Biomedical subjects

Klaus Huse

Publications and source records attributed to Klaus Huse.

24 records · Page 2Linked to original sources

PubMed2004

Locking of 3' ends of single-stranded DNA templates for improved Pyrosequencing performance.

In Pyrosequencing, a DNA strand complementary to a single-stranded DNA (ssDNA) template is synthesized, whereby each incorporated nucleotide yields detectable light, and the light intensity is proportional to the incorporated nucleotides. Correct data interpretation (i.e., signal-to-noise ratio of light intensities) is hampered by artifacts due to the formation of secondary structures of single-stranded templates. Critical among these is the looping back of the template's nonbiotinylated 3' end to itself In the resulting structure, the 3' end functions as a primer, the extension of which results in background signals. We present two ways of preventing the self-priming of a template's 3' end: (i) the use of a modified oligonucleotide, called blOligo, which is complementary to the template's 3' end and (ii) the extension of the template's 3' end with a ddNMP. In contrast to unprotected 3' ends of ssDNA templates, causing inconsistent results, we show that protecting the 3' end of an ssDNA template using either blOligos or ddNMP enables the correct interpretation of signals and results in reliable quantification.

Alleles↗

PubMed2003

Haplotype structure and association to Crohn's disease of CARD15 mutations in two ethnically divergent populations.

Current debate focuses on the relevance of linkage disequilibrium (LD), ethnicity and underlying haplotype structure to the search for genes involved in complex disorders. The recently described association between single nucleotide polymorphisms (SNPs) of the CARD15 (NOD2) gene and Crohn's disease (CD) in populations of north-European descent provides a test case that we have subjected to detailed SNP haplotype based analyses. We examined 23 SNPs spanning 290 kb, including CARD15, in large North-European and Korean samples of patients with Crohn's disease and normal controls. In Europeans we confirmed that the three disease-associated SNPs occur independently but share a common background haplotype. This suggests a common origin and the possibility of an undiscovered more strongly predisposing mutation. Korean CD patients present a phenotype identical to the European patients and have not previously been screened for CARD15. The three disease-associated SNPs were absent and there was no evidence of association between CARD15 and CD. Consequently, the disease-associated mutations in the Europeans, which are rare, have arisen recently (after the Asian-European split). Our results highlight important issues relevant to mapping the genes that predispose to complex disorders. First, although ethnically divergent populations may present identical phenotypes they do not necessarily share the same set of predisposing genes. Second, although single-locus tests of association showed consistent association with markers throughout the gene, pair-wise LD between markers (r(2) and D') yielded very little information about actual disease-association. Third, a population comparative approach allowed refining of the marker set through the examination of shared polymorphisms and common LD-groups. This approach, in conjunction with the examination of the mutational steps in a haplotype network, allows unambiguous identification of the potentially causative mutations.

Asian People↗

PubMed2002

Purification of antibodies by affinity chromatography.

This review focusses on affinity purification of immunoglobulins, a methodology which is a powerful tool to obtain pure and intact antibodies. Affinity techniques allow antibody purification both in a single step chromatographic procedure as well as in complex purification protocols depending on the intention to use the target antibody. The purification strategies for antibodies by interaction with affinity ligands such as antibodies and Fe receptors or low molecular weight compounds are described.

Animals↗

PubMed2002

Aldosterone and D-glucose stimulate the proliferation of human cardiac myofibroblasts in vitro.

The renin-angiotensin-aldosterone-system appears to be involved in the development of cardiac fibrosis in rodents, characterized by nonepithelial cell proliferation and changes in the extracellular matrix. The aim of our study was to investigate the effect of high aldosterone concentrations on the proliferation of human cardiac interstitial cells in vitro. In addition, the effect of D-glucose as another risk factor for fibrosis, eg, in the diabetic heart, was investigated. Human cardiac myofibroblast cultures were established, and growth rates were measured by WST-1 assay in fetal calf serum-free Dulbecco's modified Eagle's medium (DMEM). Cells in culture showed a significant increase in number between 24 to 72 hours of cultivation under basal conditions (DMEM, 10% fetal calf serum). Aldosterone at high concentrations (10(-8) and 10(-7) mol/L) significantly (P<0.01) increased the proliferation of cultured cardiac myofibroblasts. Comparable effects were observed after incubation of the cells with high D-glucose concentrations (15 and 25 mmol/L, P<0.01). No additive growth stimulation was evident when the cells were incubated in medium containing both aldosterone and D -glucose. These results suggest a role for aldosterone and glucose in mediating the cardiac fibrosis through stimulation of myofibroblast growth in patients with dysregulated renin-angiotensin-aldosterone-system (especially hyperaldosteronism) and impaired glucose homeostasis.

Actins↗

PubMed2002

Mutation detection and physical mapping of the CD11 gene cluster in association with inflammatory bowel disease.

A genetic component in the etiology of inflammatory bowel disease (IBD) has clearly been demonstrated by epidemiological and genetic linkage studies. Linkage to IBD on proximal Chromosome (Chr) 16p is well established and replicated. A stratification experiment showed that the recent identification of a disease gene on the q arm does not interfere with the approach on the p arm, and the linkage peak is still significant. Here we present a candidate gene study of the alpha integrins (CD11A-D) on Chr 16. The alpha integrins play a key role in inflammatory processes, including leukocyte adhesion and migration. Their genes are located on the p arm of Chr 16, and therefore represent excellent positional and functional candidates. Since the assignment of the CD11 genes in the genome was not clear, we performed physical, radiation hybrid, and fluorescent in situ hybridization mapping of the gene family. All CD11 genes map on Chr 16p11-12. CD11B-D are arranged in a gene cluster within 300 kb and CD11A is located about 2.5 Mb telomeric. Thirteen new single nucleotide polymorphisms (SNPs) and eight SNPs from databases were identified through full-length sequencing. Case-control statistics demonstrated an association lead in the CD11 gene cluster, which was not confirmed in further family based association/linkage analyses using single markers and haplotypes. It is unlikely that the CD11 genes play an important role in the pathogenesis of IBD. The marginally significant results could indicate a disease gene in the vicinity of the gene cluster.

CD11 Antigens↗

PubMed2001

Evidence for a NOD2-independent susceptibility locus for inflammatory bowel disease on chromosome 16p.

Heritable predisposition to inflammatory bowel disease (IBD) has been demonstrated by epidemiological and genetic analysis. Linkage of IBD to broad regions of chromosome 16 has been established by analysis of multiple populations. NOD2, located on proximal 16q, was recently identified as an IBD gene. As the linkage regions on chromosome 16 are large, we have investigated the possibility that NOD2 is not the only IBD gene located on this chromosome. A high-density experiment using 39 microsatellite markers was performed to identify additional regions of association, and to indicate areas of interest for further investigation. A triple-peaked configuration of the linkage curve with peak logarithm of odds (lod) scores of 2.7, 3.2, and 3.1 was observed on proximal 16p, proximal 16q, and central 16q, respectively. The cohort was stratified by coding individuals carrying the NOD2 single nucleotide polymorphism (SNP)8 and SNP13 "unknown." Significance at the central peak, corresponding to the genomic location of NOD2, then decreased from 3.2 to 1.2. The maximal lod scores on the proximal p-arm (lod = 2.1) and central q-arm (lod = 2.6) changed only moderately. An exploratory association analysis (TRANSMIT) yielded a strong lead at D16S3068 (P = 0.00028). The region around this marker was further investigated by using anonymous SNPs. An associated haplotype containing three SNPs was identified (peak significance P = 0.00027, IBD phenotype). On stratification based on NOD2 genotype, this significance increased to P = 0.0001. These results confirm the importance of NOD2 and provide evidence for a second IBD gene located on chromosome 16p.

Carrier Proteins↗