Genome-wide analysis of genetic determinants of circulating factor VII-activating protease (FSAP) activity.

Essentials Knowledge of genetic regulators of plasma factor VII activating protease (FSAP) levels is limited. We performed a genome-wide analysis of variants influencing FSAP activity in Scandinavian cohorts. We replicated an association for Marburg-1 and identified an association for a HABP2 stop variant. We identified a novel locus near ADCY2 as a potential additional regulator of FSAP activity. SUMMARY: Background Factor VII-activating protease (FSAP) has roles in both coagulation and fibrinolysis. Recent data indicate its involvement in several other processes, such as vascular remodeling and inflammation. Plasma FSAP activity is highly variable among healthy individuals and, apart from the low-frequency missense variant Marburg-I (rs7080536) in the FSAP-encoding gene HABP2, determinants of this variation are unclear. Objectives To identify novel genetic variants within and outside of the HABP2 locus that influence circulating FSAP activity. Patients/Methods We performed an exploratory genome-wide association study (GWAS) on plasma FSAP activity amongst 3230 Swedish subjects. Directly genotyped rare variants were also analyzed with gene-based tests. Using GWAS, we confirmed the strong association between the Marburg-I variant and FSAP activity. HABP2 was also significant in the gene-based analysis, and remained significant after exclusion of Marburg-I carriers. This was attributable to a rare HABP2 stop variant (rs41292628). Carriers of this stop variant showed a similar reduction in FSAP activity as Marburg-I carriers, and this finding was replicated. A secondary genome-wide significant locus was identified at a 5p15 locus (rs35510613), and this finding requires future replication. This common variant is located upstream of ADCY2, which encodes a protein catalyzing the formation of cAMP. Results and Conclusions This study verified the Marburg-I variant to be a strong regulator of FSAP activity, and identified an HABP2 stop variant with a similar impact on FSAP activity. A novel locus near ADCY2 was identified as a potential additional regulator of FSAP activity.

Exome array analysis of ischaemic stroke: results from a southern Swedish study.

BACKGROUND AND PURPOSE: Genome-wide association (GWA) studies have identified a few risk loci for ischaemic stroke, but these variants explain only a small part of the genetic contribution to the disease. Coding variants associated with amino acid substitutions or premature termination of protein synthesis could have a large effect on disease risk. We performed an exome array analysis for ischaemic stroke. METHODS: Patients with ischaemic stroke (n = 2385) and control subjects (n = 6077) from three Swedish studies were genotyped with the Illumina HumanOmniExpressExome BeadChip. Single-variant association analysis and gene-based tests were performed of exome variants with minor allele frequency of < 5%. A separate GWA analysis was also performed, based on 700 000 genotyped common markers and subsequent imputation. RESULTS: No exome variant or gene was significantly associated with all ischaemic stroke after Bonferroni correction (all P > 1.8 × 10-6 for single-variant and >4.15 × 10-6 for gene-based analysis). The strongest association in single-variant analysis was found for a missense variant in the DNAH11 gene (rs143362381; P = 5.01 × 10-6 ). In gene-based tests, the strongest association was for the ZBTB20 gene (P = 7.9 × 10-5 ). The GWA analysis showed that the sample was homogenous (median genomic inflation factor = 1.006). No genome-wide significant association with overall ischaemic stroke risk was found. However, previously reported associations for the PITX2 and ZFHX3 gene loci with cardioembolic stroke subtype were replicated (P = 7 × 10-15 and 6 × 10-3 ). CONCLUSIONS: This exome array analysis did not identify any single variants or genes reaching the pre-defined significance level for association with ischaemic stroke. Further studies on exome variants should be performed in even larger, well-defined and subtyped samples.

Structure, function and evolution of the Archaeal class I fructose-1,6-bisphosphate aldolase.

FBPA (fructose-1,6-bisphosphate aldolase) catalyses the reversible aldol condensation of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate to form fructose 1,6-bisphosphate. Two classes of FBPA, which rely on different reaction mechanisms, have so far been discovered, class I mainly found in Eucarya and class II mainly in Bacteria. Only recently were genes encoding proteins with FBPA activity identified in Archaea. Archaeal FBPAs do not share any significant overall sequence identity with members of the traditional classes of FBPAs, raising the interesting question of whether they have evolved independently by convergent evolution or diverged from a common ancestor. Biochemical characterization of FBPAs of the two hyperthermophilic Archaea Thermoproteus tenax and Pyrococcus furiosus showed that the enzymes use a Schiff-base mechanism and thus belong to the class I aldolases. The crystal structure of the archaeal FBPA from T. tenax revealed that the protein fold, as for the classical FBPA I and II, is that of a parallel (betaalpha)(8) barrel. A substrate-bound crystal structure allowed detailed active-site comparisons which showed the conservation of six important catalytic and substrate-binding residues between the archaeal and the classical FBPA I. This observation provides further evidence that the two sequence families of proteins share a common evolutionary origin. Furthermore, structure and sequence analysis indicate that the class I FBPA shares a common evolutionary origin with several other enzyme superfamilies of the (betaalpha)(8) barrel fold.

Replication-competent herpes simplex virus type 1 mutant expressing an autofluorescent glycoprotein H fusion protein.

The heterocomplex of glycoproteins H (gH) and L (gL) of herpes simplex virus type 1 (HSV-1) is essential for viral infectivity and is involved in viral penetration, cell-to-cell spread, and syncytium formation. We constructed an HSV-1 mutant expressing a gH-EGFP (enhanced green fluorescent protein) fusion protein under the control of the gH true late promoter. The EGFP coding sequence was cloned after the gH signal peptide into the HSV-1 genome. Superinfection of transfected, gH-nontranscomplementing cells with gH-negative HSV-1 resulted in a replication-competent recombinant virus. Cells infected with the recombinant virus exhibited strong and stable EGFP-specific fluorescence late in infection, and autofluorescence was detected in purified virions. The recombinant genotype of the mutant was confirmed by PCR. The 140-kD gH-EGFP fusion protein showed an N-linked glycosylation pattern similar to gH-1, was recognized by the conformation-dependent gH-specific monoclonal antibodies 52S and LP11 and formed a heterocomplex with gL which was transported to the cell surface and integrated into the viral envelope. Infectivity of the gH-EGFP mutant was neutralized by antibodies 52S and LP11. To our knowledge, this is the first replication-competent HSV-1 mutant expressing an autofluorescent essential glycoprotein which will be a versatile tool for studies of penetration, late gene expression, transport and tissue spread.

Quantitation of human cytomegalovirus genomes in the brain of AIDS patients.

Human cytomegalovirus (HCMV) is one of the major pathogens causing neurologic disease in the immunocompromised host. A competitive nested polymerase chain reaction (PCR) was used to determine DNA load, distribution, and sequence variability of HCMV genomes in the brain of AIDS patients with and without HCMV encephalitis confirmed by histology and immunocytochemistry. By quantitative PCR, HCMV genomes were found to be distributed diffusely in the central nervous system (CNS) of all five patients with histologically proven HCMV encephalitis, but also in the brain of five of eight AIDS patients without neuropathological evidence of HCMV encephalitis. The viral DNA load in cases with HCMV encephalitis was increased 10- to 1,000-fold as compared to patients without evidence of encephalitis. A viral load above 6,000 copies HCMV/10(6) copies beta-globin was found to be highly suggestive for HCMV encephalitis. Characterization of PCR products by temperature gradient gel electrophoresis (TGGE) and direct sequencing allowed us to detect a sequence variability of the amplified fragment of HCMV glycoprotein B (gB) among different patients, but also among different HCMV foci within the same patient. Furthermore, two of five AIDS patients with HCMV encephalitis most likely experienced double infections with different HCMV strains. The experimental procedure described in this study should also be applicable to the detection of significant HCMV DNA levels in biopsy samples.

In vitro expression of UL56 gene of herpes simplex virus type 1; detection of UL56 gene product in infected cells and in virions.

In order to investigate the functional properties of the UL56 gene of herpes simplex virus type 1 (HSV-1), it was necessary to express the UL56 protein in vitro. The DNA sequences corresponding to the open reading frame of the UL56 gene of HSV-1 strain F were amplified from genomic viral DNA by PCR using primers corresponding to the translational start and termination regions of the UL56 ORF. The PCR product (705 bp) was inserted into the EcoRI/XbaI recognition sites of the bacterial expression vector pMal-c2. This procedure allowed the expression of the viral UL56 gene fused to the maltose-binding protein (MBP) of Escherichia coli, and subsequent cleavage of the fusion protein with the specific protease factor Xa. The induced fusion protein was purified by affinity chromatography using amylose columns. The apparent molecular weight of the fusion protein was about 70 kDa. Factor Xa cleaves the fusion protein into two subfragments of 42 kDa (MBP) and 30 kDa (UL56). Rabbit antisera induced against recombinant UL56 protein were used for detection of the UL56 gene product during the infection cycles of HSV-1. The presence of the UL56 protein was detected in infected cells and in HSV-1 virions by Western blot experiments and by immunofluorescence assays. A strong and increasing cytoplasmic fluorescence was observed in RC-37 cells infected with HSV-1 strain F between 6 and 16 h post-infection. In addition it was found that human HSV-1 IgM/IgG positive convalescent sera recognized the recombinant UL56 protein.

[Evaluation of trainee assistant education]. / Vurdering af praksisreservelaegeuddannelsen.

All of the trainee assistant appointments in general practice in Denmark were assessed by means of a questionnaire investigation during a period of two years. Particular attention was paid to the quality of training and whether there were particular characteristics in the practice which provide the best training. The material consists of 360 questionnaires sent to trainee assistants in general practice one month before conclusion of the appointment. The percentage of replies was 89. The average number of years after graduation was 6.5, which is considerably higher than aimed at in future plans. The investigation reveals that the educational quality and training by the tutor did not appear to be influenced by the number of postgraduate years although there was no doubt that younger trainees required more training and supervision. The quality of training is best in one-man practices and this is followed closely by small partnerships. The investigation reveals that training by the tutor is of decisive significance. It is therefore recommended that trainees in general practice should be more closely associated with one of the practitioners responsible for training. Three fourths of the trainees found that trainee assistant appointment was better than hospital appointments in general. The investigation indicates that following should be standard in every practice: 1) own consultation room, 2) typed records, 3) an introductory leaflet and that training should be arranged with: 1) introduction, 2) half-way assessments, 3) daily conferences about individual patients and 4) weekly conferences about particular subjects related to the practice.

In vitro cleavage of HIV-1 vif RNA by a synthetic ribozyme.

Using the method of Haseloff and Gerlach, we constructed a ribozyme specifically targeted against the virion infectivity factor (vif) of HIV-1. Both, the vif gene and an oligonucleotide representing the catalytic RNA sequence were cloned into pSPT19 downstream of the T7 promoter and transcribed with T7 RNA polymerase. Efficient cleavage of vif RNA by the synthetic ribozyme occurred at pH 7.5 and 37 degrees C in the presence of magnesium ions in vitro. No measurable activity was observed with a vif antisense RNA. A deletion in the hybridizing region of the ribozyme decreased the cleavage rate, while a mutation in the consensus cleavage domain abolished its catalytic activity. Thus, we could demonstrate an in-vitro activity of a specifically designed ribozyme against HIV-1 vif RNA.

Identification of herpes simplex virus type 1 glycoproteins interacting with the cell surface.

To investigate the interaction of herpes simplex virus type 1 (HSV-1) with the cell surface, we studied the formation of complexes by HSV-1 virion proteins with biotinylated cell membrane components. HSV-1 virion proteins reactive with surface components of HEp-2 and other cells were identified as gC, gB, and gD. Results from competition experiments suggested that binding of gC, gB, and gD occurred in a noncooperative way. The observed complex formation could be specifically blocked by monospecific rabbit antisera against gB and gD. The interaction of gD with the cell surface was also inhibited by monoclonal antibody IV3.4., whereas other gD-specific monoclonal antibodies, despite their high neutralizing activity, were not able to inhibit this interaction. Taken together, these data provide direct evidence that at least three of the seven known HSV-1 glycoproteins are able to form complexes with cellular surface structures.