Mycolactone-dependent depletion of endothelial cell thrombomodulin is strongly associated with fibrin deposition in Buruli ulcer lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin's substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells' ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone's effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tisischemia could contribute to the development of the tissue necrosis seen in BU lesions

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During one intensive week in October 2005, the authors were gathered to discuss the impact that globalisation has on the Organic Food Systems and the opportunities that globalisation opens up for developing these systems. The meeting took place as a Ph.D. course under the auspices of the Research School of Organic Farming and Food Systems (SOAR; www.soar.dk). All participants research within Organic Agriculture and Food Production in one way or another

The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature.

Endothelial cells actively maintain an anti-thrombotic environment; loss of this protective function may lead to thrombosis and systemic coagulopathy. The transcription factor ERG is essential to maintain endothelial homeostasis. Here, we show that inducible endothelial ERG deletion (ErgiEC-KO) in mice is associated with spontaneous thrombosis, hemorrhages and systemic coagulopathy. We find that ERG drives transcription of the anticoagulant thrombomodulin (TM), as shown by reporter assays and chromatin immunoprecipitation. TM expression is regulated by shear stress (SS) via Krüppel-like factor 2 (KLF2). In vitro, ERG regulates TM expression under low SS conditions, by facilitating KLF2 binding to the TM promoter. However, ERG is dispensable for TM expression in high SS conditions. In ErgiEC-KO mice, TM expression is decreased in liver and lung microvasculature exposed to low SS but not in blood vessels exposed to high SS. Our study identifies an endogenous, vascular bed-specific anticoagulant pathway in microvasculature exposed to low SS

DNA repair kinetics after low doses of X-rays. A comparison of results obtained by the unwinding and nucleoid sedimentation methods.

A re-examination of some of our previously published and our more recent data has led us to reconsider the interpretation of the kinetics of the repair of DNA single-strand breaks in mammalian cells. We investigated the detailed kinetics of this repair after low doses of X-rays in mouse Ehrlich ascites tumour cells grown in suspension cultures, using two different methods that measure strand breaks in DNA: the unwinding method and the 'nucleoid' sedimentation method. The results thus obtained, either under weak alkaline conditions (unwinding method) or under neutral conditions ('nucleoid' sedimentation method) both showed an initial fast repair component, with a half time, t( 1/2 ) of 5-6 min, followed by a component between 10 and 30 min after irradiation, in which the kinetic curves levelled off or turned upwards, indicating the possibility of the introduction of new breaks into the DNA. We propose that these additional DNA strand breaks may arise by incision of the DNA by endonucleases at base-damaged sites, and we therefore suggest that the previous interpretation of the kinetics of DNA single-strand break repair in terms of two or more first-order repair components may have been an approximation. The measured kinetics of strand break repair after X-irradiation probably represent a mixture of break ligation and incision, and therefore correspondingly deviate from first order, especially 10-30 min after exposure to X-rays

Fluorochromes and heterochromatin. A study of the chromosomes of Microtus agrestis L.

Fluorescent patterns of the chromosomes of the female Microtus agrestis, following binding to quinacrine mustard dihydrochloride or quinacrine dihydrochloride, have been studied. In the X chromosomes constitutive and facultative heterochromatin or euchromatin were found to exhibit different intensities of fluorescence. In addition, the two X chromosomes could be distinguished from each other by the different patterns of fluorescence. Almost all the 24 pairs of autosomes show characteristic fluorescent patterns that are consistent and reproducible and can be identified. The probable relationship between heterochromatin and differential binding of fluorochromes is discussed