  No major side effects regarding liver function and blood pressure regulation were noted. One patient died of sudden death elsewhere. Most patients were in New York Heart Association (NYHA) functional class III. Clinical improvement was about one NYHA class (mean 2·8 ± 0·4–1·6 ± 0·8, P = 0·001), which was associated with an increase of transcutaneous oxygen saturation (89·9 ± 9·9–92·3 ± 7·1%; P = 0·037), maximum oxygen uptake (18·1 ± 6·8–22·8 ± 10·4 mL kg−1 * min; P = 0·043), and 6‐minute walking distance (351 ± 58–451 ± 119 m; P = 0·039). Mean pulmonary arterial pressure measured invasively decreased (62 ± 12–46 ± 18 mmHg; P = 0·041).

Conclusions  In our patient group, a combination of oral bosentan and sildenafil proved to be safe and effective. Clearly, randomized, double‐blind, placebo‐controlled studies are warranted to define the role and type of combination therapies in PAH.
DIFFUSION WITHIN SOIL MICROSTRUCTURE A STRUCTURAL PARAMETER FOR SOILS
Authors: CURRIE J. A.
Published: August 2006

To estimate the diffusion coefficient of gases, Dc, within soil crumbs gaseous diffusion was measured first on a packing of dry 1–2 mm crumbs, then again after the crumb pores, but not the inter‐crumb pores, had been rendered nonconducting by saturating with an inert liquid. Using equations for analogous electrical properties of heterogeneous materials, the range of values of Dc/Do was from 0.025 to 0.156 for eight soils with crumb porosities in the range 0.25 to 0.41. For each soil, single value parameters for soil complexity were calculated from Dc/Do and the appropriate crumb porosity, and these were in accord with known behaviour in the field: the better the structure, the smaller is the complexity parameter. The validity of the method was checked by measuring the diffusion coefficient, D, by standard methods on blocks of soil and also by the indirect method on crumbs obtained by crushing the block. Comparison of these values for D and Dc, ideally the same, confirmed the adequacy of the method.
Phosphate reactions with natural allophane, ferrihydrite and goethite
Authors: PARFITT R. L.
Published: August 2006

The reactions of phosphate with natural samples of allophane, ferrihydrite, hematite and goethite were measured for up to 30 d. The amount of phosphate sorbed on allophane showed the biggest increase with time whereas the amount sorbed on goethite showed the least increase with time. The total amount of phosphate sorbed either at high levels of phosphate addition or after 10 d followed the order hematite < goethite < ferrihydrite < allophane and was probably related to the specific surface. Si was desorbed as phosphate was adsorbed on the minerals.

The reactions of phosphate on allophane involved rapid, strong adsorption, probably at defect sites, followed by weaker adsorption, followed, probably, by disruption of the allophane structure together with precipitation of aluminium phosphates. Previous suggestions either of diffusive penetration of phosphate into surfaces or about the formation of aluminium phosphate coatings, are unlikely to hold for allophane, if all the Al is at the surface and if the structure can be ruptured.

The reactions of phosphate with iron oxides involved a rapid, strong ligand exchange, followed by weaker ligand exchange, and, probably, by a relatively slow penetration at defect sites and pores. Highly crystalline goethite has virtually no slow reaction and therefore solid‐state diffusion of phosphate does not readily occur. The extent of phosphate uptake during the slow penetration reactions probably depends on the degree of crystallinity or porosity of iron oxides.

The most reactive adsorbents, such as allophane, ferrihydrite and Al‐humus complexes do not have planar surfaces, and this needs to be considered when modelling phosphate reactions.
Patchiness of eggs, larvae and juveniles of European hake Merluccius merluccius from the NW Mediterranean
Authors: MAYNOU F. , OLIVAR M. P. , EMELIANOV M.
Published: August 2006

We estimated patchiness for different life stages (egg, larvae and juveniles) of European hake (Merluccius merluccius) from two ichthyoplankton surveys (September and November 1999) and four bottom‐trawl surveys (November 1998, February, June and September 1999). Lloyd's patchiness P = 1 + 1/k, where k is the dispersion parameter of the negative binomial distribution, was calculated. Juveniles showed lower indices of patchiness than early ontogenetic stages. In juveniles, patchiness was highest for smaller size classes (3.5–5 cm TL) settling to their near‐bottom habitat and was very low for those larger than 5 cm up to the 30–35 cm size class (approximately 3 yr of age, 50% mature individuals). Patchiness of settling juveniles was especially high in the June and September surveys, coinciding with peak recruitment to the bottom. Both eggs and yolk sac larvae showed patchiness values <10, increasing in flexion stage larvae from 12 to 20. This pattern is different to that observed in other studies for pelagic species, where patchiness of early‐stage eggs is very high, but coincides with results obtained for other demersal species with pelagic eggs. Factors that may explain these patterns are discussed in light of oceanographic conditions, adult distribution and larval ontogenesis.
Leaf breakdown in streams differing in catchment land use
Authors: PAUL MICHAEL J. , MEYER JUDY L. , COUCH CAROL A.
Published: August 2006

1. The impact of changes in land use on stream ecosystem function is poorly understood. We studied leaf breakdown, a fundamental process of stream ecosystems, in streams that represent a range of catchment land use in the Piedmont physiographic province of the south‐eastern United States.

2. We placed bags of chalk maple (Acer barbatum) leaves in similar‐sized streams in 12 catchments of differing dominant land use: four forested, three agricultural, two suburban and three urban catchments. We measured leaf mass, invertebrate abundance and fungal biomass in leaf bags over time.

3. Leaves decayed significantly faster in agricultural (0.0465 day−1) and urban (0.0474 day−1) streams than in suburban (0.0173 day−1) and forested (0.0100 day−1) streams. Additionally, breakdown rates in the agricultural and urban streams were among the fastest reported for deciduous leaves in any stream. Nutrient concentrations in agricultural streams were significantly higher than in any other land‐use type. Fungal biomass associated with leaves was significantly lower in urban streams; while shredder abundance in leaf bags was significantly higher in forested and agricultural streams than in suburban and urban streams. Storm runoff was significantly higher in urban and suburban catchments that had higher impervious surface cover than forested or agricultural catchments.

4. We propose that processes accelerating leaf breakdown in agricultural and urban streams were not the same: faster breakdown in agricultural streams was due to increased biological activity as a result of nutrient enrichment, whereas faster breakdown in urban streams was a result of physical fragmentation resulting from higher storm runoff.
A semi‐parametric gap‐filling model for eddy covariance CO2 flux time series data
Authors: STAUCH VANESSA J. , JARVIS ANDREW J.
Published: August 2006

This paper introduces a method for modelling the deterministic component of eddy covariance CO2 flux time series in order to supplement missing data in these important data sets. The method is based on combining multidimensional semi‐parametric spline interpolation with an assumed but unstated dependence of net CO2 flux on light, temperature and time. We test the model using a range of synthetic canopy data sets generated using several canopy simulation models realized for different micrometeorological and vegetation conditions. The method appears promising for filling large systematic gaps providing the associated missing data do not overerode critical information content in the conditioning data used for the model optimization.
Quantifying components of risk for European woody species under climate change
Authors: OHLEMÜLLER RALF , GRITTI EMMANUEL S. , SYKES MARTIN T. , THOMAS CHRIS D.
Published: August 2006

Estimates of species extinction risk under climate change are generally based on differences in present and future climatically suitable areas. However, the locations of potentially suitable future environments (affecting establishment success), and the degree of climatic suitability in already occupied and new locations (affecting population viability) may be equally important determinants of risk. A species considered to be at low risk because its future distribution is predicted to be large, may actually be at high risk if these areas are out of reach, given the species' dispersal and migration rates or if all future suitable locations are only marginally suitable and the species is unlikely to build viable populations in competition with other species. Using bioclimatic models of 17 representative European woody species, we expand on current ways of risk assessment and suggest additional measures based on (a) the distance between presently occupied areas and areas predicted to be climatically suitable in the future and (b) the degree of change in climatic suitability in presently occupied and unoccupied locations. Species of boreal and temperate deciduous forests are predicted to face higher risk from loss of climatically suitable area than species from warmer and drier parts of Europe by 2095 using both the moderate B1 and the severe A1FI emission scenario. However, the average distance from currently occupied locations to areas predicted suitable in the future is generally shorter for boreal species than for southern species. Areas currently occupied will become more suitable for boreal and temperate species than for Mediterranean species whereas new suitable areas outside a species' current range are expected to show greater increases in suitability for Mediterranean species than for boreal and temperate species. Such additional risk measures can be easily derived and should give a more comprehensive picture of the risk species are likely to face under climate change.
Analysis of fat body transcriptome from the adult tsetse fly, Glossina morsitans morsitans
Authors: Attardo G. M. , Strickler‐Dinglasan P. , Perkin S. A. H. , Caler E. , Bonaldo M. F. , Soares M. B. , El‐Sayeed N. , Aksoy S.
Published: August 2006

Tsetse flies (Diptera: Glossinidia) are vectors of pathogenic African trypanosomes. To develop a foundation for tsetse physiology, a normalized expressed sequence tag (EST) library was constructed from fat body tissue of immune‐stimulated Glossina morsitans morsitans. Analysis of 20 257 high‐quality ESTs yielded 6372 unique genes comprised of 3059 tentative consensus (TC) sequences and 3313 singletons (available at http://aksoylab.yale.edu). We analysed the putative fat body transcriptome based on homology to other gene products with known functions available in the public domain. In particular, we describe the immune‐related products, reproductive function related yolk proteins and milk‐gland protein, iron metabolism regulating ferritins and transferrin, and tsetse's major energy source proline biosynthesis. Expression analysis of the three yolk proteins indicates that all are detected in females, while only the yolk protein with similarity to lipases, is expressed in males. Milk gland protein, apparently important for larval nutrition, however, is primarily synthesized by accessory milk gland tissue.
Differential expression of two glutathione S‐transferases identified from the American dog tick,Dermacentor variabilis
Authors: Dreher‐Lesnick S. M. , Mulenga A. , Simser J. A. , Azad A. F.
Published: August 2006

Reciprocal signalling and gene expression play a cardinal role during pathogen–host molecular interactions and are prerequisite to the maintenance of balanced homeostasis. Gene expression repertoire changes during rickettsial infection and glutathione‐S‐transferases (GSTs) were among the genes found up‐regulated inRickettsia‐infectedDermacentor variabilis. GSTs are well known to play an important part in cellular stress responses in the host. We have cloned two full‐length GSTs fromD. variabilis(DvGST1 and DvGST2). Comparison of these two DvGST molecules with those of other species indicate that DvGST1 is related to the mammalian class theta and insect class delta GSTs, while DvGST2 does not seem to fall in the same family. Northern blotting analyses revealed differential expression patterns, where DvGST1 and DvGST2 transcripts are found in the tick gut, with DvGST2 transcripts also present in the ovaries. Both DvGST transcripts are up‐regulated upon tick feeding. Challenge of fed adult ticks withEscherichia coliinjection showed decreased transcript amounts compared with ticks injected with phosphate‐buffered saline (sham) and naïve ticks.
A female‐specific desaturase gene responsible for diene hydrocarbon biosynthesis and courtship behaviour in Drosophila melanogaster
Authors: Chertemps T. , Duportets L. , Labeur C. , Ueyama M. , Wicker‐Thomas C.
Published: August 2006

Drosophila melanogaster shows sexually dimorphic cuticular hydrocarbons, with monoenes produced in males and dienes produced in females. Here we describe a female‐specific desaturase gene, desatF. RNAi knock‐down led to a dramatic decrease in female dienes and increase in monoenes paralleled with an increase in copulation latency and a decrease in courtship index and copulation attempts by the males. The desatF gene was also expressed in females from D. sechellia, rich in dienes, but not D. simulans, which produce only monoenes. When hydrocarbons were feminized in D. melanogaster males by targeted expression of the transformer gene, the expression of desatF occurred. These results strongly suggest that desatF is a crucial enzyme for female pheromone biosynthesis and courtship behaviour in D. melanogaster.
Cloning and characterization of protease inhibitor‐like cDNAs from the Hessian fly mayetiola destructor (SAY)
Authors: Maddur A. A. , Liu X. , Zhu Y. C. , Fellers J. P. , Oppert B. , Park Y. , Bai J. , Wilde G. E. , Chen M.‐S.
Published: August 2006

Analysis of transcriptomes from the salivary glands and midgut of Hessian fly larvae Mayetiola destructor (say) identified a set of diverse cDNAs that encode proteins with a relatively high percentage (over 10%) of cysteinyl residues. Structural comparison of these putative proteins with known sequences in GenBank revealed that the positions of the cysteinyl residues in the identified proteins were highly conserved within a family of proteinase inhibitors despite very little overall sequence similarity. Phylogenetic analysis sorted this set of cDNAs into five different groups. To determine if these cDNAs indeed encode proteinase inhibitors, recombinant proteins were generated with two cDNAs from two different groups. Biochemical analysis of the recombinant proteins against commercial and insect gut proteinases demonstrated that the recombinant proteins are strong proteinase inhibitors with different specificities. Northern blot and real‐time PCR analysis revealed that the different genes were expressed at different developmental stages and in different tissues. The overall results indicated that M. destructor contains a complex of genes that code for proteinase inhibitors which may regulate proteinase activities in different regulatory pathways. The GenBank accession numbers for the cDNAs in this paper were DQ232690 to DQ232718.
A tapeworm molecule manipulates vitellogenin expression in the beetle Tenebrio molitor
Authors: Warr E. , Meredith J. M. , Nimmo D. D. , Basu S. , Hurd H. , Eggleston P.
Published: August 2006

Metacestodes of Hymenolepis diminuta secrete a molecule that decreases vitellogenin (Vg) synthesis in the beetle host, Tenebrio molitor. The 5608 bp T. molitor Vg cDNA represents a single‐copy gene encoding a single open reading frame of 1821 amino acids with a predicted molecular mass of 206 kDa. Northern blot analysis revealed detectable levels of transcripts only in adult females. In vivo, Vg mRNA abundance was significantly higher in fat bodies from infected females compared with control females at all but the earliest time point. In vitro, Vg mRNA abundance was significantly increased in fat bodies incubated with live stage I–II parasites. The apparent conflict between increased Vg mRNA abundance and decreased Vg protein in fat bodies from infected females is discussed.
High yield and rapid growth of Neoparamoeba pemaquidensis in co‐culture with a rainbow trout gill‐derived cell line RTgill‐W1
Authors: Lee L E J , Van Es S J , Walsh S K , Rainnie D J , Donay N , Summerfield R , Cawthorn R J
Published: August 2006

Neoparamoeba pemaquidensis is an ubiquitous amphizoic marine protozoan and has been implicated as the causative agent for several diseases in marine organisms, most notably amoebic gill disease (AGD) in Atlantic salmon. Despite several reports on the pathology of AGD, relatively little is known about the protozoan and its relationship to host cells. In this study, an in vitro approach using monolayers of a rainbow trout gill cell line (RTgill‐W1, ATCC CRL‐2523) was used to rapidly grow large numbers of N. pemaquidensis (ATCC 50172) and investigate cell–pathogen interactions. Established cell lines derived from other tissues of rainbow trout and other fish species were also evaluated for amoeba growth support. The amoebae showed preference and highest yield when grown with RTgill‐W1 over nine other tested fish cell lines. Amoeba yields could reach as high as 5 × 105 cells mL−1 within 3 days of growth on the gill cell monolayers. The amoebae caused visible focal lesions in RTgill‐W1 monolayers within 24 h of exposure and rapidly proliferated and spread with cytopathic effects destroying the neighbouring pavement‐like cells within 48–72 h after initial exposure in media above 700 mOsm kg−1. Disruption of the integrity of the gill cell monolayers could be noted within 30 min of exposure to the amoeba suspensions by changes in transepithelial resistance (TER) compared with control cell monolayers maintained in the exposure media. This was significantly different by 2 h (P < 0.05) compared with control cells and remained significantly different (P < 0.01) for the remaining 72 h that the TER was monitored. The RTgill‐W1 cell line is thus a convenient model for growing N. pemaquidensis and for studying host–pathogen interactions in AGD.
Towards unbiased parentage assignment: combining genetic, behavioural and spatial data in a Bayesian framework
Authors: HADFIELD J. D. , RICHARDSON D. S. , BURKE T.
Published: August 2006

Inferring the parentage of a sample of individuals is often a prerequisite for many types of analysis in molecular ecology, evolutionary biology and quantitative genetics. In all but a few cases, the method of parentage assignment is divorced from the methods used to estimate the parameters of primary interest, such as mate choice or heritability. Here we present a Bayesian approach that simultaneously estimates the parentage of a sample of individuals and a wide range of population‐level parameters in which we are interested. We show that joint estimation of parentage and population‐level parameters increases the power of parentage assignment, reduces bias in parameter estimation, and accurately evaluates uncertainty in both. We illustrate the method by analysing a number of simulated test data sets, and through a re‐analysis of parentage in the Seychelles warbler, Acrocephalus sechellensis. A combination of behavioural, spatial and genetic data are used in the analyses and, importantly, the method does not require strong prior information about the relationship between nongenetic data and parentage.
FibA and PilA act cooperatively during fruiting body formation of Myxococcus xanthus
Authors: Bonner Pamela J. , Black Wesley P. , Yang Zhaomin , Shimkets Lawrence J.
Published: August 2006

The extracellular matrix (ECM) of Myxococcus xanthus is essential for social (S‐) motility and fruiting body formation. An ECM‐bound protein, FibA, is homologous to M4 zinc metalloproteases and is important for stimulation by a phosphatidylethanolamine (PE) chemoattractant and for formation of discrete aggregation foci. In this work, we demonstrate that a correlation exists between a reduced ability to respond to PE and the observed defects in fruiting body morphogenesis. Furthermore, the fibA aggregation defect is accentuated by the absence of either PilA, the structural subunit of type IV pili, or DifD, a chemosensory response regulator. The inability to form fruiting bodies is not due to a loss of S‐motility, but rather the loss of PilA and pili as pilT fibA mutants form fruiting bodies. The FibA active site residue E342 is important for fruiting body morphogenesis in the absence of PilA. Mutants exhibiting defects in fruiting body morphogenesis also produce fewer viable spores. It is proposed that FibA and PilA act as extracellular sensors for developmental signals.
rag genes: novel components of the RamR regulon that trigger morphological differentiation in Streptomyces coelicolor
Authors: Paolo Salvatore San , Huang Jianqiang , Cohen Stanley N. , Thompson Charles J.
Published: August 2006

The filamentous bacterium, Streptomyces coelicolor, undergoes a complex cycle of growth and development in which morphological differentiation coincides with the activation of the orphan response regulator RamR and the biosynthesis of a morphogenic peptide called SapB. SapB is a lantibiotic‐like molecule derived from the product of the ramS gene that promotes formation of aerial hyphae by breaking the aqueous tension on the surface of the substrate mycelium. A ramR‐disrupted mutant is delayed in aerial hyphae formation while constitutive overexpression of ramR accelerates aerial hyphae formation in the wild‐type strain and restores SapB biosynthesis and aerial hyphae formation in all developmental mutants (bld) tested. Using DNA microarrays to globally identify S. coelicolor genes whose transcription was affected by ramR mutation or overexpression, we discovered a ramR‐activated locus of contiguous cotranscribed developmental genes that modulate both aerial hyphae formation and sporulation. The genes of this cluster of ramR‐activated genes (rag), which are chromosomally distant from previously known RamR‐regulated genes, include: ragA (sco4075) and ragB (sco4074), which encode two subunits of an ABC transporter, ragK (sco4073), a putative histidine kinase, and ragR (sco4072), a ramR paralogue. Promoter mapping and protein–DNA binding experiments indicate that RamR activates ragABKR transcription directly, by binding to three sequence motifs in the ragABKR promoter region. A constructed ragABKR null mutant was able to synthesize SapB and erect aerial hyphae; however, these hyphae were unusually branched, reminiscent of substrate hyphae. Subsequent stages of differentiation, septation and sporogenesis were delayed. The role of ragABKR in aerial hyphae formation was shown both by epistasis (ragR‐activated aerial hyphae formation in bld mutants) and extracellular complementation (ragR‐induced synthesis of an activity allowing aerial hyphae formation in bld mutants) experiments. In conclusion, the ragABKR locus activates a SapB‐independent developmental pathway that is involved in both aerial hyphae formation and sporulation, serving to integrate sequential morphogenic changes.
Phospholipid directed motility of surface‐motile bacteria
Authors: Bonner Pamela J. , Shimkets Lawrence J.
Published: August 2006

Myxococcus xanthus is a surface‐motile bacterium that has adapted at least one chemosensory system to allow directed movement towards the slowly diffusible lipid phosphatidylethanolamine (PE). The Dif chemosensory pathway is remarkable because it has at least three inputs coupled to outputs that control extracellular matrix (ECM) production and lipid chemotaxis. The methyl‐accepting chemotaxis protein, DifA, has two different sensor inputs that have been localized by mutagenesis. The Dif chemosensory pathway employs a novel protein that slows adaptation. Lipid chemotaxis may play important roles in the M. xanthus life cycle where prey‐specific and development‐specific attractants have been identified. Lipid chemotaxis may also be an important mechanism for locating nutrients by lung pathogens such as Pseudomonas aeruginosa.
Construction and analysis of chromosomal Clostridium difficile mutants
Authors: O'Connor Jennifer R. , Lyras Dena , Farrow Kylie A. , Adams Vicki , Powell David R. , Hinds Jason , Cheung Jackie K. , Rood Julian I.
Published: August 2006

Clostridium difficile is an emerging nosocomial pathogen of increasing importance and virulence but our ability to study the molecular mechanisms underlying the pathogenesis of C. difficile‐associated disease has been limited because of a lack of tools for its genetic manipulation. We have now developed a reproducible method for the targeted insertional inactivation of chromosomal C. difficile genes. The approach relies on the observation that an Escherichia coli–Clostridium perfringens shuttle vector is unstable in C. difficile and can be used as a form of conditional lethal vector to deliver gene constructs to the chromosome. We have used this methodology to insertionally inactivate two putative response regulator genes, rgaR and rgbR, which encode proteins with similarity to the toxin gene regulator, VirR, from C. perfringens. Transcriptomic analysis demonstrated that the C. difficile RgaR protein positively regulated four genes, including a putative agrBD operon. The RgaR protein was also purified and shown to bind specifically to sites that contained two consensus VirR boxes located just upstream of the putative promoters of these genes. The development of this methodology will significantly enhance our ability to use molecular approaches to develop a greater understanding of the ability of C. difficile to cause disease.
Identification of isolate‐specific and partial resistance to septoria tritici blotch in 238 European wheat cultivars and breeding lines
Authors: Arraiano L. S. , Brown J. K. M.
Published: August 2006

From a total of 238 European cultivars and breeding lines screened for isolate‐specific resistance to septoria tritici blotch (STB) with eight Mycosphaerella graminicola isolates from five different countries, 142 lines were resistant to Ethiopian isolate IPO88004, and 43 lines were specifically resistant to IPO323, with little or no leaf area bearing pycnidia of M. graminicola. These lines probably all have the resistance gene Stb6. Specific resistances to isolates CA30JI, IPO001, IPO89011, IPO92006 and ISR398 were less common. Seventy‐three per cent of the lines were specifically resistant to at least one isolate and 36 lines were resistant to more than one isolate. The line with the greatest number of specific resistances was the spring cultivar Raffles, with five. The most resistant line in which no specific resistance was identified was the Italian landrace Rieti, an ancestor of many modern European wheat cultivars. There was also a wide range of partial resistance among the lines tested, expressed in detached seedling leaves. Information about the resistance of wheat lines to M. graminicola isolates will assist breeders to choose parents of crosses from which progeny with superior resistance to STB may be selected.
Mapped Ds/T‐DNA launch pads for functional genomics in barley
Authors: Zhao Tiehan , Palotta Margaret , Langridge Peter , Prasad Manoj , Graner Andreas , Schulze‐Lefert Paul , Koprek Thomas
Published: August 2006

A system for targeted gene tagging and local saturation mutagenesis based on maize transposable elements (Ac/Ds) was developed in barley (Hordeum vulgare L.). We generated large numbers of transgenic barley lines carrying a single copy of the non‐autonomous maize Ds element at defined positions in the genome. Independent Ds lines were either generated by activating Ds elements in existing single‐copy lines after crossing with AcTPase‐expressing plants or by Agrobacterium‐mediated transformation. Genomic DNA flanking Ds and T‐DNA insertion sites from over 200 independent lines was isolated and sequenced, and was used for a sequence based mapping strategy in a barley reference population. More than 100 independent Ds insertion sites were mapped and can be used as launch pads for future targeted tagging of genes in the vicinity of the insertion sites. Sequence analysis of Ds and T‐DNA flanking regions revealed a sevenfold preference of both mutagens for insertion into non‐redundant, gene‐containing regions of the barley genome. However, whilst transposed Ds elements preferentially inserted adjacent to regions with a high number of predicted and experimentally validated matrix attachment regions (nuclear MARs), this was not the case for T‐DNA integration sites. These findings and an observed high transposition frequency from mapped launch pads demonstrate the future potential of gene tagging for functional genomics and gene discovery in barley.
Enhanced degradation in soil of tri‐allate and other carbamate pesticides following application of tri‐allate
Authors: COTTERILL E. G. , OWEN P. G.
Published: August 2006

Tri‐allate degraded faster in soil from a site (T1) that had received 1·7 kg ha−1 of tri‐allate annually for 23 years than in soil from an adjacent site (TO) that had received no pesticide application. Soil from the untreated site, which had been removed to a glasshouse and treated three times per annum with tri‐allate at 1·7 kg ha−1 for 7 years (T2), also showed faster degradation. Soil previously treated with tri‐allate showed an increased degradation rate for carbofuran and EPTC but not for aldicarb. A further experiment, 2 years after the last treatment with tri‐allate, showed that the enhanced degradation effect was still present. Degradation rates were always in the order T1 > T2 > T0 for tri‐allate, EPTC and carbofuran. Half‐life for degradation was reduced for tri‐allate and carbofuran by approximately 40% in the previously treated soils and for EPTC by approximately 80% when compared with the previously untreated soil.
Distinct features of chronic rhinosinusitis with and without nasal polyps
Authors: Polzehl D. , Moeller P. , Riechelmann H. , Perner S.
Published: August 2006

Background:  Based on the presence of nasal polyps on endoscopy, chronic rhinosinusitis (CRS) may be clinically divided in CRS with nasal polyps and CRS without nasal polyps. It is unclear, whether CRS with nasal polyps and CRS without nasal polyps represent different disease entities or just different stages of one single disease. In case of one disease, only minor histopathological differences between CRS with small early‐stage polyps (CRSNP(+)) and CRS without nasal polyps (CRSNP−) were expected.

Methods:  Patients with CRSNP(+) confined to the infundibular region or CRSNP− were selected. Histochemical and immunohistochemical characterization of ethmoidal mucosa was performed on formalin‐fixed and paraffin‐embedded tissue specimens. Frequency and distribution of eosinophils, neutrophils, mast cells, IgE+ cells, macrophages, B‐ and T‐cell subsets, natural killer cells, plasma cells and goblet cells were assessed. In addition, the thickness of the basal membrane was evaluated.

Results:  Nine CRS patients without detectable polyps, and 11 patients with small early‐stage polyps confined to the infundibular region were selected. Despite adjacent polyp stage, the amount of round cell infiltration (P < 0.05), number of eosinophils (P < 0.05), and plasma cells (P < 0.01) significantly differed in the ethmoidal specimens from patients of the two groups.

Conclusion:  Substantial histopathological differences were observed in ethmoidal mucosa of CRSNP(+) and CRSNP− patients. Thus, the results of this investigation support the concept that CRS with nasal polyps and CRS without nasal polyps are two different disease entities rather than different stages of one single disease, but may also be interpreted as a higher degree of inflammation.