Postmortem metabolic rate and calpain system activities on beef longissimus tenderness classifications

In this study, we adopted a model of tenderness classification in order to determine the factors affecting the tenderness and tenderization characteristics of beef longissimus, using cluster analysis on the basis of Warner-Bratzler shear force and myofibril fragmentation index, at 1, 7, and 14 d. The rate of tenderization was effectively differentiated by pH, R-values, mu-calpain activity, and calpastatin activity. Differences among tenderness classes were generally detected at 3 and 9 h postmortem for metabolic rate, and at 9 and 24 h for the activities of mu-calpain and calpastatin. Early postmortem metabolic rate and calpain system activities were verified as important factors with regard to longissimus tenderization.     

Postmortem Metabolic Rate and Calpain System Activities on Beef Longissimus Tenderness Classifications

In this study, we adopted a model of tenderness classification in order to determine the factors affecting the tenderness and tenderization characteristics of beef longissimus, using cluster analysis on the basis of Warner-Bratzler shear force and myofibril fragmentation index, at 1, 7, and 14 d. The rate of tenderization was effectively differentiated by pH, R-values, μ-calpain activity, and calpastatin activity. Differences among tenderness classes were generally detected at 3 and 9 h postmortem for metabolic rate, and at 9 and 24 h for the activities of μ-calpain and calpastatin. Early postmortem metabolic rate and calpain system activities were verified as important factors with regard to longissimus tenderization.     

Cytochrome c translocation does not lead to caspase activation in maitotoxin-treated SH-SY5Y neuroblastoma cells

Cytosolic cytochrome c elevation has been associated with activation of caspase-3-like proteases. In this study, we demonstrate that treatment with the neurotoxin and potent calcium channel opener maitotoxin (MTX) induces cytochrome c release from the mitochondria that is not accompanied by caspase activation. Cytochrome c translocation in MTX-treated SH-SY5Y cells was readily apparent after 30 min and peaked at 2.5h. We assayed caspase activity by acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Ac-DEVD-AMC) hydrolysis and by immunoblotting for caspase-3 processing and proteolysis of alphaII-spectrin and PARP. In contrast, treatment with pro-apoptosis agent staurosporine (STS) induced both cytochrome c release and caspase-3 activation after 2h. In addition, with MTX treatment, we found no evidence of caspase activation at any time point or MTX concentration used. Instead, we observed that caspase-9, Apaf-1 and caspase-3 were all partially truncated by calpain under these conditions. These combined effects likely contribute to the lack of caspase activation cascade in MTX-treated cells, despite the presence of cytochrome c in the cytosol.     

Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis.

Caspases are key mediators of apoptosis. Using a novel expression cloning strategy we recently developed to identify cDNAs encoding caspase substrates, we isolated the intermediate filament protein vimentin as a caspase substrate. Vimentin is preferentially cleaved by multiple caspases at distinct sites in vitro, including Asp85 by caspases-3 and -7 and Asp259 by caspase-6, to yield multiple proteolytic fragments. Vimentin is rapidly proteolyzed by multiple caspases into similar sized fragments during apoptosis induced by many stimuli. Caspase cleavage of vimentin disrupts its cytoplasmic network of intermediate filaments and coincides temporally with nuclear fragmentation. Moreover, caspase proteolysis of vimentin at Asp85 generates a pro-apoptotic amino-terminal fragment whose ability to induce apoptosis is dependent on caspases. Taken together, our findings suggest that caspase proteolysis of vimentin promotes apoptosis by dismantling intermediate filaments and by amplifying the cell death signal via a pro-apoptotic cleavage product.     

Apoptosis during ectromelia orthopoxvirus infection is DEVDase dependent: in vitro and in vivo studies

Ectromelia virus (EV), which causes mousepox, is a member of the orthopoxviruses that are defined as being able to suppress apoptosis. Caspase-3 is one of the key effector proteases which regulates the apoptotic cascade and which is responsible for DNA fragmentation observed during apoptosis. It is well known that viruses, especially poxviruses, can inhibit caspase activity. Here, we report that EV can regulate apoptosis in vitro, suppressing the activity of caspases recognizing the DEVD (Asp-Glu-Val-Asp) motif (caspase-3 and -7) before successful virus replication is completed. Caspase-3 activity measurement showed that an increase in caspase-3 activity preceded the peak of DNA fragmentation demonstrated by TUNEL staining of L929 and RK-13 cells. By using specific caspase inhibitors (Ac-DEVD-CHO, Ac-IETD-CHO and zVAD-fmk), we showed that caspase-3 and -7 (DEVDases) are major effector caspases during EV-induced apoptosis in permissive L929 and RK-13 cell cultures. Apoptosis in vivo seems to play an important role during viraemia as well as during the clearance of EV from genetically susceptible BALB/c (H-2(d)) mice. However, as shown by measurement of caspase-3 activity, caspase-3 protein detection and M30-antibody staining, both DEVDases seem to play an important role during EV clearance from draining lymph nodes and conjunctivae at 15 days p.i. up to 20 days p.i., whereas in the liver and spleen DNA fragmentation coexisted with viral multiplication and secondary viraemia. Apoptosis was DEVDase dependent only in the liver, while spleen DNA fragmentation observed between 5 and 10 days p.i. was caspase independent. Therefore, we conclude that DEVDase- (caspase-3- and caspase-7-) dependent apoptosis is an important mechanism regulating the resolution of EV infection.     

Effects of serum deprivation on myofibrillar proteolysis in chick myotube cultures

We examined the effects of serum deprivation on myofibrillar proteolysis in chick myotubes. Myotubes were incubated with serum-free medium for 24 hours. N(tau)-methylhistidine release, as an index of myofibrillar proteolysis, as well as protease activities such as calpain, proteasome, and cathepsins (B+L and D) activities were increased by serum deprivation. These results indicate that serum deprivation induces calpain, proteasome, and cathepsins activities, resulting in an increase in myofibrillar proteolysis in chick myotubes.     

Caspase-3 can be pseudo-activated by a Ca2+-dependent proteolysis at a non-canonical site

We have shown previously that calcium could trigger nuclear fragmentation, which was associated with a caspase 3 (C3)-like activity [Juin, P., Pelletier, M., Oliver, L., Tremblais, K., Gregoire, M., Meflah, K. and Vallette, F.M. (1998) Induction of a caspase-3-like activity by calcium in normal cytosolic extracts triggers nuclear apoptosis in a cell-free system. J. Biol. Chem. 273, 17559]. Here, we report that this activation is associated with a non-canonical truncation of C3, which induces a weak DEVDase activity. The cleavage of C3 via calcium-dependent proteolysis is independent of caspase 9; lysate exposure to calcium prevents further cleavage and activation by the cytochrome c and dATP pathway. Altogether, our data suggest that calcium could favour a necrotic mechanism by inducing the generation of a form of C3 insensitive to mitochondrial activation.     

Resistance to caspase-8 and -9 fragments in a malignant pleural mesothelioma cell line with acquired cisplatin-resistance

Apoptotic cysteine–aspartate proteases (caspases) are essential for the progression and execution of apoptosis, and detection of caspase fragmentation or activity is often used as markers of apoptosis. Cisplatin (cis-diamminedichloroplatinum (II)) is a chemotherapeutic drug that is clinically used for the treatment of solid tumours. We compared a cisplatin-resistant pleural malignant mesothelioma cell line (P31res1.2) with its parental cell line (P31) regarding the consequences of in vitro acquired cisplatin-resistance on basal and cisplatin-induced (equitoxic and equiapoptotic cisplatin concentrations) caspase-3, -8 and -9 fragmentation and proteolytic activity. Acquisition of cisplatin-resistance resulted in basal fragmentation of caspase-8 and -9 without a concomitant increase in proteolytic activity, and there was an increased basal caspase-3/7 activity. Similarly, cisplatin-resistant non-small-cell lung cancer cells, H1299res, had increased caspase-3 and -9 content compared with the parental H1299 cells. In P31 cells, cisplatin exposure resulted in caspase-9-mediated caspase-3/7 activation, but in P31res1.2 cells the cisplatin-induced caspase-3/7 activation occurred before caspase-8 or -9 activation. We therefore concluded that in vitro acquisition of cisplatin-resistance rendered P31res1.2 cells resistant to caspase-8 and caspase-9 fragments and that cisplatin-induced, initiator-caspase independent caspase-3/7 activation was necessary to overcome this resistance. Finally, the results demonstrated that detection of cleaved caspase fragments alone might be insufficient as a marker of caspase activity and ensuing apoptosis induction.     

Nitric oxide-mediated protection of A. actinomycetemcomitans-infected murine macrophages against apoptosis.

We investigated apoptotic cell death in murine macrophage cell line J774.1 following Actinobacillus actinomycetemcomitans infection. Infected macrophages generally kill bacteria within phagosomes with nitric oxide (NO). Our previous study demonstrated that DNA fragmentation in infected cells increased significantly on addition of S-Methylisothiourea (SMT), a selective inhibitor of inducible NO synthetase (iNOS). The purpose of the present study was to determine the mechanism via which NO affects apoptosis of infected macrophages. J774.1 cells were infected with A. actinomycetemcomitans Y4 at a bacterium/cell ratio of 500:1. The infected cells were then cultured in the presence or absence of SMT (400 microM). Culture supernatant was removed 21 h after the infection to measure LDH activity. Additionally, cellular proteins were extracted from the infected cells and measured for histone-associated DNA fragmentation and caspase-1, -3, -5, -6, -8, -9 activities. LDH activity and DNA fragmentation were significantly elevated by the infection; moreover, levels increased further on addition of SMT. Caspase activity of infected cells, particularly caspase-3, was significantly higher than that of uninfected cells. Furthermore, caspase activity increased on addition of SMT. These findings indicate that NO protects infected J774.1 cells, at least in part, against apoptotic cell death via a decrease in caspase activity.     

Different apoptotic mechanisms are involved in the antiproliferative effects of 7beta-hydroxysitosterol and 7beta-hydroxycholesterol in human colon cancer cells

Plant sterols are found in fruits and vegetables. Their cholesterol-lowering effect is well documented. Our study aimed at comparing antiproliferative effects of 7beta-hydroxysitosterol (7beta-OHsito) versus 7beta-hydroxycholesterol (7beta-OHchol) on the human colon cancer cell line Caco-2. When cells were exposed for 32 h to 60 microM 7beta-OHsito or to 30 microM 7beta-OHchol, both compounds caused 50% growth inhibition. Cells treated with 7beta-OHsito showed enhanced caspase-9 and -3 activities followed by DNA fragmentation. In contrast, 7beta-OHchol did not activate caspase-3 and activation of caspase-9 and DNA fragmentation were delayed. The treatment of cells with the caspase inhibitor Z-VAD.fmk retarded the 7beta-OHsito-induced apoptotic process but not that triggered by 7beta-OHchol. Our data suggest that the two compounds in spite of their structural similarities target different cellular pathways, which lead to cell death.     

Changes in proteolytic enzymes mRNAs and proteins relevant for meat quality during myogenesis and hypoxia of primary bovine satellite cells

The current study was conducted to evaluate the functions of μ-calpain (CAPN1), calpastatin, HSPs (heat shock proteins), and caspases during myogenesis and cell death induced by sodium azide (NaN(3)) hypoxia. The cell samples were divided into three groups: satellite cells formed at confluent monolayer (stage 1), stage 1 cells fusion into myotubes on d eight post-differentiation (stage 2), and stage 2 cells treated with 1 mM NaN(3) for 24 h (stage 3). Real-time RT-PCR showed that stage 2 cells had increased CAPN1, calpastatin, caspase 7, and CARD9 (Caspase activation and recruitment domain 9) mRNA expressions compared to stage 1 cells (*p < 0.05). By Western blotting caspase 3, caspase 7, caspase 8, and caspase 9 protein levels increased in cells at stage 2 compared to cells at stage 1 (*p < 0.05). Real-time RT-PCR showed that stage 3 cells had increased CAPN1, calpastatin, caspase 7, HSP70 (70 kDA heat shock proteins), and HSP90 (90 kDA heat shock proteins-alpha) and decreased CARD9 mRNA expression compared to stage 2 cells (*p < 0.05). Stage 3 samples had increase caspase 7 and caspase 12 activities compared to stage 2 samples, and by Western blotting protein levels of both HSP70 and HSP90 expressions, increased significantly under hypoxia condition (*p < 0.05). Here, we conclude that CAPN1, calpastatin, caspase 3, caspase 7, caspase 8, and CARD9 have important roles for satellite cell myogenesis; and that caspase 7, 12, HSP70, and HSP90 are involved in the process of apoptotic cell death under hypoxia conditions and we speculate that these proteins may be involved in early postmortem proteolysis and meat tenderization.     

High cytosolic free calcium level signals apoptosis through mitochondria-caspase mediated pathway in rat eggs cultured in vitro

The present study was aimed to find out whether an increase of cytosolic free calcium level induces egg apoptosis through mitochondria-caspase mediated pathway. To increase cytosolic free calcium level and morphological apoptotic changes, ovulated eggs were cultured in Ca²⁺/Mg²⁺ free media-199 with or without various concentrations of calcium ionophore (0.5, 1, 2, 3, 4 μM) for 3 h in vitro. The morphological apoptotic changes, cytosolic free calcium level, hydrogen peroxide (H₂O₂) concentration, catalase activity, cytochrome c concentration, caspase-9 and caspase-3 activities and DNA fragmentation were analyzed. Calcium ionophore induced morphological apoptotic features in a concentration-dependent manner followed by degeneration at higher concentrations (3 and 4 μM). Calcium ionophore increased cytosolic free calcium level, induced generation of hydrogen peroxide (H₂O₂) and inhibited catalase activity in treated eggs. The increased H₂O₂ concentration was associated with increased cytochrome c concentration, caspase-9 and caspase-3 activities that resulted in the induction of morphological features characteristic of egg apoptosis. The increased caspase-3 activity finally induced DNA fragmentation as evidenced by TUNEL positive staining in calcium ionophore-treated eggs. These findings suggest that high cytosolic free calcium level induces generation of H₂O₂ that leads to egg apoptosis through mitochondria-caspase mediated pathway.     

Significant role of μ-calpain (CANP1) in proliferation/survival of bovine skeletal muscle satellite cells

Calpains are a family of Ca²⁺-dependent intracellular cysteine proteases, including the ubiquitously expressed μ-calpain (CANP1) and m-calpain (CANP2). The CANP1 has been found to play a central role in postmortem proteolysis and meat tenderization. However, the physiological roles of CANP1 in cattle skeletal satellite cells remain unclear. In this study, three small interference RNA sequences (siRNAs) targeting CANP1 gene were designed and ligated into pSilencer plasmid vector to construct shRNA expression constructs. Suppression of CANP1 in satellite cells was evaluated using these shRNA expressing constructs. Our results revealed that all three siRNAs could downregulate the expression of CANP1. Suppression of CANP1 significantly reduced cell viability in cell proliferation when compared with control cells. We found a crosstalk between CANP1 and caspase systems, particularly suppression of CANP1 resulted in an increase in the expressions of apoptotic caspases such as caspase-3, caspase-6, caspase-7, caspase-8, and caspase-9, as well as heat-shock protein (HSP) systems. Additionally, suppression of CANP1 led to the upregulation of other apoptosis and DNA damage-regulating genes whilst at the same time downregulating proliferation, migration, and differentiation-regulating genes. The results of our findings report for the first time that suppression of CANP1 resulted in the activation of caspase and HSP systems which might in turn regulate apoptosis through the caspase-dependent cell death pathway. This clearly demonstrates the key roles of CANP1 in regulation of cell proliferation and survival.     

Cysteine suppresses oxidative stress-induced myofibrillar proteolysis in chick myotubes

The effects of cysteine as an antioxidant nutrient on change in protein modification and myofibrillar proteolysis in chick myotubes by induction of oxidative stress by H(2)O(2) treatment were investigated. Myotubes were treated for 1 h with H(2)O(2) (1 mM). After this treatment, the H(2)O(2) was removed and the cells were cultured in cysteine (0.1 and 1 mM) containing serum-free medium for 24 h. Protein carbonyl content as an index of protein modification and N(tau)-methylhistidine release as an index of myofibrillar proteolysis were increased at 24 h after H(2)O(2) treatment, and the increment was reduced by cysteine. Calpain, proteasome and cathepsin (B+L and D) activities were increased at 24 h after H(2)O(2) treatment, and the increment was also reduced by cysteine. These results indicate that cysteine suppresses protein modification by oxidative stress, resulting in a decrease of protease acitivities, finally resulting in a decrease in myofibrillar proteolysis in chick myotubes.     

Induction of apoptosis dependent on caspase activities and growth arrest in HL-60 cells by PGA2

Prostaglandin (PG) A2 has been reported to inhibit the growth or induce apoptosis of various tumor cells. In the present study, PGA2 inhibited the growth of HL-60 cells and concomitantly-induced nuclear condensation and DNA fragmentation, characteristics of apoptosis. Down-regulation of c-myc mRNA, and activation of caspase-3 were observed in the PGA2 -treated cells. PGA2-induced DNA fragmentation was completely abolished in the presence of zVAD-Fmk or zDEVD-Fmk. But, relative cell survival was not improved up to that of untreated cells by pretreatment of caspase inhibitors, and c-myc down-regulation was not recovered by caspase inhibitors, either. Moreover, cytochrome c release and activation of caspase-9 was also observed in apoptotic cells and a specific inhibitor of caspase-9 (zLEHD-Fmk) prevented both DNA fragmentation and activation of caspase-3, but not relative cell survival, implying the upstream mitochondrial event of caspase-3 activation. In addition, antagonistic Fas antibody (ZB4) exerted no effect on the apoptosis. Taken together, these results suggest that PGA2 may induce the apoptosis as well as growth inhibition in HL-60 cells, and cytochrome c release and caspase activation seem to play a critical role in this apoptosis which might be independent or downstream of growth inhibition associated with c-myc down-regulation.     

Role of caspases in murine limb bud cell death induced by 4-hydroperoxycyclophosphamide,an activated analog of cyclophosphamide

BACKGROUND: Caspases play a pivotal role in the regulation and execution of apoptosis, an essential process during limb development. Caspase 8 activation is usually downstream of the Fas/FasL death receptors, whereas caspase 9 mediates the mitochondrial signaling pathway of apoptosis. Caspase 3 is an effector caspase. Previous studies have shown that the exposure of embryonic murine limbs in vitro to 4-hydroperoxycyclophosphamide (4-OOHCPA), an activated analog of the anticancer alkylating agent, cyclophosphamide, induced limb malformations and apoptosis. The goal of this study was to determine the role of caspases in mediating apoptosis in this model system. METHODS: Limb buds from gestational day 12 CD-1 mice were excised and cultured in roller bottles in a chemically defined medium for up to 6 days in the absence or presence of 4-OOHCPA. Apoptosis was indicated by internucleosomal DNA fragmentation, as detected by TUNEL staining. The profile of caspase activation was characterized by Western blot analysis and immunohistochemistry of control and treated limbs. To determine the consequences to limb morphology of inhibiting caspase activation, DEVD-CHO, a caspase-3 inhibitor, was added to the cultures. RESULTS: Limbs cultured in the presence of 4-OOHCPA were growth retarded and malformed; apoptosis was increased in the apical ectodermal ridge and interdigital areas. Western blot analysis showed that 4-OOHCPA exposure did not activate procaspases 8 or 9 in limbs. In contrast, procaspase-3 cleavage was increased in a concentration and time-dependent manner after exposure of limbs to 4-OOHCPA. Immunoreactive activated caspase-3 was localized in the interdigital areas and the apical ectodermal ridge region in control limbs; staining in these areas and in the interdigital areas was increased dramatically in limbs exposed to 4-OOHCPA. Inhibition of caspase 3 activation with DEVD-CHO partially protected limbs from insult with 4-OOHCPA. CONCLUSION: Caspase-dependent and caspase-independent pathways of cell death are both important is mediating the abnormal limb development triggered by insult with 4-OOHCPA.     

Activity and expression of different members of the caspase family in the rat corpus luteum during pregnancy and postpartum

Studies were designed to examine the expression and activity of four caspases that contribute to the initial (caspases-2, -8, and -9) and final (caspase-3) events in apoptosis in the rat corpus luteum (CL) during pregnancy (days 7, 17, 19, and 21 of gestation), postpartum (days 1 and 4), and after injection (0, 8, 16, 24, and 36 h) of the physiological luteolysin PGF2alpha. In addition, the temporal relationship of caspase expression/activity relative to steroid production and luteal regression was evaluated. During pregnancy, the activity of all four caspases was significantly greater on day 19, before a decline in CL progesterone (P) and CYP11A1 levels at day 21 of gestation. The levels of the caspase-3 active fragment (p17, measured by Western blot) also increased at days 19 and 21 of pregnancy. Immunohistochemical analyses detected specific staining for the caspases in luteal cells (large and small) as well as in endothelial cells. However, the percentage of apoptotic cells did not increase in the CL until postpartum. Following PGF2alpha injection, there was a significant decrease in CL P by 24 h, although the activity of all four caspases did not increase until 36 h posttreatment. The active p17 fragment of caspase-3 also significantly increased at 36 h post-PGF2alpha. These results suggest that an increase in the activity of caspases-2, -8, -9, and -3 is associated with the early events of natural luteolysis at the end of pregnancy. Also, the exogenous administration of the luteolysin PGF2alpha may regulate members of the caspase family.