Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Somatic CTG•CAG repeat instability in a mouse model for myotonic dystrophy type 1 is associated with changes in cell nuclearity and DNA ploidy

Background  

Trinucleotide instability is a hallmark of degenerative neurological diseases like Huntington's disease, some forms of spinocerebellar ataxia and myotonic dystrophy type 1 (DM1). To investigate the effect of cell type and cell state on the behavior of the DM1 CTG•CAG repeat, we studied a knock-in mouse model for DM1 at different time points during ageing and followed how repeat fate in cells from liver and pancreas is associated with polyploidization and changes in nuclearity after the onset of terminal differentiation.     

Omeprazole inhibits proliferation and modulates autophagy in pancreatic cancer cells

Background

Omeprazole has recently been described as a modulator of tumour chemoresistance, although its underlying molecular mechanisms remain controversial. Since pancreatic tumours are highly chemoresistant, a logical step would be to investigate the pharmacodynamic, morphological and biochemical effects of omeprazole on pancreatic cancer cell lines.

Methodology/Principal Findings

Dose-effect curves of omeprazole, pantoprazole, gemcitabine, 5-fluorouracil and the combinations of omeprazole and 5-fluorouracil or gemcitabine were generated for the pancreatic cancer cell lines MiaPaCa-2, ASPC-1, Colo357, PancTu-1, Panc1 and Panc89. They revealed that omeprazole inhibited proliferation at probably non-toxic concentrations and reversed the hormesis phenomena of 5-fluorouracil. Electron microscopy showed that omeprazole led to accumulation of phagophores and early autophagosomes in ASPC-1 and MiaPaCa-2 cells. Signal changes indicating inhibited proliferation and programmed cell death were found by proton NMR spectroscopy of both cell lines when treated with omeprazole which was identified intracellularly. Omeprazole modulates the lysosomal transport pathway as shown by Western blot analysis of the expression of LAMP-1, Cathepsin-D and β-COP in lysosome- and Golgi complex containing cell fractions. Acridine orange staining revealed that the pump function of the vATPase was not specifically inhibited by omeprazole. Gene expression of the autophagy-related LC3 gene as well as of Bad, Mdr-1, Atg12 and the vATPase was analysed after treatment of cells with 5-fluorouracil and omeprazole and confirmed the above mentioned results.

Conclusions

We hypothesise that omeprazole interacts with the regulatory functions of the vATPase without inhibiting its pump function. A modulation of the lysosomal transport pathway and autophagy is caused in pancreatic cancer cells leading to programmed cell death. This may circumvent common resistance mechanisms of pancreatic cancer. Since omeprazole use has already been established in clinical practice these results could lead to new clinical applications.     

SOME ELECTRON MICROSCOPICAL OBSERVATIONS ON LIQUID-CRYSTALLINE PHASES IN LIPID-WATER SYSTEMS

OsO₄ fixation preserves some liquid-crystalline phases of soaps and phospholipids to an extent that it is possible to observe their structure in electron micrographs of thin sections. Good agreement exists between the structure observed directly and that deduced from x-ray diffraction studies of the same systems.     

Untersuchungen zum cytochemischen Glykogennachweis

Zusammenfassung Es wurden vier Pikrinsäuregemische an elf verschiedenen Objekten auf ihre Eignung als topochemische Glykogenfixierer geprüft. Die Fixationslösungen wurden jeweils bei +20° sowie bei — 7° benutzt. Die Ergebnisse wurden mit den Resultaten der Carnoyfixierung bei — 7° verglichen.Die wäßrigen Lösungen (Bouin, Bouin-Allen) haben sich als unbrauchbar bzw. als stark unterlegen erwiesen. Die alkoholischen Pikrinsäuregemische (Gendre, Rossman) sind im Hinblick auf quantitative Ausbeute und Lokalisationstreue wesentlich bessere Fixierer als das Carnoysche Gemische. Die Glykogenkonservierung wird durch Kaltfixation günstig beeinflußt. An einigen embryonalen Objekten konnte eine voll befriedigende Glykogenerhaltung nicht erzielt werden.

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Summary Four mixtures based on picric acid have been tested against eleven distinct histological objects with respect to their reliability for the topochemical fixation of glycogen. The solutions were used at 20° C and — 7° C respectively. The results were compared with those of Carnoy fixation at — 7° C.The aqueous solutions (Bouin, Bouin-Allen) proved inadequate, or at least inferior. The alcoholic picric acid mixtures (Gendre, Rossman) are essentially better fixatives than Carnoys fluid, with regard to the quantitative results and to the precision of localization. The preservation of glycogen is favourably influenced by cold fixation. In some embryonic objects a fully satisfactory preservation of glycogen could not be obtained by these methods.

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Mit 8 Textabbildungen

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

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Frau Prof. Dr.Carla Zawisch (Graz) zum 70. Geburtstag gewidmet.