Localization and organization of protein factors involved in chromosome inheritance in Dictyostelium discoideum

Heterochromatin protein 1 (HP1) proteins are highly conserved heterochromatin components required for genomic integrity. We have previously shown that the two HP1 isoforms expressed in Dictyostelium, HcpA and HcpB, are mainly localized to (peri-)centromeric heterochromatin and have largely overlapping functions. However, they cause distinct phenotypes when overexpressed. We show here that these isoforms display quantitative differences in dimerization behavior. Dimerization preference, as well as the mutant phenotype in overexpression strains, depends on the C-terminus containing the hinge and chromo shadow domains. Both Hcp proteins are targeted to distinct subnuclear regions by different chromo shadow domain-dependent and -independent mechanisms. In addition, both proteins bind to DNA and RNA in vitro and binding is independent of the chromo shadow domain. Thus, this DNA and/or RNA binding activity may contribute to protein targeting. To further characterize heterochromatin, we cloned the Dictyostelium homolog of the origin recognition complex subunit 2 (OrcB). OrcB localizes to distinct subnuclear foci that were also targeted by HcpA. In addition, it is associated with the centrosome throughout the cell cycle. The results indicate that, similar to Orc2 homologs from other organisms, it is required for different processes in chromosome inheritance.     

Mutation in the Scyl1 gene encoding amino-terminal kinase-like protein causes a recessive form of spinocerebellar neurodegeneration

Here, we show that the murine neurodegenerative disease mdf (autosomal recessive mouse mutant 'muscle deficient') is caused by a loss-of-function mutation in Scyl1, disrupting the expression of N-terminal kinase-like protein, an evolutionarily conserved putative component of the nucleocytoplasmic transport machinery. Scyl1 is prominently expressed in neurons, and enriched at central nervous system synapses and neuromuscular junctions. We show that the pathology of mdf comprises cerebellar atrophy, Purkinje cell loss and optic nerve atrophy, and therefore defines a new animal model for neurodegenerative diseases with cerebellar involvement in humans.     

Structural and functional analyses of methyl-lysine binding by the malignant brain tumour repeat protein Sex comb on midleg

Sex comb on midleg (Scm) is a member of the Polycomb group of proteins involved in the maintenance of repression of Hox and other developmental control genes in Drosophila. The two malignant brain tumour (MBT) repeats of Scm form a domain that preferentially binds to monomethylated lysine residues either as a free amino acid or in the context of peptides, while unmodified or di- or trimethylated lysine residues are bound with significantly lower affinity. The crystal structure of a monomethyl-lysine-containing histone tail peptide bound to the MBT repeat domain shows that the methyl-lysine side chain occupies a binding pocket in the second MBT repeat formed by three conserved aromatic residues and one aspartate. Insertion of the monomethylated side chain into this pocket seems to be the main contributor to the binding affinity. Functional analyses in Drosophila show that the MBT domain of Scm and its methyl-lysine-binding activity are required for repression of Hox genes.     

HCMV-infection in a human arterial organ culture model: effects on cell proliferation and neointimal hyperplasia

Background

The impact of infections with the human cytomegalovirus (HCMV) for the development of atherosclerosis and restenosis is still unclear. Both a clear correlation and no correlation at all have been reported in clinical, mostly serological studies. In our study we employed a human non-injury ex vivo organ culture model to investigate the effect of an in vitro permissive HCMV-infection on cell proliferation and neointimal hyperplasia for a period of 56 days.

Results

During routine-nephrectomies parts of renal arteries from 71 patients were obtained and prepared as human organ cultures. Cell free HCMV infection was performed with the fibroblast adapted HCMV strain AD169, the endotheliotropic strain TB40E, and a clinical isolate (AN 365). After 3, 7, 14, 21, 28, 35, and 56 days in culture staining of HCMV-antigens was carried out and reactive cell proliferation and neointimal thickening were analysed. Successful HCMV-infection was accomplished with all three virus strains studied. During the first 21 days in organ culture no cell proliferation or neointimal hyperplasia was detected. At day 35 and day 56 moderate cell proliferation and neointimal hyperplasia was found both in HCMV-infected segments and mock infected controls. Neointimal hyperplasia in productively HCMV-infected segments was lower than in non infected at day 35 and day 56, but relatively higher after infection with the endotheliotropic TB40E in comparison with the two other strains.

Conclusion

The data do not support the hypothesis that HCMV-infection triggers restenosis via a stimulatory effect on cell proliferation and neointimal hyperplasia in comparison to non infected controls. Interestingly however, even after lytic infection, a virus strain specific difference was observed.     

Response of cells on surface-induced nanopatterns: fibroblasts and mesenchymal progenitor cells

Ultrathin films of a poly(styrene)-block-poly(2-vinylpyrindine) diblock copolymer (PS-b-P2VP) and poly(styrene)-block-poly(4-vinylpyrindine) diblock copolymer (PS-b-P4VP) were used to form surface-induced nanopattern (SINPAT) on mica. Surface interaction controlled microphase separation led to the formation of chemically heterogeneous surface nanopatterns on dry ultrathin films. Two distinct nanopatterned surfaces, namely, wormlike and dotlike patterns, were used to investigate the influence of topography in the nanometer range on cell adhesion, proliferation, and migration. Atomic force microscopy was used to confirm that SINPAT was stable under cell culture conditions. Fibroblasts and mesenchymal progenitor cells were cultured on the nanopatterned surfaces. Phase contrast and confocal laser microscopy showed that fibroblasts and mesenchymal progenitor cells preferred the densely spaced wormlike patterns. Atomic force microscopy showed that the cells remodelled the extracellular matrix differently as they migrate over the two distinctly different nanopatterns.     

Selective enrichment of tryptophan-containing peptides from protein digests employing a reversible derivatization with malondialdehyde and solid-phase capture on hydrazide beads

A method for the selective enrichment of tryptophan-containing peptides from complex peptide mixtures such as protein digests is presented. It is based on the reversible reaction of tryptophan with malondialdehyde and trapping of the derivatized Trp-peptides on hydrazide beads via the free aldehyde group of the modified peptides. The peptides are subsequently recovered in their native form by specific cleavage reactions for further (mass spectrometric) analysis. The method was optimized and evaluated using a tryptic digest of a mixture of 10 model proteins, demonstrating a significant reduction in sample complexity while still allowing the identification of all proteins. The applicability of the tryptophan-specific enrichment procedure to complex biological samples is demonstrated for a total yeast cell lysate. Analysis of the processed fraction by 1D-LC-MS/MS confirms the specificity of the enrichment procedure, as more than 85% of the peptides recovered from the enrichment step contained tryptophan. The reduction in sample complexity also resulted in the identification of additional proteins in comparison to the untreated lysate.     

The microbial metabolic activity on carbohydrates and polymers impact the biodegradability of landfilled solid waste

Biodegradation - Biological waste degradation is the main driving factor for landfill emissions. In a 2-year laboratory experiment simulating different landfill in-situ aeration scenarios, the...     

Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure

Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, we analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. We show that Mst77F on its own causes severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F binds DNA via electrostatic interaction. This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm. Based on these findings we postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure.