Infectious salmon anemia virus (ISAV) genomic segment 3 encodes the viral nucleoprotein (NP), an RNA-binding protein with two monopartite nuclear localization signals (NLS)

Infectious salmon anemia virus (ISAV) is the type species of the genus Isavirus belonging to the Orthomyxoviridae, and causes serious disease in Atlantic salmon (Salmo salar). This study presents the expression and functional analysis of the ISAV genome segment 3, and provides further evidence that it encodes the viral nucleoprotein (NP). The encoded protein was expressed in a baculovirus system, and Western blot analysis showed that it corresponds to the 66-71 kDa structural protein previously found in purified ISAV preparations. RNA-binding activity was established by the interaction of viral and recombinant NP with single-stranded RNA transcribed in vitro. Immunofluorescence studies of infected cells showed the ISAV NP to be an early protein. It locates to the nucleus of infected cells before it is transported to the cytoplasm prior to virus assembly. A similar localization pattern was observed in cells transfected with the NP gene, confirming that the encoded protein has an intrinsic ability to be imported into the nucleus. Two monopartite nuclear localization signals (NLS) at amino acids (230)RPKR(233) and (473)KPKK(476) were identified by computer analysis, and validated by site-directed mutagenesis. In contrast to other orthomyxovirus-NPs, that have several NLSs that function independent of each other, both NLSs had to be present for the ISAV NP protein to be transported into the nucleus, indicating that these motifs cooperate to target the protein to the nucleus.     

Molecular basis of the functional podocin-nephrin complex: mutations in the NPHS2 gene disrupt nephrin targeting to lipid raft microdomains

Hereditary nephrotic syndrome is a heterogeneous disease, characterizedby heavy proteinuria and renal failure. Mutations of NPHS1 orNPHS2, the genes encoding for nephrin and podocin, lead to earlyonset of heavy proteinuria, and rapid progression to end-stagerenal disease, suggesting that both proteins are essential forthe integrity of the glomerular filter. Podocin is a stomatinprotein family member with a predicted hairpin-like structurelocalizing to the insertion site of the slit diaphragm of podocytes,the visceral glomerular epithelial cells of the kidney. Herewe investigate the pathomechanisms of different disease-causingpodocin mutations. We show that wild-type podocin is targetedto the plasma membrane, and forms homo-oligomers involving thecarboxy and amino terminal cytoplasmic domains. The associationof podocin with specialized lipid raft microdomains of the plasmamembrane was a prerequisite for recruitment of nephrin intorafts. In contrast, disease-causing mutations of podocin (R138Qand R138X) failed to recruit nephrin into rafts either becausethese mutants were retained in the endoplasmic reticulum (R138Q),or because they failed to associate with rafts (R138X) despitetheir presence in the plasma membrane. None of the mutants didaugment nephrin signaling, suggesting that lipid raft targetingfacilitates nephrin signaling. Our findings demonstrate thatthe failure of mutant podocin to recruit nephrin into lipidrafts may be essential for the pathogenesis of NPHS2. ^* To whom correspondence should be addressed. Tel: +49 7612703559;Fax: +49 7612706362; Email: benzing{at}med1.ukl.uni-freiburg.de      

Sleep and its homeostatic regulation in mice lacking the adenosine A1 receptor

Sleep deprivation (SD) increases extracellular adenosine levels in the basal forebrain, and pharmacological manipulations that increase extracellular adenosine in the same area promote sleep. As pharmacological evidence indicates that the effect is mediated through adenosine A1 receptors (A1R), we expected A1R knockout (KO) mice to have reduced rebound sleep after SD. Male homozygous A1R KO mice, wild-type (WT) mice, and heterozygotes (HET) from a mixed 129/C57BL background were implanted during anesthesia with electrodes for electroencephalography (EEG) and electromyography (EMG). After 1 week of recovery, they were allowed to adapt to recording leads for 2 weeks. EEG and EMG were recorded continuously. All genotypes had a pronounced diurnal sleep/wake rhythm after 2 weeks of adaptation. We then analyzed 24 h of baseline recording, 6 h of SD starting at light onset, and 42 h of recovery recording. Neither rapid eye movement sleep (REM sleep) nor non-REM sleep (NREMS) amounts differed significantly between the groups. SD for 6 h induced a strong NREMS rebound in all three groups. NREMS time and accumulated EEG delta power were equal in WT, HET and KO. Systemic administration of the selective A1R antagonist 8-cyclopentyltheophylline (8-CPT) inhibited sleep for 30 min in WT, whereas saline and 8-CPT both inhibited sleep in KO. We conclude that constitutional lack of adenosine A1R does not prevent the homeostatic regulation of sleep.     

Real-time PCR methods for independent quantitation of TTV and TLMV

There is considerable interest in the possible clinical effects of the human circoviruses TT virus (TTV) and TTV-like mini virus (TLMV). Most people appear to have at least one of these viruses replicating actively in their bodies, thus mere correlation of the presence of virus and disease states are probably less informative than a quantitative analysis of viraemia. Real-time PCR based methods, with either SYBR Green or TaqMan probe, designed to quantitate selectively TTV and TLMV are described. The suggested TaqMan-based protocols were suitable for quantitation of viruses in the range of 10(2)-10(9) copies/ml of sample; and proved, by sequencing of PCR products, to be specific for each of the two viruses.     

Paracetamol inhibits replicative DNA synthesis and induces sister chromatid exchange and chromosomal aberrations by inhibition of ribonucleotide reductase

Effects of paracetamol have been studied in a hydroxyurea (HU)-resistant mouse mammary tumour cell line TA3H2, shown to overproduce the small subunit of ribonucleotide reductase. These TA3H2 cells were much more resistant than the TA3H (wild-type) cells towards the inhibitory effect of paracetamol on cell growth, IC50 0.55 mM paracetamol for the wild-type compared to 2.7 mM for the HU-resistant cells. The reduced cell growth was due to an inhibition of replicative DNA synthesis, judged from an increased percentage of cells in S-phase measured by flow cytometry. Furthermore, in the wild-type cells, the increase in the number of cells in S phase was already observed at 0.1 mM while in the HU-resistant cell line this effect was first seen at 3.0 mM paracetamol. HU inhibits ribonucleotide reductase by destroying a tyrosyl free radical located on the small subunit of the enzyme. By electron paramagnetic resonance we demonstrate that paracetamol added to crude cell extracts of HU-resistant cells also immediately destroys this radical. These results show that paracetamol reduces DNA synthesis by a specific inhibition of ribonucleotide reductase. A concentration-dependent induction of sister chromatid exchanges was found both with paracetamol (1.0-10 mM) and HU (0.3-3 mM) in wild-type cells whereas no such increase was observed in HU-resistant cells. Paracetamol (1 mM for 2 h) also increased the number of chromosomal aberrations CAs in wild-type cells (i.e. chromatid breaks and chromatid exchanges). The frequency of CAs was not increased in HU-resistant cells at paracetamol concentrations up to 10 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genome profiles of bilateral dysgerminomas, a unilateral gonadoblastoma, and a metastasis from a 46, XY phenotypic female

We present a case report of a 16-year-old, phenotypic female with bilateral dysgerminomas, a unilateral gonadoblastoma, and a peritoneal metastasis. The patient's constitutional karyotype was 46,XY. The chromosomal copy number, examined by the comparative genomic hybridization technique, showed 3 gains in the dysgerminoma of the right ovary, 6 gains in the dysgerminoma of the left ovary, and 2 gains and 1 loss in the gonadoblastoma of the left ovary. The metastasis showed 5 gains of which 4 were also observed in the dysgerminoma of the left ovary. The DNA ploidy classifications of the gonadoblastoma and the dysgerminoma in the right ovary were tetraploid, whereas the dysgerminoma in the left ovary and the metastasis were aneuploid. We therefore propose that the metastasis most probably developed from the dysgerminoma of the left ovary.     

Expression of CD44 in effusions of patients diagnosed with serous ovarian carcinoma – diagnostic and prognostic implications

CD44 is a family of cell adhesion molecules involved in a variety of cellular functions. The present study analysed the expression of two CD44 isoforms in serous effusions of patients diagnosed with ovarian carcinoma and corresponding primary and metastatic lesions. Fifty-eight effusions, 23 primary ovarian tumours, and 44 metastatic lesions were studied for protein expression of CD44s and v3-10 using immunohistochemistry. Results were correlated with clinical parameters. CD44v3-10 was seen in carcinoma cells in the majority of cases at all sites. Malignant effusions showed an up-regulation of CD44s compared to both primary tumours and metastatic solid lesions. Mesothelial cells frequently expressed CD44s, but were rarely immunoreactive for v3-10. CD44s immunoreactivity in cancer cells in effusions was significantly more often observed in patients with FIGO stage 3 than in stage 4 patients (P = 0.045). Staining results did not correlate with age, effusion site, metastatic site, tumour grade or residual tumour mass after initial surgery. Likewise, comparison of overall and disease-free survival with expression of the CD44 isoforms studied did not reveal any statistically significant associations. The up-regulation in CD44 levels in effusions, primarily in stage 3 disease, suggests that adhesion of ovarian carcinoma cells to mesothelium may be regulated at the level of CD44s expression, and provides further evidence of phenotypic alteration in the transition from primary tumour cell clones to effusions. The similar expression profile of CD44 in carcinoma cells in peritoneal and pleural effusions supports our previous observations and the hypothesis that carcinoma cells in peritoneal effusions are truly metastatic. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation and characterization of two European strains of Ehrlichia phagocytophila of equine origin

We report the isolation and partial genetic characterization of two equine strains of granulocytic Ehrlichia of the genogroup Ehrlichia phagocytophila. Frozen whole-blood samples from two Swedish horses with laboratory-verified granulocytic ehrlichiosis were inoculated into HL-60 cell cultures. Granulocytic Ehrlichia was isolated and propagated from both horses. DNA extracts from the respective strains were amplified by PCR using primers directed towards the 16S rRNA gene, the groESL heat shock operon gene, and the ank gene. The amplified gene fragments were sequenced and compared to known sequences in the GenBank database. With respect to the 16S rRNA gene, the groESL gene, and the ank gene, the DNA sequences of the two equine Ehrlichia isolates were identical to sequences found in isolates from clinical cases of granulocytic ehrlichiosis in humans and domestic animals in Sweden. However, compared to amplified DNA from an American Ehrlichia strain of the E. phagocytophila genogroup, differences were found in the groESL gene and ank gene sequences.     

Sensitive Microplate Assay for Detection of Bactericidal Antibodies to Vibrio cholerae O139

A microplate assay for the detection of bactericidal antibodies to Vibrio cholerae O139 is described. The assay is sensitive, highly reproducible, specific, and convenient to perform. It has been used to demonstrate the induction of serum bactericidal antibodies in Vietnamese recipients of an oral, inactivated, bivalent O1/O139 vaccine, as well as in Bangladeshi patients with O139 disease. In both study groups there was a significant inverse correlation between the preexposure level of antibodies in serum and the magnitude of the subsequent bactericidal response. Although infection generated stronger responses than vaccination, the proportion of responders was similar among individuals with low background titers.