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Andreas Braun

Publications and source records attributed to Andreas Braun.

30 records · Page 2Linked to original sources
PubMed2004

Imaging the translational dynamics of CF3 in liquid helium droplets.

The translational dynamics of CF3 in liquid helium have been investigated by photodissociating CF3I dissolved in helium droplets consisting of several thousands of atoms. The velocity distribution of CF3 fragments that have escaped from the droplets has been determined using ion imaging techniques and is found to be considerably shifted to lower speeds with respect to the photodissociation of gas phase CF3I. The fragments furthermore show a speed dependent angular distribution that is isotropic for the slowest and approaches the gas phase distribution for the faster fragments. These distributions point to a nonthermal escape process in which, at least for the speeds relevant for the present experiment, the kinetic energy and momentum transfer from the fragments to the solvent appears to be governed by binary collisions with the individual helium atoms.

Journal Article↗
PubMed2004

Simultaneous quantitative and allele-specific expression analysis with real competitive PCR.

BACKGROUND: For a diploid organism such as human, the two alleles of a particular gene can be expressed at different levels due to X chromosome inactivation, gene imprinting, different local promoter activity, or mRNA stability. Recently, imbalanced allelic expression was found to be common in human and can follow Mendelian inheritance. Here we present a method that employs real competitive PCR for allele-specific expression analysis. RESULTS: A transcribed mutation such as a single nucleotide polymorphism (SNP) is used as the marker for allele-specific expression analysis. A synthetic mutation created in the competitor is close to a natural mutation site in the cDNA sequence. PCR is used to amplify the two cDNA sequences from the two alleles and the competitor. A base extension reaction with a mixture of ddNTPs/dNTP is used to generate three oligonucleotides for the two cDNAs and the competitor. The three products are identified and their ratios are calculated based on their peak areas in the MALDI-TOF mass spectrum. Several examples are given to illustrate how allele-specific gene expression can be applied in different biological studies. CONCLUSIONS: This technique can quantify the absolute expression level of each individual allele of a gene with high precision and throughput.

ATP Binding Cassette Transporter, Subfamily D, Mem↗
PubMed2004

Large-scale validation of single nucleotide polymorphisms in gene regions.

Genome-wide association studies using large numbers of bi-allelic single nucleotide polymorphisms (SNPs) have been proposed as a potentially powerful method for identifying genes involved in common diseases. To assemble a SNP collection appropriate for large-scale association, we designed assays for 226,099 publicly available SNPs located primarily within known and predicted gene regions. Allele frequencies were estimated in a sample of 92 CEPH Caucasians using chip-based MALDI-TOF mass spectrometry with pooled DNA. Of the 204,200 designed assays that were functional, 125,799 SNPs were determined to be polymorphic (minor allele frequency > 0.02), of which 101,729 map uniquely to the human genome. Many of the commonly available RefSNP annotations were predictive of polymorphic status and could be used to improve the selection of SNPs from the public domain for genetic research. The set of uniquely mapping, polymorphic SNPs is located within 10 kb of 66% of known and predicted genes annotated in LocusLink, which could prove useful for large-scale disease association studies.

Databases, Genetic↗
PubMed2003

Technology challenges in screening single gene disorders.

UNLABELLED: The completion of the human genome project and the accelerated discovery of genes responsible for single gene disorders will allow for the preventive screening of carriers and patients. Accuracy and reliability of analytic tests are major demands on technologies used in a diagnostic screening environment. The consistency of results and the potential of multiplexing suggest mass spectrometry as the method of choice for large-scale genetic screening programs. The added ability to analyze other large molecules such as peptides and proteins further underlines the versatility and usefulness of this technology. We describe the use of mass spectrometry for screening of sickle cell disease at the level of DNA and protein. CONCLUSION: Analytic methods are needed for large-scale screening and diagnostic applications that are highly accurate, fully automated and cost-effective. The precise measurement of molecular weights and the use of high fidelity enzymes to produce diagnostic products make mass spectrometry the method of choice for DNA and protein screening procedures.

Adolescent↗
PubMed2003

Amino acid variant in the kinase binding domain of dual-specific A kinase-anchoring protein 2: a disease susceptibility polymorphism.

The focus of human genetics in recent years has shifted toward identifying genes that are involved in the development of common diseases such as cancer, diabetes, cardiovascular diseases, and Alzheimer's disease. Because many complex diseases are late-onset, the frequencies of disease susceptibility alleles are expected to decrease in the healthy elderly individuals of the population at large because of their contribution to disease morbidity andor mortality. To test this assumption, we compared allele frequencies of 6,500 single-nucleotide polymorphisms (SNPs) located in approximately 5,000 genes between DNA pools of age-stratified healthy, European-American individuals. A SNP that results in an amino acid change from Ile to Val in the dual-specific A kinase-anchoring protein 2 (d-AKAP2) gene, showed the strongest correlation with age. Subsequent analysis of an independent sample indicated that the Val variant was associated with a statistically significant decrease in the length of the electrocardiogram PR interval. The IleVal SNP is located in the A-kinase-binding domain. An in vitro binding assay revealed that the Ile variant bound approximately 3-fold weaker to the protein kinase A (PKA)-RIalpha isoform than the Val variant. This decreased affinity resulted in alterations in the subcellular distribution of the recombinantly expressed PKA-RIalpha isoform. Our study suggests that alterations in PKA-RIalpha subcellular localization caused by variation in d-AKAP2 may have a negative health prognosis in the aging population, which may be related to cardiac dysfunction. Age-stratified samples appear to be useful for screening SNPs to identify functional gene variants that have an impact on health.

A Kinase Anchor Proteins↗
PubMed2003

Designing isoform-specific peptide disruptors of protein kinase A localization.

A kinase-anchoring proteins (AKAPs) coordinate cAMP-mediated signaling by binding and localizing cAMP-dependent protein kinase (PKA), using an amphipathic helical docking motif. Peptide disruptors of PKA localization that mimic this helix have been used successfully to assess the involvement of PKA in specific signaling pathways. However, these peptides were developed as disruptors for the type II regulatory subunit (RII) even though both RI and RII isoforms can bind to AKAPs and have discrete functions. To evaluate the effects of each localized isoform, we designed peptides that specifically bind to either RI or RII. Using a peptide array, we have defined the minimal binding sequence of dual specific-AKAP 2 (d-AKAP2), which binds tightly to both RI and RII. Side-chain requirements for affinity and isoform specificity were evaluated by using a peptide substitution array where each position along the A kinase binding domain of d-AKAP2 was substituted by the other 19 l-amino acids. This array comprises 513 single-site substitution analogs of the d-AKAP2 sequence. Peptides containing single and multiple mutations were evaluated in a quantitative fluorescence binding assay and a cell-based colocalization assay. This strategy has allowed us to design peptides with high affinity (K(D) = 1-2 nM) and high specificity for RIalpha versus RIIalpha. These isoform-specific peptides will be invaluable tools to evaluate functional differences between localized RI and RII PKA and are RIalpha-specific disruptors. This array-based analysis also provides a foundation for biophysical analysis of this docking motif.

Adaptor Proteins, Signal Transducing↗
PubMed2003

High-resolution SNP scan of chromosome 6p21 in pooled samples from patients with complex diseases.

We apply a high-throughput protocol of chip-based mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight; MALDI-TOF) as a method of screening for differences in single-nucleotide polymorphism (SNP) allele frequencies. Using pooled DNA from individuals with asthma, Crohn's disease (CD), schizophrenia, type 1 diabetes (T1D), and controls, we selected 534 SNPs from an initial set of 1435 SNPs spanning a 25-Mb region on chromosome 6p21. The standard deviations of measurements of time of flight at different dots, from different PCRs, and from different pools indicate reliable results on each analysis step. In 90% of the disease-control comparisons we found allelic differences of <10%. Of the T1D samples, which served as a positive control, 10 SNPs with significant differences were observed after taking into account multiple testing. Of these 10 SNPs, 5 are located between DQB1 and DRB1, confirming the known association with the DR3 and DR4 haplotypes whereas two additional SNPs also reproduced known associations of T1D with DOB and LTA. In the CD pool also, two earlier described associations were found with SNPs close to DRB1 and MICA. Additional associations were found in the schizophrenia and asthma pools. They should be confirmed in individual samples or can be used to develop further quality criteria for accepting true differences between pools. The determination of SNP allele frequencies in pooled DNA appears to be of value in assigning further genotyping priorities also in large linkage regions.

Asthma↗
PubMed2003

Subjective intensity of pain during the treatment of periodontal lesions with the Vector-system.

The aim of this study was to measure subjective intensities of pain during the treatment of periodontal lesions with the Vector-system when compared to pain occurring during the treatment with conventional methods. Twenty patients, each of whom had three teeth with comparable periodontal pocket depths, were treated using three different methods: (i) scaling and root planing with hand instruments, (ii) cleaning with a conventional ultrasonic instrument (Siroson S) and (iii) cleaning with the Vector-system. The subjective intensities of pain during the treatment were measured with an intermodal intensity comparison. A visual analog scale was used for the evaluation after the treatment. The results of the intermodal intensity comparison during treatment showed that the use of the Vector-system caused less pain than the cleaning with hand instruments or the conventional ultrasonic system (P < 0.05). The intermodal intensity comparisons of cleaning with hand instruments and cleaning with the conventional ultrasonic system were not significantly different (P > 0.05). These results could be confirmed by the visual analog scale. Using the Vector-system for cleaning periodontal lesions it is possible to reduce pain sensations compared to conventional methods. Using cleaning methods that cause less discomfort and pain, it might be possible to increase the patient's compliance during non-surgical periodontal therapy and recall.

Alveolar Bone Loss↗
PubMed2002

Symptoms in carriers of adrenoleukodystrophy relate to skewed X inactivation.

Skewing of X inactivation may contribute to the manifestation of symptoms in adrenoleukodystrophy carriers. We observed highly skewed X inactivation in 32% of 22 symptomatic and asymptomatic carriers but not in 7 related and 35 unrelated controls. Skewing of X inactivation correlated with clinical neurological scores but not with the extent of very long-chain fatty acid accumulation. Transcript analysis in cultured fibroblasts revealed that skewing could occur both in favor of the mutant and the wild-type allele. Adrenoleukodystrophy carriers are more susceptible to develop skewing of X inactivation in favor of the mutant allele being associated with the manifestation of symptoms.

Adolescent↗
PubMed2002

Multiplex minisequencing of the 21-hydroxylase gene as a rapid strategy to confirm congenital adrenal hyperplasia.

BACKGROUND: Congenital adrenal hyperplasia (CAH) is a frequent autosomal recessive disease, with a wide range of clinical manifestations, most commonly attributable to mutations in the 21-hydroxylase gene (CYP21). Large gene deletions, large gene conversions, a small 8-basepair deletion, and eight point mutations in CYP21 account for approximately 95% of all enzyme deficiencies. We developed a new strategy for a rapid CYP21 analysis. METHODS: DNA samples from 40 CAH patients previously genotyped by direct DNA sequencing were reanalyzed by allele-specific amplification of the functional CYP21 gene followed by a multiplex minisequencing reaction using 13 primers. In addition, a second PCR that amplified a part of exon 3 was used to demonstrate the presence or absence of at least one functional gene. RESULTS: The assay detected the P453S mutation and nine of the most common mutations (P30L, intron 2 splice, Delta 8bp, I172N, exon 6 cluster, V281L, F306+t, Q318X, and R356W) caused by microconversions from the CYP21P pseudogene. The concordance was 100% for detecting these mutations, including gene deletions and large gene conversions. The 40 patient DNA samples were analyzed in 1.5 working days by one technician (actual hands-on time, 3.5 h). The material cost for analyzing one sample was approximately 10.00 Euros (US $9.00). CONCLUSIONS: This novel mutation screening strategy rapidly detects 90-95% of all mutations associated with CAH and appears applicable as a tool for confirmation of increased 17-hydroxyprogesterone found in neonatal CAH screening.

Adrenal Hyperplasia, Congenital↗
PubMedDate not supplied

Mining disease susceptibility genes through SNP analyses and expression profiling using MALDI-TOF mass spectrometry.

To find genes that underlie disease susceptibilities, genome-wide single nucleotide polymorphisms (SNPs) have been analyzed using high-throughput matrix assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). As a proof-of-concept for this approach, gene regions have been identified that were previously associated by others with certain diseases or traits. On the same technology platform, accurate and absolute transcriptional profiling can be performed and applied to allele expression analysis. Here, we provide a brief review of the technology and its applications to disease gene discovery.

Alleles↗