M-calpain Activity Research Articles

Type I and III collagen contents and μ-calpain autolysis as a function of dry ageing time for eight different muscles from Hanwoo cattle.

Type I and III collagen content exert contrasting influences on meat tenderness. μ-calpain autolysis has shown a correlation with beef tenderness. Thus, the study was designed to determine the changes in these proteins. Three hundred twenty-four Hanwoo cattle, including cows and steers, and eight muscles were evaluated for proteolysis during dry ageing period. The ratios of type I and III collagen were determined by densitometric scans of bands resolved by SDS-PAGE, and μ-calpain activity was determined using casein zymography. Proteins involved in proteolysis were analysed by LC-MS/MS. The ratio of type I and III collagen in every muscle showed a significant difference with increasing ageing times (p < 0.05). In steers, the ratio decreased with increased ageing time, and in cows, except for BF and DP muscles, a similar decreasing trend was observed. Significant differences in the ratio of type I and III collagen were found between different muscles of cows at the same ageing time (p < 0.05), but no significant differences were found in steer muscles at the same ageing time (p > 0.05). Casein zymogram results showed an inverse relationship between pH values and μ-calpain autolysis in every muscle. A significant reduction in μ-calpain activity was observed in all muscles with extended ageing times, while the rate of autolysis differed greatly (p < 0.05) between muscles at the same ageing time. Interestingly, electropherogram analysis showed that cow muscles had a higher μ-calpain activity than steer muscles. Ageing time significantly influenced proteolysis, with 24 proteins showing marked changes and being identified. The ageing times significantly affect the ratio of type I and III collagen, which coincided with the rate of μ-calpain autolysis in steers. The ratio of type I and III collagen had a significant changes during the ageing period from cows, which may be related to the amount of collagen cross-linking.

Spatial memory impairment is associated with decreased dopamine-β-hydroxylase activity in the brains of rats exposed to manganese chloride

Chronic exposure to manganese compounds leads to accumulation of the manganese in the basal ganglia and hippocampus. High levels of manganese in these structures lead to oxidative stress, neuroinflammation, imbalance of brain neurotransmitters, and hyperactivation of calpains mediating neurotoxicity and causing motor and cognitive impairment. The purpose of this work was to study the effect of excess manganese chloride intake on rats’ spatial memory and on dopamine-β-hydroxylase (DβH) activity under conditions of calpain activity suppression. Rats were divided into 3 groups of 10 animals each. Group 1 received MnCl2 (30 days, 5 mg/kg/day, intranasally), group 2 received MnCl2 (30 days, 5 mg/kg/day, intranasally) and calpain inhibitor Cast (184–210) (30 days, 5 µg/kg/day, intranasally), and group 3 received sterile saline (30 days in a volume of 20 μl, intranasally). The spatial working memory was assessed using Morris water maze test. DβH activity was determined by HPLC. We have shown that in response to excessive intake of MnCl2, there was a development of cognitive impairments in rats, which was accompanied by a decrease in DβH activity in the hippocampus. The severity of cognitive impairment was reduced by inhibiting the activity of m-calpain. The protective effect of calpain inhibitors was achieved not through an effect on DβH activity. Thus, the development of therapeutic regimens for the treatment of manganism using dopaminomimetics and/or by inhibiting calpains, must be performed taking into account the manganese-induced decrease of DβH activity and the inability to influence this process with calpain inhibitors.

Effects of phosphoglycerate kinase 1 and pyruvate kinase M2 on metabolism and physiochemical changes in postmortem muscle

The objective of this work was to investigate the influence of phosphoglycerate kinase-1 (PGK1) and pyruvate kinase-M2 (PKM2) activity on glycolysis, myofibrillar proteins, calpain system, and apoptosis pathways of postmortem muscle. The activity of PGK1 and PKM2 was regulated by their inhibitors and activators to construct the postmortem glycolysis vitro model and then incubated at 4 °C for 24 h. The results showed that compared to PGK1 and PKM2 inhibitors groups, the addition of PGK1 and PKM2 activators could accelerate glycogen consumption, ATP and lactate production, while declining pH value. Moreover, the addition of PGK1 and PKM2 activators could increase desmin degradation, μ-calpain activity, and caspase-3 abundance. Interestingly, troponin-T degradation was significantly increased both in PKM2 inhibitor and activator groups. It was suggested that PGK1 and PKM2 might be used as robust indicators to regulate meat quality by affecting the glycolysis, myofibrillar proteins, μ-calpain and apoptosis pathways in postmortem muscle.

Foxp3-mediated blockage of ryanodine receptor 2 underlies contact-based suppression by regulatory T cells.

The suppression mechanism of Tregs remains an intensely investigated topic. As our focus has shifted toward a model centered on indirect inhibition of DCs, a universally applicable effector mechanism controlled by the transcription factor forkhead box P3 (Foxp3) expression has not been found. Here, we report that Foxp3 blocked the transcription of ER Ca2+-release channel ryanodine receptor 2 (RyR2). Reduced RyR2 shut down basal Ca2+ oscillation in Tregs, which reduced m-calpain activities that are needed for T cells to disengage from DCs, suggesting a persistent blockage of DC antigen presentation. RyR2 deficiency rendered the CD4+ T cell pool immune suppressive and caused it to behave in the same manner as Foxp3+ Tregs in viral infection, asthma, hypersensitivity, colitis, and tumor development. In the absence of Foxp3, Ryr2-deficient CD4+ T cells rescued the systemic autoimmunity associated with scurfy mice. Therefore, Foxp3-mediated Ca2+ signaling inhibition may be a central effector mechanism of Treg immune suppression.

Impacts of long-time transportation on whiteleg shrimp (Penaeus vannamei) muscle quality and underlying biochemical mechanisms.

Shrimp is widely consumed around the world. Since muscle is the primary edible component of shrimp, muscle quality (particularly texture) has a direct impact on the economic value of shrimp products. However, reports on the shrimp muscle quality influenced by transportation are rather limited, and the underlying mechanism remains unknown. During the simulated transportation, the water pH and total ammonia-nitrogen content and un-ionized ammonia contents were elevated. Furthermore, reductions in shrimp muscle water-holding capacity, hardness, and shear value with intensive myofibrillar protein degradation were detected. Simulated transportation decreased the pH and glycogen content of shrimp muscle while increasing lactic dehydrogenase activity and lactate content, resulting in an elevated level of free calcium ions and increased μ-calpain and general proteolytic activities. Water exchange could improve the water quality and reduce the mortality of shrimp during transportation, as well as decrease muscle textural softening by alleviating these stress responses. Maintaining water quality and, in particular, reducing ammonia are critical to improving shrimp survival and muscle quality during live transportation. This study is of great significance for the better maintenance of the textural properties of shrimp meat. © 2023 Society of Chemical Industry.

Inhibition of extranodal NK/T-cell lymphoma by Chiauranib through an AIF-dependent pathway and its synergy with L-asparaginase

Extranodal NK/T-cell lymphoma (NKTL) is a rare and aggressive form of extranodal lymphoma with a poor prognosis. Currently, there are very limited treatment options for patients with advanced-stage disease or those with relapsed/recurrent disease. Here we show that Chiauranib, an orally small molecule inhibitor of select serine-threonine kinases (aurora B, VEGFRs, PDGFR, CSF1R, c-Kit), inhibited NKTL cell proliferation, induced cell cycle arrest, as well as suppressed the microvessel density in vitro and in vivo similar as in other types of cancer cells. Surprisingly, Chiauranib unfolded a new effect to induce apoptosis of NKTL cells by triggering AIF-dependent apoptosis other than the traditional cyt-c/caspase mitochondrial apoptosis pathway. The knockdown of AIF in vitro and in vivo dramatically blocked the efficacy of Chiauranib on NKTL. Mechanistically, the release of AIF from mitochondria is due to the upregulation of VDAC1 by the AKT-GSK3β pathway and activation of calcium-dependent m-calpain, which promotes the cleavage of VDAC1 and therefore permits the release of AIF. Notably, the low expression of Bax in both NKTL cells and patient tissues restrained the cyt-c release. It resulted in the inhibition of cyt-c/caspase mitochondrial pathway, suggesting that drugs targeting this traditional pathway may not be effective in NKTL. Furthermore, we found that L-asparaginase triggered CD95 (Fas/Apo-1)-caspase 8-caspase 3 apoptotic pathway in NKTL cells, and combination of Chiauranib and L-asparaginase exhibited a synergistic effect, suggesting a feasibility to combine these two drugs for effective treatment of NKTL. This study demonstrates Chiauranib’s positive efficacy toward NKTL through the activation of the AIF-dependent apoptosis pathway for the first time. The novel and multi-targets of Chiauranib and the synergistic effect with L-asparaginase may provide a promising therapy for NKTL patients.

Region-specific changes in expression and activity of calpains in the CNS of native rats.

Introduction and Aim: It has been proposed that µ-calpain is responsible for neuronal survival, while m-calpain – for the degeneration. It can be assumed that the "susceptibility" to the damage factor for neurons in different CNS regions depends on the content/activity of calpain isoforms. We analyzed the mRNA levels and the activity of µ-and m-calpain in the different CNS structures of rats. Materials and Methods: After decapitation intact male Wistar rats the prefrontal cortex, striatum, hippocampus, midbrain, brainstem, cerebellum, and spinal cord were removed. Each structure was divided into two parts: casein zymography was performed to determine the activity and real-time RT–PCR - to determine the level of expression mRNA of µ-and m-calpains. Results: We have shown that m-calpain mRNA predominates in the striatum, midbrain and brainstem, while µ-calpain mRNA enrichment was noticed for the hippocampus and cerebellum. The highest µ-calpain activity was in the cervical spinal cord, the lowest - in the striatum. The m-calpain activity was relatively high in the midbrain, striatum, hippocampus and brainstem, while in the cervical spinal cord and cerebellum it was moderate. Conclusion: The selective neuronal death observed during neurodegeneration can be partially determined by the initial level of calpains expression and/or activity.

Alpha-1 Antitrypsin Therapy Modifies Neutrophil Adhesion in Patients with Obstructive Lung Disease.

Alpha-1 antitrypsin deficiency (AATD) is characterized by neutrophil-dominated inflammation resulting in emphysema. The cholesterol-rich neutrophil outer plasma membrane plays a central role in adhesion and subsequent transmigration to underlying tissues. This study aimed to investigate mechanisms of increased neutrophil adhesion in AATD and whether alpha-1 antitrypsin (AAT) augmentation therapy abrogates this effect. Plasma and blood neutrophils were donated by healthy controls (n = 20), AATD (n = 30), and AATD patients after AAT augmentation therapy (n = 6). Neutrophil membrane protein expression was investigated using liquid chromatography-tandem mass spectrometry. The effect of once-weekly intravenous AAT augmentation therapy was assessed by calcium fluorometric, μ-calpain, and cell adhesion assays. Decreased neutrophil plasma membrane cholesterol content (P = 0.03), yet increased abundance of integrin α-M (fold change 1.91), integrin α-L (fold change 3.76), and cytoskeletal adaptor proteins including talin-1 (fold change 4.04) were detected on AATD neutrophil plasma membrane fractions. The described inflammatory induced structural changes were a result of a more than twofold increased cytosolic calcium concentration (P = 0.02), leading to significant calcium-dependent μ-calpain activity (3.5-fold change; P = 0.005), resulting in proteolysis of the membrane cholesterol trafficking protein caveolin-1. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased caveolin-1 and membrane cholesterol content (111.8 ± 15.5 vs. 64.18 ± 7.8 μg/2 × 107 cells before and after treatment, respectively; P = 0.02), with concurrent decreased neutrophil integrin expression and adhesion. Results demonstrate an auxiliary benefit of AAT augmentation therapy, evident by a decrease in circulating inflammation and controlled neutrophil adhesion.

Verdiperstat attenuates acute lung injury by modulating MPO/μ-calpain/β-catenin signaling

Verdiperstat, a myeloperoxidase (MPO) inhibitor, is a well-known drug used for the treatment of multisystem atrophy. However, its therapeutic effect on acute lung injury (ALI) remains to be elucidated. In this study, the effect of verdiperstat on lipopolysaccharide (LPS)-induced two-hit rat ALI model was studied in vivo. Subsequently, to explore the anti-ALI mechanism of verdiperstat, an LPS-induced injury in human pulmonary microvascular endothelial cells (HMs) was studied in vitro. The continuous administration of verdiperstat at 120 mg/kg for 3 days exerted a protective effect on the LPS-induced two-hit rat ALI model, as reflected by the change in the lung coefficient and lung pathology scores from 0.72 to 0.61 and 6.08 to 4.37, respectively. Furthermore, the values of protective adhesion protein VE-cadherin and tight junction protein claudin 5 changed from 0.42 to 0.97 and 0.25 to 0.72, but MPO, the ratio of N-μ-calpain to μ-calpain, and the distribution of β-catenin in the nucleus changed from 3.04 to 2.17, 0.62 to 0.38 and 2.25 to 0.76, respectively. LPS-induced HMs in vitro also showed similar results, including lower MPO and the distribution of β-catenin in the nucleus, but higher VE-cadherin claudin 5 and N-μ-calpain. Moreover, MPO inhibition resulted in lower μ-calpain activation and lower β-catenin in the nucleus. Our cumulative results suggest that verdiperstat alleviates ALI by strengthening VE-cadherin and claudin 5 through the inhibition of MPO/μ-calpain/β-catenin activation.

Effect of energy metabolism and proteolysis on the toughness of intermediate ultimate pH beef

The objective of this study was to identify the tenderness and energy metabolism attributes in normal ultimate pH (pHu, 5.4–5.8, NpHu), intermediate pHu (5.8–6.2, IpHu) and high pHu (> 6.2, HpHu) Longissimus lumborum from beef. During 21 days of aging, the IpHu group exhibited a higher Warner-Bratzler shear force, lower activity of μ-calpain, and a higher content of heat shock protein 27 and greater protein oxidation. Metabolomics analysis revealed that 24 differential metabolites were detected during the first 24 h postmortem between pH groups, and these were mainly the products of glycolysis, the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. The relatively higher adenosine triphosphate content in the IpHu group could delay tenderization by being used as key substrate for protein phosphorylation. In addition, the relationship between guanosine triphosphate, diphosphate and monophosphate, and beef tenderness was worthy to be verified in the future study.

COL-3-Induced Molecular and Ultrastructural Alterations in K562 Cells.

Tetracycline-3 (4-dedimethylamino sancycline, COL-3) is a non-antibiotic tetracycline derivative. COL-3 exerts potent anti-metalloproteinase activity and its antitumor effects have been reported both in vitro and in vivo. In this study, we investigated the mechanisms of COL-3-induced cytotoxicity in a chronic myeloid leukemia cell line, K562, characterized by the BCR–ABL fusion protein. COL-3 induced K562 cell death in a concentration-dependent manner with an IC50 of 10.8 µg/mL and exhibited features of both apoptosis and necrosis. However, flow cytometry analysis revealed that necrotic cells dominated over the early and late apoptotic cells upon treatment with COL-3. Transmission electron microscopy analysis in combination with Western blotting (WB) analysis revealed early mitochondrial swelling accompanied by the early release of cytochrome c and truncated apoptosis inducing factor (tAIF). In addition, ultrastructural changes were detected in the endoplasmic reticulum (ER). COL-3 affected the levels of glucose-regulated protein-94 (GRP94) and resulted in m-calpain activation. DNA double strand breaks as a signature for DNA damage was also confirmed using an antibody against γH2AX. WB analyses did not demonstrate caspase activation, while Bcl-xL protein remained unaffected. In conclusion, COL-3-induced cell death involves DNA damage as well as mitochondrial and ER perturbation with features of paraptosis and programmed necrosis.

Hydroxynonenal causes Langerhans cell degeneration in the pancreas of Japanese macaque monkeys.

For their functions of insulin biosynthesis and glucose- and fatty acid- mediated insulin secretion, Langerhans β-cells require an intracellular milieu rich in oxygen. This requirement makes β-cells, with their constitutively low antioxidative defense, susceptible to the oxidative stress. Although much progress has been made in identifying its molecular basis in experimental systems, whether the oxidative stress due to excessive fatty acids plays a crucial role in the Langerhans cell degeneration in primates is still debated. Focusing on Hsp70.1, which has dual functions as molecular chaperone and lysosomal stabilizer, the mechanism of lipotoxicity to Langerhans cells was studied using macaque monkeys after the consecutive injections of the lipid peroxidation product 'hydroxynonenal'. Based on the 'calpain-cathepsin hypothesis' formulated in 1998, calpain activation, Hsp70.1 cleavage, and lysosomal integrity were studied by immunofluorescence histochemistry, electron microscopy, and Western blotting. Light microscopy showed more abundant vacuole formation in the hydroxynonenal-treated islet cells than the control cells. Electron microscopy showed that vacuolar changes, which were identified as enlarged rough ER, occurred mainly in β-cells followed by δ-cells. Intriguingly, both cell types showed a marked decrease in insulin and somatostatin granules. Furthermore, they exhibited marked increases in peroxisomes, autophagosomes/autolysosomes, lysosomal and peroxisomal membrane rupture/permeabilization, and mitochondrial degeneration. Disrupted peroxisomes were often localized in the close vicinity of degenerating mitochondria or autolysosomes. Immunofluorescence histochemical analysis showed an increased co-localization of activated μ-calpain and Hsp70.1 with the extralysosomal release of cathepsin B. Western blotting showed increases in μ-calpain activation, Hsp70.1 cleavage, and expression of the hydroxynonenal receptor GPR109A. Taken together, these data implicate hydroxynonenal in both oxidation of Hsp70.1 and activation of μ-calpain. The calpain-mediated cleavage of the carbonylated Hsp70.1, may cause lysosomal membrane rupture/permeabilization. The low defense of primate Langerhans cells against hydroxynonenal and peroxisomally-generated hydrogen peroxide, was presumably overwhelmed to facilitate cell degeneration.

Changes in activity of µ- and m-calpains and signs of neuroinflammation in the hippocampus and striatum of rats after single intraperitoneal injection of subseptic dose of endotoxin.

Some mechanisms of neuronal degeneration in endotoxinemia are already well described, but need to be detailed. In this study, we tested the effect of a single intraperitoneal injection of a LPS sub-septic dose (1mg/kg of animal weight) on calpain activity in the striatum and hippocampus. We showed, that in the hippocampus the day after LPS administration an increase in production of IL-1β and TNF-α mRNA, followed by elevated mRNA expression and activity of µ- and m-calpains without signs of microglia activation is observed. In striatal cells, the day after LPS injection an increase in expression of IL-1β, TNF-α, IBA-1, m-calpain and calpastatin mRNA is revealed, which only intensifies over time. The elicited changes are accompanied by a decrease in motor behavior, which can be considered as a sign of sickness behavior. In the hippocampus, 180days after LPS administration expression of TNF-α, content and activity of µ-calpain are increased. In the striatum, elevation in expression of TNF-α, IBA-1, µ- and m-calpain mRNA, with hyperactivation of only m-calpain, is observed. Significantly reduced motor activity can be a consequence of LPS-induced neuronal death. A long-lasting endotoxin activates microglia that damage neurons via proinflammation cytokines and calpain hyperactivation. The endotoxin hypothesis of neurodegeneration is unproven, but if correct, then neurodegeneration may be reduced by decreasing endotoxin-induced neuroinflammation and m-calpain hyperactivation. Therefore, the drugs, that decrease endotoxin-induced neuroinflammation and differently inhibit µ- or m-calpain, can be used to prevent or reduce the severity of neurodegeneration.

μ-Calpain oxidation and proteolytic changes on myofibrillar proteins from Coregonus Peled in vitro

The purpose of this study was to investigate the structural properties of μ-calpain induced by hydroxyl radical oxidation and its effect on the degradation of myofibrillar protein (MP) from the dorsal muscles of Coregonus peled. The carbonyl and sulfhydryl content of μ-calpain changed significantly after oxidation. The content of α-helix in the secondary structure decreased from 0.825 to 0.232 and the changes in intrinsic fluorescence and ultraviolet (UV) absorption spectra indicated that oxidation could cause the expansion and aggregation of µ-calpain molecules. Changes in µ-calpain structure could improve the activity of µ-calpain, reaching the highest value at 0.5 mM H2O2. The highest µ-calpain activity facilitate the degradation of unoxidized MP, while the degradation of oxidized MP was facilitated at the 1 mM H2O2. Thus, our results provide a scientific basis for the interaction mechanism among hydroxyl radical oxidation, µ-calpain, and MP degradation.

Lysosomal Fe2+contributes to myofibrillar protein degradation through mitochondrial-dysfunction-induced apoptosis

To verify the role of lysosomal Fe2+ in mitochondrial-dysfunction-mediated apoptosis and postmortem tenderization of bovine muscles, mitochondrial apoptotic factors, myofibrillar protein degradation, and μ-calpain activity were evaluated in the control group and the iron chelator desferrioxamine (DFO) group. The control group showed 1) increased mitochondrial swelling and lipid peroxidation by 47% and 36% at 72 h, respectively (P < 0.05), significantly higher than the DFO group at 12–24 h and 72–168 h (P < 0.05); 2) significantly increased mitochondrial Ca2+ influx at 12–24 h (P < 0.01); 3) a 15%, 24%, and 30% lower cytochrome c reduction level than the DFO group at 6–24 h (P < 0.01, P < 0.05); 4) a significantly increased caspase-3 expression level at 0–120 h (P < 0.05), where the caspase-3 activity was 30% and 49% higher than that of the DFO group at 12–24 h, respectively (P < 0.01); 5) an increased μ-calpain activity by 56% at 24–168 h (P < 0.05), and 6) 75% (P < 0.01) and 34% (P < 0.05) higher degradation of desmin and troponin-T than the DFO group at 168 h, respectively. In summary, lysosomal Fe2+ facilitates postmortem tenderization by promoting the mitochondrial-dysfunction-induced apoptosis of bovine muscle.

Calpain activity in plasma of patients with essential tremor and Parkinson’s disease: a pilot study

ABSTRACT Background : Essential tremor (ET) and Parkinson’s disease (PD) are the two most common movement disorders in adults with similar clinical symptoms, which is hinting towards existence of coincident pathogenesis steps. Objectives : The objective of this report is to characterize the relationship between ET and PD severity and the activity of calcium-dependent proteases calpain in plasma. Methods : The study enrolled 12 volunteers for each condition: ET, PD, healthy. We evaluated the stage of PD on the H&Y scale in patients with PD, and the severity of tremor in patients with ET on the FTMS scale. IL-1β, TNFα, IL6, IL10 were determined in plasma using ELISA. Calpain activity was measured using fluorescent substrate and zymography methods. Results : We demonstrated that the activity of calpains in plasma of patients with PD and ET increased 5.1 and 4.3 times, respectively. The increase of calpain activity in plasma of PD patients correlated with the content of IL-1β, for ET such a connection was not found. At the advanced stages of PD calpain activity in plasma was significantly higher than that of the PD group at the early stage, and this increase was mediated by the increase in m-calpain activity. The increase in the tremor severity in ET did not lead to an increase in the activity of calpains in plasma. Conclusions : We observed general increase in the activity of calpains in plasma of both PD and ET patients that hints towards presence of the common steps in the pathogenesis of these diseases.

The investigation of calpain in human placenta with fetal growth restriction.

The mechanism of fetal growth restriction (FGR) is not fully understood. In this study, we explored the contribution of the calpain-calpastatin system and the activated states of calpains in human FGR placenta. The placentas were collected from patients of FGR (n=17) and controls (n=23) at elective cesarean sections in Nagoya City University Hospital and used for experiments upon informed consent. The existence and the expression of calpains and calpastatin in human placenta were compared between FGR and controls using immunohistochemistry, SDS-PAGE, and Western blotting. Staining of calpains (pre-, post-μ-calpain, pre-, post-m-calpain, and calpain-6) and calpastatin was observed in cytoplasm of trophoblast cells, both in FGR and control placenta. Pre-μ-calpain was located in the cytoplasm, and post-μ-calpain was located mainly in proximity to the cytoplasmic membrane. The expression of pre-μ-calpain was significantly higher (P<.001) and calpain-6 was significantly lower (P=.01) in FGR placentas. The inactive μ-calpain (80kDa) was significantly elevated (P<.01), and active μ-calpain (76kDa) was significantly decreased (P=.01) in FGR placentas. The results demonstrate that activation of μ-calpain is suppressed in FGR placentas and that calpain-6 in human placenta is involved in the pathology of FGR.

Influence of oxidation on heat shock protein 27 translocation, caspase-3 and calpain activities and myofibrils degradation in postmortem beef muscles

The objective of this study was to investigate the influence of oxidation on heat shock protein 27 (HSP27) and cytochrome c translocation, myofibrils degradation and endogenous enzymes activities, perfecting tenderization mechanism after slaughter. Bovine muscle (longissimus thoracis) was obtained at 30 min postmortem. Bovine muscle was cut and exposed to saline solution with or without H2O2 at 4 °C for 0.25, 1, 3 and 5 days, followed by detection of proteins degradation, location and enzymes activities. Results showed that oxidation promoted the translocation of HSP27 and cytochrome c from the cytoplasm to the cell membrane, which reduced µ-calpain activity, but increased caspase-3 activity through mediating the interaction with the two enzymes. Oxidation retarded troponin-T degradation, but accelerated desmin degradation, which is probably because oxidative modification of myofibrils induced different susceptibility to proteolysis. Therefore, oxidation leads to different regulatory mechanism on µ-calpain and caspase-3, as well as the degree of degradation of myofibrillar proteins, possibly through mediating HSP27 and cytochrome c.

Excessive amount and activity of μ-calpain affects apoptotic machinery in chronic B-cell leukemia cells and influences the course of the disease.

B-cell Chronic Lymphocytic Leukemia (B-CLL) is the most common hematological disorder among middle-aged/elderly people in the Western countries. We have shown earlier that B-CLL cells exhibit elevated total amount and available activity of µ-calpain, belonging to a family of ubiquitous, strongly Ca-dependent proteases, involved in the control of proliferation and apoptosis. In this study we attempted to estimate a potential clinical value of μ-calpain in relation to B-CLL clinical staging in patients with extremely high lymphocytosis and studied the molecular mechanisms associating calpain activity with clinical progress of the disease. We observed significant correlations between the amounts of intracellular μ-calpain and clinical staging of the disease, with RAI stage 1 corresponding to the highest calpain amounts in the leukemic cells. There was also a positive, statistically significant correlation between the amount of μ-calpain and phosphorylated (p)ZAP-70 in B-CLL lymphocytes. Calpain activity in the B-CLL cells is associated with decreased activities of pro-apoptotic caspases -3 and -9, and reciprocally with an increased amount of anti-apoptotic Bcl-2. Together, all of these findings make calpain activity in B-CLL cells a promising target modifying the properties of these cells and facilitating therapy. Finally, the proportion of CD19+ B cells with elevated μ-calpain and pZap-70 was markedly reduced in patients after successful therapy.

Effects of µ-calpain oxidation on Coregonus peled myofibrillar protein degradation in vitro.

The aim of this study was to evaluate the effect of µ-calpain oxidation on Coregonus peled myofibrillar protein degradation. In the present study, a hydroxyl radical oxidation system was selected to investigate oxidative modification on µ-calpain activity and its degradation on C. peled myofibrillar protein. When subjected to oxidation, the carbonyl content of µ-calpain significantly increased with the increasing of oxidation levels, and oxidation modification promoted the µ-calpain activity. Incubation of C. peled myofibrillar protein with oxidized µ-calpain resulted in the enhanced degradation of myosin heavy chains, actin, and troponin T, but the degradation of desmin at higher levels of oxidation was slightly inhibited, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. This study suggests that oxidation treatment of µ-calpain could accelerate myofibrillar proteolysis through regulating the enzyme activity during postmortem aging. PRACTICAL APPLICATION: Endogenous proteases, especially µ-calpain, are reported to be involved in fish softening during early postmortem storage, which is critical to muscle quality. The cysteine residues of proteins are particularly sensitive to oxidation. The investigation of the effect of oxidation on µ-calpain (a cysteine protease) activity allows for the monitoring of its role in the postmortem proteolysis of fish myofibrils and the associated softening of fish meat, in an attempt to minimize this softening.