Thomas Winckler

The genome of the social amoeba Dictyostelium discoideum

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal–fungal lineage after the plant–animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

Sequence and analysis of chromosome 2 of Dictyostelium discoideum

The genome of the lower eukaryote Dictyostelium discoideum comprises six chromosomes. Here we report the sequence of the largest, chromosome 2, which at 8 megabases (Mb) represents about 25% of the genome. Despite an A + T content of nearly 80%, the chromosome codes for 2,799 predicted protein coding genes and 73 transfer RNA genes. This gene density, about 1 gene per 2.6 kilobases (kb), is surpassed only by Saccharomyces cerevisiae (one per 2 kb) and is similar to that of Schizosaccharomyces pombe (one per 2.5 kb). If we assume that the other chromosomes have a similar gene density, we can expect around 11,000 genes in the D. discoideum genome. A significant number of the genes show higher similarities to genes of vertebrates than to those of other fully sequenced eukaryotes. This analysis strengthens the view that the evolutionary position of D. discoideum is located before the branching of metazoa and fungi but after the divergence of the plant kingdom, placing it close to the base of metazoan evolution.

Bivalent β-Carbolines as Potential Multitarget Anti-Alzheimer Agents

Alzheimers disease (AD) is a prevalent neurodegenerative disorder with multifactorial causes that requires multitargeted treatment. Inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) improve cholinergic signaling in the central nervous system and thus AChE inhibitors are well established in the therapy of AD to improve memory disturbances and other cognitive symptoms. On the other hand, AD patients benefit from reduction of pathologic glutamate-induced, Ca(2+)-mediated excitotoxicity by the N-methyl-d-aspartate receptor (NR) antagonist memantine. New drugs that simultaneously affect both cholinergic transmission and glutamate-induced excitotoxicity may further improve AD treatment. While connecting beta-carboline units by alkylene spacers in two different series of compounds and subsequent evaluation of their AChE/BChE-inhibitory potential, we found that several of these bivalent beta-carbolines were potent NR blockers. The most promising compound was a N(9)-homobivalent beta-carboline with a nonylene spacer, which displayed IC(50) values of 0.5 nM for AChE, 5.7 nM for BChE, and 1.4 microM for NR, respectively.

Beta and gamma carboline derivatives as potential anti-Alzheimer agents: A comparison.

Nine novel β- and γ-carboline derivatives bearing either methyl-, propargyl- or phenethyl-residues at the indole nitrogen were synthesized and tested as potential anti-Alzheimer drugs. Antagonism of recombinantly expressed NMDA receptors, inhibition of cholinesterases, and radical scavenging properties were determined for all compounds. Some were additionally tested in vivo for their ability to reverse scopolamine-induced cognitive impairment in an 8-arm radial maze experiment with rats. For the most promising candidates, the interaction with muscarinic M1 receptors was also investigated. With this set of compounds assays the influence of the scaffold itself and the substituents can be investigated separately. 5-Methyl-γ-carboline (6) was the most potent (0.25 μmol/100 g b.w.) compound in the in vivo test and might be a good starting point for the development of novel anti-Alzheimer drugs.

The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling.

Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into early eukaryote evolution. We describe extensive use of histidine kinase-based two-component systems and tyrosine kinase signaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases in Acanthamoeba and Physarum as representatives of two distantly related subdivisions of Amoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfer.

Virulence determinants of the human pathogenic fungus Aspergillus fumigatus protect against soil amoeba predation

Filamentous fungi represent classical examples for environmentally acquired human pathogens whose major virulence mechanisms are likely to have emerged long before the appearance of innate immune systems. In natural habitats, amoeba predation could impose a major selection pressure towards the acquisition of virulence attributes. To test this hypothesis, we exploited the amoeba Dictyostelium discoideum to study its interaction with Aspergillus fumigatus, two abundant soil inhabitants for which we found co-occurrence in various sites. Fungal conidia were efficiently taken up by D. discoideum, but ingestion was higher when conidia were devoid of the green fungal spore pigment dihydroxynaphtalene melanin, in line with earlier results obtained for immune cells. Conidia were able to survive phagocytic processing, and intracellular germination was initiated only after several hours of co-incubation which eventually led to a lethal disruption of the host cell. Besides phagocytic interactions, both amoeba and fungus secreted cross inhibitory factors which suppressed fungal growth or induced amoeba aggregation with subsequent cell lysis, respectively. On the fungal side, we identified gliotoxin as the major fungal factor killing Dictyostelium, supporting the idea that major virulence attributes, such as escape from phagocytosis and the secretion of mycotoxins are beneficial to escape from environmental predators.

Transfer RNA gene-targeted integration: an adaptation of retrotransposable elements to survive in the compact Dictyostelium discoideum genome

Almost every organism carries along a multitude of molecular parasites known as transposable elements (TEs). TEs influence their host genomes in many ways by expanding genome size and complexity, rearranging genomic DNA, mutagenizing host genes, and altering transcription levels of nearby genes. The eukaryotic microorganism Dictyostelium discoideum is attractive for the study of fundamental biological phenomena such as intercellular communication, formation of multicellularity, cell differentiation, and morphogenesis. D. discoideum has a highly compacted, haploid genome with less than 1 kb of genomic DNA separating coding regions. Nevertheless, the D. discoideum genome is loaded with 10% of TEs that managed to settle and survive in this inhospitable environment. In depth analysis of D. discoideum genome project data has provided intriguing insights into the evolutionary challenges that mobile elements face when they invade compact genomes. Two different mechanisms are used by D. discoideum TEs to avoid disruption of host genes upon retrotransposition. Several TEs have invented the specific targeting of tRNA gene-flanking regions as a means to avoid integration into coding regions. These elements have been dispersed on all chromosomes, closely following the distribution of tRNA genes. By contrast, TEs that lack bona fide integration specificities show a strong bias to nested integration, thus forming large TE clusters at certain chromosomal loci that are hardly resolved by bioinformatics approaches. We summarize our current view of D. discoideum TEs and present new data from the analysis of the complete sequences of D. discoideum chromosomes 1 and 2, which comprise more than one third of the total genome.

Conformationally constrained NR2B selective NMDA receptor antagonists derived from ifenprodil: Synthesis and biological evaluation of tetrahydro-3-benzazepine-1,7-diols.

NR2B selective NMDA receptor antagonists with tetrahydro-3-benzazepine-1,7-diol scaffold have been designed by formal cleavage and reconstitution of the piperidine ring of the lead compound ifenprodil (1). The secondary amine 10 represents the central building block for the synthesis of more than 25 tetrahydro-3-benzazepin-1-ols. Generally 7-hydroxy derivatives display higher NR2B receptor affinities than the corresponding 7-benzyloxy compounds. A distance of four atoms (five bond lengths) between the basic amino group and the terminal aryl moiety led to highest NR2B affinity. 3-(4-Phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol (WMS-1410, 25) represents the most promising NR2B antagonist of this series showing a K(i)-value of 14nM. Compound 25 reveals excellent selectivity over more than 100 further relevant target proteins, antagonizes glutamate induced excitotoxicity (IC(50)=18.4nM) and is metabolically more stable than ifenprodil. Up to a dose of 100mg/kg 25 is well tolerated by mice and it shows dose dependent analgesic activity in the late neuropathic pain phase of the formalin assay.

Non-LTR retrotransposons with unique integration preferences downstream of Dictyostelium discoideum tRNA genes

Abstract Retrotransposable elements are genetic entities which move and replicate within host cell genomes. We have previously reported on the structures and genomic distributions of two non-long terminal repeat (non-LTR) retrotransposons, DRE and Tdd-3, in the eukaryotic microorganism Dictyostelium discoideum. DRE elements are found inserted upstream, and Tdd-3 elements downstream, of transfer RNA (tRNA) genes with remarkable position and orientation specificities. The data set currently available from the Dictyostelium Genome Project led to the characterisation of two repetitive DNA elements which are related to the D. discoideum non-LTR retrotransposon Tdd-3 in both their structural properties and genomic distributions. It appears from our data that in the D. discoideum genome tRNA genes are major targets for the insertion of mobilised non-LTR retrotransposons. This may be interpreted as the consequence of a process of coevolution, allowing a viable population of retroelements to transpose without being deleterious to the small microbial host genome which carries only short intergenic DNA sequences. A new nomenclature is introduced to designate all tRNA gene-targeted non-LTR retrotransposons (TREs) in the D. discoideum genome. TREs inserted 5′ and 3′ of tRNA genes are named TRE5 and TRE3, respectively. According to this nomenclature DRE and Tdd-3 are renamed TRE5-A and TRE3-A, respectively. The new retroelements described in this study are named TRE3-B (formerly RED) and TRE3-C.

Design, Synthesis, and Biological Evaluation of 3-Benzazepin-1-ols as NR2B-Selective NMDA Receptor Antagonists.

Cleavage and reconstitution of a bond in the piperidine ring of ifenprodil (1) leads to 7‐methoxy‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepin‐1‐ols, a novel class of NR2B‐selective NMDA receptor antagonists. The secondary amine 7‐methoxy‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepin‐1‐ol (12), which was synthesized in six steps starting from 2‐phenylethylamine 3, represents the central building block for the introduction of several N‐linked residues. A distance of four methylene units between the basic nitrogen atom and the phenyl residue in the side chain results in high NR2B affinity. The 4‐phenylbutyl derivative 13 (WMS‐1405, Ki=5.4 nM) and the conformationally restricted 4‐phenylcyclohexyl derivative 31 (Ki=10 nM) represent the most potent NR2B ligands of this series. Whereas 13 shows excellent selectivity, the 4‐phenylcyclohexyl derivative 31 also interacts with σ1 (Ki=33 nM) and σ2 receptors (Ki=82 nM). In the excitotoxicity assay the phenylbutyl derivative 13 inhibits the glutamate‐induced cytotoxicity with an IC50 value of 360 nM, indicating that 13 is an NMDA antagonist.