Centromere fragmentation is a common mitotic defect of S and G2 checkpoint override

DNA damaging agents, including those used in the clinic, activate cell cycle checkpoints, which blocks entry into mitosis. Given that checkpoint override results in cell death via mitotic catastrophe, inhibitors of the DNA damage checkpoint are actively being pursued as chemosensitization agents. Here we explored the effects of gemcitabine in combination with Chk1 inhibitors in a panel of pancreatic cancer cell lines and found variable abilities to override the S phase checkpoint. In cells that were able to enter mitosis, the chromatin was extensively fragmented, as assessed by metaphase spreads and Comet assay. Notably, electron microscopy and high-resolution light microscopy showed that the kinetochores and centromeres appeared to be detached from the chromatin mass, in a manner reminiscent of mitosis with unreplicated genomes (MUGs). Cell lines that were unable to override the S phase checkpoint were able to override a G₂ arrest induced by the alkylator MMS or the topoisomerase II inhibitors doxorubicin or etoposide. Interestingly, checkpoint override from the topoisomerase II inhibitors generated fragmented kinetochores (MUGs) due to unreplicated centromeres. Our studies show that kinetochore and centromere fragmentation is a defining feature of checkpoint override and suggests that loss of cell viability is due in part to acentric genomes. Furthermore, given the greater efficacy of forcing cells into premature mitosis from topoisomerase II-mediated arrest as compared with gemcitabine-mediated arrest, topoisomerase II inhibitors maybe more suitable when used in combination with checkpoint inhibitors.     

The action of fish peptide Orpotrin analogs on microcirculation

In order to investigate the relationship between the primary structure of Orpotrin, a vasoactive peptide previously isolated from the freshwater stingray Potamotrygon gr. orbignyi, and its microcirculatory effects, three Orpotrin analogs were synthesized. The analogs have a truncated N‐terminal with a His residue deletion and two substituted amino acid residues, where one Nle is substituted for one internal Lys residue and the third analog has a substitution of a Pro for an Ala (Orp‐desH¹, Orp‐Nle and Orp‐Pro/Ala, respectively). Only Orp‐desH¹ could induce a lower vasoconstriction effect compared with the natural Orpotrin, indicating that besides the N‐terminal, the positive charge of Lys and the Pro residues located at the center of the amino acid chain is crucial for this vasoconstriction effect. Importantly, the suggestions made with bioactive peptides were based on the molecular modeling and dynamics of peptides, the presence of key amino acids and shared activity in microcirculation, characterized by intravital microscopy. Moreover, this study has demonstrated that even subtle changes in the primary structure of Orpotrin alter the biological effects of this native peptide significantly, which could be of interest for biotechnological applications. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Contribution of cathepsin L to secretome composition and cleavage pattern of mouse embryonic fibroblasts

The endolysosomal cysteine endoprotease cathepsin L is secreted from cells in a variety of pathological conditions such as cancer and arthritis. We compared the secretome composition and extracellular proteolytic cleavage events in cell supernatants of cathepsin L-deficient and wild-type mouse embryonic fibroblasts (MEFs). Quantitative proteomic comparison of cell conditioned media indicated that cathepsin L deficiency affects, albeit in a limited manner, the abundances of extracellular matrix (ECM) components, signaling proteins, and further proteases as well as endogenous protease inhibitors. Immunodetection corroborated that cathepsin L deficiency results in decreased abundance of the ECM protein periostin and elevated abundance of matrix metalloprotease (MMP)-2. While mRNA levels of MMP-2 were not affected by cathepsin L ablation, periostin mRNA levels were reduced, potentially indicating a downstream effect. To characterize cathepsin L contribution to extracellular proteolysis, we performed terminal amine isotopic labeling of substrates (TAILS), an N-terminomic technique for the identification and quantification of native and proteolytically generated protein N-termini. TAILS identified >1500 protein N-termini. Cathepsin L deficiency predominantly reduced the magnitude of collagenous cleavage sites C-terminal to a proline residue. This contradicts cathepsin L active site specificity and indicates altered activity of further proteases as a result of cathepsin L ablation.     

Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines

Hepatitis C virus (HCV) is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model.     

Differential modulation of Beta-adrenergic receptor signaling by trace amine-associated receptor 1 agonists

Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes.     

Identification of bilateral changes in TID1 expression in the 6-OHDA rat model of Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra and the aggregation of α-synuclein into Lewy bodies. Existing therapies address motor dysfunction but do not halt progression of the disease. A still unresolved question is the biochemical pathway that modulates the outcome of protein misfolding and aggregation processes in PD. The molecular chaperone network plays an important defensive role against cellular protein misfolding and has been identified as protective in experimental models of protein misfolding diseases like PD. Molecular mechanisms underlying chaperone-neuroprotection are actively under investigation. Current evidence implicates a number of molecular chaperones in PD including Hsp25, Hsp70 and Hsp90, however their precise involvement in the neurodegenerative cascade is unresolved. The J protein family (DnaJ or Hsp40 protein family) has long been known to be important in protein conformational processes.We assessed sensory and motor function of control and PD rats and then evaluated the brain region-specific expression levels of select J proteins by Western analysis. Surprisingly, we observed a widespread 26 kDa breakdown product of the J protein, TID1, (tumorous imaginal discs, mtHsp40 or DnaJ3) in a 6-hydroxydopamine (6-OHDA) rat model of PD in which food handling, gait symmetry and sensory performance were impaired. Greater behavioral deficits were associated with lower TID1 expression. Furthermore, direct application of either 6-OHDA or MPP+ (1-methyl-4-phenylpyridinum) to CAD (CNS-derived catecholinaminergic neuronal cell line) cell cultures, reduced TID1 expression levels.Our results suggest that changes in cellular TID1 are a factor in the pathogenesis of PD by impeding functional and structural compensation and exaggerating neurodegenerative processes. In contrast, no changes were observed in CSPα, Hsp40, Hsp70, Hsc70 and PrP(C) levels and no activation of caspase3 was observed. This study links TID1 to PD and provides a new target for therapeutics that halts the PD progression.     

The Pseudomonas aeruginosa chemotaxis methyltransferase CheR1 impacts on bacterial surface sampling

The characterization of factors contributing to the formation and development of surface-associated bacterial communities known as biofilms has become an area of intense interest since biofilms have a major impact on human health, the environment and industry. Various studies have demonstrated that motility, including swimming, swarming and twitching, seems to play an important role in the surface colonization and establishment of structured biofilms. Thereby, the impact of chemotaxis on biofilm formation has been less intensively studied. Pseudomonas aeruginosa has a very complex chemosensory system with two Che systems implicated in flagella-mediated motility. In this study, we demonstrate that the chemotaxis protein CheR1 is a methyltransferase that binds S-adenosylmethionine and transfers a methyl group from this methyl donor to the chemoreceptor PctA, an activity which can be stimulated by the attractant serine but not by glutamine. We furthermore demonstrate that CheR1 does not only play a role in flagella-mediated chemotaxis but that its activity is essential for the formation and maintenance of bacterial biofilm structures. We propose a model in which motility and chemotaxis impact on initial attachment processes, dispersion and reattachment and increase the efficiency and frequency of surface sampling in P. aeruginosa.     

LOCAS--a low coverage assembly tool for resequencing projects

Motivation

Next Generation Sequencing (NGS) is a frequently applied approach to detect sequence variations between highly related genomes. Recent large-scale re-sequencing studies as the Human 1000 Genomes Project utilize NGS data of low coverage to afford sequencing of hundreds of individuals. Here, SNPs and micro-indels can be detected by applying an alignment-consensus approach. However, computational methods capable of discovering other variations such as novel insertions or highly diverged sequence from low coverage NGS data are still lacking.

Results

We present LOCAS, a new NGS assembler particularly designed for low coverage assembly of eukaryotic genomes using a mismatch sensitive overlap-layout-consensus approach. LOCAS assembles homologous regions in a homology-guided manner while it performs de novo assemblies of insertions and highly polymorphic target regions subsequently to an alignment-consensus approach. LOCAS has been evaluated in homology-guided assembly scenarios with low sequence coverage of Arabidopsis thaliana strains sequenced as part of the Arabidopsis 1001 Genomes Project. While assembling the same amount of long insertions as state-of-the-art NGS assemblers, LOCAS showed best results regarding contig size, error rate and runtime.

Conclusion

LOCAS produces excellent results for homology-guided assembly of eukaryotic genomes with short reads and low sequencing depth, and therefore appears to be the assembly tool of choice for the detection of novel sequence variations in this scenario.