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Among the primary threats to human health worldwide, nonsmall cell lung cancer (NSCLC) remains a significant factor and is a leading cause of cancer-related deaths. Due to subtle early symptoms, NSCLC patients are diagnosed at advanced stages, resulting in low survival rates. Herein, novel Au-Se bond nanoprobes (NPs) designed for the specific detection of Calpain-2 (CAPN2) and Human Neutrophil Elastase (HNE), pivotal biomarkers in NSCLC, were developed. The NPs demonstrated exceptional specificity and sensitivity toward CAPN2 and HNE, enabling dual-color fluorescence imaging to distinguish between NSCLC cells and normal lung cells effectively. The NPs' performance was consistent across a wide pH range (6.2 to 8.0), and it exhibited remarkable resistance to biological thiol interference, indicating its robustness in complex physiological environments. These findings suggest the nanoprobe is a promising tool for early NSCLC diagnosis, offering a novel approach for enhancing the accuracy of cancer detection.

This study investigated the effect of superchilled storage (-4 °C) on protein degradation and structural changes of beef steaks from M. longissimus lumborum compared with traditional chilling (2 °C) and frozen storage (-18 °C). Traditional chilling induced significantly greater degradation of troponin T and desmin, and more rapid loss of calpain activity, compared to superchilled or frozen storage treatments. The proteolysis of key myofibrillar proteins resulted in a sharp decline of WBSF values during traditional chilled storage. For frozen beef samples, no major changes were observed with respect to protein degradation or muscle structure during storage. However, superchilled samples exhibited wider gaps between muscle fibers at 12 weeks storage, associated with muscle fiber shrinkage., Competing Interests: Declaration of Competing Interest The authors declare that there are no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Calpain activation induces retinal ganglion cell (RGC) death, while calpain inhibition suppresses RGC death, in animal studies. However, the role of calpain in human retinal disease is unclear. This study investigated a new strategy to study the role of calpain based on real-time imaging. We synthesized a novel fluorescent probe for calpain, acetyl-l-leucyl-l-methionine-hydroxymethyl rhodamine green (Ac-LM-HMRG) and used it for real-time imaging of calpain activation. The toxicity of Ac-LM-HMRG was evaluated with a lactate dehydrogenase cytotoxicity assay, retinal sections, and electroretinograms. Here, we performed real-time imaging of calpain activation in a rat model. First, we administered N -methyl-d-aspartate (NMDA) to induce retinal injury. Twenty minutes later, we administered an intravitreal injection of Ac-LM-HMRG. Real-time imaging was then completed with a noninvasive confocal scanning laser ophthalmoscope. The inhibitory effect of SNJ-1945 against calpain activation was also examined with the same real-time imaging method. Ac-LM-HMRG had no toxic effects. The number of Ac-LM-HMRG-positive cells in real-time imaging significantly increased after NMDA injury, and SNJ-1945 significantly lowered the number of Ac-LM-HMRG-positive cells. Real-time imaging with Ac-LM-HMRG was able to quickly quantify the NMDA-induced activation of calpain and the inhibitory effect of SNJ-1945. This technique, used as a companion diagnostic system, may aid research into the development of new neuroprotective therapies.

Visualizing biomolecular and cellular processes inside intact living organisms is a major goal of chemical biology. However, existing molecular biosensors, based primarily on fluorescent emission, have limited utility in this context due to the scattering of light by tissue. In contrast, ultrasound can easily image deep tissue with high spatiotemporal resolution, but lacks the biosensors needed to connect its contrast to the activity of specific biomolecules such as enzymes. To overcome this limitation, we introduce the first genetically encodable acoustic biosensors-molecules that 'light up' in ultrasound imaging in response to protease activity. These biosensors are based on a unique class of air-filled protein nanostructures called gas vesicles, which we engineered to produce nonlinear ultrasound signals in response to the activity of three different protease enzymes. We demonstrate the ability of these biosensors to be imaged in vitro, inside engineered probiotic bacteria, and in vivo in the mouse gastrointestinal tract.

Meat tenderness is considered as the most important criterion for meat quality by consumers and can be improved by the actions of endogenous proteases, mainly calpains, during postmortem storage at 0-5°C. The purpose of this study, therefore, was to examine the postmortem calpain activation and proteolysis in breast (BM) and leg and thigh (LM) muscles of White Roman goose. BM and LM were taken from goose carcasses (n = 15) at 0 (10-15 min postmortem), 1, 3, and 7 days of storage at 5°C. The decrease in postmortem pH, calpain-1 and -11 activities, and contents of the calpain-1 80 kDa subunit and desmin was more rapid (p < .05) in BM than in LM. Our results show that postmortem proteolysis was more extensive in BM than in LM of White Roman goose, not only because the difference in fiber type composition between two muscles, but because the rate and extent of calpain activation were greater in BM as well. These results may provide useful information to optimize meat processing for different muscles in goose industry., (© 2020 Japanese Society of Animal Science.)

The objective of this study was to create various pH/temp decline rates in hot-boned bull beef M. longissimus lumborum (LL) through a combination of electrical stimulation (ES) and pre-rigor holding temperature. The relationship between the pre-rigor interventions, the activities of µ-calpain and small heat shock proteins (sHSP), and the impacts on meat product quality were determined. Paired LL loins from 13 bulls were hot-boned within 40 min of slaughter, immediately ES and subjected to various holding temperatures (5, 15, 25, and 35°C) for 3 hr. The rate of muscle pH decline, sarcomere length, shear force, and proteolysis of muscle proteins were measured. ES-25°C had a longer sarcomere length compared to non-electrical stimulation samples. ES-25°C and ES-35°C samples had lower shear force values, higher µ-calpain activity and higher desmin, troponin-T, and sHSP degradation. The above findings suggest that pH/temp decline rates created in hot-boned muscle impacted muscle protein proteolysis by increasing the activity of proteases and degradation of sHSP., (© 2019 Japanese Society of Animal Science.)

Calpain 1 is a proinflammatory calcium-activated cysteine protease, which can be partly externalized. Extracellular calpains limit inflammatory processes and promote tissue repair, through cell proliferation and migration. Toll like receptor (TLR) 2 has been identified as a target of extracellular calpains in lymphocytes. The aim was to investigate the externalization of calpain 1 and the release of soluble TLR2 during tumor progression of pulmonary lepidic predominant adenocarcinoma (LPA). Extracellular calpain 1, soluble fragment of TLR2 and cytokines were analyzed by ELISA in bronchoalveolar lavage fluid (BALF) supernatants from patients with LPA (n=68). Source of calpain was analyzed by immunohistochemistry and soluble TLR2 by flow cytometry on polymorphonuclear neutrophils (PMN) and human lung cancer cell lines. Extracellular calpain 1, secreted by tumor cells, was associated to tumor progression, neutrophilic inflammation, with a poor prognostic factor on survival (P=0.003). TLR2 was expressed on PMN and tumor cells and decreased after calpain exposure. Soluble fragment of TLR2 in BALF supernatants was correlated to the extracellular calpain 1 concentration (r=0.624; P<0.001), and its high level was associated with tumor progression and a pro-inflammatory environment. Extracellular calpain 1 secreted by tumor cells, could participate in inflammatory microenvironment and tumor progression through TLR2 in LPA., (Copyright © 2018 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

This study aimed to determine the effect of frozen-then-chilled storage on free Ca 2+ , proteolytic enzyme activity of calpains and the proteasome, water-holding capacity and shear force of porcine longissimus thoracis et lumborum muscle. Pork loins were subjected to either chilled storage at 2 ± 1 °C for 1, 2, 4, 6 and 9 days, or frozen-then chilled storage (-20 ± 1 °C for 1 week followed by thawing overnight). Free Ca 2+ increased with chilled storage in the non-frozen group. Frozen-then-chilled storage increased free Ca 2+ concentration, followed by a faster decrease of calpain-1 activity and activation of around 50% of calpain-2. Proteasome activity was reduced by around 40% following freezing-thawing. Purge loss increased and water-holding capacity of myofibrils decreased in the frozen-thawed group, suggesting considerable denaturation of myofibrillar proteins. Shear force was not affected by freezing-thawing, and we speculate that the tenderizing effect of calpain activation was counteracted by loss of proteasome activity and substantial exudate loss., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Filaggrin is central to the pathogenesis of atopic dermatitis (AD). The cheeks are a common initiation site of infantile AD. Regional and temporal expression of levels of filaggrin degradation products [natural moisturizing factors (NMFs)], activities of filaggrin-processing enzymes [bleomycin hydrolase (BH) and calpain-1 (C-1)] and plasmin, and corneocyte envelope (CE) maturity in early life are largely unknown., Objectives: We conducted a cross-sectional, observational study investigating regional and age-dependent variations in NMF levels, activity of proteases and CE maturity in stratum corneum (SC) from infants to determine whether these factors could explain the observed predilection sites for AD in early life., Methods: We measured NMF using a tape-stripping method at seven sites in the SC of 129 children (aged < 12 months to 72 months) and in three sites in 56 neonates and infants (< 48 h to 3 months). In 37 of these neonates and infants, corneocyte size, maturity, BH, C-1 and plasmin activities were determined., Results: NMF levels are low at birth and increase with age. Cheek SC, compared with elbow flexure and nasal tip, has the lowest NMF in the first year of life and is the slowest to reach stable levels. Cheek corneocytes remain immature. Plasmin, BH and C-1 activities are all elevated by 1 month of age in exposed cheek skin, but not in elbow skin., Conclusions: Regional and temporal differences in NMF levels, CE maturity and protease activities may explain the predilection for AD to affect the cheeks initially and are supportive of this site as key for allergen priming in early childhood. These observations will help design early intervention and treatment strategies for AD., (© 2018 The Authors British Journal of Dermatology published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.)

The objective of this study was to determine associations of small heat shock proteins (sHSPs) in tenderness development of loins from callipyge and normal genotype lambs. Loins (M. longissimus lumborum) from sixteen lambs across four genotypes were collected throughout 9 days of postmortem aging. The loins from callipyge lambs had more intact desmin and troponin T throughout aging periods, as well as less μ-calpain autolysis and more calpastatin compared to loins from other genotypes (P < 0.05). Delayed onset of apoptosis was found in the callipyge loins indicated by less cytochrome c and more inactive procaspase-3 compared to normal lamb loins (P < 0.05). Less degraded HSP27 was also consistently found in the callipyge loins compared with loins from normal lambs (P < 0.001). The results found up-regulation of anti-apoptotic activities coincided with toughness in callipyge loins, which suggest apoptosis is likely involved in postmortem proteolysis and subsequent meat tenderization., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Nuclear forkhead box protein P1 (N-FOXP1) expression in invasive breast cancer has been documented in the literature. However, the FOXP1 expression patterns at different stages of breast cancer progression are largely unknown, and the significance of cytoplasmic FOXP1 (C-FOXP1) expression in breast cancer has not been well illustrated. The aims of this study were to investigate FOXP1 expression patterns in invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS), atypical ductal hyperplasia (ADH) and usual ductal hyperplasia (UDH), and to analyze the clinicopathological relevance of C-FOXP1 and its prognostic value in IDC., Methods: N-FOXP1 and C-FOXP1 expression in cases of IDC, DCIS, ADH and UDH was determined using immunohistochemistry. The correlation between C-FOXP1 expression and clinicopathological parameters as well as the overall survival (OS) and disease-free survival (DFS) rates of patients with IDC were analyzed., Results: Exclusive N-FOXP1 expression was found in 85.0% (17/20), 40.0% (8/20), 12.2% (5/41) and 10.8% (9/83) of UDH, ADH, DCIS, and IDC cases, respectively, and exclusive C-FOXP1 expression was observed in 0% (0/20), 0% (0/20), 4.9% (2/41), and 31.3% (26/83) of the cases, respectively. Both N- and C-FOXP1 staining were observed in 15.0% (3/20), 60.0% (12/20), 82.9% (34/41) and 48.2% (40/83) of the above cases, respectively, while complete loss of FOXP1 expression was observed in only 9.6% (8/83) of IDC cases. Estrogen receptor (ER) expression in C-FOXP1-positive IDC cases (31/66, 47.0%) was significantly lower than that in C-FOXP1-negative cases (13/17, 76.5%) (p = 0.030). Calpain II expression was observed in 83.3% (55/66) of C-FOXP1-positive IDC cases, which was significantly higher than that in C-FOXP1-negative cases (9/17, 52.9%) (p = 0.007). Calpain II was significantly associated with pAKT (p = 0.029), pmTOR (p = 0.011), p4E-BP1 (p < 0.001) and p-p70S6K (p = 0.003) expression levels. The 10-year OS and DFS rates of the C-FOXP1-positive patients were 60.5% and 48.7%, respectively, both of which were lower than those of the C-FOXP1-negative patients (93.3, 75.3%). The OS curve showed a dramatic impact of C-FOXP1 status on OS (p = 0.045)., Conclusions: Cytoplasmic relocalization of FOXP1 protein was a frequent event in breast IDC. Calpain II might play an important role in nucleocytoplasmic trafficking of FOXP1 and the AKT pathway might be involved in this process. C-FOXP1 expression was inversely associated with ER expression and might be a predictor of poor OS in patients with IDC.

In the past few yr, an emerging muscle abnormality termed wooden breast (WB) was found to affect broilers' Pectoralis major muscles. Although different studies have been performed in order to evaluate the effect of WB on meat quality, there is no evidence concerning its impact on the proteolytic processes taking place during meat aging. Thus, this study aimed at investigating the effect of a 7-day storage of broiler breast fillets on free calcium concentration, calpain activity, and proteolysis. Both the superficial and the deep layers of the Pectoralis major muscles were considered. Although similar electrophoretic profiles were observed by comparing the corresponding sampling positions, an evident lack of a high-molecular weight protein band, ascribed to nebulin, was found in the superficial layer of the WB fillets at 10 h postmortem. Compared to normal fillets (NB), both the superficial and the deep layer of WB exhibited a significantly higher amount of free calcium at 168 h postmortem (96 and 88 vs. 20 and 53 μM; P ≤ 0.001). Casein zymograms evidenced the presence of μ/m-calpain and its autolyzed form migrating as a doublet within the gel. Interestingly, neither the occurrence of WB nor the intra-fillet sampling position exerted any relevant effect on calpain activity. Indeed, a significant reduction (P ≤ 0.05) in the unautolyzed μ/m-calpain activity coupled with a remarkable increase (P ≤ 0.05) in the autolyzed form activity was observed during storage. Concurrently, if compared to NB, a significantly larger (P ≤ 0.05) amount of desmin was detected in both the superficial and the deep layers of the WB samples at 10 h postmortem. Then, a sharp decrease of the intact desmin band coupled with a progressive accumulation of its 39-kDa degradation fragment was observed without any significant difference among groups. In conclusion, the increased hardness that typically affects the WB cases seemed not to be exclusively attributable to differences in the proteolytic processes taking place within the postmortem period.

The objective of this study was to investigate the role of calpain isotypes, especially poultry-specific μ/m-calpain in the proteolysis and meat quality changes of chicken breast muscle during postmortem storage. Calpain activity was detected by casein zymography, while the degradation of titin, desmin and Troponin-T was analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and western blot. Meat quality indicators such as water holding capacity and tenderness were also studied. The correlation analysis between calpain activity, proteolysis and the changes in meat quality indicators indicated that there were strong correlations for μ-calpain during the first 12 h of storage, while such strong correlations for μ/m-calpain were only found in samples stored from 12 h to 7 days. Our study suggested that μ-calpain played a major role in meat quality changes while μ/m-calpain could also be involved but played a limited role in the proteolysis and meat quality changes during 12 h to 7 days postmortem storage of chicken breast muscle., (© 2017 Japanese Society of Animal Science.)

Miltefosine is the only orally administrable drug for the treatment of leishmaniasis. But in recent years, a decline in its efficacy points toward the emergence of resistance to this drug. Knowledge of biomarkers for miltefosine resistance may be beneficial for proper selection of treatment regimen. Splenic aspirates were collected and parasites cultured from patients relapsed after initial cure ( N = 15) and successfully treated ( N = 15) with miltefosine. Differential expression of genes in miltefosine-resistant strains was examined by DNA microarray and validated by real-time reverse transcription polymerase chain reaction and Western blotting. Of 669 upregulated genes, the cysteine protease-like protein of calpain family (GenBank: CBZ34784) was found to be significantly overexpressed in resistant parasite strains and only anti-calpain antibodies showed its presence in the sera of relapse patients through Western blotting. Calpain family cysteine protease-like protein can be useful as a potential biomarker of miltefosine unresponsiveness.

Lowering dietary cation-anion differences (DCAD) can enhance responsiveness to Ca-homeostatic hormones and increase Ca availability, which might have potential to activate a Ca-dependent protease, calpain, and to enhance postmortem myofibrillar proteolysis. In this study, we investigated the effects of DCAD manipulation on calpain activity and beef tenderness in Japanese Black cattle which are characterized by their high marbling. Thirty-six Japanese Black steers were allotted to one of two treatments: (i) control (CON; DCAD +6.09 mEq/100 g of dry matter (DM)) or (ii) negative DCAD (NEGD; DCAD -8.27 mEq/100 g DM) for 70 days before slaughter. Lowering DCAD decreased DM and energy intake (P < 0.01) even though it did not negatively affect the growth performance or carcass characteristics. In NEGD, urine pH was decreased by acidification caused by the negative DCAD (P < 0.01). Calpain activities tended to be improved in NEGD (P = 0.09), but Warner-Bratzler shear force values were not affected by treatment. Although calpain activities tended to improve, lowering DCAD to -8.27 for 70 days before slaughter was insufficient to enhance beef tenderness in Japanese Black steers., (© 2017 Japanese Society of Animal Science.)

Impaired apoptosis is one of the hallmarks of cancer. Caspase-3 and -8 are key regulators of the apoptotic response and have been shown to interact with the calpain family, a group of cysteine proteases, during tumorigenesis. The current study sought to investigate the prognostic potential of caspase-3 and -8 in breast cancer, as well as the prognostic value of combinatorial caspase and calpain expression. A large cohort (n = 1902) of early stage invasive breast cancer patients was used to explore the expression of caspase-3 and -8. Protein expression was examined using standard immunohistochemistry on tissue microarrays. High caspase-3 expression, but not caspase-8, is significantly associated with adverse breast cancer-specific survival (P = 0.008 and P = 0.056, respectively). Multivariate analysis showed that caspase-3 remained an independent factor when confounding factors were included (hazard ratio (HR) 1.347, 95% confidence interval (CI) 1.086-1.670; P = 0.007). The analyses in individual subgroups demonstrated the significance of caspase-3 expression in clinical outcomes in receptor positive (ER, PR or HER2) subgroups (P = 0.001) and in non-basal like subgroup (P = 0.029). Calpain expression had been previously assessed. Significant association was also found between high caspase-3/high calpain-1 and breast cancer-specific survival in the total patient cohort (P = 0.005) and basal-like subgroup (P = 0.034), as indicated by Kaplan-Meier analysis. Caspase-3 expression is associated with adverse breast cancer-specific survival in breast cancer patients, and provides additional prognostic values in distinct phenotypes. Combinatorial caspase and calpain expression can predict worse prognosis, especially in basal-like phenotypes. The findings warrant further validation studies in independent multi-centre patient cohorts., Competing Interests: The authors have declared no conflicts of interest.

Ethnopharmacological Relevance: Tongxinluo (TXL) is a multifunctional traditional Chinese medicine and has been widely used in the treatment of cardiovascular and cerebrovascular diseases. Numerous studies demonstrate that TXL is a novel neuroprotective drug, however, the mechanisms are largely unknown., Aim of the Study: we aimed to demonstrate the protective effect of TXL on cerebral ischemia/reperfusion (I/R) injury and provide the evidence for the involvement of Connexin 43/Calpain II/ Bax/Caspase-3 pathway in TXL-mediated neuroprotection., Methods: Focal cerebral I/R injury were induced by transient middle cerebral artery occlusion (MCAO, for 90min) in adult male Sprague-Dawley rats. We estimated the effects of TXL on I/R injury including neurological deficit assessment and cerebral infarct volume measurement via TTC staining, and detected the protein expression of Connexin 43 (Cx43) by western blot. Furthermore, after the intracerebroventricular injection of carbenoxolone (CBX, the inhibitor of Cx43) at 30min before MCAO surgery, Calpain II, Bax and cleaved Caspased-3 immunoreactivity in ischemic penumbra region was detected by immunofluorescent staining, and cell apoptosis was detected by TUNEL staining., Results: TXL treatment greatly improved neurological deficit and reduced the infarction volume compared to MCAO with buffer treatment (P<0.05), and TXL pre-post treatment showed better results than TXL pre-treatment. TXL pre-post treatment significantly up-regulated Cx43 protein expression at 3d, 7d and 14d post-injury compared to MCAO with buffer treatment (P<0.05). Meanwhile, the immunoreactivity of Calpain II, Bax and cleaved Caspase-3 in ischemic penumbra region was obviously decreased by TXL pre-post treatment compared to MCAO group (P<0.05). However, with the treatment of the Cx43 inhibitor, CBX, the down-regulated effect of TXL on Calpain II, Bax and cleaved Caspase-3 immunoreactivity was abolished (P<0.05). Moreover, the protective effect of TXL against neuron apoptosis in penumbra region was conteracted by CBX (P<0.05)., Conclusions: TXL could effectively protect against I/R injury and reduced cell death via Cx43/Calpain II/Bax/Caspase-3 pathway, which contribute to I/R injury prevention and therapy., (Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.)

Hornerin (HRNR) shares numerous features with filaggrin, a key contributor to the epidermal barrier functions. The two proteins display a related structural organization, are expressed by the granular keratinocytes as a large precursor processed by proteolysis, and are cross-linked to the cornified cell envelopes. Two main steps in the metabolism of filaggrin are its deimination and calpain-1 cleavage. Here, using ion-exchange chromatography and two-dimensional gel electrophoresis of human epidermis extracts, we determined that HRNR is deiminated in vivo. Accordingly, cornified envelopes, purified from plantar and abdominal human skin, were shown to contain deiminated proteins. A recombinant form of HRNR (HRNR His ) deiminated in vitro was shown to be a better substrate for transglutaminases 1 and 3 than the unmodified form. Our data also indicated that calpain-1 may be involved in the proteolytic processing of HRNR, because calpain-1 was co-located with HRNR in the cytoplasm of granular keratinocytes. Using Western blotting and mass spectrometry analysis, HRNR His was shown to be cleaved by calpain-1 in vitro, its deimination enhancing its proteolysis. In HRNR full sequence, four calpain-1 cleavage sites were identified. Altogether, these data allowed a new role to be deciphered for deimination during cornification and provided further characterization of HRNR metabolism., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Casein zymography has become one of the gold standard assays for monitoring mammalian calcium-activated proteases (calpains) in purified enzyme, cell, or tissue samples. This calpain zymography method takes advantages of (1) casein is an excellent substrate for major isoforms of calpains (Calpain-1, 2 and 3), (2) the embedded casein is digested into small peptides where the calpain bands are located, thus creating a clear zone upon Commassie blue gel staining, and (3) the calpain isoforms have different gel mobility under native gel conditions. Casein zymography is also useful in studying reversibility of inhibitor binding to calpains.

The effect of S-nitrosylation on the autolysis and catalytic ability of μ-calpain in vitro in the presence of 50μM Ca(2 +) was investigated. μ-Calpain was incubated with different concentrations of nitric oxide donor S-nitrosoglutathione (GSNO) and subsequently reacted with purified myofibrils. Results showed that the amount of 80kDa μ-calpain subunit significantly decreased as GSNO increased from 0 to 300μM, but increases of GSNO to 300, 500 and 1000μM did not result in further inhibition. The catalytic ability of nitrosylated μ-calpain to degrade titin, nebulin, troponin-T and desmin was significantly reduced when the GSNO concentration was higher than 300μM. The cysteine residues of μ-calpain at positions 49, 351, 384, and 592 in the catalytic subunit and at 142 in small subunit were S-nitrosylated, which could be responsible for decreased μ-calpain activity. Thus, S-nitrosylation can negatively regulate the activation of μ-calpain resulting in decreased proteolytic ability on myofibrils., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: To study the effect of leptin on the expression of calcium-activated neutral protease 1 (calpain-1) and B cell lymphoma-2 (Bcl-2) and apoptosis in the myocardial tissue of neonatal rats after asphyxia., Methods: A total of 48 neonatal rats were randomly and equally divided into normal control group, asphyxia group, leptin treatment groups, and calpain-1 inhibitor (CAI-1) group. The neonatal rat model of asphyxia under normal atmospheric condition was established in all groups except the control group. For the leptin treatment groups, rats received 20, 80, and 160 μg/kg leptin by intraperitoneal injection immediately after model establishment, respectively. For the CAI-1 group, rats received 10 mg/kg CAI-1 by intraperitoneal injection immediately after model establishment. For all the groups, the myocardial tissue was collected at 2 hours after model establishment. Immunohistochemistry was used to measure the expression of calpain-1 and Bcl-2. The TUNEL method was used to evaluate apoptosis of myocardial cells., Results: The expression of calpain-1 and Bcl-2 and apoptosis index (AI) were significantly higher in the asphyxia group than in the normal control group (P˂0.05). The leptin treatment groups and the CAI-1 group had significantly lower expression of calpain-1, significantly lower AI, and significantly higher expression of Bcl-2 than the asphyxia group (P˂0.05). The CAI-1 group had the largest changes in all the indices compared with the asphyxia group. However, there were no significant differences in all indices between the 160 μg/kg leptin treatment group and the CAI-1 group. After asphyxia, the expression of calpain-1 was positively correlated with AI, while the expression of Bcl-2 was negatively correlated with AI and the expression of calpain-1 (P˂0.05)., Conclusions: Leptin reduces apoptosis of myocardial cells in asphyxiated neonatal rats by the inhibition of calpain-1 activation and upregulation of Bcl-2 expression.

Aneurysms of the ascending aorta are an outstanding challenge to clinicians as they may persist asymptomatic until they present with dissection or rupture. Intensive research is performed to reveal the molecular mechanisms causing aneurysm formation. Calpains are ubiquitous non-lysosomal cysteine proteases which are classically activated by calcium signaling. The two major forms of the calpain-family are calpain-I and calpain-II. Calpastatin specifically inhibits the proteolytic activity of calpain-I and -II. Recently it has been demonstrated in aneurysm tissues from ascending aortas obtained from Marfan syndrome patients that calpain-II expression is increased and calpastatin expression is decreased. Thus, we were interested in the probable role of calpains in aneurysms of ascending aorta in non-Marfan patients. Therefore, ascending aortic samples of dilated and non-dilated aortas were analyzed according to their calpain-I, -II and calpastatin content as well as the expression levels of MMPs and elastin as well as the infiltration of inflammatory cells. We have found significant differences in calpain-I and calpastatin protein expression and serum levels in patients with aneurysm of the ascending aorta. Furthermore, MMP-1 and MMP-3 expression levels correlate with calpain-I protein levels. Due to our findings we conclude that calpain-1 seems to be related to fibrotic alteration in aortic aneurysm tissue in our experimental group. The change in calpain-1 modulates the structure of aortic tissue causing alteration in elastin structure, thus enabling macrophage infiltration and elevation of MMP levels. Circulating levels of calpain-1 may be used as a prognostic marker in the future if further correlation analyses are done.

The activities of calpain and caspase systems during ageing in Longissimus lumborum (LL) and Infraspinatus (IS) muscles of Italian Simmental young bulls (Bos taurus) were assessed. Samples from 10 animals were collected within 20 min of exsanguination (T0), after 48 h (T1) and 7 days (T2) post mortem. Calpain and caspase activity were evaluated based on the formation of αII spectrin cleavage products of 145 kDa (SBDP145) and 120 kDa (SBDP120), respectively. Caspase activity was also assessed by the presence of poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1) cleavage product. At T0, LL showed higher levels of SBDP145 than IS (P < 0.01), while SBDP120 and PARP-1 degradation products were similar between muscles. At T1, no difference was found in the level of SBDP145 between muscles, while SBDP120 and PARP-1 cleavage products were not detected. At T2 neither αII spectrin nor PARP-1 cleavage products were found. LL and IS showed different proteolysis after slaughter that was influenced more by calpain than caspase activity, which was detectable only in the early post mortem period., (© 2015 Japanese Society of Animal Science.)

The objective of this study was to examine the differences in calpain system, desmin degradation, pH values and water holding capacity (WHC) between muscles of commercial Meishan and Duroc × Landrace × Yorkshire crossbred pigs. Meishan pork presented better WHC evidenced by lower purge loss at days 1 and 3 and less centrifugation loss at day 1 post mortem (P < 0.05). pH values at 45 min post mortem in Meishan pork were significantly higher compared to crossbred pork (P < 0.05). Calpain-1 messenger RNA (mRNA) expression was lower in Meishan pork compared to that from crossbred pork (P < 0.05). Additionally, calpain-1 activity, the ratio of calpain-1 to calpastatin activity and desmin degradation were lower in Meishan pork compared to those from crossbred pork samples (P < 0.05). The results indicate that the calpain system including mRNA expression and activity were different between commercial Meishan and crossbred pork resulting in difference in the degree of desmin degradation during post mortem aging. pH values at 45 min and 24 h post mortem rather than calpain activity and desmin degradation could explain the higher water holding capacity in commercial Meishan pork., (© 2015 Japanese Society of Animal Science.)

Calpain, calcineurin (CaN), and nuclear factor of activated T cell (NFAT) play a key role in the development of atrial fibrillation. Patients with valvular heart disease (VHD) are prone to develop atrial fibrillation (AF). Thus, our current study was aimed at investigating whether activation of calpain-CaN-NFAT pathway is associated with the incidence of AF in the patients with VHD and diabetes. The expressions of calpain 2 and alpha- and beta-isoforms of CaN catalytic subunit (CnA) as well as NFAT-c3 and NFAT-c4 were quantified by quantitative reverse transcription-polymerase chain reaction in atrial tissues from 77 hospitalized patients with VHD and diabetes. The relevant protein content was measured by Western blot and calpain 2 in human atrium was localized by immunohistochemistry. We found that the expressions of calpain 2, CnA alpha and CnA beta, and NFAT-c3 but not NFAT-c4 were significantly elevated in the samples from patients with AF compared to those with sinus rhythm (SR). Elevated protein levels of calpain 2 and CnA were observed in patients with AF, and so was the enhanced localization of calpain 2. We thereby concluded that CaN together with its upstream molecule, calpain 2, and its downstream effector, NFAT-c3, might contribute to the development of AF in patients with VHD and diabetes.

Calpain-3 is an intracellular cysteine protease, belonging to Calpain superfamily and predominantly expressed in skeletal muscle. In human melanoma cell lines and biopsies, we previously identified two novel splicing variants (hMp78 and hMp84) of Calpain-3 gene (CAPN3), which have a significant lower expression in vertical growth phase melanomas and, even lower, in metastases, compared to benign nevi. In the present study, in order to investigate the pathophysiological role played by the longer Calpain-3 variant, hMp84, in melanoma cells, we over-expressed it in A375 and HT-144 cells. In A375 cells, the enforced expression of hMp84 induces p53 stabilization, and modulates the expression of a few p53- and oxidative stress-related genes. Consistently, hMp84 increases the intracellular production of ROS (Reactive Oxygen Species), which lead to oxidative modification of phospholipids (formation of F2-isoprostanes) and DNA damage. Such events culminate in an adverse cell fate, as indicated by the decrease of cell proliferation and by cell death. To a different extent, either the antioxidant N-acetyl-cysteine or the p53 inhibitor, Pifithrin-α, recover cell viability and decrease ROS formation. Similarly to A375 cells, hMp84 over-expression causes inhibition of cell proliferation, cell death, and increase of both ROS levels and F2-isoprostanes also in HT-144 cells. However, in these cells no p53 accumulation occurs. In both cell lines, no significant change of cell proliferation and cell damage is observed in cells over-expressing the mutant hMp84C42S devoid of its enzymatic activity, suggesting that the catalytic activity of hMp84 is required for its detrimental effects. Since a more aggressive phenotype is expected to benefit from down-regulation of mechanisms impairing cell growth and survival, we envisage that Calpain-3 down-regulation can be regarded as a novel mechanism contributing to melanoma progression.

In the intraerythrocytic trophozoite stages of Plasmodium falciparum, the calcium-dependent cysteine protease calpain (Pf-calpain) has an important role in the parasite calcium modulation and cell development. We established specific conditions to follow by confocal microscopy and spectrofluorimetry measurements the intracellular activity of Pf-calpain in live cells. The catalytic activity was measured using the fluorogenic Z-Phe-Arg-MCA (where Z is carbobenzoxy and MCA is 4-methylcoumaryl-7-amide). The calmodulin inhibitor calmidazolium and the sarcoplasmic reticulum calcium ATPase inhibitor thapsigargin were used for modifications in the cytosolic calcium concentrations that persisted in the absence of extracellular calcium. The observed calcium-dependent peptidase activity was greatly inhibited by specific cysteine protease inhibitor E-64 and by the selective calpain inhibitor ALLN (N-acetyl-l-leucyl-l-leucyl-l-norleucinal). Taken together, we observed that intracellular Pf-calpain can be selectively detected and is the main calcium-dependent protease in the intraerythrocytic stages of the parasite. The method described here can be helpful in cell metabolism studies and antimalarial drug screening., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In the membrane fraction of mouse parotid gland (PG), the protein level of aquaporin 5 (AQP5), a member of the water channel family, was increased by injection (ip) of isoproterenol (IPR), a β-adrenergic agonist, at 1 h, and stayed at high levels until 6 h; this change occurred simultaneously as amylase secretion. The AQP5 level then decreased and returned toward the original level at 12-48 h. After IPR injection, the AQP5 mRNA gradually increased and reached a maximum at 24 h. The facts suggest a rapid appearance of AQP5 at plasma membrane by IPR and subsequent degradation/metabolism by activation of proteolytic systems. Pretreatment of animals with two calpain inhibitors, N-Ac-Leu-Leu-methininal (ALLM) and calpeptin, as well as a protein synthesis inhibitor, cycloheximide (CHX), significantly suppressed the IPR-induced AQP5 degradation in the PG membrane fraction; such suppression was not observed by two proteasome inhibitors, MG132 and lactacystin, or the lysosome denaturant chloroquine, although most of these inhibitors increased AQP5 protein levels in unstimulated mice. The AQP5 protein was also degraded by μ-calpain in vitro. Furthermore, we demonstrated that μ-calpain was colocalized with AQP5 in the acinar cells by immunohistochemistry, and its activity in the PG was increased at 6 h after IPR injection. These results suggest that the calpain system was responsible for IPR-induced AQP5 degradation in the parotid gland and that such a system was coupled to the secretory-restoration cycle of amylase in the PG.

This experiment was conducted to determine the influence of low voltage electrical stimulation (ES) on the tenderness development of beef round muscles. Eight steers were slaughtered, and ES applied to one side of each carcass within 90 min of exsanguination. Steaks from M. longissimus dorsi, semimembranosus, adductor, and gracilis were vacuum packaged and aged at 4 °C for 9 d. Star probe, sensory evaluation, Western blot assays of troponin-T and μ-calpain autolysis and 2D-DIGE were conducted. ES resulted in accelerated (P<0.05) pH decline of the longissimus in the first 24h postmortem. ES did not influence (P>0.05) proteolysis and tenderness, but did alter the predominance of metabolic proteins in the soluble fraction of muscle. Aging for 9 d improved tenderness (P<0.05). The data confirmed that low voltage ES at 90 min of exsanguination had no effect on proteolysis and tenderness development in the longissimus dorsi, semimembranosus, adductor or gracilis in beef., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

High-pressure processing (HPP) is a technology of growing interest for food preservation, due to its ability to control the activity of degradative enzymes. The effect of three variables (pressure levels of 100, 250, and 400 MPa; pressure holding times of 0, 5, 15, and 30 min; and pressurization rates of 8 and 14 MPa/s) on the activity of the enzymes acid phosphatase, cathepsins (B and D), lipase, and calpains was studied using sea bass fillets as a case study model. Additionally, the effect of HPP on sarcoplasmic proteins was studied by SDS-PAGE and isoelectric focusing electrophoreses. The increase in pressure level and holding time decreased the protein concentration in sarcoplasmic extracts, and also the activity of calpains. As compared to nontreated samples, acid phosphatase activity was lower at 400 MPa, and for cathepsin D lower activities were observed at 100 and 400 MPa. The increase in pressurization rate increased the activity of cathepsin D, lipase, and calpains, although it was not always significant. In contrast, cathepsin B and lipase activities were less affected by HPP treatments. Electrophoresis separation of sarcoplasmic proteins showed that the intensity of many protein bands changed mainly due to pressure level and holding time. The results of this study suggest that HPP causes lysosomes disruption and also denaturation, aggregation, and fragmentation of sarcoplasmic proteins, and this evidence might be related to the decrease in enzymes activity especially at 400 MPa. In conclusion, HPP can be a potential tool to control the activity of degradative enzymes, which might prevent the softening of sea bass muscle due to autolytic reactions.

The application of high pressure offers some interesting opportunities in the processing of muscle-based food products. It is well known that high-pressure processing can prolong the shelf life of meat products in addition to chilling but the pressure-labile nature of protein systems limits the commercial range of applications. High pressure can affect the texture and gel-forming properties of myofibrillar proteins and, hence, has been suggested as a physical and additive-free alternative to tenderize and soften or restructure meat and fish products. However, the rate and magnitude at which pressure and temperature effects take place in muscles are variable and depend on a number of circumstances and conditions that are still not precisely known. This review provides an overview of the current knowledge of the effects of high pressure on muscle tissue over a range of temperatures as it relates to meat texture, microstructure, color, enzymes, lipid oxidation, and pressure-induced gelation of myofibrillar proteins.

Background: Pancreatic cancer, including cancer of the ampulla of Vater and bile duct, is very aggressive and has a poor five year survival rate; improved methods of patient stratification are required., Methods: We assessed the expression of calpain-1, calpain-2 and calpastatin in two patient cohorts using immunohistochemistry on tissue microarrays. The first cohort was composed of 68 pancreatic adenocarcinomas and the second cohort was composed of 120 cancers of the bile duct and ampulla., Results: In bile duct and ampullary carcinomas an association was observed between cytoplasmic calpastatin expression and patient age (P = 0.036), and between nuclear calpastatin expression and increased tumour stage (P = 0.026) and the presence of vascular invasion (P = 0.043). In pancreatic cancer, high calpain-2 expression was significantly associated with improved overall survival (P = 0.036), which remained significant in multivariate Cox-regression analysis (hazard ratio = 0.342; 95% confidence interva l = 0.157-0.741; P = 0.007). In cancers of the bile duct and ampulla, low cytoplasmic expression of calpastatin was significantly associated with poor overall survival (P = 0.012), which remained significant in multivariate Cox-regression analysis (hazard ratio = 0.595; 95% confidence interval = 0.365-0.968; P = 0.037)., Conclusion: The results suggest that calpain-2 and calpastatin expression is important in pancreatic cancers, influencing disease progression. The findings of this study warrant a larger follow-up study.

The septal and temporal poles of the hippocampus differ markedly in their anatomical organization, but whether these distinct regions exhibit differential neurochemical profiles underlying lead (Pb(2+)) neurotoxicity remains to be determined. In the present study, we examined changes in the expression of Ca(2+)/calmodulin-dependent enzymes, including calpain, calcineurin, phospho-CaMKII (Thr286) and neuronal nitric oxide synthase (nNOS), in the rat dorsal and ventral hippocampus (DH and VH) after acute Pb(2+) exposure. Five days after Pb(2+) exposure, we observed constitutively active forms of calcineurin (45 kDa and 48 kDa) in ventral portions of the hippocampus, a result consistent with the observed calpain activation that is indicated by the breakdown of spectrin in this region. Our data demonstrate that nNOS expression is significantly higher in the ventral region of the hippocampus when compared to the dorsal region, whereas phosphorylation of CaMKII (Thr286) is less pronounced in the ventral portion of the hippocampus and more pronounced in dorsal regions after acute Pb(2+) exposure. Thus, it appears likely that the ventral region of hippocampus is more vulnerable to the neurotoxic effects of Pb(2+) than the dorsal region. Taken together, the present data suggest that acute lead exposure leads to differential expression patterns of Ca(2+)/calmodulin-dependent enzymes along the dorsoventral axis of the hippocampus., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Proteolysis affects every protein at some point in its life cycle. Many biomarkers of disease or cancer are stable proteolytic fragments in biological fluids. There is great interest and a challenge in proteolytically modified protein study to identify physiologic protease-substrate relationships and find potential biomarkers. In this study, two human hepatocellular carcinoma (HCC) cell lines with different metastasis potential, MHCC97L, and HCCLM6, were researched with a high-throughput and sensitive PROTOMAP platform. In total 391 proteins were found to be proteolytically processed and many of them were cleaved into persistent fragments instead of completely degraded. Fragments related to 161 proteins had different expressions in these two cell lines. Through analyzing these significantly changed fragments with bio-informatic tools, several bio-functions such as tumor cell migration and anti-apoptosis were enriched. A proteolysis network was also built up, of which the CAPN2 centered subnetwork, including SPTBN1, ATP5B, and VIM, was more active in highly metastatic HCC cell line. Interestingly, proteolytic modifications of CD44 and FN1 were found to affect their secretion. This work suggests that proteolysis plays an important role in human HCC metastasis., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Limb girdle muscular dystrophy (LGMD) type 2A is caused by mutations in the CAPN3 gene and complete lack of functional calpain 3 leads to the most severe muscle wasting. Calpain 3 is suggested to be involved in maturation of contractile elements after muscle degeneration. The aim of this study was to investigate how mutations in the four functional domains of calpain 3 affect muscle regeneration., Methods: We studied muscle regeneration in 22 patients with LGMD2A with calpain 3 deficiency, in five patients with LGMD2I, with a secondary reduction in calpain 3, and in five patients with Becker muscular dystrophy (BMD) with normal calpain 3 levels. Regeneration was assessed by using the developmental markers neonatal myosin heavy chain (nMHC), vimentin, MyoD and myogenin and counting internally nucleated fibers., Results: We found that the recent regeneration as determined by the number of nMHC/vimentin-positive fibers was greatly diminished in severely affected LGMD2A patients compared to similarly affected patients with LGMD2I and BMD. Whorled fibers, a sign of aberrant regeneration, was highly elevated in patients with a complete lack of calpain 3 compared to patients with residual calpain 3. Regeneration is not affected by location of the mutation in the CAPN3 gene., Conclusions: Our findings suggest that calpain 3 is needed for the regenerative process probably during sarcomere remodeling as the complete lack of functional calpain 3 leads to the most severe phenotypes.

Messenger RNA (mRNA) expression of calpain-1 (µ-calpain), -2 (m-calpain), -3 (p94), small subunit (calpain-4; 28 kDa), and three types of calpastatin (CSTN) isoform were investigated for 10 skeletal muscles of Holstein cattle by real-time and/or semi-quantitative reverse transcription polymerase chain reaction. Noticeably, effect of muscle type was observed on 28 kDa expression (P < 0.001) with a tendency of higher 28 kDa expression in myosin heavy chain (MyHC)-2x-rich muscles compared to MyHC-slow-rich muscles. The CSTN-I and -III expression in Longissimus thoracis (LT) showed the lowest value among the muscles tested. Moreover, 28 kDa/CSTN-I ratio was higher in the diaphragm (DP), psoas major (PM), and LT than those in the lingual muscles (TN), masseter (MS) and pectoralis (PP) (P < 0.05). Calpain-1/CSTN I, calpain-2/CSTN I in LT and PM were higher than that in TN (P < 0.05). Calpain-3/CSTN-I and -III in LT and/or PM showed higher values than that in TN (P < 0.05). These results indicated that the calpain and CSTN expressions are regulated by muscle type, suggesting especially by muscle fiber type. Calpains/CSTN-I ratios, especially 28 kDa/CSTN-I, may account for higher extent of post mortem proteolysis previously observed in LT and PM muscles., (© 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.)

Thymidylate synthase (TS) plays a major role in the response to 5-fluorouracil (5-FU) by binding directly to the 5-FU metabolite, 5-fluoro-dUMP (FdUMP). The change in the TS expression levels after 5-FU administration was examined in parallel to 5-FU responsiveness in six human gastric adenocarcinoma cell lines to elucidate the source of variability of 5-FU sensitivity. MKN-1, SH-10-TC and MKN-74 cells were more resistant to 5-FU than MKN-28, KATO III and MKN-45 cells. Western blotting analysis revealed that the 5-FU sensitivity of these cells did not correlate with the basal TS expression levels but did correlate with rapid detection of the TS-FdUMP complex after exposure to 5-FU. In 5-FU-resistant cells, very low levels of the TS-FdUMP complex early after 5-FU exposure were elevated by pretreatment with calpain inhibitors such as benzyloxycarbonyl-leucyl-leucinal (ZLLH), benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLH) and ALLN, but not by other protease inhibitors. In contrast, ONO-3403, which causes calpain activation, stimulated downregulation of the TS-FdUMP complex in 5-FU-sensitive cells. The expression levels of calpastatin, an endogenous calpain inhibitor, were higher in 5-FU-sensitive cells than in 5-FU-resistant cells. ZLLH increased the 5-FU sensitivity of 5-FU-resistant cells, whereas ONO-3403 decreased the sensitivity of 5-FU-sensitive cells. In addition, knockdown of m-calpain by siRNA increased the 5-FU sensitivity in 5-FU-resistant cells, while knockdown of calpastatin reduced the sensitivity in 5-FU-sensitive cells. These results suggest that calpain might reduce the chemosensitivity of human gastric cancer cells to 5-FU possibly by rapid degradation of the TS-FdUMP complex, a finding that is considered to have novel therapeutic implications., (© 2011 Japanese Cancer Association.)

Highly toxic organophosphorus (OP) nerve agents, sarin and soman act by inhibiting acetylcholinesterase (AChE) function at neuronal synapses and cause many toxic effects including death within minutes. The effect of nerve agents on protein oxidation, calpain, and cytoskeletal protein levels was not well known. In the present study we investigated these parameters after subcutaneous injection of sarin (120 μg/kg) and soman (80 μg/kg) in the rat brain. Results indicate that several rat brain proteins were intensely oxidized after nerve agent poisoning. Immunoreactivity levels of μ-calpain were significantly elevated in cerebral cortex and cerebellum regions of rat brain from 2.5 h to 30 days. Alpha tubulin levels reduced from 1 to 7 days in the supernatant and 1 to 3 days in the pellet fractions of cerebellum and cerebral cortex, where as phosphorylation of high molecular weight neurofilament (pNF-H) was increased significantly in nerve agent intoxicated rat brains as compared to control rats. AChE activity was inhibited up to 3 days after nerve agent exposure in plasma and brain. Results suggest that altered protein oxidation, calpain and cytoskeletal protein levels are due to multiple mechanisms of nerve agents actions and these changes might be involved in nerve agent induced complex neurotoxicity., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objective: Controlled mechanical ventilation results in diaphragmatic dysfunction, and oxidative stress has been shown to be an important contributor to ventilator-induced diaphragm dysfunction. We hypothesized that the administration of an antioxidant, N-acetylcysteine, would restore the redox balance in the diaphragm and prevent against the deleterious effects of controlled mechanical ventilation., Design: Randomized, controlled experiment., Settings: Basic science animal laboratory., Subjects: Male Wistar rats, 14 wks old., Interventions: Anesthetized rats were submitted for 24 hrs to either spontaneous breathing receiving 150 mg/kg N-acetylcysteine (SBNAC) or saline (SBSAL) or to controlled mechanical ventilation receiving 150 mg/kg N-acetylcysteine (MVNAC) or saline (MVSAL)., Measurements and Main Results: After 24 hrs of controlled mechanical ventilation, diaphragmatic force production was significantly lower in MVSAL compared with all groups. Importantly, administration of N-acetylcysteine completely abolished this controlled mechanical ventilation-induced diaphragmatic contractile dysfunction. Diaphragmatic protein oxidation was significantly increased after 24 hrs of controlled mechanical ventilation (+53%, p < .01) in MVSAL animals, whereas administration of N-acetylcysteine prevented this controlled mechanical ventilation-induced oxidative stress. Diaphragmatic 20S proteasome activity was increased in MVSAL (+62%, p < .05). Further, compared with SBSAL, diaphragm caspase-3 activity was significantly increased in MVSAL (+279%, p < .001), and N-acetylcysteine treatment provided partial protection against caspase-3 activation. Diaphragmatic calpain activity was significantly increased after controlled mechanical ventilation (+137%, p < .001) in MVSAL animals, but N-acetylcysteine treatment protected against this event. Finally, significant negative correlations existed between calpain activity and diaphragm force production (r from -0.56 to -0.49, p < .05)., Conclusions: These data show that the administration of N-acetylcysteine protects the diaphragm from the deleterious effects of controlled mechanical ventilation. Specifically, N-acetylcysteine prevents against controlled mechanical ventilation-induced diaphragmatic oxidative stress and proteolysis and abolishes controlled mechanical ventilation-induced diaphragmatic contractile dysfunction.

Rationale: Diaphragmatic function is a major determinant of the ability to successfully wean patients from mechanical ventilation (MV). Paradoxically, MV itself results in a rapid loss of diaphragmatic strength in animals. However, very little is known about the time course or mechanistic basis for such a phenomenon in humans., Objectives: To determine in a prospective fashion the time course for development of diaphragmatic weakness during MV; and the relationship between MV duration and diaphragmatic injury or atrophy, and the status of candidate cellular pathways implicated in these phenomena., Methods: Airway occlusion pressure (TwPtr) generated by the diaphragm during phrenic nerve stimulation was measured in short-term (0.5 h; n = 6) and long-term (>5 d; n = 6) MV groups. Diaphragmatic biopsies obtained during thoracic surgery (MV for 2-3 h; n = 10) and from brain-dead organ donors (MV for 24-249 h; n = 15) were analyzed for ultrastructural injury, atrophy, and expression of proteolysis-related proteins (ubiquitin, nuclear factor-κB, and calpains)., Measurements and Main Results: TwPtr decreased progressively during MV, with a mean reduction of 32 ± 6% after 6 days. Longer periods of MV were associated with significantly greater ultrastructural fiber injury (26.2 ± 4.8 vs. 4.7 ± 0.6% area), decreased cross-sectional area of muscle fibers (1,904 ± 220 vs. 3,100 ± 329 μm²), an increase of ubiquitinated proteins (+19%), higher expression of p65 nuclear factor-κB (+77%), and greater levels of the calcium-activated proteases calpain-1, -2, and -3 (+104%, +432%, and +266%, respectively) in the diaphragm., Conclusions: Diaphragmatic weakness, injury, and atrophy occur rapidly in critically ill patients during MV, and are significantly correlated with the duration of ventilator support.

Calpains, also called calcium activated neutral proteases (CANP), are expressed ubiquitously. They are intracellular, non-lysosomal cytoplasmic cysteine endopeptidases. Calcium is required for their activation. Their endogenous specific inhibitor is calpastatin, which is expressed ubiquitously and coexists within cells besides calpain. When calcium is present, calpastatin and calpain attach to each other inhibiting the protease. The calpain system plays an important role in many processes including apoptosis, necrosis, ischemia formation and exocytosis. So far, many reports exist on studies about the influence of calpains in different tumors (skin, breast, renal cell and prostate cancers). The role of calpains in pathogenesis or further tumor progression has always been proved in related studies, but their exact function could not be demonstrated. So far, no studies on calpains being involved in the pathogenesis of ovarian cancer have been published. In our study we focused on the expression of the enzymes calpain 1, calpain 2 and their inhibitor calpastatin in normal and malign ovarian tissue. Therefore, we performed immunohistochemical stainings of paraffin slices and evaluated staining intensity (SI), percentage of positive cells (PP) and immunoreactive score (IRS). We evaluated the correlation between enzyme expression in malign and benign ovarian tissues. In malignant ovarian tissue, we found decreased expression, staining intensity and immunoreactive score of calpastatin. With higher grading of the ovarian carcinoma, staining intensity and immunoreactive score of calpain 1 decreased. Staining intensity of calpain 2 in ovarian carcinoma decreased with increasing lymph node status. We clearly demonstrated differences between enzyme expressions in malign and benign tissue. This study could not find any specific function of calpains. Only few studies in the literature have been found that deal with calpain evaluation of ovarian cancer. Additional studies including more patients are required to elucidate the functional role and impact of calpain in tumors in detail.

The present study investigated the effects of eccentric muscle contractions (ECC) on the content of myofibrillar proteins (my-proteins) and the catalytic activity of myofibrillar ATPase (my-ATPase) in skeletal muscles. Rat extensor digitorum longus and tibialis anterior muscles were exposed to 200-repeated ECC or isometric contractions (ISC) and used for measures of force output and for biochemical analyses, respectively. Whereas in ISC-treated muscles, full restoration of tetanic force was attained after 2 days of recovery, force developed by ECC-treated muscles remained depressed (P < 0.05) after 6 days. The total my-protein content and the relative content of myosin heavy chain (MHC) in total my-proteins were unaltered during 4 days of recovery after ECC, but fell (P < 0.05) to 55.9 and 63.4% after 6 days of recovery, respectively. my-ATPase activity expressed on a my-protein weight basis was unaltered immediately after ECC. However, it decreased (P < 0.05) to 75.3, 45.3, and 49.3% after 2, 4 and 6 days of recovery, respectively. Total maximal calpain activity measured at 5 mM Ca(2+) was significantly augmented (P < 0.05) after 2 days of recovery, reaching a level of threefold higher after 6 days. These alterations were specific for ECC and not observed for ISC. These results suggest that depressions in my-ATPase activity contribute to ECC-induced decreases in force and power which can take a number of days to recover.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a genetic disease that is caused by mutations in the calpain 3 gene (CAPN3), which encodes the skeletal muscle-specific calpain, calpain 3 (also known as p94). However, the precise mechanism by which p94 functions in the pathogenesis of this disease remains unclear. Here, using p94 knockin mice (termed herein p94KI mice) in which endogenous p94 was replaced with a proteolytically inactive but structurally intact p94:C129S mutant protein, we have demonstrated that stretch-dependent p94 distribution in sarcomeres plays a crucial role in the pathogenesis of LGMD2A. The p94KI mice developed a progressive muscular dystrophy, which was exacerbated by exercise. The exercise-induced muscle degeneration in p94KI mice was associated with an inefficient redistribution of p94:C129S in stretched sarcomeres. Furthermore, the p94KI mice showed impaired adaptation to physical stress, which was accompanied by compromised upregulation of muscle ankyrin-repeat protein-2 and hsp upon exercise. These findings indicate that the stretch-induced dynamic redistribution of p94 is dependent on its protease activity and essential to protect muscle from degeneration, particularly under conditions of physical stress. Furthermore, our data provide direct evidence that loss of p94 protease activity can result in LGMD2A and molecular insight into how this could occur.

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. In this video, calpain activity in fixed and living murine 32 Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32 Dkit cells are shown to have elevated activity compared to normal splenocytes.

Background: Calpain 3 (Capn3), also named p94, is a skeletal muscle tissue-specific protein known to be responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Recent experimental studies have hypothesized a pro-apoptotic role of Capn3 in some melanoma cell lines. So far the link between calpain3 and tumors comes from in vitro studies. The objective of this study was to describe Capn3 activation in naturally occurring urothelial tumors of the urinary bladder in cattle., Methods and Findings: Here we describe, for the first time in veterinary and comparative oncology, the activation of Capn3 in twelve urothelial tumor cells of the urinary bladder of cattle. Capn3 protein was initially identified with nanoscale liquid chromatography coupled with tandem mass spectrometry (nano LC-MS/MS) in a co-immunoprecipitation experiment on E2F3, known to be a transcription factor playing a crucial role in bladder carcinogenesis in humans. Capn3 expression was then confirmed by reverse transcription polymerase chain reaction (RT-PCR). Finally, the Ca(2+)-dependent proteolytic activity of Capn3 was assayed following ion exchange chromatography. Morphologically, Capn3 expression was documented by immunohistochemical methods. In fact numerous tumor cells showed an intracytoplasmic immunoreactivity, which was more rarely evident also at nuclear level. In urothelial tumors, bovine papillomavirus type 2 (BPV-2) DNA was amplified by PCR and the expression of E5 protein, the major oncogenic protein of BVP-2, was detected by western blotting, immunohistochemistry, and immunofluorescence. E2F3 overexpression and pRb protein downregulation were shown by western blotting., Conclusion: The role of capn3 protein in urothelial cancer of the urinary bladder remains to be elucidated: further studies would be required to determine the precise function of this protease in tumor development and progression. However, we suggest that activated Capn3 may be involved in molecular pathways leading to the overexpression of E2F3, which in turn could be responsible for urothelial tumor cell proliferation also in cattle, though other mechanisms are likely to exist. If further studies corroborate the important role of Capn3 in urothelial tumors of the urinary bladder, cattle with urinary tumors may prove useful as animal model for bladder carcinogenesis.

Pf-calpain, a cysteine protease of Plasmodium falciparum, is believed to be one of the central mediators for essential parasitic activity. However, the roles of calpain on parasitic activity have not been determined in P. falciparum. In the present study, the localization of Pf-calpain was investigated using polyclonal antibodies (anti-Pf-calpain antibody A and B) against peptides that distinguished it from human calpain-7 and rat calpain-10 protein. Recombinant Pf-calpain (rPf-calpain) was identified as a 46 kDa protein using an anti-Pf-calpain antibody A, which can recognize the Pf-calpain binding site. Confocal microscopy revealed calpain within cytoplasmic localized parasites in the erythrocytic cycle. The findings suggested that the expression of Pf-calpain would be proportional to all different parasites in the erythrocytic cycle. On the other hand, anti-human calpain-7 antibody detected Pf-calpain in schizonts, and the immunofluorescence was stronger than with anti-rat calpain-10 antibody. However, the antibodies reacted with calpains in human red blood cells. These results show that anti-Pf-calpain antibody A and B specifically recognize only Pf-calpain. Taken together, the results suggest that Pf-calpain is expressed in all erythrocytic stages. In particular, the expression of Pf-calpain is increased much more when the late ring matures into the early trophozoite. Moreover, anti-Pf-calpain antibody A and B against synthetic peptides of the catalytic domain of Pf-calpain are useful to specifically detect Pf-calpain in all erythrocytic stages, while human and rat calpain antibody are not useful., (Copyright 2009 Elsevier Inc. All rights reserved.)

Neural progenitor cells play an essential role in both the developing embryonic nervous system and in the adult brain, where the capacity for self-renewal would be important for normal brain functions. In the present study, we used embryonic cortical neural progenitor cells to investigate the effects of trimethyltin chloride (TMT) on the survival of neural progenitor cells. In cultures of cortical neural progenitor cells, the formation of round neurospheres was observed in the presence of epidermal growth factor and basic fibroblast growth factor within 9 days in vitro. The neurospheres were then harvested for subsequent replating and culturing for assessment of cell viability in either the presence or absence of TMT at the concentration of 5microM. Lasting exposure to TMT produced not only nuclear condensation in the cells in a time-dependent manner over a period of 6-24h, but also the release of lactate dehydrogenase into the culture medium. Immunoblot and immunocytochemical analyses revealed that TMT had the ability to activate both caspase-3 and calpain, as well as to cause nuclear translocation of deoxyribonuclease II, which is located within cytoplasm in intact cells. Additionally, treatment with a calpain inhibitor [trans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane] and a caspase inhibitor [Z-Val-Ala-Asp(OMe)-CH2F] produced a significant reduction in damaged cells induced by TMT. Taken together, our data indicate that neural progenitor cells are highly susceptible to TMT in undergoing cell death via the activation of 2 parallel pathways, ones involving calpain and the other, caspase-3.

Inclusion body myositis (IBM) is an inflammatory disease of skeletal muscle of unknown cause. To further understand the nature of the tissue injury in this disease, we developed methods for large-scale detection and quantitation of proteins in muscle biopsy samples and analyzed proteomic data produced by these methods together with histochemical, immunohistochemical, and microarray data. Twenty muscle biopsy samples from patients with inflammatory myopathies (n = 17) or elderly subjects without neuromuscular disease (n = 3) were profiled by proteomic studies using liquid chromatographic separation of peptides followed by mass spectrometry. Thirteen of the diseased samples additionally underwent microarray studies. Seventy muscle specimens from patients with a range of neuromuscular disorders were examined by ATPase histochemical methods. Smaller numbers of samples underwent immunohistochemical and immunoblot studies. Mass spectrometric studies identified and quantified approximately 300 total distinct proteins in each muscle sample. In IBM and to a lesser extent in polymyositis, proteomic studies confirmed by histochemical, immunohistochemical, and immunoblot studies showed loss of many fast-twitch specific structural proteins and glycolytic enzymes despite relative preservation of transcript levels. Increased abundance of a nuclear membrane protein, immunoglobulins, and two calpain-3 substrates were present. The atrophy present in IBM muscle is accompanied by preferential loss of fast-twitch structural proteins and glycolytic enzymes, particularly glycogen debranching enzyme, with relative preservation of the abundance of their respective transcripts. Although muscle atrophy has long been recognized in IBM, these studies are the first to report specific proteins which are reduced in quantity in IBM muscle.

Lanthanide-based luminescent sensors are noteworthy because their long-lifetime luminescence enables time-resolved fluorescence measurements. After exploring suitable antenna groups, we designed and synthesized lanthanide-based luminogenic sensors detecting protease activities. This sensor yielded strong luminescence on addition of proteases such as calpain I and leucine aminopeptidase (LAP). Since the luminescence lifetimes of the probes were very long, the sensors could be applied to time-resolved measurements that exclude background fluorescence signals derived from proteins or other impurities. This sensing principle could be applicable to general time-resolved fluorescence assays for other proteases.

The role of the unique plant calpain Defective Kernel 1 (DEK1) in development has remained unclear due to the severity of mutant phenotypes. Here, we used complementation studies of the embryo-lethal mutant to dissect DEK1 protein behavior and to show that DEK1 plays a key role in growth regulation in Arabidopsis thaliana. We show that although full-length DEK1 protein localizes to membranes, it undergoes intramolecular autolytic cleavage events that release the calpain domain into the cytoplasm. The active calpain domain alone is not only necessary for DEK1 function but is sufficient for full complementation of dek1 mutants. A novel set of phenotypes, including leaf ruffling, increased leaf thickness, and abnormalities of epidermal cell interdigitation, was caused by expression of the constitutively active calpain domain. This analysis of the novel phenotypes produced by DEK1 under- and overexpression, as well as DEK1 subcellular localization and protein processing, has revealed a fundamental role for DEK1-mediated signaling in growth regulation.