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Biomedical subjects

Theo Dingermann

Publications and source records attributed to Theo Dingermann.

At least 19 recordsLinked to original sources
PubMed2006

Overexpression, refolding, and purification of polyhistidine-tagged human retinoic acid related orphan receptor RORalpha4.

RORalpha4 is a nuclear receptor activating the transcription of genes that are important for a variety of physiological processes like muscle differentiation, lipid and bone metabolism, cerebellar development, and inflammation. Furthermore, it plays an essential role in maintaining circadian rhythmicity of the core clock in the suprachiasmatic nuclei (SCN). Here, we describe the successful overexpression and purification of human full-length RORalpha4 in Escherichia coli using a T7 expression system. The expressed protein formed inclusion bodies which were solubilized in the presence of 6M guanidinium-HCl and renatured by gradual removal of guanidinium-HCl and addition of l-arginine. The refolded protein was purified by nickel affinity chromatography due to an N-terminal polyhistidine tag which can be cleaved with thrombin subsequently. This method permitted us to obtain up to 20mg of pure and native RORalpha4 protein per liter of E. coli culture. The DNA binding activity of the refolded protein was demonstrated by electrophoretic mobility shift assay (EMSA) using an oligonucleotide comprising the ROR-response element (RORE) motif (A/G)GGTCA. In addition, we developed a new monoclonal antibody to human RORalpha in mice with high sensitivity and specificity.

Animals↗
PubMed2006

Macromolecular immunosuppressants.

Recombinant immunosuppressants have come of age and represent a significant class of quite diverse drugs. They target extracellular molecules, and either label or inhibit them. Those targets are soluble factors or membrane proteins almost all of which are components of a very complex molecular network of communication and amplification. Notably, many recombinant immunosuppressants have been developed in a rather short period of time. This is due to the fact that developing the actual drug is left to nature, so to speak; either it is a human protein as is the case for Anakinra or it is an antibody or antibody derivative "developed" by the immune system of mice or rats following exposure to the antigen. The challenge for developing recombinant immunosuppressants is to identify relevant targets. It is no longer very difficult to generate proteins to targets, once those are identified. The clinical use of recombinant immunosuppressants has yet to show which targets are truly relevant and which drugs prove effective.

Animals↗
PubMed2006

Genomic DNA of leukemic patients: target for clinical diagnosis of MLL rearrangements.

Genomic DNA is the optimal resource to analyze questions concerning genetic changes that are related to oncogenesis. This article tries to summarize recent efforts to analyze chromosomal changes that trigger the development of human acute myeloid and lymphoblastic leukemias. The aim of this study was to establish an universal method that enables the identification and characterization of chromosomal translocations of the human MLL gene at the genomic nucleotide level. Chromosomal translocations of the MLL gene are the result of illegitimate recombination events in hematopoietic stem or precursor cells, strictly associated with the onset of highly malignant leukemic diseases. The applied technology was able to identify specific fusion alleles that were generated by chromosomal translocations, chromosomal deletions, chromosomal inversions and partial tandem duplications. Moreover, it allowed us to investigate even highly complex genetic changes by applying systematic breakpoint analyses. On the basis of these analyses, patient-specific molecular markers were established that turned out to be a very good source for monitoring minimal residual disease (MRD). MRD analyses control the efficiency and efficacy of current treatment protocols and have become a very sensitive molecular tool to monitor therapeutic success or failure in individual leukemia patients.

Chromosome Mapping↗
PubMed2004

Diagnostic tool for the identification of MLL rearrangements including unknown partner genes.

Approximately 50 different chromosomal translocations of the human MLL gene are currently known and associated with high-risk acute leukemia. The large number of different MLL translocation partner genes makes a precise diagnosis a demanding task. After their cytogenetic identification, only the most common MLL translocations are investigated by RT-PCR analyses, whereas infrequent or unknown MLL translocations are excluded from further analyses. Therefore, we aimed at establishing a method that enables the detection of any MLL rearrangement by using genomic DNA isolated from patient biopsy material. This goal was achieved by establishing a universal long-distance inverse-PCR approach that allows the identification of any kind of MLL rearrangement if located within the breakpoint cluster region. This method was applied to biopsy material derived from 40 leukemia patients known to carry MLL abnormalities. Thirty-six patients carried known MLL fusions (34 with der(11) and 2 with reciprocal alleles), whereas 3 patients were found to carry novel MLL fusions to ACACA, SELB, and SMAP1, respectively. One patient carried a genomic fusion between MLL and TIRAP, resulting from an interstitial deletion. Because of this interstitial deletion, portions of the MLL and TIRAP genes were deleted, together with 123 genes located within the 13-Mbp interval between both chromosomal loci. Therefore, this previously undescribed diagnostic tool has been proven successful for analyzing any MLL rearrangement including previously unrecognized partner genes. Furthermore, the determined patient-specific fusion sequences are useful for minimal residual disease monitoring of MLL associated acute leukemias.

DNA-Binding Proteins↗
PubMed2004

Interaction of AF4 wild-type and AF4.MLL fusion protein with SIAH proteins: indication for t(4;11) pathobiology?

The human AF4 (ALL-1 fused gene on chromosome 4) gene (4q11) is recurrently involved in reciprocal translocations to the MLL (mixed lineage leukemia) gene (11q23), correlated with high-risk acute lymphoblastic leukemia (ALL) in infants and early childhood. The t(4;11) translocation is one of the most frequent MLL translocations known today. In general, MLL translocations are the result of an illegitimate recombination process leading to reciprocal fusions of unrelated translocation partner (TP) genes with the MLL gene. Owing to the constant presence of the derivative (11) product, it was hypothesised that only MLL.TP fusion genes are responsible for the leukemogenic process. This concept has been successfully tested for some known MLL fusions, while other MLL fusions failed. Here, we demonstrate growth-transforming potential of AF4 wild-type and the AF4.MLL fusion protein. The underlying oncogenic mechanism involves the two E3 ubiquitin ligases SIAH1 and SIAH2, the N-terminal portion of AF4 and the protection of the AF4.MLL fusion protein against proteosomal degradation.

Animals↗
PubMed2003

MLL-mediated transcriptional gene regulation investigated by gene expression profiling.

The human mixed lineage leukemia (MLL) gene is involved in about 50 different chromosomal translocations, associated with the disease phenotype of acute leukemia. However, the normal function of MLL is less understood. Homozygous knockouts of murine Mll were embryonal lethal, while heterozygous disruption led to aberrant hox gene expression associated with skeletal malformations, growth retardation, and impaired hematopoiesis. To understand MLL functions on the molecular level, gene expression profiling experiments were performed with a pair of murine cell lines (MLL(+/+) and MLL(-/-)). Microarray hybridization experiments revealed 197 potential target genes that are differentially expressed, providing new and important clues about MLL functions.

Animals↗