Cystatins as calpain inhibitors: Engineered chicken cystatin- and stefin B-kininogen domain 2 hybrids support a cystatin-like mode of interaction with the catalytic subunit of μ-calpain

Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit μ- and m-calpain. In an attempt to elucidate the structural requirements of cystatins for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and Delta L110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefin B by the corresponding KD2 sequence resulted in a calpain inhibitor of K-i = 188 nM. Deletion of L110, which forms a beta -bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of mu -calpain 4- to 8-fold. All engineered cystatins were temporary inhibitors of calpain due to slow substrate-like cleavage of a single peptide bond corresponding to Gly9-Ala10 in chicken cystatin. Biomolecular interaction analysis revealed that, unlike calpastatin, the cystatin-type inhibitors do not bind to the calmodulin-like domain of the small subunit of calpain, and their interaction with the mu -calpain heterodimer is completely prevented by a synthetic peptide comprising subdomain B of calpastatin domain 1. Based on these results we propose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatin-like mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold

Reduction of myocardial infarction by postischemic administration of the calpain inhibitor A-705253 in comparison to the Na(+)/H(+) exchange inhibitor Cariporide (R) in isolated perfused rabbit hearts

The calpain inhibitor A-705253 and the Na(+)/H(+) exchange inhibitor Cariporide (R) were studied in isolated perfused rabbit hearts subjected to 60 min occlusion of the ramus interventricularis of the left coronary artery (below the origin of the first diagonal branch), followed by 120 min of reperfusion. The inhibitors were added to the perfusion fluid solely or in combination at the beginning of reperfusion. Hemodynamic monitoring and biochemical analysis of perfusion fluid from the coronary outflow were performed. Myocardial infarct size and area at risk (transiently not perfused myocardium) were determined from left ventricular slices after a special staining procedure with Evans blue and 2,3,5-triphenyltetrazolium chloride. The infarcted area (dead myocardium) was 72.7 +/- 4.0% of the area at risk in untreated controls, but was significantly smaller in the presence of the inhibitors. The largest effect was observed with 10(-6) M A-705253, which reduced the infarcted area to 49.2 +/- 4.1% of the area at risk, corresponding to a reduction of 33.6%. Cariporide (R) at 10(-6) M reduced the infarct size to the same extent. The combination of both inhibitors, however, did not further improve cardioprotection. No significant difference was observed between the experimental groups in coronary perfusion, left ventricular pressure, heart rate, or in the release of lactate dehydrogenase and creatine kinase from heart muscle

Tranexamic acid for spontaneous intracerebral hemorrhage: a randomized controlled pilot trial (ISRCTN50867461)

Background: Spontaneous intracerebral hemorrhage (ICH) can be devastating, particularly if hematoma expansion (HE) occurs. Tranexamic acid (TA), an antifibrinolytic drug, significantly reduced mortality in bleeding patients after trauma in the large CRASH-2 trial. The CRASH-2 ICH substudy found that TA nonsignificantly reduced mortality and dependency in traumatic ICH. The aim of this study was to assess the feasibility of performing a randomized controlled trial of tranexamic acid in spontaneous ICH, ahead of a definitive study. Methods: We performed a single-center, prospective, randomized (2:1), double-blind, placebo-controlled blinded endpoint trial of TA (intravenous 1 g bolus, 1 g infusion/8 h) in acute (<24 hours) spontaneous ICH. The primary objective was to test the feasibility of recruiting to the trial. Other objectives included tolerability (adverse events) and the effect of TA on HE and death and dependency. Results: The trial was feasible, with 24 patients enrolled (TA, n 5 16; placebo, n 5 8) between March 2011 and March 2012, and acceptable—only 3 patients declined to participate. All patients received the correct randomized treatment; 1 patient in the TA group did not complete the infusion because of neurologic deterioration. There were no significant differences in secondary outcomes including adverse events, HE, death, and dependency. One patient in the TA group had a deep vein thrombosis. Conclusions: This, the first randomized controlled trial of TAin ICH, found that the protocol could be delivered on schedule (2 patients/mo) and was feasible. Larger studies are needed to assess safety and efficacy of TA in ICH

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

Using a 3D virtual muscle model to link gene expression changes during myogenesis to protein spatial location in muscle

Background: Myogenesis is an ordered process whereby mononucleated muscle precursor cells (myoblasts) fuse into multinucleated myotubes that eventually differentiate into myofibres, involving substantial changes in gene expression and the organisation of structural components of the cells. To gain further insight into the orchestration of these structural changes we have overlaid the spatial organisation of the protein components of a muscle cell with their gene expression changes during differentiation using a new 3D visualisation tool: the Virtual Muscle 3D (VMus3D)

Gene Expression Profiling in Limb-Girdle Muscular Dystrophy 2A

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in calpain 3 (CAPN3). Calpain 3 plays different roles in muscular cells, but little is known about its functions or in vivo substrates. The aim of this study was to identify the genes showing an altered expression in LGMD2A patients and the possible pathways they are implicated in. Ten muscle samples from LGMD2A patients with in which molecular diagnosis was ascertained were investigated using array technology to analyze gene expression profiling as compared to ten normal muscle samples. Upregulated genes were mostly those related to extracellular matrix (different collagens), cell adhesion (fibronectin), muscle development (myosins and melusin) and signal transduction. It is therefore suggested that different proteins located or participating in the costameric region are implicated in processes regulated by calpain 3 during skeletal muscle development. Genes participating in the ubiquitin proteasome degradation pathway were found to be deregulated in LGMD2A patients, suggesting that regulation of this pathway may be under the control of calpain 3 activity. As frizzled-related protein (FRZB) is upregulated in LGMD2A muscle samples, it could be hypothesized that β-catenin regulation is also altered at the Wnt signaling pathway, leading to an incorrect myogenesis. Conversely, expression of most transcription factor genes was downregulated (MYC, FOS and EGR1). Finally, the upregulation of IL-32 and immunoglobulin genes may induce the eosinophil chemoattraction explaining the inflammatory findings observed in presymptomatic stages. The obtained results try to shed some light on identification of novel therapeutic targets for limb-girdle muscular dystrophies

Comprehensive Analysis of Transcript Start Sites in Ly49 Genes Reveals an Unexpected Relationship with Gene Function and a Lack Of Upstream Promoters

Comprehensive analysis of the transcription start sites of the Ly49 genes of C57BL/6 mice using the oligo-capping 5′-RACE technique revealed that the genes encoding the “missing self” inhibitory receptors, Ly49A, C, G, and I, were transcribed from multiple broad regions in exon 1, in the intron1/exon2 region, and upstream of exon -1b. Ly49E was also transcribed in this manner, and uniquely showed a transcriptional shift from exon1 to exon 2 when NK cells were activated in vitro with IL2. Remarkably, a large proportion of Ly49E transcripts was then initiated from downstream of the translational start codon. By contrast, the genes encoding Ly49B and Q in myeloid cells, the activating Ly49D and H receptors in NK cells, and Ly49F in activated T cells, were predominantly transcribed from a conserved site in a pyrimidine-rich region upstream of exon 1. An ∼200 bp fragment from upstream of the Ly49B start site displayed tissue-specific promoter activity in dendritic cell lines, but the corresponding upstream fragments from all other Ly49 genes lacked detectable tissue-specific promoter activity. In particular, none displayed any significant activity in a newly developed adult NK cell line that expressed multiple Ly49 receptors. Similarly, no promoter activity could be found in fragments upstream of intron1/exon2. Collectively, these findings reveal a previously unrecognized relationship between the pattern of transcription and the expression/function of Ly49 receptors, and indicate that transcription of the Ly49 genes expressed in lymphoid cells is achieved in a manner that does not require classical upstream promoters

Getting the feel of food structure with atomic force microscopy

This article describes the progress in the development of the atomic force microscope as an imaging tool and a force transducer, with particular reference to applications in food science. Use as an imaging tool has matured and emphasis is placed on the novel insights gained from the use of the technique to study food macromolecules and food colloids, and the subsequent applications of this new knowledge in food science. Use as a force transducer is still emerging and greater emphasis is given on the methodology and analysis. Where available, applications of force measurements between molecules or between larger colloidal particles are discussed, where they have led to new insights or solved problems related to food science. The future prospects of the technique in imaging or through force measurements are discussed

Characterisation and expression of calpain family members in relation to nutritional status, diet composition and flesh texture in gilthead sea bream (Sparus aurata).

Calpains are non-lysosomal calcium-activated neutral proteases involved in a wide range of cellular processes including muscle proteolysis linked to post-mortem flesh softening. The aims of this study were (a) to characterise several members of the calpain system in gilthead sea bream and (b) to examine their expression in relation to nutritional status and muscle tenderisation. We identified the complete open reading frame of gilthead sea bream calpains1-3, sacapn1, sacapn2, sacapn3, and two paralogs of the calpain small subunit1, sacapns1a and sacapns1b. Proteins showed 63-90% sequence identity compared with sequences from mammals and other teleost fishes, and the characteristic domain structure of vertebrate calpains. Transcripts of sacapn1, sacapn2, sacapns1a and sacapns1b had a wide tissue distribution, whereas sacapn3 was almost exclusively detected in skeletal muscle. Next, we assessed transcript expression in skeletal muscle following alteration of nutritional status by (a) fasting and re-feeding or (b) feeding four experimental diets with different carbohydrate-to-protein ratios. Fasting significantly reduced plasma glucose and increased free fatty acids and triglycerides, together with a significant increase in sacapns1b expression. Following 7 days of re-feeding, plasma parameters returned to fed values and sacapn1, sacapn2, sacapns1a and sacapns1b expression was significantly reduced. Furthermore, an increase in dietary carbohydrate content (11 to 39%) diminished growth but increased muscle texture, which showed a significant correlation with decreased sacapn1 and sacapns1a expression, whilst the other calpains remained unaffected. This study has demonstrated that calpain expression is modulated by nutritional status and diet composition in gilthead sea bream, and that the expression of several calpain members is correlated with muscle texture, indicating their potential use as molecular markers for flesh quality in aquaculture production