bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcription of histone gene cluster by differential core-promoter factors
Authors: Isogai Yoh , Keles Sündüz , Prestel Matthias , Hochheimer Andreas , Tjian Robert
Published: November 2007

The 100 copies of tandemly arrayed Drosophila linker (H1) and core (H2A/B and H3/H4) histone gene cluster are coordinately regulated during the cell cycle. However, the molecular mechanisms that must allow differential transcription of linker versus core histones prevalent during development remain elusive. Here, we used fluorescence imaging, biochemistry, and genetics to show that TBP (TATA-box-binding protein)-related factor 2 (TRF2) selectively regulates the TATA-less Histone H1 gene promoter, while TBP/TFIID targets core histone transcription. Importantly, TRF2-depleted polytene chromosomes display severe chromosomal structural defects. This selective usage of TRF2 and TBP provides a novel mechanism to differentially direct transcription within the histone cluster. Moreover, genome-wide chromatin immunoprecipitation (ChIP)-on-chip analyses coupled with RNA interference (RNAi)-mediated functional studies revealed that TRF2 targets several classes of TATA-less promoters of >1000 genes including those driving transcription of essential chromatin organization and protein synthesis genes. Our studies establish that TRF2 promoter recognition complexes play a significantly more central role in governing metazoan transcription than previously appreciated.
A small RNA regulates multiple ABC transporter mRNAs by targeting C/A-rich elements inside and upstream of ribosome-binding sites
Authors: Sharma Cynthia M. , Darfeuille Fabien , Plantinga Titia H. , Vogel Jörg
Published: November 2007

The interactions of numerous regulatory small RNAs (sRNAs) with target mRNAs have been characterized, but how sRNAs can regulate multiple, structurally unrelated mRNAs is less understood. Here we show that Salmonella GcvB sRNA directly acts on seven target mRNAs that commonly encode periplasmic substrate-binding proteins of ABC uptake systems for amino acids and peptides. Alignment of GcvB homologs of distantly related bacteria revealed a conserved G/U-rich element that is strictly required for GcvB target recognition. Analysis of target gene fusion regulation in vivo, and in vitro structure probing and translation assays showed that GcvB represses its target mRNAs by binding to extended C/A-rich regions, which may also serve as translational enhancer elements. In some cases (oppA, dppA), GcvB repression can be explained by masking the ribosome-binding site (RBS) to prevent 30S subunit binding. However, GcvB can also effectively repress translation by binding to target mRNAs at upstream sites, outside the RBS. Specifically, GcvB represses gltI mRNA translation at the C/A-rich target site located at positions −57 to −45 relative to the start codon. Taken together, our study suggests highly conserved regions in sRNAs and mRNA regions distant from Shine-Dalgarno sequences as important elements for the identification of sRNA targets.
FoxO transcription factors suppress Myc-driven lymphomagenesis via direct activation of Arf
Authors: Bouchard Caroline , Lee Soyoung , Paulus-Hock Viola , Loddenkemper Christoph , Eilers Martin , Schmitt Clemens A.
Published: November 2007

FoxO transcription factors play critical roles in cell cycle control and cellular stress responses, and abrogation of FoxO function promotes focus formation by Myc in vitro. Here we show that stable introduction of a dominant-negative FoxO moiety (dnFoxO) into Eμ-myc transgenic hematopoietic stem cells accelerates lymphoma development in recipient mice by attenuating Myc-induced apoptosis. When expressed in Eμ-myc; p53+/− progenitor cells, dnFoxO alleviates the pressure to inactivate the remaining p53 allele in upcoming lymphomas. Expression of the p53 upstream regulator p19Arf is virtually undetectable in most dnFoxO-positive Myc-driven lymphomas. We find that FoxO proteins bind to a distinct site within the Ink4a/Arf locus and activate Arf expression. Moreover, constitutive Myc signaling induces a marked increase in nuclear FoxO levels and stimulates binding of FoxO proteins to the Arf locus. These data demonstrate that FoxO factors mediate Myc-induced Arf expression and provide direct genetic evidence for their tumor-suppressive capacity.
Sequence-based estimation of minisatellite and microsatellite repeat variability
Authors: Legendre Matthieu , Pochet Nathalie , Pak Theodore , Verstrepen Kevin J.
Published: November 2007

Variable tandem repeats are frequently used for genetic mapping, genotyping, and forensics studies. Moreover, variation in some repeats underlies rapidly evolving traits or certain diseases. However, mutation rates vary greatly from repeat to repeat, and as a consequence, not all tandem repeats are suitable genetic markers or interesting unstable genetic modules. We developed a model, “SERV,” that predicts the variability of a broad range of tandem repeats in a wide range of organisms. The nonlinear model uses three basic characteristics of the repeat (number of repeated units, unit length, and purity) to produce a numeric “VARscore” that correlates with repeat variability. SERV was experimentally validated using a large set of different artificial repeats located in theSaccharomyces cerevisiae URA3gene. Further in silico analysis shows that SERV outperforms existing models and accurately predicts repeat variability in bacteria and eukaryotes, including plants and humans. Using SERV, we demonstrate significant enrichment of variable repeats within human genes involved in transcriptional regulation, chromatin remodeling, morphogenesis, and neurogenesis. Moreover, SERV allows identification of known and candidate genes involved in repeat-based diseases. In addition, we demonstrate the use of SERV for the selection and comparison of suitable variable repeats for genotyping and forensic purposes. Our analysis indicates that tandem repeats used for genotyping should have a VARscore between 1 and 3. SERV is publicly available fromhttp://hulsweb1.cgr.harvard.edu/SERV/.
Confirmation study of prostate cancer risk variants at 8q24 in African Americans identifies a novel risk locus
Authors: Robbins Christiane , Torres Jada Benn , Hooker Stanley , Bonilla Carolina , Hernandez Wenndy , Candreva Angela , Ahaghotu Chiledum , Kittles Rick , Carpten John
Published: November 2007

Prostate cancer is a common complex disease that disproportionately affects men of African descent. Recently, several different common variants on chromosome 8q24 have been shown to be associated with prostate cancer in multiple studies and ethnic groups. The objective of this study was to confirm the association of 8q24 markers with prostate cancer in African Americans. We genotyped 24 markers along 8q24 and 80 unlinked ancestry informative markers in a hospital-based case-control sample of 1057 African American men (490 prostate cancer cases and 567 controls). Association analyses of 8q24 markers with prostate cancer risk were adjusted for both global and local 8q24 admixture stratification using estimates from ancestry informative markers. We report that rs7008482, which maps to the 8q24.13 region, is an additional independent prostate cancer risk variant (P = 5 × 10−4), and we also replicate the association of rs16901979 with prostate cancer (P = 0.002). Other published risk variants in the region such as rs1447295 and rs6983267 showed a similar direction and magnitude of effect, but were not significant in our population. Both rs7008482 and rs16901979 independently predicted risk and remained significant (P < 0.001) after controlling for each other. Our data combined with additional replications of 8q24 markers provide compelling support for multiple regions of risk for prostate cancer on 8q24.
Quantitation of DNA and RNA
Authors: Barbas Carlos F. , Burton Dennis R. , Scott Jamie K. , Silverman Gregg J.
Published: November 2007

INTRODUCTION

There are several ways to quantitate solutions of nucleic acids. If the solution is pure, one can use a spectrophotometer to measure the amount of ultraviolet radiation absorbed by the bases. DNA can also be quantified by measuring the UV-induced emission of fluorescence from intercalated ethidium bromide. This method is useful if there is not enough DNA to quantify with a spectrophotometer, or if the DNA solution is contaminated. Strategies for accurately quantifying nucleic acids using these approaches are discussed here.
Preparation of Reversed-Phase Microcolumns: Figure 1.
Authors: Larsen Martin R.
Published: November 2007

INTRODUCTION

One versatile strategy for sample cleanup prior to MALDI-MS analysis uses microscale columns designed for direct sample elution onto the MALDI target plate. This protocol describes the fabrication of a reversed-phase microcolumn designed for this purpose. The microcolumns are prepared from GELoader tips. This protocol has been optimized for sample cleanup prior to MALDI-MS. However, with slight modifications, it works equally well with samples destined for ESI-MS.
Phosphopeptide Purification by IMAC with Fe(III) and Ga(III)
Authors: Steen Hanno , Stensballe Allan , Jensen Ole N.
Published: November 2007

INTRODUCTION

Immobilized metal ion affinity chromatography (IMAC) makes use of matrix-bound metals to affinity-purify phosphoproteins and phosphopeptides. Commonly used metals in early studies such as Ni2+, Co2+, Zn2+, and Mn2+ were shown to bind strongly to proteins with a high density of histidines. More recently, immobilized Fe3+, Ga3+, and Al3+ metal ions have been used for the selective enrichment of phosphopeptides from complex proteolytic digest mixtures containing both phosphorylated and nonphosphorylated components. The use of a nitrilotriacetic acid (NTA) matrix over iminodiacetic-acid-modified matrices has been reported to provide an advantage in selectivity. The development of elution conditions that are directly compatible with MS analysis of the enriched phosphopeptide samples provides the option to interface IMAC and MS online. This protocol describes the enrichment of phosphopeptides by IMAC using Fe3+- and Ga3+-NTA resin.
Offline Micro-IMAC Enrichment of Phosphoproteins
Authors: Corthals Garry L. , Wasinger Valerie C. , Goodlett David R.
Published: November 2007

INTRODUCTION

Immobilized metal affinity chromatography (IMAC) can be performed offline or online with μLC-ESI-MS/MS. The online configuration has been used successfully despite suboptimal low-level analysis due to the relative disparity between IMAC and μLC flow rates. With IMAC performed offline from μLC-ESI-MS, sample volumes from 1 to 100 μL can be loaded using a sample pressure vessel or an autosampling system equipped with a sample-trapping cartridge. This protocol describes the construction and use of a micro-IMAC column for the enrichment of phosphoproteins.
Imaging Mouse Embryos after Whole-Mount In Situ Hybridization
Authors: Wakamiya Maki
Published: November 2007

INTRODUCTION

Embryos that are processed for whole-mount in situ hybridization can be photodocumented after the staining procedure depending on the region of the embryo of interest. The embryos can be photodocumented immediately after the color reaction or after clearing in glycerol solutions. This protocol describes methods for embryo clearing and imaging.
Sectioning Mouse Embryos after Whole-Mount In Situ Hybridization
Authors: Shen Michael
Published: November 2007

INTRODUCTION

Embryos that have been processed for whole-mount in situ hybridization are sectioned frozen or embedded in wax to visualize which tissues express the gene transcripts.
Handling Mouse Blastocysts for Fixation
Authors: Nagy Andras , Gertsenstein Marina , Vintersten Kristina , Behringer Richard
Published: November 2007

INTRODUCTION

Whole blastocysts or sections of blastocysts can be fixed and used for histological studies, including in situ hybridization and immunohistochemistry. As described in this protocol, different handling techniques are required for whole versus sectioned embryos. For sectioning, it is more convenient to transfer blastocysts into the ampulla of an oviduct prior to fixation. The oviduct serves as a carrier that is easy to handle during subsequent processing for histology.
Embedding Mouse Embryos and Tissues in Wax
Authors: Nagy Andras , Gertsenstein Marina , Vintersten Kristina , Behringer Richard
Published: November 2007

INTRODUCTION

This protocol describes how to embed mouse tissues and embryos (large and small) in wax. The specimens must be dehydrated prior to embedding. Embedded samples are subsequently sectioned and used for in situ hybridization and immunohistochemistry experiments.
Preparing Glass Slides and Coverslips for In Situ Hybridization
Authors: Nagy Andras , Gertsenstein Marina , Vintersten Kristina , Behringer Richard
Published: November 2007

INTRODUCTION

Precleaned glass slides are of high enough quality for both in situ and immunohistochemical techniques. However, for in situ hybridization, the slides need to be treated with diethyl pyrocarbonate (DEPC) so that any RNase attached to them is destroyed. The slides also need to be coated with 3-triethoxysilylpropylamine (TESPA) or poly-L-lysine so that the sections adhere tightly and do not detach during subsequent extensive washing procedures. This protocol describes techniques for coating slides with TESPA and poly-L-lysine. There are advantages and disadvantages to each coating method. TESPA-treated slides can be stored for a long time, but the sections do not adhere tightly until after drying. Poly-L-lysine-coated slides need to be made fresh, but the sections adhere immediately on contact with the surface. This protocol also describes how to prepare coverslips for in situ hybrization by coating them in a siliconizing solution.
In Situ Hybridization of Mouse Embryo and Tissue Sections with Radiolabeled RNA Probes
Authors: Nagy Andras , Gertsenstein Marina , Vintersten Kristina , Behringer Richard
Published: November 2007

INTRODUCTION

This protocol describes in situ hybridization of embryo and tissue sections with 35S-labeled, single-stranded, antisense RNA probes (riboprobes). Protocols have also been developed for in situ hybridization to tissue sections using nonradiolabeled RNA probes that can be detected with antibodies coupled to alkaline phosphatase and a chromogenic substrate. Nonradioactive methods have the advantage that the results can be obtained relatively quickly, but the sensitivity is probably lower than with radioactive probes. In addition, care must be taken to optimize the amount of each probe used in the hybridization reaction.
In Situ Hybridization of Whole-Mount Mouse Embryos with RNA Probes: Preparation of Embryos and Probes
Authors: Lufkin Thomas
Published: November 2007

INTRODUCTION

This is the first of two protocols describing how to perform in situ hybridization on whole mouse embryos. Here we describe how to fix and permeabilize embryos with detergent and how to synthesize single-stranded RNA probes (riboprobes) in vitro using UTP labeled with the plant steroid digoxigenin (DIG-11-UTP). Antisense digoxigenin-labeled riboprobes are synthesized as run-off transcripts from linearized plasmid templates using bacteriophage RNA polymerases (T3, T7, SP6) under standard conditions. The probe is precipitated with ethanol in the presence of NaCl, and after resuspension in DEPC-treated H2O is stable at −20°C for many months. Probes ranging from 250 to 1500 bp have been used successfully. The optimal probe concentration is in the 100 ng to 1 μg/mL range and should be determined empirically.
In Situ Hybridization of Whole-Mount Mouse Embryos with RNA Probes: Hybridization, Washes, and Histochemistry
Authors: Lufkin Thomas
Published: November 2007

INTRODUCTION

This is the second of two protocols describing how to perform in situ hybridization on whole mouse embryos. Here we describe the hybridization procedure, and the localization of the DIG-labeled RNA using a conjugate of anti-DIG Fab antibody and calf intestinal alkaline phosphatase. Enzyme activity of the reporter is detected by a color reaction, resulting in the formation of a water-insoluble purple/blue precipitate.
Imaging Real-Time Gene Expression in Living Yeast
Authors: Zenklusen Daniel , Wells Amber L , Condeelis John S. , Singer Robert H.
Published: November 2007

INTRODUCTION

This protocol describes the application of the MS2 system to the yeast Saccharomyces cerevisiae. ASH1 mRNA tagged with six MS2 repeats (6MBSs) is used to follow the localization of the ASH1 mRNA particles to the bud tip of a haploid yeast cell. W303 yeast cells transformed with pG14-MS2-GFP and pGAL-lacZ-MS2-ASH1 are grown on select medium lacking tryptophan and leucine. RNA expression is induced by the addition of galactose, and a time-lapse movie is then acquired.
Imaging Real-Time Gene Expression in Living Systems with Single-Transcript Resolution: Image Analysis of Single mRNA Transcripts
Authors: Wells Amber L. , Condeelis John S. , Singer Robert H. , Zenklusen Daniel
Published: November 2007

INTRODUCTION

This protocol describes a method for establishing a green fluorescent protein (GFP) calibration curve using dilutions of recombinant GFP and blue fluorescent beads. The total fluorescence intensity (TFI) per mRNA molecule is first calculated by imaging serial dilutions of purified enhanced GFP (eGFP) to determine the TFI within a specific volume. A calibration curve of fluorescence intensity in a given voxel per molecule of GFP is then used to determine the number of GFP molecules in the sample of formaldehyde fixed cells to be imaged. This is followed by a method for detection of single molecules in formaldehyde-fixed and live cells. These cells have been cotransfected with mRNA reporter and MCP-xFP plasmids, where MCP-xFP refers to a fluorescent protein fused to the MS2 capsid protein. It is important to collect micrographs and establish the calibration curve on the same day that the cells are imaged, using the same equipment configuration, camera settings, and image acquisition parameters.
Micrococcal Nuclease-Southern Blot Assay: I. MNase and Restriction Digestions: Figure 1.
Authors: Carey Michael , Smale Stephen T.
Published: November 2007

INTRODUCTION

This is the first phase of a two-part assay for exploring whether an endogenous control region of interest is assembled into nucleosomes or is devoid of nucleosomes. This assay can also be used to determine if nucleosomes are similarly positioned at the locus in every cell within a population. Micrococcal nuclease (MNase) is unique among nucleases in its relative ability to induce double-strand breaks within nucleosomal linker regions, but only single-stranded nicks within the nucleosome itself. Because of this property, micrococcal nuclease can be used to determine whether a DNA fragment of interest is nucleosomal. In this phase of the assay, the cells are lysed and nuclei are isolated. Limiting concentrations of micrococcal nuclease are added to the nuclei, resulting in cleavage at nucleosome linker regions (preferably cleavage at two sites per DNA molecule). The cleavage reactions are stopped, and then the genomic DNA is purified and digested with restriction enzymes.
Micrococcal Nuclease-Southern Blot Assay: II. Capillary Transfer and Hybridization: Figure 1.
Authors: Carey Michael , Smale Stephen T.
Published: November 2007

INTRODUCTION

This is the second phase of a two-part assay for exploring whether an endogenous control region of interest is assembled into nucleosomes or is devoid of nucleosomes. This assay can also be used to determine if nucleosomes are similarly positioned at the locus in every cell within a population. In this second part of the assay, the restricted genomic DNA is separated by agarose gel electrophoresis and stained with ethidium bromide, resulting in a ladder of bands corresponding in size to multiples of the nucleosome core plus linker (~200 bp). To determine whether a DNA fragment of interest is nucleosomal, the genomic DNA is subjected to Southern blot analysis. If a probe derived from the DNA fragment hybridizes to the ladder of nucleosomal bands, the fragment may indeed be assembled into nucleosomes.
Fate-Mapping Technique: Targeted Whole-Embryo Electroporation of DNA Constructs into the Germ Layers of Mouse Embryos 7-7.5 Days Post-coitum
Authors: Khoo P.-L. , Franklin V.J. , Tam P.P.L.
Published: November 2007

INTRODUCTION

Fate maps reveal body plan organization and presage the expression of molecular characteristics of cell lineages and formation of body parts. This protocol targets DNA expression constructs into the germ layers of gastrula-stage mouse embryos by focal electroporation. Plasmids utilizing a promoter that drives widespread, non-lineage-restricted expression of transgenes are introduced to cells in defined germ layer regions by whole-embryo electroporation. Germ-layer cells are exposed to the DNA by microinjecting the plasmids into the proamniotic cavity (ectoderm) or directly into the intercellular space of the mesenchyme (mesoderm), or by incubating the embryo in the DNA solution (endoderm). Electroporation is performed on whole embryos in vitro by electric current-mediated permeation of the cell membrane, which allows DNA adsorbed to cell surfaces to enter the cells. A point electrode is used to focus the electric field to the intended site of electroporation and a plate electrode is used to generate the current at an effective voltage low enough to minimize damage to the embryonic tissue. Expression of the transgene can be used to track the fate and movement of cells and the cDNA to study the functional consequences of overexpression of genes during embryonic development in vitro.
Combined Flow Cytometric Measurement of Two Cell-Surface Antigens and DNA-RNA Content: Figure 1.
Authors: Schmid Ingrid
Published: November 2007

INTRODUCTION

Flow cytometry is frequently used to assess nucleic acid content in individual cells. Based on DNA content alone, however, cells in the quiescent G0 phase cannot be discriminated from cells in the proliferative G1 phase, as DNA content remains constant until S-phase entry. In contrast, by measuring RNA content in addition to DNA content, cells can be assigned to G0 and cell-cycle subcompartments of G1. Assessing phenotype at the same time as nucleic acid content allows determination of the cell-cycle status of subpopulations in mixed-cell preparations. This protocol describes an optimized method for combining dual-color cell-surface immunofluorescent staining with staining for DNA-RNA, adapted for a basic dual-laser flow cytometer with blue (488-nm) and red (633- or 647-nm) excitation. DNA is stained at low pH in the presence of saponin with 7-aminoactinomycin D (7-AAD), and RNA is stained with pyronin Y (PY). Both dyes are used at low concentration, and 7-AAD is exchanged with nonfluorescent actinomycin D to minimize fluorochrome-fluorochrome interactions, which can negatively affect detection of cell-surface antigen staining.
Imaging Real-Time Gene Expression in Living Systems with Single-Transcript Resolution: Construct Design and Imaging System Setup
Authors: Wells Amber L. , Condeelis John S. , Singer Robert H. , Zenklusen Daniel
Published: November 2007

INTRODUCTION

The most common way for a cell to respond to internal and external signals is to change its gene expression pattern. This requires the synchronization of regulatory steps along the expression pathway. Biological imaging techniques can be used to visualize and measure such processes in individual live cells in real time. This article discusses the use of a fluorescent RNA-binding protein system that allows real-time analysis of gene expression with single-transcript resolution.
Genetic diversity and population structure of indigenous yellow cattle breeds of China using 30 microsatellite markers
Authors: Zhang G. X. , Wang Z. G. , Chen W. S. , Wu C. X. , Han X. , Chang H. , Zan L. S. , Li R. L. , Wang J. H. , Song W. T. , Xu G. F. , Yang H. J. , Luo Y. F.
Published: November 2007

Twenty‐seven domesticated yellow cattle breeds of China and three introduced cattle breeds were analysed by means of 30 microsatellite markers to determine the level of genetic variation within and among populations as well as the population structure. In all, 480 microsatellite alleles were observed across the 30 breeds with the mean number of alleles per locus of 9.093 for native breeds and 6.885 for the three introduced breeds. Mean F‐statistics (0.08) for Chinese native cattle breeds implied that 92% of the total genetic variation was from genetic differentiation within each breed and 8% of the genetic variation existed among breeds. A phylogenetic tree was constructed based on Nei's genetic distances, and three clusters were obtained. According to the tree, the three introduced breeds were distinct from the 27 native breeds. The indigenous cattle breeds were divided into two clusters, one cluster including five humpless breeds and the other cluster containing 22 humped breeds. This study identifies multiple origins of yellow cattle of China from Bos taurus and Bos indicus. Furthermore, population structure analysis implies that there are possibly five independent original domestications for yellow cattle in China. Four of five origins were four different Bos indicus types, mainly in areas of the Chang Jiang, the Zhu Jiang River basin, the Yellow River and the Huai River basin. The other origin was for Bos taurus type of Mongolian descent, mainly located in Northwestern China, the Mongolian plateau and Northeastern China or north of the Great Wall.
Anti‐myeloperoxidase and anti‐cathepsin G antibodies in sulphonamide hypersensitivity
Authors: Lavergne S. N. , Drescher N. J. , Trepanier L. A.
Published: November 2007

BackgroundAnti‐neutrophil cytoplasmic antibodies (ANCA) are associated with vasculitis in humans. Sulphonamide antimicrobials cause drug hypersensitivity (HS) reactions with some clinical signs that are suggestive of vasculitis.

ObjectiveThe purpose of this study was to determine whether sulphonamide HS is associated with anti‐neutrophil antibodies, using the dog as a spontaneous clinical model.

MethodsThirty‐four sulphonamide‐HS dogs, 11 sulphonamide‐‘tolerant’ dogs, and nine healthy sulphonamide‐naïve dogs were evaluated for anti‐neutrophil antibodies using a commercial ELISA against human myeloperoxidase (MPO), a commercial human ANCA Western blot protocol, and immunoblotting against whole canine neutrophils.

ResultsUsing ELISA, anti‐MPO antibodies were found with an apparent higher frequency in HS dogs (50%), compared with ‘tolerant’ dogs (18%), which also showed significantly lower absorbances. Among HS dogs, anti‐MPO antibodies were significantly more common, with significantly higher absorbances, in dogs that did not survive the HS reaction (78%) compared with survivors (35%). Using immunoblotting, ANCA were detected with similar overall frequencies in HS and ‘tolerant’ dogs. However, one protein targeted by several HS dogs, but no ‘tolerant’ dogs, was identified as cathepsin G.

ConclusionThese data indicate that anti‐MPO antibodies and anti‐cathepsin G antibodies are associated with sulphonamide HS. Anti‐MPO antibodies have been shown to be pathogenic bothin vitroandin vivo, leading to vasculitis lesions and vasculitis‐like syndromes. The present study therefore suggests that vasculitis might be one mechanism of tissue damage in this sulphonamide HS. Furthermore, the evaluation of ANCA, and its relationship to disease severity and clinical outcome, should be considered in human patients with sulphonamide drug HS.
Relationship between early intestinal colonization, mucosal immunoglobulin A production and systemic immune development
Authors: Martino D. J. , Currie H. , Taylor A. , Conway P. , Prescott S. L.
Published: November 2007

Background
Variations in early intestinal colonization patterns have been implicated in the predispostion to allergic disease through effects on mucosal and systemic immune function.

Methods
Colonization patterns and mucosal IgA production at 6 months of age were examined in relation to early exposures, systemic immune development and early allergic outcomes. This was performed in a cohort (n=189) who had received either Lactobacillus acidophilus or a placebo since birth.

Results
Children who developed sensitization to one or more allergens by 12 months of age had significantly higher total (but not secretory) IgA levels at 6 months (P=0.024), but this was independent of probiotic treatment. There were no relationships between colonization patterns and IgA (total or secretory IgA) levels or allergic outcomes (sensitization, atopic dermatitis or food allergies). Breastfeeding, and the use of yoghurt or probiotic supplements were independently associated with significantly higher proportions of ‘favourable’ lactobacilli and bifidobacteria species. Caesarean delivery and the use of antibiotics during the first 6 months were not associated with significant variations in colonization. Higher proportions of ‘favourable’ bacteria (notably bifidobacteria) were associated with reduced production of most cytokine response to allergens, but increased expression of regulatory cell markers (P=0.009) and transforming growth factor‐β (P=0.016).

Conclusion
‘Favourable’ colonization, particularly with bifidobacteria, was associated with effects on cellular immune function, but this was not associated with mucosal immunity (secretory IgA) or reduced risk of allergic disease.
Effect of stress on basophil function in chronic idiopathic urticaria
Authors: Dyke S. M. , Carey B. S. , Kaminski E. R.
Published: November 2007

Background
Chronic idiopathic urticaria (CIU) is a distressing skin condition involving recurrent itchy hives lasting 6 weeks or longer. The mechanism involves mast cell and basophil degranulation, which releases inflammatory mediators including histamine. In our clinical practice, we have observed that the onset of CIU is often preceded by a major life event.

Objective
To investigate the role of the hormones of the hypothalamic–pituitary–adrenal (HPA) axis in the link between psychological stress and CIU.

Methods
Thirty people with CIU and 30 normal controls were recruited. A flow cytometric CD63 expression assay was used to quantify basophil activation, and serum cortisol concentrations were measured as an indication of stress.

Results
Both corticotrophin releasing factor (CRF) and adrenocorticotrophic hormone (ACTH) were shown to activate basophils. There was no significant difference between numbers of CIU patients and normal controls responding to CFR, ACTH or cortisol. However, the responses in the CIU patients were stronger than those in normal controls. There was also a trend towards higher serum cortisol concentrations in CIU patients. The basophil response to CRF and ACTH correlated with the serum cortisol concentration in normal controls, but not in CIU patients.

Conclusions
Although our data have not supported the hypothesis that stress makes a major contribution to CIU, the heightened basophil response to CFR and ACTH and higher levels of serum cortisol do suggest a derangement of the HPA axis in CIU.
Beta‐sitosterolaemia: a new nonsense mutation in the ABCG5 gene
Authors: Mannucci L. , Guardamagna O. , Bertucci P. , Pisciotta L. , Liberatoscioli L. , Bertolini S. , Irace C. , Gnasso A. , Federici G. , Cortese C.
Published: November 2007

Background  Sitosterolaemia is a rare autosomal recessive disorder characterised by elevated plasma levels of plant sterols and cholesterol. Sitosterolaemia is caused by gene mutations in either of two ATP‐binding cassette (ABC) half transporters, ABCG5 and ABCG8. The plasma sterol profile and genetic analysis of a 10‐year‐old girl who had tuberous xanthomas is the subject of this report.

Materials and methods  Genomic DNA was isolated from white blood cells from the proband, her family and a control group of healthy people. All exons of ABCG5 and ABCG8 were sequenced. Plasma cholesterol and triglycerides were measured by routine methods. All other plasma sterols were measured by Gas Chromatography coupled to Mass Spectrometry.

Results  The proband was found to be homozygous for a single nucleotide mutation in exon 10 of the ABCG5 gene, consisting of a C to T transition at nucleotide 1336 of the coding sequence, which results in the premature termination of the ABCG5 protein at amino acid 446 (Arg446X). Her mother and brother were also homozygous for the same mutation and all had elevated plasma beta‐sitosterol levels. The father was heterozygous and showed normal beta‐sitosterol levels. This mutation was not found in healthy normolipidaemic subjects.

Conclusions  We describe a novel nonsense mutation in exon 10 of the ABCG5 gene in a 10‐year‐old girl showing clinical and biochemical features of sitosterolaemia. This family study broadens the spectrum of the ABCG5/ABCG8 mutations causing sitosterolaemia and helps highlight the correlations between such gene mutations, biochemical phenotype and the development of cardiovascular disease.
Declining trends in conception rates in recent birth cohorts of native Danish women: a possible role of deteriorating male reproductive health
Authors: Jensen Tina Kold , Sobotka Tomáš , Hansen Martin A. , Pedersen Anette Tønnes , Lutz Wolfgang , Skakkebæk Niels E.
Published: November 2007

Recent findings of poor semen quality among at least 20% of normal young men in Denmark prompted us to use unique Danish registers on births and induced abortions to evaluate a possible effect of the poor male fecundity on pregnancy rates among their presumed partners – the younger cohorts of women. We have analysed data from the Danish birth and abortion registries as well as the Danish registry for assisted reproduction (ART) and defined a total natural conception rate (TNCR), which is equal to fertility rate plus induced abortion rate minus ART conception rate. A unique personal identification number allowed the linkage of these databases. Our database included 706 270 native Danish women born between 1960 and 1980. We used projections to estimate the fertility of the later cohorts of women who had not yet finished their reproduction. We found that younger cohorts had progressively lower TNCR and that in terms of their total fertility rate, the declining TNCR is compensated by an increasing use of ART. Our hypothesis of an ongoing birth cohort‐related decline in fecundity was also supported by our finding of increasing and substantial use of ART in the management of infertility of relatively young couples in the later cohorts. Furthermore, the lower rates of induced abortion among the younger birth cohorts, often viewed as a success of health education programs, may not be fully explained by improved use of contraception. It seems more likely that decreased fecundity because of widespread poor semen quality among younger cohorts of otherwise normal men may explain some of the observed decline in conception rates. This may imply increasing reproductive health problems and lower fertility in the future, which is difficult to reverse in the short term. The current and projected widespread use of ART in Denmark may be a sign of such an emerging public health problem.
The restoration of ecological interactions: plant–pollinator networks on ancient and restored heathlands
Authors: Forup Mikael Lytzau , Henson Kate S. E. , Craze Paul G. , Memmott Jane
Published: November 2007

Attempts to restore damaged ecosystems usually emphasize structural aspects of biodiversity, such as species richness and abundance. An alternative is to emphasize functional aspects, such as patterns of interaction between species. Pollination is a ubiquitous interaction between plants and animals. Patterns in plant–pollinator interactions can be analysed with a food web or complex‐systems approach and comparing pollination webs between restored and reference sites can be used to test whether ecological restoration has taken place.

Using an ecological network approach, we compared plant–pollinator interactions on four pairs of restored and ancient heathlands 11 and 14 years following initiation of restoration management. We used the network data to test whether visitation by pollinators had been restored and we calculated pollinator importance indices for each insect species on the eight sites. Finally, we compared the robustness of the restored and ancient networks to species loss.

Plant and pollinator communities were established successfully on the restored sites. There was little evidence of movement of pollinators from ancient sites onto adjacent restored sites, although paired sites correlated in pollinator species richness in both years. There was little insect species overlap within each heathland between 2001 and 2004.

A few widespread insect species dominated the communities and were the main pollinators. The most important pollinators were typically honeybees (Apis mellifera), species of bumblebee (Bombus spp.) and one hoverfly species (Episyrphus balteatus). The interaction networks were significantly less complex on restored heathlands, in terms of connectance values, although in 2004 the low values might reflect the negative relationship between connectance and species richness. Finally, there was a trend of restored networks being more susceptible to perturbation than ancient networks, although this needs to be interpreted with caution.

Synthesis and applications. Ecological networks provide a powerful tool for assessing the outcome of restoration programmes. Our results indicate that heathland restoration does not have to occur immediately adjacent to ancient heathland for functional pollinator communities to be established. Moreover, in terms of restoring pollinator interactions, heathland managers need only be concerned with the most common insect species. Our focus on pollination demonstrates how a key ecological service can serve as a yardstick for judging restoration success.
Do small mammals avoid roads because of the traffic?
Authors: McGregor Rachelle L , Bender Darren J , Fahrig Lenore
Published: November 2007

Roads can act as barriers to animal movement, which may reduce population persistence by reducing recolonization of empty habitats and limiting immigration. Appropriate mitigation of this barrier effect (e.g. seasonal road closures, location and design of wildlife over‐ or underpasses) depends upon whether the animals avoid the road itself or the traffic on the road. Empirical studies of road avoidance to date do not generally differentiate between these.

We conducted short‐ and long‐distance translocations and trapping studies of white‐footed mice (Peromyscus leucopus) and eastern chipmunks (Tamias striatus) near two‐lane paved roads, which differed widely in traffic amount, from 47 to 15 433 vehicles per day.

In the trapping study (13 sites) only five animals moved across a road, in comparison to 36 animals that moved the same distance without an intervening road (P < 0·0001). In the short‐distance translocations (15 sites), 51% of the small mammals that were translocated across roads returned, in comparison to a return rate of 77% of animals that were translocated a similar distance with no intervening road (P = 0·009).

In the long‐distance translocation study (24 sites) we found that each intervening road reduced the probability of successful return by about 50%.

We found no significant effects of traffic amount on return rates in either the short‐distance or the long‐distance translocations studies.

Small mammal densities were not lower near roads and we found no evidence for a decrease in density near roads with increasing traffic amount.

Synthesis and applications. Our results suggest that small mammals avoid the road itself, and not emissions such as noise from the traffic on the roads. Our results imply that the barrier effect of roads on these species cannot be mitigated by measures aimed at reducing traffic amount; other measures such as wildlife passages would be needed.
Land‐use change and subalpine tree dynamics: colonization of Larix decidua in French subalpine grasslands
Authors: Albert Cécile H. , Thuiller Wilfried , Lavorel Sandra , Davies Ian D. , Garbolino Emmanuel
Published: November 2007

In many places in Europe, trees are currently far below their theoretical altitudinal limit because of past land use. However, under the current crisis affecting mountain agriculture, trees are likely to recolonize the subalpine belt. Addressing the societal issues associated with such landscape change requires predictive tools to assess vegetation dynamics in relation to management strategies. This study aims to analyse the factors determining tree dynamics at the subalpine ecotone and to evaluate the impact of land‐use change on landscape vegetation patterns.

We developed a hierarchical scaling approach and applied it to a European larch (Larix decidua Mill.) colonization in the French Alps. Our landscape case study focused on subalpine south‐facing grasslands of Villar d’Arêne (France). First, we used a habitat‐suitability model to delineate the climatic suitability habitats of larch in the French Alps. Then we used a landscape model (LaMoS) to determine whether larch is able to colonize these grasslands predicted to be climatically suitable, considering land use and local vegetation. The sensitivity of landscape colonization patterns to land‐use scenarios (abandonment, delayed mowing or traditional mowing) and biological attributes of larch were analysed with a factorial simulation experiment.

The accuracy of the habitat‐suitability model at the French Alps scale allowed for the prediction that the grasslands of Villar d’Arêne were highly suitable for larch.

Simulations highlighted the effect of land use on larch establishment at the local but not at the landscape scale. On the other hand, larch attributes such as dispersal capacity, juvenile tolerance to light interaction (competition and facilitation) and its capacity to survive on infertile soil were shown as essential for tree spatial dynamics.

 Synthesis and applications. Combining a habitat‐suitability model with a spatially and temporally explicit landscape model enhances the capacity to account for environmental factors acting at different overlapping scales. This modelling strategy increases the robustness and accuracy of predictions, a prerequisite for landscape management in a global change context. We conclude that ongoing and future agri‐environmental policies have to be quickly adapted to protect biodiversity and ecosystem services provided by subalpine grasslands.
Wild bee pollinators provide the majority of crop visitation across land‐use gradients in New Jersey and Pennsylvania, USA
Authors: Winfree Rachael , Williams Neal M. , Gaines Hannah , Ascher John S. , Kremen Claire
Published: November 2007

Concern about a global decline in wild pollinators has increased interest in how pollinators are affected by human land use, and how this, in turn, affects crop pollination.

We measured wild bee visitation to four summer vegetable crops, and investigated associations between flower visitation rates and land‐use intensity at local and landscape scales. We studied 29 farms in New Jersey and Pennsylvania, USA. Over 2 years we recorded >7400 bee visits to crop flowers and identified 54 species of wild bees visiting crops.

Wild bees were the dominant flower visitors at three of the four crops studied; domesticated honeybees, Apis mellifera L., provided the remainder of visits.

Ordination of the two best studied crops showed that the wild bee species visiting tomato, Solanum lycopersicum L., were distinct from those visiting watermelon, Citrullus lanatus (Thunb.) Matsum. & Nakai.

Crop visitation by wild bees was not associated with organic farming, nor with natural habitat cover at either local or landscape scale.

Synthesis and applications. The extent of crop visitation by wild bees observed in this study is among the highest recorded. In contrast to previous studies of crop visitation by wild bees, we did not find negative effects of conventional farming or natural habitat loss. In our study system, organic and conventional farms differ little in field size, crop diversity and weedy flower diversity, unlike some systems where organic farms have smaller fields with greater crop and weed diversity. Such variables may be more important than organic vs. conventional farming practices. Second, small patches of natural habitat are dispersed throughout our entire study system, and this habitat heterogeneity may support high bee abundance even in landscapes with a low proportion of natural habitat overall. Our findings suggest that agri‐environment schemes and similar programmes that work through local habitat restoration should target farms in intensively agricultural (homogeneous) landscapes to gain maximum conservation benefits.
Testing the accuracy of synthetic stellar libraries
Authors: Martins Lucimara P. , Coelho Paula
Published: November 2007

One of the main ingredients of stellar population synthesis models is a library of stellar spectra. Both empirical and theoretical libraries are used for this purpose, and the question about which one is preferable is still debated in the literature. Empirical and theoretical libraries are being improved significantly over the years, and many libraries have become available lately. However, it is not clear in the literature what are the advantages of using each of these new libraries, and how far behind models are compared to observations. Here we compare in detail some of the major theoretical libraries available in the literature with observations, aiming at detecting weaknesses and strengths from the stellar population modelling point of view. Our test is twofold: we compared model predictions and observations for broad-band colours and for high-resolution spectral features. Concerning the broad-band colours, we measured the stellar colour given by three recent sets of model atmospheres and flux distributions, and compared them with a recent UBVRIJHK calibration which is mostly based on empirical data. We found that the models can reproduce with reasonable accuracy the stellar colours for a fair interval in effective temperatures and gravities. The exceptions are (1) the U−B colour, where the models are typically redder than the observations, and (2) the very cool stars in general (V−K≳ 3). Castelli & Kurucz is the set of models that best reproduce the bluest colours (U−B, B−V) while Gustafsson et al. and Brott & Hauschildt more accurately predict the visual colours. The three sets of models perform in a similar way for the infrared colours. Concerning the high-resolution spectral features, we measured 35 spectral indices defined in the literature on three high-resolution synthetic libraries, and compared them with the observed measurements given by three empirical libraries. The measured indices cover the wavelength range from ∼3500 to ∼8700 Å. We found that the direct comparison between models and observations is not a simple task, given the uncertainties in parameter determinations of empirical libraries. Taking that aside, we found that in general the three libraries present similar behaviours and systematic deviations. For stars with Teff≤ 7000 K, the library by Coelho et al.is the one with best average performance. We detect that lists of atomic and molecular line opacities still need improvement, specially in the blue region of the spectrum, and for the cool stars (Teff≲ 4500 K).