Intracranial aneurysms in Finnish families: confirmation of linkage and refinement of the interval to chromosome 19q13.3

We recently reported a two-stage genomewide screen of 48 sib pairs affected with intracranial aneurysms (IAs) that revealed suggestive linkage to chromosome 19q13, with a LOD score of 2.58. The region supporting linkage spanned ~22 cM. Here, we report a follow-up study of the locus at 19q13, with a sample size expanded to 139 affected sib pairs, along with 83 other affected relative pairs (222 affected relative pairs in total). Suggestive linkage was observed in both independent sample sets, and linkage was significant in the combined set at 70 cM (LOD score 3.50; P=.00006) and at 80 cM (LOD score 3.93; P=.00002). Linkage was highly significant at 70 cM (LOD score 5.70; P=.000001) and at 80 cM (LOD score 3.99; P=.00005) when a covariate measuring the number of affected individuals in the nuclear family was included. To evaluate further the contribution to the linkage signal from families with more than two affected relatives, we performed model-based linkage analysis with a recessive model and a range of penetrances, and we obtained maximum linkage at 70 cM (LOD score 3.16; P=.00007) with a penetrance of 0.3. We then estimated location by using GENEFINDER. The most likely location for a gene predisposing to IAs in the Finnish population is in a region with a 95% confidence interval of 11.6 cM (P=.00007) centered 2.0 cM proximal to D19S246.     

Detection of Streptococcus pneumoniae DNA by using polymerase chain reaction and microwell hybridization with Europium-labelled probes

The present paper describes a novel modification of polymerase chain reaction (PCR) for the detection of Streptococcus pneumoniae DNA in clinical specimens. PCR was based on the detection of a 209-base pair segment of the S. pneumoniae pneumolysin gene. For the demonstration of the amplification product, microwell hybridization with a Europium-labelled oligonucleotide probe complementary to a biotinylated strand of the PCR product was performed, and the presence of the PCR product was monitored by time-resolved fluorescence (TRF) of the Europium chelate. The sensitivity of the assay for purified S. pneumoniae DNA was 50 fg DNA corresponding to 20 genome equivalents of S. pneumoniae DNA. The efficiency of the hybridization step was monitored by using known amounts of synthetic target oligonucleotides as standards. Sensitivity of 3×10⁸ molecules per individual reaction well was achieved with a 30-min attachment time and a 3-h hybridization time.

Detection of PCR-amplified products by the microwell hybridization technique and TRF was compared to agarose gel electrophoresis in 50 middle ear fluid samples obtained from children with acute otitis media. The agarose gel and TRF detection methods identified all culture-positive samples, but both were also positive for 55% of the culture-negative samples. The results suggest that the detection of amplified PCR products by microwell hybridization using Europium-labelled oligonucleotides is a reliable method for the demonstration of the pneumolysin gene fragment. Furthermore, the method is suitable for automation and, thus, for testing high numbers of samples. The clinical significance of the PCR findings remains to be studied.     

Monoclonal antibodies, immunofluorometric assay, and detection of human semenogelin in male reproductive tract: no association with in vitro fertilizing capacity of sperm

Semenogelin plays an important role in sperm clotting and is degraded into smaller fragments by prostate-specific antigen (PSA) during clot liquefaction. Semenogelin and its fragments inhibit sperm motility in vitro. We studied the expression of semenogelin I mRNA and its localization in various tissues of the male genital tract. We also studied semenogelin concentrations with respect to sperm parameters and the outcome of in vitro fertilization. Semenogelin protein was detected by immunohistochemical staining and semenogelin I mRNA was detected by Northern blot analysis in the seminal vesicles and ampullary part of the vas deferens, whereas specimens from the prostate, epididymis, testis, and the female genital tract were negative. Using monoclonal antibodies against semenogelin, an immunofluorometric assay was developed to measure semenogelin levels in seminal plasma and to evaluate possible correlations with sperm parameters and fertilization in vitro. No correlation was found between the semenogelin concentration and the volume of the ejaculate, sperm concentration, sperm motility, or in vitro fertilization rate. Semenogelin levels were positively correlated with the total protein concentration in seminal plasma, and there was an inverse correlation between the concentration of semenogelin and that of PSA. The levels of semenogelin appear to bear no relationship to the in vitro fertilization capacity of the spermatozoa.     

Gelsolin and functionally similar actin-binding proteins are regulated by lysophosphatidic acid.

An extensive survey was carried out for compounds capable of regulating actin-binding proteins in a manner similar to phosphatidylinositol 4,5 bisphosphate (PI 4,5-P2). For this purpose we developed a sensitive assay involving release of radioactively phosphorylated actin from the fragminP-actin complex. We found that the structurally simplest lysophospholipid, lysophosphatidic acid (LPA), dissociated the complex between fragminP and actin, whereas other lysophospholipids or sphingosine-1-phosphate were inactive. Furthermore, LPA inhibited the F-actin severing activity of human gelsolin, purified from plasma or as recombinant protein, mouse adseverin and Physarum fragminP. Dissociation of actin-containing complexes by LPA analyzed by gelfiltration indicated that LPA is active as a monomer, in contrast to PI 4,5-P2. We further show that binding of LPA to these actin-regulatory proteins promotes their phosphorylation by pp60(c-src). A PI 4,5-P2-binding peptide counteracted the effects mediated by LPA, suggesting that LPA binds to the same target region in these actin-binding proteins. When both LPA and PI 4,5-P2 were used in combination we found that LPA reduced the threshold concentration at which PI 4,5-P2 was active. Significantly, LPA promoted the release of gelsolin from barbed actin filaments in octylglucoside-permeabilized human platelets. These results suggest that lysophosphatidic acid could act as an intracellular modulator of actin-binding proteins. Our findings can also explain agonist-induced changes in the actin cytoskeleton that are not mediated by polyphosphoinositides.     

Ribonuclease insolubilization using diazotized silk

Ribonuclease (pancreatic) (EC 3.1.27.5) and aspartate aminotransferase (l-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) have been immobilized on woven silk using the diazo method; the optimum conditions for coupling the enzyme to woven silk were established. The stability of each silk-enzyme system was examined. The kinetic constants and the effect of pH on the reaction rate of free and immobilized enzymes were compared.     

Modulation of murine phenobarbital-inducible CYP2A5, CYP2B10 and CYP1A enzymes by inhibitors of protein kinases and phosphatases.

Phenobarbital causes a multitude of effects in hepatocytes, including increased cell proliferation, inhibition of apoptosis and upregulation of xenobiotic and endobiotic metabolizing enzymes. In this study, the involvement of several protein kinase and phosphatase pathways on constitutive and phenobarbital-induced activities of CYP2A5, CYP2B10 and CYP1A1/2 in primary mouse hepatocytes was determined using well-defined chemical modulators of intracellular protein phosphorylation and desphosphorylation events. A 48-h treatment of the hepatocytes with 2-aminopurine, a nonspecific serine/threonine kinase inhibitor, elicited dose-dependent increases in both basal and phenobarbital-induced CYP2A5 catalytic activity (assayed as coumarin 7-hydroxylation), the maximal induction being 60-fold greater than the control value upon cotreatment with 1.5 mM phenobarbital and 10 mM 2-aminopurine. In contrast, phenobarbital induction of CYP2B10 (pentoxyresorufin O-deethylase) and CYP1A1/2 (ethoxyresorufin O-deethylase) activities were blocked by 2-aminopurine. Increases in CYP2A5 activity were also observed after exposure of the hepatocytes to other protein kinase inhibitors affecting the cell cycle, i.e. roscovitine, K-252a and rapamycin. Inhibitors of protein kinases A and C, as well as tyrosine kinases, did not appreciably affect CYP2A5 activity levels. The serine/threonine phosphatase inhibitors tautomycin, calyculin A and okadaic acid all reduced both basal and phenobarbital-induced CYP2A5, CYP2B10 and CYP1A1/2 activities. These results further strengthen the concept that hepatic CYP2A5 is regulated in a unique way compared with CYP2B10 and CYP1A.     

Functional analysis of immune response genes in Drosophila identifies JNK pathway as a regulator of antimicrobial peptide gene expression in S2 cells

The templates of innate immunity have ancient origins. Thus, such model animals as the fruit fly, Drosophila melanogaster, can be used to identify gene products that also play a key role in the innate immunity in mammals. We have used oligonucleotide microarrays to identify genes that are responsive to gram-negative bacteria in Drosophila macrophage-like S2 cells. In total, 53 genes were induced by greater than threefold in response to Escherichia coli. The induction of all these genes was peptidoglycan recognition protein LC (PGRP-LC) dependent. Twenty-two genes including 10 of the most strongly induced genes are also known to be up-regulated by septic injury in vivo. Importantly, we identified 31 genes that are not known to respond to bacterial challenge. We carried out targeted dsRNA treatments to assess the functional importance of these gene products for microbial recognition, phagocytosis and antimicrobial peptide release in Drosophila S2 cells in vitro. RNAi targeting three of these genes, CG7097, CG15678 and beta-Tubulin 60D, caused altered antimicrobial peptide release in vitro. Our results indicate that the JNK pathway is essential for normal antimicrobial peptide release in Drosophila in vitro.     

UniProt archive

UniProt Archive (UniParc) is the most comprehensive, non-redundant protein sequence database available. Its protein sequences are retrieved from predominant, publicly accessible resources. All new and updated protein sequences are collected and loaded daily into UniParc for full coverage. To avoid redundancy, each unique sequence is stored only once with a stable protein identifier, which can be used later in UniParc to identify the same protein in all source databases. When proteins are loaded into the database, database cross-references are created to link them to the origins of the sequences. As a result, performing a sequence search against UniParc is equivalent to performing the same search against all databases cross-referenced by UniParc. UniParc contains only protein sequences and database cross-references; all other information must be retrieved from the source databases.     

B-Cell Responses to Human Bocaviruses 1–4: New Insights from a Childhood Follow-Up Study

Human bocaviruses (HBoVs) 1–4 are recently discovered, antigenically similar parvoviruses. We examined the hypothesis that the antigenic similarity of these viruses could give rise to clinically and diagnostically important immunological interactions. IgG and IgM EIAs as well as qPCR were used to study ~2000 sera collected from infancy to early adolescence at 3–6-month intervals from 109 children whose symptoms were recorded. We found that HBoV1-4-specific seroprevalences at age 6 years were 80%, 48%, 10%, and 0%, respectively. HBoV1 infections resulted in significantly weaker IgG responses among children who had pre-existing HBoV2 IgG, and vice versa. Furthermore, we documented a complete absence of virus type-specific immune responses in six viremic children who had pre-existing IgG for another bocavirus, indicating that not all HBoV infections can be diagnosed serologically. Our results strongly indicate that interactions between consecutive HBoV infections affect HBoV immunity via a phenomenon called “original antigenic sin”, cross-protection, or both; however, without evident clinical consequences but with important ramifications for the serodiagnosis of HBoV infections. Serological data is likely to underestimate human exposure to these viruses.     

The asthma candidate gene <Emphasis Type="Italic">NPSR1</Emphasis> mediates isoform specific downstream signalling

Background

Neuropeptide S Receptor 1 (NPSR1, GPRA, GPR154) was first identified as an asthma candidate gene through positional cloning and has since been replicated as an asthma and allergy susceptibility gene in several independent association studies. In humans, NPSR1 encodes two G protein-coupled receptor variants, NPSR1-A and NPSR1-B, with unique intracellular C-termini. Both isoforms show distinct expression pattern in asthmatic airways. Although NPSR1-A has been extensively studied, functional differences and properties of NPSR1-B have not yet been clearly examined. Our objective was to investigate downstream signalling properties of NPSR1-B and functional differences between NPSR1-A and NPSR1-B.

Methods

HEK-293 cells transiently overexpressing NPSR1-A or NPSR1-B were stimulated with the ligand neuropeptide S (NPS) and downstream signalling effects were monitored by genome-scale affymetrix expression-arrays. The results were verified by NPS concentration-response and time series analysis using qRT-PCR, cAMP and Ca²⁺ assays, and cAMP/PKA, MAPK/JNK and MAPK/ERK pathway specific reporter assays.

Results

NPSR1-B signalled through the same pathways and regulated the same genes as NPSR1-A, but NPSR1-B yielded lower induction on effector genes than NPSR1-A, with one notable exception, CD69, a marker of regulatory T cells.

Conclusions

We conclude that NPSR1-B is regulating essentially identical set of genes as NPSR1-A, with few, but possibly important exceptions, and that NPSR1-A induces stronger signalling effects than NPSR1-B. Our findings suggest an isoform-specific link to pathogenetic processes in asthma and allergy.