Genetic characterization of a herd of the endangered Danish Jutland cattle.

In this paper we present results from a genetic characterization of a herd of the Danish Jutland cattle breed named the Kortegaard herd (n = 135; 57 males and 78 females). The herd is genotyped on the Bovine HD BeadChip microarray with 697,548 evenly spaced SNP across the bovine genome. The aim of the study was to characterize the genetic profile of the Kortegaard herd, which has been closed for several generations, by quantifying the degree of genetic homogeneity within the herd and to compare its genetic profile to that of other cattle breeds. A total of 868 animals from the Angus, Belgian Blue, Charolais, Friesian, Hereford, Holstein, Holstein-Friesian crosses, Limousin, and Simmental breeds was used for genetic profile comparisons. The level of genetic variation within the breeds were quantified by the expected heterozygosity (H(E)), observed heterozygosity (H(O)), average minor allele frequency (MAF), the degree of polymorphism, and runs of homozygosity (ROH), which are contiguous lengths of homozygous genotypes of varying length. Interestingly, the Kortegaard herd had the lowest within-breed genetic variation (lowest H(E), H(O), and MAF), showed moderate levels of short ROH (<5 Mb), and had the highest mean long ROH (>5 Mb) compared to all the other breeds. This is possibly due to recent consanguineous matings, a strong founder effect, and a lack of gene flow from other herds and breeds. We further examined whether the observed genetic patterns in the Kortegaard herd can be used to design breeding strategies for the preservation of the genetic pool by focusing on a subset of SNP outside homozygote regions. By calculating the pairwise identical-by-state between all possible matings, we designed a breeding plan that maximized heterozygosity in the short term. The benefits and limitations of such a breeding strategy are discussed.

X-ray tomography using the full complex index of refraction.

We report on x-ray tomography using the full complex index of refraction recorded with a grating-based x-ray phase-contrast setup. Combining simultaneous absorption and phase-contrast information, the distribution of the full complex index of refraction is determined and depicted in a bivariate graph. A simple multivariable threshold segmentation can be applied offering higher accuracy than with a single-variable threshold segmentation as well as new possibilities for the partial volume analysis and edge detection. It is particularly beneficial for low-contrast systems. In this paper, this concept is demonstrated by experimental results.

Molecular X-ray computed tomography of myelin in a rat brain.

In this work we demonstrate the feasibility of applying small-angle X-ray scattering computed tomography (SAXS-CT) for non-invasive molecular imaging of myelin sheaths in a rat brain. Our results show that the approach yields information on several quantities, including the relative myelin concentration, its periodicity, the total thickness of the myelin sheaths, and the relative concentration of cytoskeletal neurofilaments. For example the periodicity of the myelin sheaths varied in the range from 17.0 to 18.2 nm around an average of 17.6 (±0.3) nm. We believe that imaging, i.e., spatially resolved measuring these quantities could provide general means for understanding the relation to a number of neurodegenerative diseases.

Cell shape and spreading of stromal (mesenchymal) stem cells cultured on fibronectin coated gold and hydroxyapatite surfaces.

In order to identify the cellular mechanisms leading to the biocompatibility of hydroxyapatite implants, we studied the interaction of human bone marrow derived stromal (mesenchymal) stem cells (hMSCs) with fibronectin-coated gold (Au) and hydroxyapatite (HA) surfaces. The adsorption of fibronectin was monitored by Quartz Crystal Microbalance with Dissipation (QCM-D) at two different concentrations, 20 µg/ml and 200 µg/ml, and the fibronectin adsorption experiments were complemented with antibody measurements. The QCM-D results show that the surface mass uptake is largest on the Au surfaces, while the number of polyclonal and monoclonal antibodies directed against the cell-binding domain (CB-domain) on the fibronectin (Fn) is significantly larger on the (HA) surfaces. Moreover, a higher number of antibodies bound to the fibronectin coatings formed from the highest bulk fibronection concentration. In subsequent cell studies with hMSC's we studied the cell spreading, cytoskeletal organization and cell morphology on the respective surfaces. When the cells were adsorbed on the uncoated substrates, a diffuse cell actin cytoskeleton was revealed, and the cells had a highly elongated shape. On the fibronectin coated surfaces the cells adapted to a more polygonal shape with a well-defined actin cytoskeleton, while a larger cell area and roundness values were observed for cells cultured on the coated surfaces. Among the coated surfaces a slightly larger cell area and roundness values was observed on HA as compared to Au. Moreover, the results revealed that the morphology of cells cultured on fibronectin coated HA surfaces were less irregular. In summary we find that fibronectin adsorbs in a more activated state on the HA surfaces, resulting in a slightly different cellular response as compared to the fibronectin coated Au surfaces.

Low pathogenic avian influenza (H7N1) transmission between wild ducks and domestic ducks.

This article describes a virological investigation in a mixed flock of ducks and geese following detection of avian influenza virus antibodies in domestic geese. Low pathogenic H7N1 was found in both domestic and wild birds, indicating that transmission of virus was likely to have taken place between these. The importance of implementing and maintaining appropriate biosecurity measures is re-emphasized.

Diversity and stability of Aleutian mink disease virus during bottleneck transitions resulting from eradication in domestic mink in Denmark.

Aleutian mink disease (plasmacytosis) virus (AMDV) in domestic mink (Neovison vison) has been subject to eradication in Denmark since 1976. In 2001, approximately 5% of Danish mink farms were still infected and all were located in the northern part of the peninsula of Jutland. In the present study a total of 274 Danish isolates of AMDV collected during the two seasons of 2004 and 2005 were characterized by partial sequencing of the coding region of the non-structural (NS) proteins. Older AMDV isolates from Denmark, available, were also included. The Danish isolates represent a very homogenous cluster compared with Swedish, Finnish and Dutch isolates and seem to represent a minor fraction of the genetic diversity previously found in Denmark. Stability of nucleotide deviations reveals that the purifying selection of bottlenecks imposed on the AMDV population in Denmark by the stamping out policy for more than 6 years exceeds the rate of mutation driven diversity. Among the isolates from farms in northern Jutland two distinct types could be identified and within each of them a number of sub-types which were all useful in tracking spread of infections. Infection at a farm the preceding season was a predisposing risk parameter for disease outbreak at a farm, and strain identity substantiates the suggestion that inadequate disinfection is involved in the recurrence of outbreaks. In cases of new introductions to farms it is indicated that contact including transport between farms played a most significant role.

Dynamics in shear flow studied by X-ray Photon Correlation Spectroscopy.

X-ray Photon Correlation Spectroscopy was used to measure the diffusive dynamics of colloidal particles in a shear flow. The results presented here show how the intensity autocorrelation functions measure both the diffusive dynamics of the particles and their flow-induced, convective motion. However, in the limit of low flow/shear rates, it is possible to obtain the diffusive component of the dynamics, which makes the method suitable for the study of the dynamical properties of a large class of complex soft-matter and biological fluids. An important benefit of this experimental strategy over more traditional X-ray methods is the minimization of X-ray-induced beam damage. While the method can be applied also for photon correlation spectroscopy in the visible domain, our analysis shows that the experimental conditions under which it is possible to measure the diffusive dynamics are easier to achieve at higher q values (with X-rays).

Global gene expression analysis in fetal mouse ovaries with and without meiosis and comparison of selected genes with meiosis in the testis.

In order to identify novel genes involved in early meiosis and early ovarian development in the mouse, we used microarray technology to compare transcriptional activity in ovaries without meiotic germ cells at embryonic age 11.5 (E11.5) and E13.5 ovaries with meiosis. Overall, 182 genes were differentially expressed; 134 were known genes and 48 were functionally uncharacterized. A comparison of our data with the literature associated, for the first time, at least eight of the known genes with female meiosis/germ cell differentiation (Aldh1a1, C2pa, Tex12, Stk31, Lig3, Id4, Recql, Piwil2). These genes had previously only been described in spermatogenesis. The microarray also detected an abundance of vesicle-related genes of which four were upregulated (Syngr2, Stxbp1, Ric-8, SytIX) and one (Myo1c) was downregulated in E13.5 ovaries. Detailed analysis showed that the temporal expression of SytIX also coincided with the first meiotic wave in the pubertal testis. This is the first time that SytIX has been reported in non-neuronal tissue. Finally, we examined the expression of one of the uncharacterized genes and found it to be gonad-specific in adulthood. We named this novel transcript "Gonad-expressed transcript 1" (Get-1). In situ hybridization showed that Get-1 was expressed in meiotic germ cells in both fetal ovaries and mature testis. Get-1 is therefore a novel gene in both male and female meiosis.

Transcriptome analysis of FSH and FSH variant stimulation in granulosa cells from IVM patients reveals novel regulated genes.

FSH is crucial for oocyte maturation and fertility and is the main component in infertility treatment in assisted reproduction. The granulosa cells expressing the FSH receptor interact with the oocyte and provide nourishing substrates controlling the oocyte maturation. Thus, transcriptome analysis of granulosa cells stimulated by FSH is of major importance in understanding the communication between oocytes and granulosa cells. In this study, gene expression profiles were assessed in human granulosa cells from normal cycling in vitro maturation (IVM) patients using oligonucleotide gene chips. Granulosa cells were stimulated for 2 h with either FSH or a previously generated glycosylated FSH variant (FSH1208) that exhibited increased in vivo activity because of prolonged half-life. The analysis identified 74 significantly FSH/FSH1208 regulated genes. Amongst these were well known FSH regulated genes as well as genes not previously described to be important in the FSH signalling pathway. These novel FSH regulated genes include transcription factors [cAMP responsive element modulator (CREM)/inducible cAMP early repressors (ICER), GATA 6, ZFN 361, Bcl11a, CITED1 and TCF 8] and other regulatory proteins and enzymes (IGF-BP3, syntaxin and PCK1) possibly important for oocyte/granulosa cell interaction and function. Array data were validated for 13 genes by northern blots or RT-PCR. Furthermore, no significant differences in gene regulation were detected between the two FSH analogs. This work uncovers novel data important for understanding the folliculogenesis. Furthermore, the results suggest that FSH1208 has a gene expression profile like FSH and thus, in the light of known prolonged in vivo activity, might be a candidate for improved infertility treatment.

Lactic acidosis in the rectal lumen of patients with septic shock measured by luminal equilibrium dialysis.

BACKGROUND: Gut ischaemia may contribute to morbidity in sepsis, but little is known about the metabolic state of the gut mucosa in such patients. METHODS: Nine patients with abdominal septic shock treated with norepinephrine, and ten healthy subjects, were subjected to equilibrium dialysis with a rectal balloon. pH, PCO(2) and concentrations of L-lactate were measured by auto-analyser. RESULTS: In rectal dialysis fluid from patients with septic shock, acidosis was present (pH 7.23, 95% CI 7.11-7.36) and concentrations of L-lactate were approximately five times greater than controls (2.5-5.8 vs 0.5-1.2 mmol litre(-1)). The lactate concentration was related to the dose of norepinephrine (P<0.001). In contrast, values of dialysate PCO(2) did not differ significantly between patients and controls (6.4-11.0 vs 8.9-13.8 kPa). CONCLUSIONS: The results suggest that, either lactic acidosis in rectal mucosa is related to shock severity, or that norepinephrine causes mucosal ischaemia. In any case, metabolic dysfunction is present in the rectal mucosa in patients with abdominal septic shock treated with norepinephrine.

Variations in the severity of phocid herpesvirus type 1 infections with age in grey seals and harbour seals.

The data recorded during an outbreak of phocid herpesvirus type 1 infection among 19 harbour seals and 29 grey seals being nursed in a seal rehabilitation centre in The Netherlands in 1998 were used, together with data from similar outbreaks in previous years, to compare the clinical signs observed in the two species at different ages. The severity of the disease was inversely correlated with age in the harbour seals, and the infected harbour seals generally developed more severe clinical signs than the infected grey seals.

The DECD box putative ATPase Sub2p is an early mRNA export factor.

Nuclear mRNA metabolism relies on the interplay between transcription, processing, and nuclear export. RNA polymerase II transcripts experience major rearrangements within the nucleus, which include alterations in the structure of the mRNA precursors as well as the addition and perhaps even removal of proteins prior to transport across the nuclear membrane. Such mRNP-remodeling steps are thought to require the activity of RNA helicases/ATPases. One such protein, the DECD box RNA-dependent ATPase Sub2p/UAP56, is involved in both early and late steps of spliceosome assembly. Here, we report a more general function of Saccharomyces cerevisiae Sub2p in mRNA nuclear export. We observe a rapid and dramatic nuclear accumulation of poly(A)(+) RNA in strains carrying mutant alleles of sub2. Strikingly, an intronless transcript, HSP104, also accumulates in nuclei, suggesting that Sub2p function is not restricted to splicing events. The HSP104 transcripts are localized in a single nuclear focus that is suggested to be at or near their site of transcription. Intriguingly, Sub2p shows strong genetic and functional interactions with the RNA polymerase II-associated DNA/DNA:RNA helicase Rad3p as well as the nuclear RNA exosome component Rrp6p, which was independently implicated in the retention of mRNAs at transcription sites. Taken together, our data suggest that Sub2p functions at an early step in the mRNA export process.

Quality control of mRNA 3'-end processing is linked to the nuclear exosome.

An emerging theme in messenger RNA metabolism is the coupling of nuclear pre-mRNA processing events, which contributes to mRNA quality control. Most eukaryotic mRNAs acquire a poly(A) tail during 3'-end processing within the nucleus, and this is coupled to efficient export of mRNAs to the cytoplasm. In the yeast Saccharomyces cerevisiae, a common consequence of defective nuclear export of mRNA is the hyperadenylation of nascent transcripts, which are sequestered at or near their sites of transcription. This implies that polyadenylation and nuclear export are coupled in a step that involves the release of mRNA from transcription site foci. Here we demonstrate that transcripts which fail to acquire a poly(A) tail are also retained at or near transcription sites. Surprisingly, this retention mechanism requires the protein Rrp6p and the nuclear exosome, a large complex of exonucleolytic enzymes. In exosome mutants, hypo- as well as hyperadenylated mRNAs are released and translated. These observations suggest that the exosome contributes to a checkpoint that monitors proper 3'-end formation of mRNA.

Long-term results after cemented revision of the femoral component in total hip arthroplasty.

The need for revision total hip arthroplasty has been increasing. The early results have been poor, and different revision techniques have been introduced. We report our results of 84 consecutive cemented first-time revisions of femoral components performed from 1981 through 1988 using a long-stem revision component. The average time to follow-up was 11.4 years (range, 7.9-15.0 years). Patients with 47 revisions had died; 2 of these had been rerevised. Two additional patients were lost to follow-up for other reasons. Of the living patients, 12 had been rerevised, leaving 23 patients (23 hips) for complete follow-up evaluation, including clinical and radiographic assessment. Of 23 patients, 15 reported no pain, 4 had only slight pain, and 4 had more severe pain. In 4 cases, there were definite radiographic signs of loosening of the femoral component. Kaplan-Meier survivorship analysis showed an overall 10-year survival of the femoral component of 77.9%. Using rerevision because of aseptic loosening or definite radiographic loosening as endpoint, the 10-year survival was 80.7%. Simple recementation is well indicated in elderly patients with only minor bone loss.

A block to mRNA nuclear export in S. cerevisiae leads to hyperadenylation of transcripts that accumulate at the site of transcription.

Several factors contribute to nuclear mRNA export in Saccharomyces cerevisiae, including Mex67p, Mtr2p, Gle1p, Nup159p, Dbp5p, and Rip1p. Strains carrying mutations in these factors show rapid and dramatic nuclear accumulation of poly(A)(+) RNA. We have characterized two heat shock mRNAs, SSA4 and HSP104, in these mutant backgrounds; each transcript concentrates in a single intranuclear focus. Evidence suggests that it coincides with the site of transcription. Interestingly, all detectable SSA4 transcripts have undergone 3'-end formation, indicating that RNAs in the foci are no longer nascent. Poly(A) tails of the transcripts are also dramatically longer in all of these export mutants. Based on all of the data, we suggest that very early mRNA maturation events determine transcript export competence.

[Emergency room patients and alcohol consumption]. / Skadestuepatienter og alkoholforbrug.

To investigate the consumption of alcohol among emergency room patients, we included 395 patients above 18 years old entering the emergency room during a period of ten days. The patients completed a questionnaire about alcohol consumption, smoking habits and medication. In our investigation 56% of the men and 25% of the women had a daily consumption above the limits recommended by the Ministry of Health (three drinks daily for men and two for women), while 41% men and 14% women consumed at least five drinks daily or 35 per week. Trauma seen in male patients with high alcohol consumption was characterised by excessive damage to the head, trauma of the lower limb and blows from an object, person or an animal. The alcoholic women were characterised by excessive chemical injuries, incisions and stab wounds, and trauma of the upper limb. In conclusion a surprisingly large amount of emergency room patients can be defined as alcohol abusers and drinking is found to be associated with excessive damage.

Identification of novel Saccharomyces cerevisiae proteins with nuclear export activity: cell cycle-regulated transcription factor ace2p shows cell cycle-independent nucleocytoplasmic shuttling.

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiae proteins not previously known to have nuclear export activity. These proteins are the 5' RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G(1)-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G(1) but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner.

The specificity of the CRM1-Rev nuclear export signal interaction is mediated by RanGTP.

Nuclear export of intron-containing human immunodeficiency virus type 1 (HIV-1) RNA is mediated by the viral Rev protein that contains both an RNA binding domain specific for the viral Rev response element (RRE) and a nuclear export signal (NES). The cellular CRM1 (Exportin1) protein functions as a nuclear export receptor for proteins carrying a Rev-like NES in a process that also requires the GTP bound form of the Ran GTPase. Using purified recombinant factors, we show by co-precipitation, gel mobility shift and protein footprinting assays that full-length Rev protein interacts directly with CRM1 in vitro independently of both the integrity of the characteristic leucine residues of the NES and the presence of the cytotoxin leptomycin B (LMB). Addition of RanGTP induces the formation of an RRE-Rev-CRM1-RanGTP complex that is sensitive to LMB, NES mutations, and Ran being charged with GTP. Within this complex, CRM1 is readily cross-linked to Cys89 near the NES of Rev. By protein footprinting, we demonstrate that the NES of Rev and two regions in CRM1 become inaccessible to endoproteinases upon binding suggesting that these regions are involved in protein-protein interactions. Our data are consistent with a model in which CRM1 is the nuclear export receptor for the Rev-RRE ribonucleoprotein complex and that RanGTP binds to a preformed Rev-CRM1 complex and specifies a functional interaction with the NES.

HIV-1 rev nuclear export signal binding peptides isolated by phage display.

The human immunodeficiency virus type 1 (HIV-1) Rev protein is absolutely essential in the viral replication cycle, where it induces the production of viral structural proteins. Rev functions in part by inducing the nuclear export of incompletely spliced mRNA species specified by the presence of an RNA element, the Rev response element (RRE). Several proteins implicated in RNA processing and nucleo-cytoplasmic transport have been shown to interact with Rev, however, their exact roles remain unknown. To map potential protein recognition sites within the Rev structure, we have screened a phage library, displaying random 15-mer peptides, and isolated clones exhibiting similar sequences that specifically interact with Rev. The binding sites on Rev of the corresponding synthetic peptides were characterised by protein footprinting, involving partial proteolysis of radioactively end-labelled Rev protein. Two of the peptides produced a significant footprint within the nuclear export signal of Rev, raising the possibility that they mimic the binding of cellular protein factors implicated in nuclear export.

Probing the structure of HIV-1 Rev by protein footprinting of multiple monoclonal antibody-binding sites.

Human immunodeficiency virus type 1 (HIV-1) Rev is a small RNA-binding protein which is essential for viral replication. To investigate the structure of Rev we have mapped the binding sites of a panel of monoclonal antibodies (mAb) by protein footprinting and identified a mAb protecting amino acids within both the N- and C-terminal parts of Rev. Our mapping results support a previously proposed structure (Auer et al., Biochemistry, 33 (1994) 2988-2996) predicting that a helix-loop-helix motif in Rev brings the termini of the protein into proximity. Furthermore, we demonstrate that the binding sites mapped by protein footprinting are in agreement with conventional epitope mapping results and that this technique provides an advantageous strategy for mapping discontinuous sites.