Biomedical subjects

Johannes Schuchhardt

Publications and source records attributed to Johannes Schuchhardt.

7 recordsLinked to original sources

PubMed2006

Mycobacterium tuberculosis gene expression profiling within the context of protein networks.

As one of the world's most successful intracellular pathogens, Mycobacterium tuberculosis, the causative agent of human tuberculosis, is responsible for two to three million deaths annually. The pathogenicity of M. tuberculosis relies on its ability to survive and persist within host macrophage cells during infection. It is of central importance, therefore, to identify genes and pathways that are involved in the survival and persistence of M. tuberculosis within these cells. Utilizing genome-wide DNA arrays we have identified M. tuberculosis genes that are specifically induced during macrophage infection. To better understand the cellular context of these differentially expressed genes, we have also combined our array analyses with computational methods of protein network identification. Our combined approach reveals certain signatures of M. tuberculosis residing within macrophage cells, including the induction of genes involved in DNA damage repair, fatty acid degradation, iron metabolism, and cell wall metabolism.

Amino Acids↗

PubMed2006

Differential binding studies applying functional protein microarrays and surface plasmon resonance.

A variety of different in vivo and in vitro technologies provide comprehensive insights in protein-protein interaction networks. Here we demonstrate a novel approach to analyze, verify and quantify putative interactions between two members of the S100 protein family and 80 recombinant proteins derived from a proteome-wide protein expression library. Surface plasmon resonance (SPR) using Biacore technology and functional protein microarrays were used as two independent methods to study protein-protein interactions. With this combined approach we were able to detect nine calcium-dependent interactions between Arg-Gly-Ser-(RGS)-His6 tagged proteins derived from the library and GST-tagged S100B and S100A6, respectively. For the protein microarray affinity-purified proteins from the expression library were spotted onto modified glass slides and probed with the S100 proteins. SPR experiments were performed in the same setup and in a vice-versa approach reversing analytes and ligands to determine distinct association and dissociation patterns of each positive interaction. Besides already known interaction partners, several novel binders were found independently with both detection methods, albeit analogous immobilization strategies had to be applied in both assays.

Biosensing Techniques↗

PubMed2006

Unique transcriptome signature of Mycobacterium tuberculosis in pulmonary tuberculosis.

Although tuberculosis remains a substantial global threat, the mechanisms that enable mycobacterial persistence and replication within the human host are ill defined. This study represents the first genome-wide expression analysis of Mycobacterium tuberculosis from clinical lung samples, which has enabled the identification of M. tuberculosis genes actively expressed during pulmonary tuberculosis. To obtain optimal information from our DNA array analyses, we analyzed the differentially expressed genes within the context of computationally inferred protein networks. Protein networks were constructed using functional linkages established by the Rosetta stone, phylogenetic profile, conserved gene neighbor, and operon computational methods. This combined approach revealed that during pulmonary tuberculosis, M. tuberculosis actively transcribes a number of genes involved in active fortification and evasion from host defense systems. These genes may provide targets for novel intervention strategies.

Bacterial Proteins↗

PubMed2005

SPOT synthesis: reliability of array-based measurement of peptide binding affinity.

Peptide arrays prepared by the SPOT synthesis technology have emerged as a proteomic tool to study molecular recognition and identify biologically active peptides. However, it was previously not clear how accurately signal intensities obtained by probing peptide arrays for protein binding really reflect the dissociation constants of the protein-peptide complexes. Using the monoclonal antibody CB4-1 as a model system, we systematically compared dissociation constants of antibody-peptide complexes with signal intensities obtained using the SPOT technology. By analyzing a set of peptides possessing different affinities to the antibody, we determined the strengths of the SPOT screening method. The accuracy of the measured results was improved by taking regional trends in the membrane surface into account. A model based on the mass action law compares well with the experimental results. Interestingly, the applied concentrations of the binding partners do not directly correspond to the effective concentrations in the assay. We show that the SPOT technology is an accurate method for assigning the spots' measured signal intensities to three different binding affinity classes. The dissociation constants of the intermediate region were found to be between pK(dis)=5 and pK(dis)=7. Altering the experimental parameters causes a directed change of this region.

Antibodies, Monoclonal↗

PubMed2004

High reproducibility of large-gel two-dimensional electrophoresis.

Two-dimensional gel electrophoresis (2-DE) facilitates the separation of thousands of proteins from highly complex protein mixtures and has become a central method in proteomics in recent years. In the present study, we examined the technical variability of large 2-DE gels with respect to sample preparation, electrophoresis procedure, data acquisition, and biological variation by analyzing a disease (Huntington's disease) and control state with a commercially available software package, PROTEOMWEAVER trade mark. Scatter plots and correlation coefficients were obtained to quantify both technical and biological variation. Even 2-DE gels run separately in both dimensions yielded correlation coefficients around 0.88 and deviations from the mean close to 20% for low-intensity spots. This indicates a high technical reproducibility of the 2-DE procedure developed in our laboratory. Variability within a biological condition was low and comparable to technical variation (at least 0.87). Two-dimensional (2-D) gels obtained from samples of different biological conditions (health vs. disease) achieved a variability similar to intracondition and technical variability. These findings highlight the importance of multiple gel and spot-by-spot comparisons to identify biological significant changes. Minor errors introduced by technical and biological variation allow a comparison of all gels within a study which facilitates the tackling of complex biological problems.

Animals↗

PubMed2002

Oligodb--interactive design of oligo DNA for transcription profiling of human genes.

MOTIVATION: The increasing use of customized oligo-DNA-microarrays has created a demand for convenient design of oligo nucleotides. The oligo-nucleotides have to serve as specific templates in the hybridization reaction. RESULTS: We have devised a web-based system for interactive design of suitable oligos for transcription profiling experiments with user-defined parameters. Oligodb is based on predicted human gene transcripts from the ENSEMBL project. The web interface allows the user to retrieve oligos for human transcripts in a flexible manner. Criteria for selecting the oligos are low similarity to all other known human genes, the absence of stable secondary structures, the length of the oligo, and the absence of low complexity regions. Furthermore, the user may select oligos corresponding to the desired position in the 5'-3' direction of the transcript, and based on the melting temperature. All data and results are tightly web-linked to the corresponding data entries of ENSEMBL project.

Algorithms↗

PubMed2001

Extracting information from cDNA arrays.

High-density DNA arrays allow measurements of gene expression levels (messenger RNA abundance) for thousands of genes simultaneously. We analyze arrays with spotted cDNA used in monitoring of expression profiles. A dilution series of a mouse liver probe is deployed to quantify the reproducibility of expression measurements. Saturation effects limit the accessible signal range at high intensities. Additive noise and outshining from neighboring spots dominate at low intensities. For repeated measurements on the same filter and filter-to-filter comparisons correlation coefficients of 0.98 are found. Next we consider the clustering of gene expression time series from stimulated human fibroblasts which aims at finding co-regulated genes. We analyze how preprocessing, the distance measure, and the clustering algorithm affect the resulting clusters. Finally we discuss algorithms for the identification of transcription factor binding sites from clusters of co-regulated genes. (c) 2001 American Institute of Physics.

Journal Article↗