Protein C Mutation (A267T) Results in ER Retention and Unfolded Protein Response Activation

BACKGROUND: Protein C (PC) deficiency is associated with a high risk of venous thrombosis. Recently, we identified the PC-A267T mutation in a patient with PC deficiency and revealed by in vitro studies decreased intracellular and secreted levels of the mutant. The aim of the present study was to characterize the underlying mechanism(s). METHODOLOGY/PRINCIPAL FINDINGS: CHO-K1 cells stably expressing the wild-type (PC-wt) or the PC mutant were generated. In order to examine whether the PC mutant was subjected to increased intracellular degradation, the cells were treated with several inhibitors of various degradation pathways and pulse-chase experiments were performed. Protein-chaperone complexes were analyzed by treating the cells with a cross-linker followed by Western blotting (WB). Expression levels of the immunoglobulin-binding protein (BiP) and the phosphorylated eukaryotic initiation factor 2α (P-eIF2α), both common ER stress markers, were determined by WB to examine if the mutation induced ER stress and unfolded protein response (UPR) activation. We found no major differences in the intracellular degradation between the PC variants. The PC mutant was retained in the endoplasmic reticulum (ER) and had increased association with the Grp-94 and calreticulin chaperones. Retention of the PC-A267T in ER resulted in UPR activation demonstrated by increased expression levels of the ER stress markers BiP and P-eIF2α and caused also increased apoptotic activity in CHO-K1 cells as evidenced by elevated levels of DNA fragmentation. CONCLUSIONS/SIGNIFICANCE: The reduced intracellular level and impaired secretion of the PC mutant were due to retention in ER. In contrast to other PC mutations, retention of the PC-A267T in ER resulted in minor increased proteasomal degradation, rather it induced ER stress, UPR activation and apoptosis

An Analysis of the Ireviken event in the Boree Creek Formation, New South Wales, Australia

Conodont data from a section through the Boree Creek Formation in New South Wales, the best sequence in Australia extending through the Llandovery–Wenlock boundary, represent strata of the Ireviken Event. A significant number of conodont datum planes within the sequence indicating step-wise extinctions can be recognised and correlated with the sequence through the Visby Beds, in Gotland, Sweden, the most comprehensively analysed Ireviken Event sequence globally. This intercontinental correlation, involving both north and south hemispheres, adds to the data set concerning cyclicity in Silurian marine strata and postulated models concerning causality.16 page(s

Facies development across the Late Silurian Lau Event based on temperate carbonates of the Prague Basin (Czech Republic)

The facies development through the stratigraphical interval of the Silurian (late Ludlow) Lau Event and the associated major δ13C record excursion have been studied in the Prague Basin. The investigated sections consist of temperate-water carbonates (Kopanina Formation) that were deposited in shallow as well as deeper parts of the basin. In both areas, the facies evolution reflects a major shallowing of sea-level during the early part of the event, which resulted in weathering (karstification) and erosion in the shallower parts of the basin, leading to stratigraphic gaps there. Paleokarst features and dolomitization are associated with these strata in the vicinity of Prague, where the δ13C record displays a strong diagenetic overprint and palynomorphs are very poorly preserved. A transition from deep-water shale to subtidal, thick-bedded limestone facies in the deeper parts of the basin, close to Kosov, signals the same sea-level drop, indicating that it was basin-wide. Detailed lithologic descriptions for the studied sections are provided and the development in the Prague Basin is compared with the coeval succession on Gotland, Sweden, which was located in the tropical realm at this times