The calpastatin-derived calpain inhibitor CP1B reduces mRNA expression of matrix metalloproteinase-2 and-9 and invasion by leukemic THP-1 cells

The ubiquitous proteases μ- and m-calpain are Ca2+-dependent cysteine endopeptidases. Besides involvement in a variety of physio(patho)logical processes, recent studies suggest a pivotal role of calpains in differentiation of hematopoietic cells and tumor cell invasion. However, the precise actions of calpains and their endogenous inhibitor, calpastatin, in these processes are only partially understood. Here we have studied the role of the calpain/calpastatin system in the invasion of leukemic cells under basal and differentiationstimulating conditions. To further differentiate the human leukaemic cell line THP-1 (monocytic), the cells were treated for 24 hours with the differentiationstimulating reagents phorbol 12-myristate 13-acetate (PMA) and dimethyl sulfoxide (DMSO). Macrophage and granulocytelike differentiation was confirmed by induction of vimentin expression as well as by microscopic and fluorescence assisted cytometric analysis. Extracellular matrix (ECM) invasion of both the basal and differentiation stimulated cells in a Matrigel assay was inhibited by preincubation of the cells with the specific calpain inhibitor CP1B for 24 hours. Inhibition of invasiveness correlated with decreased mRNA expression and secretion of the matrix metalloproteinases MMP-2 and MMP-9. In contrast, addition of CP1B only during the invasion process did neither influence transmigration nor MMP release. This is the first report showing that the calpain/calpastatin system mediates MMPmRNA expression of the leukemic THP-1 cells and as a consequence their invasiveness

Human μ-calpain: Simple isolation from erythrocytes and characterization of autolysis fragments

Heterodimeric μ-calpain, consisting of the large (80 kDa) and the small (30 kDa) subunit, was isolated and purified from human erythrocytes by a highly reproducible four-step purification procedure. Obtained material is more than 95% pure and has a specific activity of 6 - 7 mU/mg. Presence of contaminating proteins could not be detected by HPLC and sequence analysis. During storage at -80 °C the enzyme remains fully activatable by Ca²⁺, although the small subunit is partially processed to a 22 kDa fragment. This novel autolysis product of the small subunit starts with the sequence (60)RILG and is further processed to the known 18 kDa fragment. Active forms and typical transient and stable autolysis products of the large subunit were identified by protein sequencing. In casein-zymograms only the activatable forms 80 kDa+30 kDa, 80 kDa+22 kDa and 80 kDa+18 kDa displayed caseinolysis

Competitive nationalism:state, class, and the forms of capital in devolved Scotland

Devolved government in Scotland actively reconstitutes the unequal conditions of social class reproduction. Recognition of state-led class reconstitution draws upon the social theory of Bourdieu. Our analysis of social class in devolved Scotland revisits theories that examine the state as a `power container'. A range of state-enabling powers regulate the legal, economic, social, and cultural containers of class relations as specific forms of what Bourdieu called economic, social, and cultural `capital'. The preconditions of class reproduction are structured in direct ways by the Scottish state as a wealth container but also, more indirectly, as a cultural container and a social container. Competitive nationalism in the devolved Scottish state enacts neoliberal policies as a class- specific worldview but, at the same time, discursively frames society as a panclass national fraternity in terms of distinctive Scottish values of welfare nationalism. Nationalism is able to express this ambiguity in symbolic ways in which the partisan language of social class cannot

Does a gluten-free diet improve quality of life and sleep in patients with non-coeliac gluten/wheat sensitivity?

Introduction: The role of a gluten-free diet (GFD) in Non-Coeliac Gluten/Wheat Sensitivity (NCGWS) is unclear. We present the largest study comparing adherence to a GFD in patients with Coeliac Disease (CD) and NCGWS and assess its impact on quality of life (QoL) and sleep in patients with NCGWS. Methods: Patients with NCGWS at a tertiary centre completed the Coeliac Disease Adherence Test (CDAT), Coeliac Symptom Index (CSI) and Sleep Condition Indicator (SCI). Higher CDAT scores indicate worse adherence, higher CSI scores indicate poorer QoL, and higher SCI scores indicate better sleep. CDAT scores were correlated with CSI and SCI scores. A second group of patients with CD completed the CDAT questionnaire only. Results were compared with the CDAT responses from the NCGWS group. Results: For the NCGWS cohort (n = 125), the median CDAT score was 17/35, indicating poor adherence. The median CSI score was 44/80, with 40% of scores associated with a poor QoL. The median SCI score was 14/32, and DSM-V criteria for insomnia was met by 42% of patients. There was a positive correlation between CSI and CDAT scores (r = 0.59, p < 0.0001) and a negative correlation between SCI and CDAT scores (r = −0.37, p = 0.0002). In the CD cohort (n = 170), the median CDAT score was 13/35. Patients with NCGWS had poorer adherence compared to CD (CDAT: 17.0 vs. 13.0, respectively, p = 0.0001). Conclusion: Patients with NCGWS adhere to a GFD less than those with CD. Poorer adherence to a GFD in patients with NCGWS correlates with a worse QoL and sleep performance

Calpain Cleavage Prediction Using Multiple Kernel Learning

Calpain, an intracellular -dependent cysteine protease, is known to play a role in a wide range of metabolic pathways through limited proteolysis of its substrates. However, only a limited number of these substrates are currently known, with the exact mechanism of substrate recognition and cleavage by calpain still largely unknown. While previous research has successfully applied standard machine-learning algorithms to accurately predict substrate cleavage by other similar types of proteases, their approach does not extend well to calpain, possibly due to its particular mode of proteolytic action and limited amount of experimental data. Through the use of Multiple Kernel Learning, a recent extension to the classic Support Vector Machine framework, we were able to train complex models based on rich, heterogeneous feature sets, leading to significantly improved prediction quality (6% over highest AUC score produced by state-of-the-art methods). In addition to producing a stronger machine-learning model for the prediction of calpain cleavage, we were able to highlight the importance and role of each feature of substrate sequences in defining specificity: primary sequence, secondary structure and solvent accessibility. Most notably, we showed there existed significant specificity differences across calpain sub-types, despite previous assumption to the contrary. Prediction accuracy was further successfully validated using, as an unbiased test set, mutated sequences of calpastatin (endogenous inhibitor of calpain) modified to no longer block calpain's proteolytic action. An online implementation of our prediction tool is available at http://calpain.org