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Sample records for calpain

  1. Inhibiting calpain, rescuing cells.

    OpenAIRE

    Robinson, A.

    1996-01-01

    Drs. John Elce and Peter Davies, biochemists at Queen's University, Kingston, Ont., are investigating the molecular structure of calpain, an enzyme that has been implicated in the cellular damage that occurs after such events as myocardial infarction and stroke. This damage is precipitated by an imbalance in the regulation of calpain that arises as an indirect result of ischemia. Elce and Davies hope that their research, which involves techniques such as recombinant DNA technology and x-ray c...

  2. Calpain 3 is important for muscle regeneration

    DEFF Research Database (Denmark)

    Hauerslev, Simon; Sveen, Marie-Louise; Duno, Morten;

    2012-01-01

    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 wa...... was to investigate how mutations in the four functional domains of calpain 3 affect muscle regeneration....

  3. Calpain Activator Dibucaine Induces Platelet Apoptosis

    Directory of Open Access Journals (Sweden)

    Jun Liu

    2011-03-01

    Full Text Available Calcium-dependent calpains are a family of cysteine proteases that have been demonstrated to play key roles in both platelet glycoprotein Ibα shedding and platelet activation and altered calpain activity is associated with thrombotic thrombocytopenic purpura. Calpain activators induce apoptosis in several types of nucleated cells. However, it is not clear whether calpain activators induce platelet apoptosis. Here we show that the calpain activator dibucaine induced several platelet apoptotic events including depolarization of the mitochondrial inner transmembrane potential, up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation and phosphatidylserine exposure. Platelet apoptosis elicited by dibucaine was not affected by the broad spectrum metalloproteinase inhibitor GM6001. Furthermore, dibucaine did not induce platelet activation as detected by P-selectin expression and PAC-1 binding. However, platelet aggregation induced by ristocetin or α-thrombin, platelet adhesion and spreading on von Willebrand factor were significantly inhibited in platelets treated with dibucaine. Taken together, these data indicate that dibucaine induces platelet apoptosis and platelet dysfunction.

  4. Identification of active Plasmodium falciparum calpain to establish screening system for Pf-calpain-based drug development

    Directory of Open Access Journals (Sweden)

    Soh Byoung

    2013-02-01

    Full Text Available Abstract Background With the increasing resistance of malaria parasites to available drugs, there is an urgent demand to develop new anti-malarial drugs. Calpain inhibitor, ALLN, is proposed to inhibit parasite proliferation by suppressing haemoglobin degradation. This provides Plasmodium calpain as a potential target for drug development. Pf-calpain, a cysteine protease of Plasmodium falciparum, belongs to calpain-7 family, which is an atypical calpain not harboring Ca2+-binding regulatory motifs. In this present study, in order to establish the screening system for Pf-calpain specific inhibitors, the active form of Pf-calpain was first identified. Methods Recombinant Pf-calpain including catalytic subdomain IIa (rPfcal-IIa was heterologously expressed and purified. Enzymatic activity was determined by both fluorogenic substrate assay and gelatin zymography. Molecular homology modeling was carried out to address the activation mode of Pf-calpain in the aspect of structural moiety. Results Based on the measurement of enzymatic activity and protease inhibitor assay, it was found that the active form of Pf-calpain only contains the catalytic subdomain IIa, suggesting that Pf-calpain may function as a monomeric form. The sequence prediction indicates that the catalytic subdomain IIa contains all amino acid residues necessary for catalytic triad (Cys-His-Asn formation. Molecular modeling suggests that the Pf-calpain subdomain IIa makes an active site, holding the catalytic triad residues in their appropriate orientation for catalysis. The mutation analysis further supports that those amino acid residues are functional and have enzymatic activity. Conclusion The identified active form of Pf-calpain could be utilized to establish high-throughput screening system for Pf-calpain inhibitors. Due to its unique monomeric structural property, Pf-calpain could be served as a novel anti-malarial drug target, which has a high specificity for malaria parasite

  5. Rational Design of Calpain Inhibitors Based on Calpastatin Peptidomimetics.

    Science.gov (United States)

    Low, Kristin E; Ler, Spencer; Chen, Kevin J; Campbell, Robert L; Hickey, Jennifer L; Tan, Joanne; Scully, Conor C G; Davies, Peter L; Yudin, Andrei K; Zaretsky, Serge

    2016-06-01

    Our previously reported structures of calpain bound to its endogenous inhibitor calpastatin have motivated the use of aziridine aldehyde-mediated peptide macrocyclization toward the design of cyclic peptides and peptidomimetics as calpain inhibitors. Inspired by nature's hint that a β-turn loop within calpastatin forms a broad interaction around calpain's active site cysteine, we have constructed and tested a library of 45 peptidic compounds based on this loop sequence. Four molecules have shown reproducibly low micromolar inhibition of calpain-2. Further systematic sequence changes led to the development of probes that displayed increased potency and specificity of inhibition against calpain over other cysteine proteases. Calculated Ki values were in the low micromolar range, rivaling other peptidomimetic calpain inhibitors and presenting an improved selectivity profile against other therapeutically relevant proteases. Competitive and mixed inhibition against calpain-2 was observed, and an allosteric inhibition site on the enzyme was identified for a noncompetitive inhibitor.

  6. Involvement of calpains in adult neurogenesis: implications for stroke

    OpenAIRE

    Vanessa Mendes Machado; Maria Inês Morte; Bruno Pereira Carreira; Maria Manuela Azevedo; Jiro eTakano; Nobuhisa eIwata; Saido, Takaomi C; Hannelore eAsmussen; Alan Rick Horwitz; Caetana Monteiro Carvalho; Inês Maria Araújo

    2015-01-01

    Calpains are ubiquitous proteases involved in cell proliferation, adhesion and motility. In the brain, calpains have been associated with neuronal damage in both acute and neurodegenerative disorders, but their physiological function in the nervous system remains elusive. During brain ischemia, there is a large increase in the levels of intracellular calcium, leading to the activation of calpains. Inhibition of these proteases has been shown to reduce neuronal death in a variety of stroke mod...

  7. Calpain-10 and insulin resistance in human skeletal muscle

    OpenAIRE

    Norton, Luke

    2007-01-01

    Variation in the calpain-10 gene has been linked to a three-fold increased risk for type 2 diabetes in Pima Indian and some European populations. Furthermore, reduced skeletal muscle expression of calpain-10 is associated with reduced insulin mediated glucose disposal and carbohydrate oxidation. The skeletal muscle specific calpain-3 plays a key role in skeletal muscle integrity and has also been linked to insulin resistance in humans and rodents. The major aims of this thesis were to...

  8. Calpain inhibitor nanocrystals prepared using Nano Spray Dryer B-90

    OpenAIRE

    Baba, Koichi; Nishida, Kohji

    2012-01-01

    The Nano Spray Dryer B-90 offers a new, simple, and alternative approach for the production of drug nanocrystals. Among attractive drugs, calpain inhibitor that inhibits programmed cell death ‘apoptosis’ is a candidate for curing apoptosis-mediated intractable diseases such as Alzheimer’s disease and Parkinson’s disease. In this study, the preparation of calpain inhibitor nanocrystals using Nano Spray Dryer B-90 was demonstrated. The particle sizes were controlled by means of selecting mesh a...

  9. AB230. Calpain inhibition improves diabetic erectile dysfunction in rats

    Science.gov (United States)

    Li, Hao; Wang, Tao; Liu, Jihong

    2016-01-01

    Objective Diabetic erectile dysfunction is an intractable disease which results from both vascular and nervous dysfunction in penis. Calpain mediates the vascular dysfunction during hyperglycemia and is involved in some neurodegenerative diseases. This study was designed to investigate the role of calpain inhibition in improving diabetic erectile dysfunction in rats. Methods Type 1 diabetes was induced by intraperitoneal injection of streptozotocin at the dose of 60 mg/kg in rats. After 2 months, diabetic erectile dysfunction was confirmed by apomorphine test. Then the animals were divided into three groups: (I) nondiabetic control groups, (II) diabetic rats + vehicle and (III) diabetic rats + MDL28170. Two weeks later the erectile function was measured by electrical stimulation of the cavernous nerve and the ratio between intracavernosal pressure (ICP) and mean systemic arterial blood pressure (MAP) at the peak of erectile response was calculated. After that penis tissue was harvested. Calpain activity in corpus cavernosum was measured by western blot. Neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) were observed by immunohistochemistry and western blot. The endothelial content in the cavernosum was measured by immunohistochemistry. Results The calpain activity was increased in diabetic rats and inhibited by MDL28170. The erectile function was improved by MDL28170 treatment. The expression of nNOS and eNOS, as well as the content of endothelium in corpus cavernosum were also increased by inhibition of calpain. Conclusions Calpain activation may play a role in the erectile dysfunction of diabetic rats. Inhibition of calpain could improve diabetic erectile dysfunction by increasing expression of nNOS and eNOS in the corpus cavernosum. This could be a novel therapeutic target to protect the erectile function in diabetic patient.

  10. Analysis of calpain-3 protein in muscle biopsies of different muscular dystrophies from India

    OpenAIRE

    Renjini, R.; Gayathri, N.; A Nalini; Bharath, M.M. Srinivas

    2012-01-01

    Background & objectives: Calpain-3, a Ca2+-dependent protease has been implicated in the pathology of neuromuscular disorders (NMDs). The current study aimed to analyze calpain-3 expression in cases diagnosed as muscular dystrophy from the Indian population. Methods: Calpain-3 Western blot analysis in muscle biopsies of immunohistochemically confirmed cases of Duchenne muscular dystrophy (DMD) (n=10), dysferlinopathy (n=30) and sarcoglycanopathy (n=8) was carried out. Calpain-3 Western blotti...

  11. Calpain inhibitor nanocrystals prepared using Nano Spray Dryer B-90

    Science.gov (United States)

    Baba, Koichi; Nishida, Kohji

    2012-08-01

    The Nano Spray Dryer B-90 offers a new, simple, and alternative approach for the production of drug nanocrystals. Among attractive drugs, calpain inhibitor that inhibits programmed cell death `apoptosis' is a candidate for curing apoptosis-mediated intractable diseases such as Alzheimer's disease and Parkinson's disease. In this study, the preparation of calpain inhibitor nanocrystals using Nano Spray Dryer B-90 was demonstrated. The particle sizes were controlled by means of selecting mesh aperture sizes. The obtained average particle sizes were in the range of around 300 nm to submicron meter.

  12. Calpain Activity Is Generally Elevated during Transformation but Has Oncogene-Specific Biological Functions

    Directory of Open Access Journals (Sweden)

    N.O. Carragher

    2004-01-01

    Full Text Available Several oncogene and tumor-suppressor gene products are known substrates for the calpain family of cysteine proteases, and calpain is required for transformation by v-src and tumor invasion. Thus, we have now addressed whether calpain is generally associated with transformation and how calpain contributes to oncogene function. Our results demonstrate that calpain activity is enhanced upon transformation induced by the v-Src, v-Jun, v-Myc, k-Ras, and v-Fos oncoproteins. Furthermore, elevated calpain activity commonly promotes focal adhesion remodelling, disruption of actin cytoskeleton, morphological transformation, and cell migration, although proteolysis of target substrates (such as focal adhesion kinase, talin, and spectrin is differently specified by individual oncoproteins. Interestingly, v-Fos differs from other common oncoproteins in not requiring calpain activity for actin/adhesion remodelling or migration of v-Fos transformed cells. However, anchorage-independent growth of all transformed cells is sensitive to calpain inhibition. In addition, elevated calpain activity contributes to oncogene-induced apoptosis associated with transformation by v-Myc. Taken together, these studies demonstrate that calpain activity is necessary for full cellular transformation induced by common oncoproteins, but has distinct roles in oncogenic events induced by individual transforming proteins. Thus, targeting calpain activity may represent a useful general strategy for interfering with activated protooncogenes in cancer cells.

  13. Brucella infection inhibits macrophages apoptosis via Nedd4-dependent degradation of calpain2.

    Science.gov (United States)

    Cui, Guimei; Wei, Pan; Zhao, Yuxi; Guan, Zhenhong; Yang, Li; Sun, Wanchun; Wang, Shuangxi; Peng, Qisheng

    2014-11-01

    The calcium-dependent protease calpain2 is involved in macrophages apoptosis. Brucella infection-induced up-regulation of intracellular calcium level is an essential factor for the intracellular survival of Brucella within macrophages. Here, we hypothesize that calcium-dependent E3 ubiquitin ligase Nedd4 ubiquitinates calpain2 and inhibits Brucella infection-induced macrophage apoptosis via degradation of calpain2.Our results reveal that Brucella infection induces increases in Nedd4 activity in an intracellular calcium dependent manner. Furthermore, Brucella infection-induced degradation of calpain2 is mediated by Nedd4 ubiquitination of calpain2. Brucella infection-induced calpain2 degradation inhibited macrophages apoptosis. Treatment of Brucella infected macrophages with calcium chelator BAPTA or Nedd4 knock-down decreased Nedd4 activity, prevented calpain2 degradation, and resulted in macrophages apoptosis.

  14. Cleavage of desmin by cysteine proteases: Calpains and cathepsin B

    DEFF Research Database (Denmark)

    Baron, Caroline; Jacobsen, S.; Purslow, P.P.

    2004-01-01

    sequential C-terminal degradation pattern characteristic of this dipeptylpeptidase. The substrate primary structure was not found to be essential for regulation of the proteolytic activity of the cysteine peptidases studied. However, the degradation patterns obtained imply that calpains are involved in...

  15. Calpain 3 is important for muscle regeneration: Evidence from patients with limb girdle muscular dystrophies

    Directory of Open Access Journals (Sweden)

    Hauerslev Simon

    2012-03-01

    Full Text Available Abstract 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.

  16. Calpain I Inhibition prevents atrial structural remodeling in a canine model with atrial fibrillation

    Institute of Scientific and Technical Information of China (English)

    XUE Hong-jie; SHAN Hong-bo; LIU Jie; LI Wei-min; LI Yue; GONG Yong-tai; YANG Bao-feng; JIN Cheng-luo; SHENG Li; CHU Shan; ZHANG Li

    2008-01-01

    Background Atrial fibrillation (AF) is accompanied by atrial structural remodeling. Calpain activity is induced during AR To lest a causal relationship between calpain activation and atrial structural changes, N-acetyl-Leu-Leu-Met (ALLM), a calpain inhibitor, was utilized in a canine AF model.Methods Fifteen dogs were randomly divided into 3 groups: sham-operated group, control group and calpain inhibitor group; each with 5 dogs. Sustained AF was induced by rapid right atrium pacing at 600 beats per minute for 3 weeks. ALLM was administered at a dosage of 1.0 mg-kg-1·d-1 in the calpain inhibitor group. Three weeks later, the proteolysis, protein expression of TnT and myosin, calpain l localization and expression and structural changes were examined in left atrial free walls, right atrial free walls and the interatrial septum respectively. Atrial size and contractile function were also measured by echocardiography.Results Long-term rapid atrial pacing induced marked structural changes such as enlarged atrial volume, myolysis, degradation of TnT and myosin, accumulation of glycogen and changes in mitochondrial shape and size, which were paralleled by an increase in calpain activity. The positive correlation between calpain activity and the degree of myolysis (rs=0.90 961, P<0.0001) was demonstrated. In addition to structural abnormalities, pacing-induced atrial contractile dysfunction was observed in this study. The pacing-induced atrial structural alterations and loss of contractility were partially prevented by the calpain inhibitor ALLM.Conclusions Activation of calpain represents key features in the progression towards overt structural remodeling. Calpain inhibitor, ALLM, suppressed the increased calpain activity and reversed structural remodeling caused by sustained atrial fibrillation in the present model. Calpain Inhibition may therefore provide a possibility for therapeutic Intervention in AF.

  17. Effect of Calpain inhibitor I on glucocorticoid receptor-dependent degradation and its transactivation ability

    Institute of Scientific and Technical Information of China (English)

    程晓刚; 粟永萍; 罗成基; 刘晓宏

    2004-01-01

    Objective: To investigate the effect of Calpain inhibitor I on glucocorticoid receptor-dependent proteasomal degradation and its transcriptional activity. Methods: After Raw-264.7 cells were treated with Calpain inhibitor I, dexamethasone, or both for about 12 h, the change of glucocorticoid receptor was detected by western blot analysis. COS-7 cells were transfected with PRsh-GRα expression vector and glucocorticoid-responsive receptor pMAMneo-CAT, then the effect of Calpain inhibitor I on glucocorticoid receptor transcriptional activation ability was determined by CAT activity. Results: The glucocorticoid receptor levels decreased after RAW-264.7 cells were treated with dexamethasone for 12 hours, which effect can be inhibited by Calpain inhibitor I to some extent. CAT activity assay showed that Calpain inhibitor I enhance glucocorticoid receptor transcriptional activity. Conclusion: Calpain inhibitor I can inhibit the down-regulation of dexamethasone on glucocoaicoid receptor, and enhances glucocorticoid receptor transactivation ability.

  18. Vascular smooth muscle cell spreading onto fibrinogen is regulated by calpains and phospholipase C.

    Science.gov (United States)

    Paulhe, F; Bogyo, A; Chap, H; Perret, B; Racaud-Sultan, C

    2001-11-01

    Fibrinogen deposition and smooth muscle cell migration are important causes of atherosclerosis and angiogenesis. Involvement of calpains in vascular smooth muscle cell adhesion onto fibrinogen was investigated. Using calpain inhibitors, we showed that activation of calpains was required for smooth muscle cell spreading. An increase of (32)P-labeled phosphatidic acid and phosphatidylinositol-3,4-bisphosphate, respective products of phospholipase C and phosphoinositide 3-kinase activities, was measured in adherent cells. Addition of the calpain inhibitor calpeptin strongly decreased phosphatidic acid and phosphatidylinositol-3,4-bisphosphate. However, smooth muscle cell spreading was prevented by the phospholipase C inhibitor U-73122, but poorly modified by phosphoinositide 3-kinase inhibitors wortmannin and LY-294002. Moreover, PLC was found to act upstream of the PI 3-kinase IA isoform. Thus, our data provide the first evidence that calpains are required for smooth muscle cell spreading. Further, phospholipase C activation is pointed as a key step of cell-spreading regulation by calpains.

  19. Moderation of calpain activity promotes neovascular integration and lumen formation during VEGF-induced pathological angiogenesis.

    Directory of Open Access Journals (Sweden)

    Mien V Hoang

    Full Text Available BACKGROUND: Successful neovascularization requires that sprouting endothelial cells (ECs integrate to form new vascular networks. However, architecturally defective, poorly integrated vessels with blind ends are typical of pathological angiogenesis induced by vascular endothelial growth factor-A (VEGF, thereby limiting the utility of VEGF for therapeutic angiogenesis and aggravating ischemia-related pathologies. Here we investigated the possibility that over-exuberant calpain activity is responsible for aberrant VEGF neovessel architecture and integration. Calpains are a family of intracellular calcium-dependent, non-lysosomal cysteine proteases that regulate cellular functions through proteolysis of numerous substrates. METHODOLOGY/PRINCIPAL FINDINGS: In a mouse skin model of VEGF-driven angiogenesis, retroviral transduction with dominant-negative (DN calpain-I promoted neovessel integration and lumen formation, reduced blind ends, and improved vascular perfusion. Moderate doses of calpain inhibitor-I improved VEGF-driven angiogenesis similarly to DN calpain-I. Conversely, retroviral transduction with wild-type (WT calpain-I abolished neovessel integration and lumen formation. In vitro, moderate suppression of calpain activity with DN calpain-I or calpain inhibitor-I increased the microtubule-stabilizing protein tau in endothelial cells (ECs, increased the average length of microtubules, increased actin cable length, and increased the interconnectivity of vascular cords. Conversely, WT calpain-I diminished tau, collapsed microtubules, disrupted actin cables, and inhibited integration of cord networks. Consistent with the critical importance of microtubules for vascular network integration, the microtubule-stabilizing agent taxol supported vascular cord integration whereas microtubule dissolution with nocodazole collapsed cord networks. CONCLUSIONS/SIGNIFICANCE: These findings implicate VEGF-induction of calpain activity and impairment of

  20. The calpain/calpastatin system has opposing roles in growth and metastatic dissemination of melanoma.

    Directory of Open Access Journals (Sweden)

    Quentin Raimbourg

    Full Text Available Conventional calpains are ubiquitous cysteine proteases whose activity is promoted by calcium signaling and specifically limited by calpastatin. Calpain expression has been shown to be increased in human malignant cells, but the contribution of the calpain/calpastatin system in tumorigenesis remains unclear. It may play an important role in tumor cells themselves (cell growth, migration, and a contrario cell death and/or in tumor niche (tissue infiltration by immune cells, neo-angiogenesis. In this study, we have used a mouse model of melanoma as a tool to gain further understanding of the role of calpains in tumor progression. To determine the respective importance of each target, we overexpressed calpastatin in tumor and/or host in isolation. Our data demonstrate that calpain inhibition in both tumor and host blunts tumor growth, while paradoxically increasing metastatic dissemination to regional lymph nodes. Specifically, calpain inhibition in melanoma cells limits tumor growth in vitro and in vivo but increases dissemination by amplifying cell resistance to apoptosis and accelerating migration process. Meanwhile, calpain inhibition restricted to host cells blunts tumor infiltration by immune cells and angiogenesis required for antitumor immunity, allowing tumor cells to escape tumor niche and disseminate. The development of highly specific calpain inhibitors with potential medical applications in cancer should take into account the opposing roles of the calpain/calpastatin system in initial tumor growth and subsequent metastatic dissemination.

  1. Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells

    Science.gov (United States)

    Koh, T. J.; Tidball, J. G.

    2000-01-01

    We tested the hypothesis that nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore on C(2)C(12) muscle cells, including preventing talin proteolysis and release into the cytosol and reducing loss of vinculin, cell detachment, and loss of cellular protein. These results indicate that nitric oxide inhibition of calpain protected the cells from ionophore-induced proteolysis. Calpain inhibitor I and a cell-permeable calpastatin peptide also protected the cells from proteolysis, confirming that ionophore-induced proteolysis was primarily calpain mediated. The activity of m-calpain in a casein zymogram was inhibited by sodium nitroprusside, and this inhibition was reversed by dithiothreitol. Previous incubation with the active site-targeted calpain inhibitor I prevented most of the sodium nitroprusside-induced inhibition of m-calpain activity. These data suggest that nitric oxide inhibited m-calpain activity via S-nitrosylation of the active site cysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.

  2. Growth and development of skeletal muscle in mu-calpain knockout mice

    Science.gov (United States)

    The calpain system has been identified as a potential candidate in muscle growth and development due to its role in a variety of cellular processes such as cytoskeletal remodeling and myogenesis. The objective of this study was to evaluate growth and development of skeletal muscle in mu-calpain kno...

  3. Mechanism of Action of Thalassospiramides, A New Class of Calpain Inhibitors

    KAUST Repository

    Lu, Liang

    2015-03-05

    Thalassospiramides comprise a large family of lipopeptide natural products produced by Thalassospira and Tistrella marine bacteria. Here we provide further evidence of their nanomolar inhibitory activity against the human calpain 1 protease. Analysis of structure-activity relationship data supported our hypothesis that the rigid 12-membered ring containing an α,β-unsaturated carbonyl moiety is the pharmacologically active functional group, in contrast to classic electrophilic "warheads" in known calpain inhibitors. Using a combination of chemical modifications, mass spectrometric techniques, site-directed mutagenesis, and molecular modeling, we show the covalent binding of thalassospiramide\\'s α,β-unsaturated carbonyl moiety to the thiol group of calpain\\'s catalytic Cys115 residue by a Michael 1,4-addition reaction. As nanomolar calpain inhibitors with promising selectivity and low toxicity from natural sources are rare, we consider thalassospiramides as promising drug leads.

  4. Effects of arsenic poisoning on neuronal cell apoptosis and mRNA and protein expression of calpain 1,calpain 2,and cdk5/p25

    Institute of Scientific and Technical Information of China (English)

    李新

    2014-01-01

    Objective To study the effect of arsenic on neuronal cell apoptosis and the mRNA and protein expression of calpain 1,calpain 2,and cyclin-dependent kinases 5(cdk5)/p25 and to provide a scientific basis for the research on neurotoxic mechanism of arsenic trioxide(As2O3).Methods Primary cultured rat neurons were divided into untreated control group,dimethyl sulfoxide

  5. Erythropoietin Modulates Cerebral and Serum Degradation Products from Excess Calpain Activation following Prenatal Hypoxia-Ischemia.

    Science.gov (United States)

    Jantzie, Lauren L; Winer, Jesse L; Corbett, Christopher J; Robinson, Shenandoah

    2016-01-01

    Preterm infants suffer central nervous system (CNS) injury from hypoxia-ischemia and inflammation - termed encephalopathy of prematurity. Mature CNS injury activates caspase and calpain proteases. Erythropoietin (EPO) limits apoptosis mediated by activated caspases, but its role in modulating calpain activation has not yet been investigated extensively following injury to the developing CNS. We hypothesized that excess calpain activation degrades developmentally regulated molecules essential for CNS circuit formation, myelination and axon integrity, including neuronal potassium-chloride co-transporter (KCC2), myelin basic protein (MBP) and phosphorylated neurofilament (pNF), respectively. Further, we predicted that post-injury EPO treatment could mitigate CNS calpain-mediated degradation. Using prenatal transient systemic hypoxia-ischemia (TSHI) in rats to mimic CNS injury from extreme preterm birth, and postnatal EPO treatment with a clinically relevant dosing regimen, we found sustained postnatal excess cortical calpain activation following prenatal TSHI, as shown by the cleavage of alpha II-spectrin (αII-spectrin) into 145-kDa αII-spectrin degradation products (αII-SDPs) and p35 into p25. Postnatal expression of the endogenous calpain inhibitor calpastatin was also reduced following prenatal TSHI. Calpain substrate expression following TSHI, including cortical KCC2, MBP and NF, was modulated by postnatal EPO treatment. Calpain activation was reflected in serum levels of αII-SDPs and KCC2 fragments, and notably, EPO treatment also modulated KCC2 fragment levels. Together, these data indicate that excess calpain activity contributes to the pathogenesis of encephalopathy of prematurity. Serum biomarkers of calpain activation may detect ongoing cerebral injury and responsiveness to EPO or similar neuroprotective strategies. PMID:26551007

  6. Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer's disease brain.

    Science.gov (United States)

    Kurbatskaya, Ksenia; Phillips, Emma C; Croft, Cara L; Dentoni, Giacomo; Hughes, Martina M; Wade, Matthew A; Al-Sarraj, Safa; Troakes, Claire; O'Neill, Michael J; Perez-Nievas, Beatriz G; Hanger, Diane P; Noble, Wendy

    2016-03-31

    Alterations in calcium homeostasis are widely reported to contribute to synaptic degeneration and neuronal loss in Alzheimer's disease. Elevated cytosolic calcium concentrations lead to activation of the calcium-sensitive cysteine protease, calpain, which has a number of substrates known to be abnormally regulated in disease. Analysis of human brain has shown that calpain activity is elevated in AD compared to controls, and that calpain-mediated proteolysis regulates the activity of important disease-associated proteins including the tau kinases cyclin-dependent kinase 5 and glycogen kinase synthase-3. Here, we sought to investigate the likely temporal association between these changes during the development of sporadic AD using Braak staged post-mortem brain. Quantification of protein amounts in these tissues showed increased activity of calpain-1 from Braak stage III onwards in comparison to controls, extending previous findings that calpain-1 is upregulated at end-stage disease, and suggesting that activation of calcium-sensitive signalling pathways are sustained from early stages of disease development. Increases in calpain-1 activity were associated with elevated activity of the endogenous calpain inhibitor, calpastatin, itself a known calpain substrate. Activation of the tau kinases, glycogen-kinase synthase-3 and cyclin-dependent kinase 5 were also found to occur in Braak stage II-III brain, and these preceded global elevations in tau phosphorylation and the loss of post-synaptic markers. In addition, we identified transient increases in total amyloid precursor protein and pre-synaptic markers in Braak stage II-III brain, that were lost by end stage Alzheimer's disease, that may be indicative of endogenous compensatory responses to the initial stages of neurodegeneration. These findings provide insight into the molecular events that underpin the progression of Alzheimer's disease, and further highlight the rationale for investigating novel treatment

  7. μ- and m-calpain expression and activity changes following diethylstilbestrol injection in the rat anterior pituitary

    Institute of Scientific and Technical Information of China (English)

    Weijiang Zhao; Zhongfang Shi; Fang Yuan; Guilin Li; Yazhuo Zhang; Zhongcheng Wang

    2011-01-01

    Little is known about changes in calpain activity in the pituitary gland.In the present study,μ- and m-calpain activity changes were detected in the rat anterior pituitary following intraperitoneal injection of diethylstilbestrol.Double-immunofluorescence labeling confirmed colocalization of μ - and m-calpain in prolactin-secreting cells (lactotrophs).Western blot analysis revealed significantly increased expression of both calpains,which accompanied upregulated cytosol and membrane zymographic activities at 12 weeks following diethylstilbestrol injection,compared with rats injected with sunflower oil.Moreover,following estrogen injection,pituitary gland pathological damage gradually worsened with increasing time.Results demonstrated that estrogen regulated calpain expression and activity,and both calpains participated in the pathophysiological processes of the pituitary gland.Ubiquitous calpain expression could serve as an effective target for anti-estrogen drugs.

  8. Effect of protein S-nitrosylation on autolysis and catalytic ability of μ-calpain.

    Science.gov (United States)

    Liu, Rui; Li, Yupin; Wang, Mengqin; Zhou, Guanghong; Zhang, Wangang

    2016-12-15

    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. PMID:27451206

  9. Calpeptin, not calpain, directly inhibits an ion channel of the inner mitochondrial membrane.

    Science.gov (United States)

    Derksen, Maria; Vorwerk, Christian; Siemen, Detlef

    2016-05-01

    The permeability transition pore (PTP) of inner mitochondrial membranes is a large conductance pathway for ions up to 1500 Da which opening is responsible for ion equilibration and loss of membrane potential in apoptosis and thus in several neurodegenerative diseases. The PTP can be regulated by the Ca(2+)-activated mitochondrial K channel (BK). Calpains are Ca(2+)-activated cystein proteases; calpeptin is an inhibitor of calpains. We wondered whether calpain or calpeptin can modulate activity of PTP or BK. Patch clamp experiments were performed on mitoplasts of rat liver (PTP) and of an astrocytoma cell line (BK). Channel-independent open probability (P o) was determined (PTP) and, taking into account the number of open levels, NPo by single channel analysis (BK). We find that PTP in the presence of Ca(2+) (200 μM) is uninfluenced by calpain (13 nM) and shows insignificant decrease by the calpain inhibitor calpeptin (1 μM). The NPo of the BK is insensitive to calpain (54 nM), too. However, it is significantly and reversibly inhibited by the calpain inhibitor calpeptin (IC50 = 42 μM). The results agree with calpeptin-induced activation of the PTP via inhibition of the BK. Screening experiments with respirometry show calpeptin effects, fitting to inhibition of the BK by calpeptin, and strong inhibition of state 3 respiration. PMID:26108743

  10. Calpain 4 is not necessary for LFA-1-mediated function in CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Sarah A Wernimont

    Full Text Available BACKGROUND: T cell activation and immune synapse formation require the appropriate activation and clustering of the integrin, LFA-1. Previous work has reported that the calpain family of calcium-dependent proteases are important regulators of integrin activation and modulate T cell adhesion and migration. However, these studies have been limited by the use of calpain inhibitors, which have known off-target effects. METHODOLOGY/PRINCIPAL FINDINGS: Here, we used a LoxP/CRE system to specifically deplete calpain 4, a small regulatory calpain subunit required for expression and activity of ubiquitously expressed calpains 1 and 2, in CD4+ T cells. CD4+ and CD8+ T cells developed normally in Capn4(F/F:CD4-CRE mice and had severely diminished expression of Calpain 1 and 2, diminished talin proteolysis and impaired casein degradation. Calpain 4-deficient T cells showed no difference in adhesion or migration on the LFA-1 ligand ICAM-1 compared to control T cells. Moreover, there was no impairment in conjugation between Capn4(F/F:CD4-CRE T cells and antigen presenting cells, and the conjugates were still capable of polarizing LFA-1, PKC-theta and actin to the immune synapse. Furthermore, T cells from Capn4(F/F:CD4-CRE mice showed normal proliferation in response to either anti-CD3/CD28 coated beads or cognate antigen-loaded splenocytes. Finally, there were no differences in the rates of apoptosis following extrinsic and intrinsic apoptotic stimuli. CONCLUSION/SIGNIFICANCE: Our findings demonstrate that calpain 4 is not necessary for LFA-1-mediated adhesion, conjugation or migration. These results challenge previous reports that implicate a central role for calpains in the regulation of T cell LFA-1 function.

  11. Cloning, expression, and polymorphism of the porcine calpain10 gene

    Institute of Scientific and Technical Information of China (English)

    Xiuqin Yang; Di Liu; Hao Yu; Lijuan Guo; Hui Liu

    2008-01-01

    Calpains are calcium-regulated protcases involved in cellular functions that include muscle proteolysis both ante- and postmortem. This study was designed to clone the complete coding sequence of the porcine calpain10 gene, CAPN10, to analyze its expression characteristics and to investigate its polymorphism. Two isoforms of the CAPN10 gene, CAPN10A and CAPN10B, were obtained by reverse transcriptionpolymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods combined with in silico cloning. RT-PCR results indicated that CAPN10 mRNA was ubiquitously expressed in all tissues examined and, with increasing age,the expression level increased in muscles at six different growth points. In the same tissues, the expression level of CAPN10A was higher than that of CAPN10B. In addition,three single nucleotide polymorphisms were detected by the PCR-single-stranded conformational polymorphism method and by comparing the sequences of Chinese Min pigs with those of Yorkshire pigs. C527T mutation was a missense mutation and led to transforming Pro into Leu at the 176th amino acid. The results of the current study provided basic molecular information for further study of the function of the porcine CAPN10 gene.

  12. Calpain system and its involvement in myocardial ischemia and reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Christiane; Neuhof; Heinz; Neuhof

    2014-01-01

    Calpains are ubiquitous non-lysosomal Ca2+-dependent cysteine proteases also present in myocardial cytosol and mitochondria.Numerous experimental studies reveal an essential role of the calpain system in myocardial injury during ischemia,reperfusion and postischemic structural remodelling.The increasing Ca2+-content and Ca2+-overload in myocardial cytosol and mitochondria during ischemia and reperfusion causes an activation of calpains.Upon activation they are able to injure the contractile apparatus and impair the energy production by cleaving structural and functional proteins of myocytes and mitochondria.Besides their causal involvement in acute myocardial dysfunction they are also involved in structural remodelling after myocardial infarction by the generation and release of proapoptotic factors from mitochondria.Calpain inhibition can prevent or attenuate myocardial injury during ischemia,reperfusion,and in later stages of myocardial infarction.

  13. LOX-1 in macrophage migration in response to ox-LDL and the involvement of calpains.

    Science.gov (United States)

    Wang, Xianwei; Ding, Zufeng; Lin, Juntang; Guo, Zhikun; Mehta, Jawahar L

    2015-11-01

    Previous studies have shown that oxidized low-density lipoprotein (ox-LDL) inhibits macrophage migration, but the precise mechanisms remain unclear. Lectin-like ox-LDL receptor-1 (LOX-1) is a scavenger receptor that is expressed in macrophages and binds ox-LDL. Calpains, a family of calcium-dependent proteases, influence several aspects of cell migration. In this study, we investigated the role of LOX-1 in macrophage migration in response to ox-LDL and the involvement of calpains in this process. Peritoneal macrophages from wild type C57BL/6 mice were exposed to different concentrations of ox-LDL (1-20 μg/mL), and expression of LOX-1 and calpain-1 and -2, cell migration and intracellular calcium (Ca(2+)in) were measured. Our results showed that ox-LDL stimulated LOX-1 and calpain-2 expression, and inhibited calpain-1 expression in a dose- and time-dependent manner. Further, ox-LDL inhibited macrophage migration and increased Ca(2+)in concentration in macrophages. To further elucidate the role of LOX-1 in ox-LDL-impaired macrophage migration, we isolated peritoneal macrophages from LOX-1 knockout mice, and treated them with ox-LDL. Interestingly, calpain-1 expression was much higher, and calpain-2 expression was lower in LOX-1 knockout macrophages than in wild-type macrophages following exposure to ox-LDL. LOX-1 deletion significantly improved macrophage migration and decreased Ca(2+)in concentration. These data indicate that LOX-1 is, at least in part, responsible for the inhibitory effect of ox-LDL on macrophage migration and this process involves calpain-1 and -2.

  14. Chronic intermittent ethanol induced axon and myelin degeneration is attenuated by calpain inhibition.

    Science.gov (United States)

    Samantaray, Supriti; Knaryan, Varduhi H; Patel, Kaushal S; Mulholland, Patrick J; Becker, Howard C; Banik, Naren L

    2015-10-01

    Chronic alcohol consumption causes multifaceted damage to the central nervous system (CNS), underlying mechanisms of which are gradually being unraveled. In our previous studies, activation of calpain, a calcium-activated neutral protease has been found to cause detrimental alterations in spinal motor neurons following ethanol (EtOH) exposure in vitro. However, it is not known whether calpain plays a pivotal role in chronic EtOH exposure-induced structural damage to CNS in vivo. To test the possible involvement of calpain in EtOH-associated neurodegenerative mechanisms the present investigation was conducted in a well-established mouse model of alcohol dependence - chronic intermittent EtOH (CIE) exposure and withdrawal. Our studies indicated significant loss of axonal proteins (neurofilament light and heavy, 50-60%), myelin proteins (myelin basic protein, 20-40% proteolipid protein, 25%) and enzyme (2', 3'-cyclic-nucleotide 3'-phosphodiesterase, 21-55%) following CIE in multiple regions of brain including hippocampus, corpus callosum, cerebellum, and importantly in spinal cord. These CIE-induced deleterious effects escalated after withdrawal in each CNS region tested. Increased expression and activity of calpain along with enhanced ratio of active calpain to calpastatin (sole endogenous inhibitor) was observed after withdrawal compared to EtOH exposure. Pharmacological inhibition of calpain with calpeptin (25 μg/kg) prior to each EtOH vapor inhalation significantly attenuated damage to axons and myelin as demonstrated by immuno-profiles of axonal and myelin proteins, and Luxol Fast Blue staining. Calpain inhibition significantly protected the ultrastructural integrity of axons and myelin compared to control as confirmed by electron microscopy. Together, these findings confirm CIE exposure and withdrawal induced structural alterations in axons and myelin, predominantly after withdrawal and corroborate calpain inhibition as a potential protective strategy against

  15. Post-mortem kinetics of meat tenderness and the components of the calpain system in bull skeletal muscle.

    Science.gov (United States)

    Thomson, B C; Dobbie, P M; Singh, K; Speck, P A

    1996-11-01

    Eight strip loins (M. longissimus dorsi) from pasture fed Friesian bulls were aged at 15 °C for a range of times from 1 to 120 h. pH declined from 6.29 (SE 0.119) one hour post slaughter to an ultimate pH of 5.48 (SE 0.013). The activities of the components of the calpain system (μ-calpain, m-calpain and calpastatin) were determined after separation on a DEAE-sephacel column. There was a dramatic decline in μ-calpain activity post slaughter with a complete disappearance within 48 h. The rates of decline in m-calpain and calpastatin activity were slower with 30% and 50% remaining 120 h post slaughter, respectively. The rapid decline in μ-calpain activity relative to the calpastatin activity is likely to reduce the degree of tenderisation and ultimate tenderness of the meat.

  16. Calpain activation induced by glucose deprivation is mediated by oxidative stress and contributes to neuronal damage.

    Science.gov (United States)

    Páramo, Blanca; Montiel, Teresa; Hernández-Espinosa, Diego R; Rivera-Martínez, Marlene; Morán, Julio; Massieu, Lourdes

    2013-11-01

    The mechanisms leading to neuronal death during glucose deprivation have not been fully elucidated, but a role of oxidative stress has been suggested. In the present study we have investigated whether the production of reactive oxygen species during glucose deprivation, contributes to the activation of calpain, a calcium-dependent protease involved in neuronal injury associated with brain ischemia and cerebral trauma. We have observed a rapid activation of calpain, as monitored by the cleavage of the cytoskeletal protein α-spectrin, after glucose withdrawal, which is reduced by inhibitors of xanthine oxidase, phospholipase A2 and NADPH oxidase. Results suggest that phospholipase A2 and NADPH oxidase contribute to the early activation of calpain after glucose deprivation. In particular NOX2, a member of the NADPH oxidase family is involved, since reduced stimulation of calpain activity is observed after glucose deprivation in hippocampal slices from transgenic mice lacking a functional NOX2. We observed an additive effect of the inhibitors of xanthine oxidase and phospholipase A2 on both ROS production and calpain activity, suggesting a synergistic action of these two enzymes. The present results provide new evidence showing that reactive oxygen species stimulate calpain activation during glucose deprivation and that this mechanism is involved in neuronal death.

  17. Calpain-like: A Ca(2+) dependent cystein protease in Entamoeba histolytica cell death.

    Science.gov (United States)

    Monroy, Virginia Sánchez; Flores, Olivia Medel; García, Consuelo Gómez; Maya, Yesenia Chávez; Fernández, Tania Domínguez; Pérez Ishiwara, D Guillermo

    2015-12-01

    Entamoeba histolytica programmed cell death (PCD) induced by G418 is characterized by the release of important amounts of intracellular calcium from reservoirs. Nevertheless, no typical caspases have been detected in the parasite, the PCD phenotype is inhibited by the cysteine protease inhibitor E-64. These results strongly suggest that Ca(2+)-dependent proteases could be involved in PCD. In this study, we evaluate the expression and activity of a specific dependent Ca(2+) protease, the calpain-like protease, by real-time quantitative PCR (RTq-PCR), Western blot assays and a enzymatic method during the induction of PCD by G418. Alternatively, using cell viability and TUNEL assays, we also demonstrated that the Z-Leu-Leu-Leu-al calpain inhibitor reduced the rate of cell death. The results demonstrated 4.9-fold overexpression of calpain-like gene 1.5 h after G418 PCD induction, while calpain-like protein increased almost two-fold with respect to basal calpain-like expression after 3 h of induction, and calpain activity was found to be approximately three-fold higher 6 h after treatment compared with untreated trophozoites. Taken together, these results suggest that this Ca(2+)-dependent protease could be involved in the executory phase of PCD.

  18. Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer’s disease brain

    OpenAIRE

    Kurbatskaya, Ksenia; Phillips, Emma Claire; Croft, Cara Louise; Dentoni, Giacomo; Hughes, Martina; Wade, Matthew Austen James; Al-Sarraj, Safa; Troakes, Claire; O'Neill, Michael; Gomez Perez-Nievas, Beatriz; Hanger, Diane Pamela; Noble, Wendy Jane

    2016-01-01

    Alterations in calcium homeostasis are widely reported to contribute to synaptic degeneration and neuronal loss in Alzheimer’s disease. Elevated cytosolic calcium concentrations lead to activation of the calcium-sensitive cysteine protease, calpain, which has a number of substrates known to be abnormally regulated in disease. Analysis of human brain has shown that calpain activity is elevated in AD compared to controls, and that calpain-mediated proteolysis regulates the activity of important...

  19. Silibinin induces apoptosis via calpain-dependent AIF nuclear translocation in U87MG human glioma cell death

    Directory of Open Access Journals (Sweden)

    Kim Yong K

    2011-04-01

    Full Text Available Abstract Background Silibinin, a natural polyphenolic flavonoid, has been reported to induce cell death in various cancer cell types. However, the molecular mechanism is not clearly defined. Our previous study showed that silibinin induces glioma cell death and its effect was effectively prevented by calpain inhibitor. The present study was therefore undertaken to examine the role of calpain in the silibinin-induced glioma cell death. Methods U87MG cells were grown on well tissue culture plates and cell viability was measured by MTT assay. ROS generation and △ψm were estimated using the fluorescence dyes. PKC activation and Bax expression were measured by Western blot analysis. AIF nuclear translocation was determined by Western blot and immunocytochemistry. Results Silibinin induced activation of calpain, which was blocked by EGTA and the calpain inhibitor Z-Leu-Leu-CHO. Silibinin caused ROS generation and its effect was inhibited by calpain inhibitor, the general PKC inhibitor GF 109203X, the specific PKCδ inhibitor rottlerin, and catalase. Silibinin-induce cell death was blocked by calpain inhibitor and PKC inhibitors. Silibinin-induced PKCδ activation and disruption of △ψm were prevented by the calpain inhibitor. Silibinin induced AIF nuclear translocation and its effect was prevented by calpain inhibitor. Transfection of vector expressing microRNA of AIF prevented the silibinin-induced cell death. Conclusions Silibinin induces apoptotic cell death through a calpain-dependent mechanism involving PKC, ROS, and AIF nuclear translocation in U87MG human glioma cells.

  20. Calpastatin exon 1B-derived peptide, a selective inhibitor of calpain: enhancing cell permeability by conjugation with penetratin.

    Science.gov (United States)

    Gil-Parrado, Shirley; Assfalg-Machleidt, Irmgard; Fiorino, Ferdinando; Deluca, Dominga; Pfeiler, Dietmar; Schaschke, Norbert; Moroder, Luis; Machleidt, Werner

    2003-03-01

    The ubiquitous calpains, mu- and m-calpain, have been implicated in essential physiological processes and various pathologies. Cell-permeable specific inhibitors are important tools to elucidate the roles of calpains in cultivated cells and animal models. The synthetic N-acetylated 27-mer peptide derived from exon B of the inhibitory domain 1 of human calpastatin (CP1B) is unique as a potent and highly selective reversible calpain inhibitor, but is poorly cell-permeant. By addition of N-terminal cysteine residues we have generated a disulfide-conjugated CP1B with the cell-penetrating 16-mer peptide penetratin derived from the third helix of the Antennapedia homeodomain protein. The inhibitory potency and selectivity of CP1B for calpain versus cathepsin B and L, caspase 3 and the proteasome was not affected by the conjugation with penetratin. The conjugate was shown to efficiently penetrate into living LCLC 103H cells, since it prevents ionomycin-induced calpain activation at 200-fold lower concentration than the non-conjugated inhibitor and is able to reduce calpain-triggered apoptosis of these cells. Penetratin-conjugated CP1B seems to be a promising alternative to the widely used cell-permeable peptide aldehydes (e.g. calpain inhibitor 1) which inhibit the lysosomal cathepsins and partially the proteasome as well or even better than the calpains. PMID:12715890

  1. Loss of Calpain 3 Proteolytic Activity Leads to Muscular Dystrophy and to Apoptosis-Associated Iκbα/Nuclear Factor κb Pathway Perturbation in Mice

    OpenAIRE

    RICHARD, Isabelle; Roudaut, Carinne; Marchand, Sylvie; Baghdiguian, Stephen; Herasse, Muriel; Stockholm, Daniel; Ono, Yasuko; Suel, Laurence; Bourg, Nathalie; Sorimachi, Hiroyuki; Lefranc, Gérard; Fardeau, Michel; Sébille, Alain; Beckmann, Jacques S.

    2000-01-01

    Calpain 3 is known as the skeletal muscle–specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. It was previously shown that defects in the human calpain 3 gene are responsible for limb girdle muscular dystrophy type 2A (LGMD2A), an inherited disease affecting predominantly the proximal limb muscles. To better understand the function of calpain 3 and the pathophysiological mechanisms of LGMD2A and also to develop an adequate model for therapy research, we...

  2. Overview of calpain-mediated regulation of bone and fat mass in osteoblasts.

    Science.gov (United States)

    Shimada, Masako

    2013-05-01

    The receptor for parathyroid hormone (PTH) and PTH-related peptide (PTH1R) belongs to the class II G protein-coupled receptor superfamily. The calpain small subunit encoded by the gene Capns1 is the second protein and the first enzyme identified by a yeast two-hybrid screen using the intracellular C-terminal tail of the rat PTH1R. The calpain regulatory small subunit forms a heterodimer with the calpain large catalytic subunit and modulates various cellular functions as a cysteine protease. To investigate a physiological role of the calpain small subunit in cells of the osteoblast lineage, we generated osteoblast-specific Capns1 knockout mouse models and characterized their bone phenotype. Molecular mechanisms by which calpain modulates cell proliferation of the osteoblast lineage were further examined in vitro. Moreover, we utilized the mutant mice as a disease model of osteoporosis accompanied with impaired bone resorptive function and suggested a possible clinical translation of our basic research finding.

  3. Tissue-Specific Expression of the Chicken Calpain2 Gene

    Directory of Open Access Journals (Sweden)

    Zeng-Rong Zhang

    2010-01-01

    Full Text Available We quantified chicken calpain 2 (CAPN2 expression in two Chinese chicken breeds (mountainous black-bone chicken breed [MB] and a commercial meat type chicken breed [S01] to discern the tissue and ontogenic expression pattern and its effect on muscle metabolism. Real-time quantitative PCR assay was developed for accurate measurement of the CAPN2 mRNA expression in various tissues from chickens of different ages (0, 2, 4, 6, 8, 10, and 12 weeks. Results showed that the breast muscle and leg muscle tissues had the highest expression of CAPN2 compared to the other tissues from the same individual (P<.05. Overall, the CAPN2 mRNA level exhibited a “rise” developmental change in all tissues. The S01 chicken had a higher expression of the CAPN2 mRNA in all tissues than the MB chicken. Our results suggest that chicken CAPN2 expression may be related to chicken breeds and tissues.

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

    Science.gov (United States)

    Díaz, B G; Gross, S; Assfalg-Machleidt, I; Pfeiler, D; Gollmitzer, N; Gabrijelcic-Geiger, D; Stubbs, M T; Fritz, H; Auerswald, E A; Machleidt, W

    2001-01-01

    Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit mu- and m-calpain. In an attempt to elucidate the structural requirements of cystatins for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and deltaL110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefin B by the corresponding KD2 sequence resulted in a calpain inhibitor of Ki = 188 nM. Deletion of L110, which forms a beta-bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of mu-calpain 4- to 8-fold. All engineered cystatins were temporary inhibitors of calpain due to slow substrate-like cleavage of a single peptide bond corresponding to Gly9-Ala10 in chicken cystatin. Biomolecular interaction analysis revealed that, unlike calpastatin, the cystatin-type inhibitors do not bind to the calmodulin-like domain of the small subunit of calpain, and their interaction with the mu-calpain heterodimer is completely prevented by a synthetic peptide comprising subdomain B of calpastatin domain 1. Based on these results we propose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatin-like mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold.

  5. Calpain inhibitor, MDL 28170 confer electrophysiological, nociceptive and biochemical improvement in diabetic neuropathy.

    Science.gov (United States)

    Kharatmal, Shivsharan B; Singh, Jitendra N; Sharma, Shyam S

    2015-10-01

    Calpain plays an important role in the pathophysiology of neurological and cardiovascular complications, but its functional association in diabetic neuropathy is not yet elucidated. Therefore, we investigated the role of calpain in modulation of tetrodotoxin-resistant sodium channels (TTX-R Na(+) channels) in dorsal root ganglion (DRG) neurons using a pharmacological approach. The effects of a calpain inhibitor, MDL 28170 (3 and 10 mg/kg, i.p.) on TTX-R Na(+) channels in DRG neurons of streptozotocin-induced diabetic rats were assessed by using whole-cell patch-clamp technique. In addition to this biochemical, functional and behavioral deficits were also measured. Diabetic rats demonstrated the mechanical allodynia and thermal hyperalgesia with reduced nerve perfusion and conduction velocity as compared to control. MDL 28170 treatments significantly recovered these functional and nociceptive deficits. Moreover, diabetic rats exhibited increased calpain activation, lipid peroxidation and proinflammatory cytokines as compared to control. Drug treatment significantly improved these biochemical deficits. Additionally, DRG neurons from diabetic rats illustrated a significant increase in TTX-R sodium current (INa) density as compared to control. MDL 28170 treatments in diabetic rats significantly blocked the altered channel kinetics with hyperpolarizing shift in voltage-dependence of steady-state activation and inactivation curves. All together, our study provides evidence that calpain activation is directly associated with alterations in TTX-R Na(+) channels and triggers functional, nociceptive and biochemical deficits in experimental diabetic neuropathy. The calpain inhibitor, MDL 28710 have shown beneficial effects in alleviating diabetic neuropathy via modulation of TTX-R Na(+) channel kinetics and reduction of oxidative stress and neuro-inflammation.

  6. Influence of early pH decline on calpain activity in porcine muscle

    DEFF Research Database (Denmark)

    Pomponio, Luigi; Ertbjerg, Per; Karlsson, Anders H;

    2010-01-01

    . The rate of pH decline early post-mortem differed between the three groups, but the ultimate pH values were similar at 24 h. Calpain activity and autolysis from 1 to 72 h post-mortem were determined using casein zymography and studied in relation to myofibrillar fragmentation. Colour and drip loss were...

  7. The Prediction of Calpain Cleavage Sites with the mRMR and IFS Approaches

    Directory of Open Access Journals (Sweden)

    Wenyi Zhang

    2013-01-01

    Full Text Available Calpains are an important family of the Ca2+-dependent cysteine proteases which catalyze the limited proteolysis of many specific substrates. Calpains play crucial roles in basic physiological and pathological processes, and identification of the calpain cleavage sites may facilitate the understanding of the molecular mechanisms and biological function. But traditional experiment approaches to predict the sites are accurate, and are always labor-intensive and time-consuming. Thus, it is common to see that computational methods receive increasing attention due to their convenience and fast speed in recent years. In this study, we develop a new predictor based on the support vector machine (SVM with the maximum relevance minimum redundancy (mRMR method followed by incremental feature selection (IFS. And we concern the feature of physicochemical/biochemical properties, sequence conservation, residual disorder, secondary structure, and solvent accessibility to represent the calpain cleavage sites. Experimental results show that the performance of our predictor is better than several other state-of- the-art predictors, whose average prediction accuracy is 79.49%, sensitivity is 62.31%, and specificity is 88.12%. Since user-friendly and publicly accessible web servers represent the future direction for developing practically more useful predictors, here we have provided a web-server for the method presented in this paper.

  8. Calpain system protein expression in carcinomas of the pancreas, bile duct and ampulla

    International Nuclear Information System (INIS)

    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. 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. 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). 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

  9. Chronic exposure to paclitaxel diminishes phosphoinositide signaling by calpain-mediated neuronal calcium sensor-1 degradation.

    Science.gov (United States)

    Boehmerle, Wolfgang; Zhang, Kun; Sivula, Michael; Heidrich, Felix M; Lee, Yashang; Jordt, Sven-Eric; Ehrlich, Barbara E

    2007-06-26

    Paclitaxel (Taxol) is a well established chemotherapeutic agent for the treatment of solid tumors, but it is limited in its usefulness by the frequent induction of peripheral neuropathy. We found that prolonged exposure of a neuroblastoma cell line and primary rat dorsal root ganglia with therapeutic concentrations of Taxol leads to a reduction in inositol trisphosphate (InsP(3))-mediated Ca(2+) signaling. We also observed a Taxol-specific reduction in neuronal calcium sensor 1 (NCS-1) protein levels, a known modulator of InsP(3) receptor (InsP(3)R) activity. This reduction was also found in peripheral neuronal tissue from Taxol treated animals. We further observed that short hairpin RNA-mediated NCS-1 knockdown had a similar effect on phosphoinositide-mediated Ca(2+) signaling. When NCS-1 protein levels recovered, so did InsP(3)-mediated Ca(2+) signaling. Inhibition of the Ca(2+)-activated protease mu-calpain prevented alterations in phosphoinositide-mediated Ca(2+) signaling and NCS-1 protein levels. We also found that NCS-1 is readily degraded by mu-calpain in vitro and that mu-calpain activity is increased in Taxol but not vehicle-treated cells. From these results, we conclude that prolonged exposure to Taxol activates mu-calpain, which leads to the degradation of NCS-1, which, in turn, attenuates InsP(3)mediated Ca(2+) signaling. These findings provide a previously undescribed approach to understanding and treating Taxol-induced peripheral neuropathy. PMID:17581879

  10. Aspirin Has Antitumor Effects via Expression of Calpain Gene in Cervical Cancer Cells

    Directory of Open Access Journals (Sweden)

    Sang Koo Lee

    2008-01-01

    Full Text Available Aspirin and other nonsteroidal anti-inflammatory drugs show efficacy in the prevention of cancers. It is known that they can inhibit cyclooxygenases, and some studies have shown that they can induce apoptosis. Our objective in this study was to investigate the mechanism by which aspirin exerts its apoptosis effects in human cervical cancer HeLa cells. The effect of aspirin on the gene expression was studied by differential mRNA display RT-PCR. Among the isolated genes, mu-type calpain gene was upregulated by aspirin treatment. To examine whether calpain mediates the antitumor effects, HeLa cells were stably transfected with the mammalian expression vector pCR3.1 containing mu-type calpain cDNA (pCRCAL/HeLa, and tumor formations were measured in nude mice. When tumor burden was measured by day 49, HeLa cells and pCR/HeLa cells (vector control produced tumors of 2126 mm3 and 1638 mm3, respectively, while pCRCAL/HeLa cells produced markedly smaller tumor of 434 mm3 in volume. The caspase-3 activity was markedly elevated in pCRCAL/HeLa cells. The increased activity levels of caspase-3 in pCRCAL/HeLa cells, in parallel with the decreased tumor formation, suggest a correlation between caspase-3 activity and calpain protein. Therefore, we conclude that aspirin-induced calpain mediates an antitumor effect via caspase-3 in cervical cancer cells.

  11. Reactive protoplasmic and fibrous astrocytes contain high levels of calpain-cleaved alpha 2 spectrin.

    Science.gov (United States)

    Kim, Jung H; Kwon, Soojung J; Stankewich, Michael C; Huh, Gi-Yeong; Glantz, Susan B; Morrow, Jon S

    2016-02-01

    Calpain, a family of calcium-dependent neutral proteases, plays important roles in neurophysiology and pathology through the proteolytic modification of cytoskeletal proteins, receptors and kinases. Alpha 2 spectrin (αII spectrin) is a major substrate for this protease family, and the presence of the αII spectrin breakdown product (αΙΙ spectrin BDP) in a cell is evidence of calpain activity triggered by enhanced intracytoplasmic Ca(2+) concentrations. Astrocytes, the most dynamic CNS cells, respond to micro-environmental changes or noxious stimuli by elevating intracytoplasmic Ca(2+) concentration to become activated. As one measure of whether calpains are involved with reactive glial transformation, we examined paraffin sections of the human cerebral cortex and white matter by immunohistochemistry with an antibody specific for the calpain-mediated αΙΙ spectrin BDP. We also performed conventional double immunohistochemistry as well as immunofluorescent studies utilizing antibodies against αΙΙ spectrin BDP as well as glial fibrillary acidic protein (GFAP). We found strong immunopositivity in selected protoplasmic and fibrous astrocytes, and in transitional forms that raise the possibility of some of fibrous astrocytes emerging from protoplasmic astrocytes. Immunoreactive astrocytes were numerous in brain sections from cases with severe cardiac and/or respiratory diseases in the current study as opposed to our previous study of cases without significant clinical conditions that failed to reveal such remarkable immunohistochemical alterations. Our study suggests that astrocytes become αΙΙ spectrin BDP immunopositive in various stages of activation, and that spectrin cleavage product persists even in fully reactive astrocytes. Immunohistochemistry for αΙΙ spectrin BDP thus marks reactive astrocytes, and highlights the likelihood that calpains and their proteolytic processing of spectrin participate in the morphologic and physiologic transition from

  12. Hippocampal Cortactin Levels are Reduced Following Spatial Working Memory Formation, an Effect Blocked by Chronic Calpain Inhibition

    Directory of Open Access Journals (Sweden)

    Mikel L. Olson

    2015-06-01

    Full Text Available The mechanism by which the hippocampus facilitates declarative memory formation appears to involve, among other things, restructuring of the actin cytoskeleton within neuronal dendrites. One protein involved in this process is cortactin, which is an important link between extracellular signaling and cytoskeletal reorganization. In this paper, we demonstrate that total hippocampal cortactin, as well as Y421-phosphorylated cortactin are transiently reduced following spatial working memory formation in the radial arm maze (RAM. Because cortactin is a substrate of the cysteine protease calpain, we also assessed the effect of chronic calpain inhibition on RAM performance and cortactin expression. Calpain inhibition impaired spatial working memory and blocked the reduction in hippocampal cortactin levels following RAM training. These findings add to a growing body of research implicating cortactin and calpain in hippocampus-dependent memory formation.

  13. A New Insight into the Role of Calpains in Post-mortem Meat Tenderization in Domestic Animals: A review

    OpenAIRE

    Lian, Ting; Wang, Linjie; Liu, Yiping

    2013-01-01

    Tenderness is the most important meat quality trait, which is determined by intracellular environment and extracellular matrix. Particularly, specific protein degradation and protein modification can disrupt the architecture and integrity of muscle cells so that improves the meat tenderness. Endogenous proteolytic systems are responsible for modifying proteinases as well as the meat tenderization. Abundant evidence has testified that calpains (CAPNs) including calpain I (CAPN1) and calpastati...

  14. Trypanosoma cruzi: a stage-specific calpain-like protein is induced after various kinds of stress.

    Science.gov (United States)

    Giese, Viviane; Dallagiovanna, Bruno; Marchini, Fabricio K; Pavoni, Daniela P; Krieger, Marco A; Goldenberg, Samuel

    2008-09-01

    Calpains are calcium-dependent cysteine proteinases found in all living organisms and are involved in diverse cellular processes. Calpain-like proteins have been reported after in silico analysis of the Tritryps genome and are believed to play important roles in cell functions of trypanosomatids. We describe the characterization of a member of this family, which is differentially expressed during the life-cycle of Trypanosoma cruzi.

  15. Changes of Calpain mRNA Level in Rat Hepatocyte after Recurrent Intraperitoneal Administration of Ceium Nitrate

    Institute of Scientific and Technical Information of China (English)

    杨维东; 王艇; 刘洁生; 龚孟濂; 雷衡毅; 杨燕生

    2001-01-01

    The effect of Ce(NO3)3 on expression of calpain in rat hepatocyte was studied by means of reverse transcription-polymerase chain reaction (RT-PCR). The result shows that high dose of Ce(NO3)3 (50 mg.kg-1) induces the increase of expression of calpain mRNA, but low dose of Ce(NO3)3 (1 mg.kg-1) does not. Possible mechanism for this phenomenon was discussed.

  16. Calpain-1 Mediated Disorder of Pyrophosphate Metabolism Contributes to Vascular Calcification Induced by oxLDL.

    Science.gov (United States)

    Tang, Futian; Chan, Erqing; Lu, Meili; Zhang, Xiaowen; Dai, Chunmei; Mei, Meng; Zhang, Suping; Wang, Hongxin; Song, Qing

    2015-01-01

    We previously reported that oxidized low density lipoprotein (oxLDL) accelerated the calcification in aorta of rats and rat vascular smooth muscle cells (RVSMCs). However, the molecular mechanism underlying the acceleration remains poorly understood. The present study aimed to investigate the role of calpain-1, Ca2+-sensitive intracellular cysteine proteases, in the vascular calcification of rats treated with both high dose of vitamin D2 and high cholesterol diet. The results showed that calpain activity significantly increased in calcified aortic tissue of rats and RVSMCs treated with oxLDL. Specific calpain inhibitor I (CAI, 0.5mg/kg, intraperitoneal) inhibited the vascular calcification in rats with hypercholesterolemia accompanied by the increase in the level of extracellular inorganic pyrophosphate (PPi), the endogenous inhibitor of vascular calcification. In addition, CAI increased the content of adenosine triphosphate (ATP), decreased the activity, mRNA and protein expression of alkaline phosphatase (ALP) and reduced the production of superoxide anion in calcified aortic tissue. CAI also increased the activity of ATP synthase as well as protein expression of ATP5D, δ subunit of ATP synthase. In the in vitro study, suppression of calpain-1 using siRNA assay inhibited the calcium deposition, increased the levels of PPi and ATP, improved the activity of ATP synthase as well as protein expression of ATP5D in RVSMCs treated with oxLDL. Calpain-1 suppression also decreased the activity, mRNA and protein expression of ALP and reduced the mitochondrial ROS (Mito-ROS) production in RVSMCs. However, mito-TEMPO, the mitochondria-targeted ROS scavenger, reduced the calcium deposition, increased the PPi in culture medium, decreased the activity, mRNA and protein expression of ALP in RVSMCs treated with oxLDL. Taken together, the results suggested that calpain-1 activation plays critical role in vascular calcification caused by oxLDL, which might be mediated by PPi

  17. Chronic administration of a leupeptin-derived calpain inhibitor fails to ameliorate severe muscle pathology in a canine model of Duchenne muscular dystrophy

    OpenAIRE

    MartinKChilders; DanielJBogan; MelanieHolder; HanselGreiner; RobertGrange

    2012-01-01

    Calpains likely play a role in the pathogenesis of Duchenne muscular dystrophy (DMD). Accordingly, calpain inhibition may provide therapeutic benefit to DMD patients. In the present study, we sought to measure benefit from administration of a novel calpain inhibitor, C101, in a canine muscular dystrophy model. Specifically, we tested the hypothesis that treatment with C101 mitigates progressive weakness and severe muscle pathology observed in young dogs with golden retriever muscular dystroph...

  18. Crystal structure of calpain-3 penta-EF-hand (PEF) domain - a homodimerized PEF family member with calcium bound at the fifth EF-hand

    Energy Technology Data Exchange (ETDEWEB)

    Partha, Sarathy K.; Ravulapalli, Ravikiran; Allingham, John S.; Campbell, Robert L.; Davies, Peter L. [Queens

    2014-08-21

    Calpains are Ca2+dependent intracellular cysteine proteases that cleave a wide range of protein substrates to help implement Ca2+ signaling in the cell. The major isoforms of this enzyme family, calpain-1 and calpain-2, are heterodimers of a large and a small subunit, with the main dimer interface being formed through their C-terminal penta-EF hand (PEF) domains. Calpain-3, or p94, is a skeletal muscle-specific isoform that is genetically linked to limb-girdle muscular dystrophy. Biophysical and modeling studies with the PEF domain of calpain-3 support the suggestion that full-length calpain-3 exists as a homodimer. Here, we report the crystallization of calpain-3's PEF domain and its crystal structure in the presence of Ca2+, which provides evidence for the homodimer architecture of calpain-3 and supports the molecular model that places a protease core at either end of the elongated dimer. Unlike other calpain PEF domain structures, the calpain-3 PEF domain contains a Ca2+ bound at the EF5-hand used for homodimer association. Three of the four Ca2+-binding EF-hands of the PEF domains are concentrated near the protease core, and have the potential to radically change the local charge within the dimer during Ca2+ signaling. Examination of the homodimer interface shows that there would be steric clashes if the calpain-3 large subunit were to try to pair with a calpain small subunit.

  19. Stable expression of calpain 3 from a muscle transgene in vivo: Immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation

    OpenAIRE

    Spencer, M.J.; Guyon, J. R.; Sorimachi, H.; Potts, A; Richard, I.; Herasse, M; Chamberlain, J.; Dalkilic, I.; Kunkel, L. M.; Beckmann, J S

    2002-01-01

    Limb-girdle muscular dystrophy, type 2A (LGMD 2A), is an autosomal recessive disorder that causes late-onset muscle-wasting, and is due to mutations in the muscle-specific protease calpain 3 (C3). Although LGMD 2A would be a feasible candidate for gene therapy, the reported instability of C3 in vitro raised questions about the potential of obtaining a stable, high-level expression of C3 from a transgene in vivo. We have generated transgenic (Tg) mice with muscle-specific overexpression of ful...

  20. Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice.

    Science.gov (United States)

    Diepenbroek, Meike; Casadei, Nicolas; Esmer, Hakan; Saido, Takaomi C; Takano, Jiro; Kahle, Philipp J; Nixon, Ralph A; Rao, Mala V; Melki, Ronald; Pieri, Laura; Helling, Stefan; Marcus, Katrin; Krueger, Rejko; Masliah, Eliezer; Riess, Olaf; Nuber, Silke

    2014-08-01

    Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (αSyn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of αSyn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here, we investigated the role of calpain cleavage of αSyn in vivo by generating two opposing mouse models. We crossed into human [A30P]αSyn transgenic (i) mice deficient for calpastatin, a calpain-specific inhibitor, thus enhancing calpain activity (SynCAST(-)) and (ii) mice overexpressing human calpastatin leading to reduced calpain activity (SynCAST(+)). As anticipated, a reduced calpain activity led to a decreased number of αSyn-positive aggregates, whereas loss of calpastatin led to increased truncation of αSyn in SynCAST(-). Furthermore, overexpression of calpastatin decreased astrogliosis and the calpain-dependent degradation of synaptic proteins, potentially ameliorating the observed neuropathology in [A30P]αSyn and SynCAST(+) mice. Overall, our data further support a crucial role of calpains, particularly of calpain 1, in the pathogenesis of PD and in disease-associated aggregation of αSyn, indicating a therapeutic potential of calpain inhibition in PD. PMID:24619358

  1. Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors.

    Science.gov (United States)

    Siklos, Marton; BenAissa, Manel; Thatcher, Gregory R J

    2015-11-01

    Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy.

  2. Carcass characteristics, the calpain proteinase system, and aged tenderness of Angus and Brahman crossbred steers.

    Science.gov (United States)

    Pringle, T D; Williams, S E; Lamb, B S; Johnson, D D; West, R L

    1997-11-01

    We used 69 steers of varying percentage Brahman (B) breeding (0% B, n = 11; 25% B, n = 13; 37% B, n = 10; 50% B, n = 12; 75% B, n = 12; 100% B, n = 11) to study the relationship between carcass traits, the calpain proteinase system, and aged meat tenderness in intermediate B crosses. Calpains and calpastatin activities were determined on fresh longissimus muscle samples using anion-exchange chromatography. The USDA yield and quality grade data (24 h) were collected for each carcass. Longissimus steaks were removed and aged for 5 or 14 d for determination of shear force and 5 d for sensory panel evaluation. Even though some yield grade factors were affected by the percentage of B breeding, USDA yield grades did not differ (P > .15) between breed types. Marbling score and USDA quality grade decreased linearly (P Brahman crosses. PMID:9374310

  3. Carcass characteristics, the calpain proteinase system, and aged tenderness of Angus and Brahman crossbred steers.

    Science.gov (United States)

    Pringle, T D; Williams, S E; Lamb, B S; Johnson, D D; West, R L

    1997-11-01

    We used 69 steers of varying percentage Brahman (B) breeding (0% B, n = 11; 25% B, n = 13; 37% B, n = 10; 50% B, n = 12; 75% B, n = 12; 100% B, n = 11) to study the relationship between carcass traits, the calpain proteinase system, and aged meat tenderness in intermediate B crosses. Calpains and calpastatin activities were determined on fresh longissimus muscle samples using anion-exchange chromatography. The USDA yield and quality grade data (24 h) were collected for each carcass. Longissimus steaks were removed and aged for 5 or 14 d for determination of shear force and 5 d for sensory panel evaluation. Even though some yield grade factors were affected by the percentage of B breeding, USDA yield grades did not differ (P > .15) between breed types. Marbling score and USDA quality grade decreased linearly (P Brahman crosses.

  4. Carbamazepine suppresses calpain-mediated autophagy impairment after ischemia/reperfusion in mouse livers

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jae-Sung, E-mail: Jae.Kim@surgery.ufl.edu; Wang, Jin-Hee, E-mail: jin-hee.wang@surgery.ufl.edu; Biel, Thomas G., E-mail: Thomas.Biel@surgery.ufl.edu; Kim, Do-Sung, E-mail: do-sung.kim@surgery.med.ufl.edu; Flores-Toro, Joseph A., E-mail: Joseph.Flores-Toro@surgery.ufl.edu; Vijayvargiya, Richa, E-mail: rvijayvargiya@ufl.edu; Zendejas, Ivan, E-mail: ivan.zendejas@surgery.ufl.edu; Behrns, Kevin E., E-mail: Kevin.Behrns@surgery.ufl.edu

    2013-12-15

    Onset of the mitochondrial permeability transition (MPT) plays a causative role in ischemia/reperfusion (I/R) injury. Current therapeutic strategies for reducing reperfusion injury remain disappointing. Autophagy is a lysosome-mediated, catabolic process that timely eliminates abnormal or damaged cellular constituents and organelles such as dysfunctional mitochondria. I/R induces calcium overloading and calpain activation, leading to degradation of key autophagy-related proteins (Atg). Carbamazepine (CBZ), an FDA-approved anticonvulsant drug, has recently been reported to increase autophagy. We investigated the effects of CBZ on hepatic I/R injury. Hepatocytes and livers from male C57BL/6 mice were subjected to simulated in vitro, as well as in vivo I/R, respectively. Cell death, intracellular calcium, calpain activity, changes in autophagy-related proteins (Atg), autophagic flux, MPT and mitochondrial membrane potential after I/R were analyzed in the presence and absence of 20 μM CBZ. CBZ significantly increased hepatocyte viability after reperfusion. Confocal microscopy revealed that CBZ prevented calcium overloading, the onset of the MPT and mitochondrial depolarization. Immunoblotting and fluorometric analysis showed that CBZ blocked calpain activation, depletion of Atg7 and Beclin-1 and loss of autophagic flux after reperfusion. Intravital multiphoton imaging of anesthetized mice demonstrated that CBZ substantially reversed autophagic defects and mitochondrial dysfunction after I/R in vivo. In conclusion, CBZ prevents calcium overloading and calpain activation, which, in turn, suppresses Atg7 and Beclin-1 depletion, defective autophagy, onset of the MPT and cell death after I/R. - Highlights: • A mechanism of carbamazepine (CBZ)-induced cytoprotection in livers is proposed. • Impaired autophagy is a key event contributing to lethal reperfusion injury. • The importance of autophagy is extended and confirmed in an in vivo model. • CBZ is a potential

  5. Tear me down: Role of calpain in the development of cardiac ventricular hypertrophy

    OpenAIRE

    Patterson, Cam; Portbury, Andrea; Schisler, Jonathan C; Willis, Monte S.

    2011-01-01

    Cardiac hypertrophy develops most commonly in response to hypertension and is an independent risk factor for the development of heart failure. The mechanisms by which cardiac hypertrophy may be reversed to reduce this risk have not been fully determined to the point where mechanism-specific therapies have been developed. Recently, proteases in the calpain family have been implicated in regulating the development of cardiac hypertrophy in preclinical animal models. In this review, we summarize...

  6. New single nucleotide polymorphisms in the mu-calpain gene in Spanish maternal beef breeds.

    Science.gov (United States)

    Avilés, C; Azor, P J; Pannier, L; Hamill, R M; Membrillo, A; Molina, A

    2009-01-01

    Calpains play an important role in the postmortem tenderization process of meat and several SNP in the mu-calpain gene (CAPN1) have been reported to be associated with tenderness in beef cattle. Our objectives were to identify the previously reported CAPN1 331G>C SNP and to detect new polymorphisms in this gene in Spanish maternal beef breeds. A fragment (exon 8 to 10) of the bovine CAPN1 gene was sequenced and genotyped in a sample of the main Spanish maternal beef breeds including Retinta, Morucha, and Avilenã Negra-Ibérica. These breeds are characterized for their high meat quality, their adaptation to adverse environmental conditions, and their good maternal aptitude. This adaptation makes it possible to rear these breeds in the south and west of Spain, where drought and feed shortages occur frequently. Six SNP in the mu-calpain gene were found, five of which (CAPN1 80C>T, 302C>G, 310G>A, 445C>T, 524A>C) have not been reported previously. Sequences obtained for these five newly found SNP were submitted to GenBank (Accessions EU386166 to EU386183).

  7. Dexamethasone enhances necrosis-like neuronal death in ischemic rat hippocampus involving μ-calpain activation.

    Science.gov (United States)

    Müller, Georg Johannes; Hasseldam, Henrik; Rasmussen, Rune Skovgaard; Johansen, Flemming Fryd

    2014-11-01

    Transient forebrain ischemia (TFI) leads to hippocampal CA1 pyramidal cell death which is aggravated by glucocorticoids (GC). It is unknown how GC affect apoptosis and necrosis in cerebral ischemia. We therefore investigated the co-localization of activated caspase-3 (casp-3) with apoptosis- and necrosis-like cell death morphologies in CA1 of rats treated with dexamethasone prior to TFI (DPTI). In addition, apoptosis- (casp-9, casp-3, casp-3-cleaved PARP and cleaved α-spectrin 145/150 and 120kDa) and necrosis-related (calpain-specific casp-9 cleavage, μ-calpain upregulation and cleaved α-spectrin 145/150kDa) cell death mechanisms were investigated by Western blot analysis. DPTI expedited CA1 neuronal death from day 4 to day 1 and increased the magnitude of CA1 neuronal death from 66.2% to 91.3% at day 7. Furthermore, DPTI decreased the overall (days 1-7) percentage of dying neurons displaying apoptosis-like morphology from 4.7% to 0.3% and, conversely, increased the percentage of neurons with necrosis-like morphology from 95.3% to 99.7%. In animals subjected to TFI without dexamethasone (ischemia-only), 7.4% of all dying CA1 neurons were casp-3-immunoreactive (IR), of which 3.1% co-localized with apoptosis-like and 4.3% with necrosis-like changes. By contrast, DPTI decreased the percentage of dying neurons with casp-3 IR to 1.4%, of which 0.3% co-localized with apoptosis-like changes and 1.1% with necrosis-like changes. Western blot analysis from DPTI animals showed a significant elevation of μ-calpain, a calpain-produced necrosis-related casp-9 fragment (25kDa) and cleavage of α-spectrin into 145/150kDa fragments at day 4, whereas in ischemia-only animals a significant increase of casp-3-cleaved PARP, cleavage of α-spectrin into 145/150 and 120kDa fragments was detected at day 7. We conclude that DPTI, in addition to augmenting and expediting CA1 neuronal death, causes a shift from apoptosis-like cell death to necrosis involving μ-calpain activation.

  8. Calpain-mediated proteolysis of polycystin-1 C-terminus induces JAK2 and ERK signal alterations

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyunho [Transplantation Research Institute, Seoul National University Medical Research Center, Seoul (Korea, Republic of); Department of Medicine, University of Maryland, Baltimore, MD (United States); Kang, Ah-Young [Transplantation Research Institute, Seoul National University Medical Research Center, Seoul (Korea, Republic of); Department of Medicine, Program of Immunology, Graduate School, Seoul National University, Seoul (Korea, Republic of); Ko, Ah-ra [Clinical Research Center, Samsung Biomedical Research Institute, Seoul (Korea, Republic of); Park, Hayne Cho [Transplantation Research Institute, Seoul National University Medical Research Center, Seoul (Korea, Republic of); Research Coordination Center for Rare Diseases, Seoul National University Hospital, Seoul (Korea, Republic of); So, Insuk [Department of Physiology, Seoul National University College of Medicine, Seoul (Korea, Republic of); Park, Jong Hoon [Department of Biological Science, Sookmyung Women’s University, Seoul (Korea, Republic of); Cheong, Hae Il [Research Coordination Center for Rare Diseases, Seoul National University Hospital, Seoul (Korea, Republic of); Department of Pediatrics, Seoul National University Children’s Hospital, Seoul (Korea, Republic of); Kidney Research Institute, Medical Research Center, Seoul National University College of Medicine, Seoul (Korea, Republic of); Hwang, Young-Hwan [Research Coordination Center for Rare Diseases, Seoul National University Hospital, Seoul (Korea, Republic of); Department of Internal Medicine, Eulji General Hospital, Eulji University College of Medicine, Seoul (Korea, Republic of); and others

    2014-01-01

    Autosomal dominant polycystic kidney disease (ADPKD), a hereditary renal disease caused by mutations in PKD1 (85%) or PKD2 (15%), is characterized by the development of gradually enlarging multiple renal cysts and progressive renal failure. Polycystin-1 (PC1), PKD1 gene product, is an integral membrane glycoprotein which regulates a number of different biological processes including cell proliferation, apoptosis, cell polarity, and tubulogenesis. PC1 is a target of various proteolytic cleavages and proteosomal degradations, but its role in intracellular signaling pathways remains poorly understood. Herein, we demonstrated that PC1 is a novel substrate for μ- and m-calpains, which are calcium-dependent cysteine proteases. Overexpression of PC1 altered both Janus-activated kinase 2 (JAK2) and extracellular signal-regulated kinase (ERK) signals, which were independently regulated by calpain-mediated PC1 degradation. They suggest that the PC1 function on JAK2 and ERK signaling pathways might be regulated by calpains in response to the changes in intracellular calcium concentration. - Highlights: • Polycystin-1 is a target of ubiquitin-independent degradation by calpains. • The PEST domain is required for calpain-mediated degradation of polycystin-1. • Polycystin-1 may independently regulate JAK2 and ERK signaling pathways.

  9. Calpain-mediated proteolysis of polycystin-1 C-terminus induces JAK2 and ERK signal alterations

    International Nuclear Information System (INIS)

    Autosomal dominant polycystic kidney disease (ADPKD), a hereditary renal disease caused by mutations in PKD1 (85%) or PKD2 (15%), is characterized by the development of gradually enlarging multiple renal cysts and progressive renal failure. Polycystin-1 (PC1), PKD1 gene product, is an integral membrane glycoprotein which regulates a number of different biological processes including cell proliferation, apoptosis, cell polarity, and tubulogenesis. PC1 is a target of various proteolytic cleavages and proteosomal degradations, but its role in intracellular signaling pathways remains poorly understood. Herein, we demonstrated that PC1 is a novel substrate for μ- and m-calpains, which are calcium-dependent cysteine proteases. Overexpression of PC1 altered both Janus-activated kinase 2 (JAK2) and extracellular signal-regulated kinase (ERK) signals, which were independently regulated by calpain-mediated PC1 degradation. They suggest that the PC1 function on JAK2 and ERK signaling pathways might be regulated by calpains in response to the changes in intracellular calcium concentration. - Highlights: • Polycystin-1 is a target of ubiquitin-independent degradation by calpains. • The PEST domain is required for calpain-mediated degradation of polycystin-1. • Polycystin-1 may independently regulate JAK2 and ERK signaling pathways

  10. In silico affinity profiling of neuroactive polyphenols for post-traumatic calpain inactivation: a molecular docking and atomistic simulation sensitivity analysis.

    Science.gov (United States)

    Kumar, Pradeep; Choonara, Yahya E; Pillay, Viness

    2015-01-01

    Calcium-activated nonlysosomal neutral proteases, calpains, are believed to be early mediators of neuronal damage associated with neuron death and axonal degeneration after traumatic neural injuries. In this study, a library of biologically active small molecular weight calpain inhibitors was used for model validation and inhibition site recognition. Subsequently, two natural neuroactive polyphenols, curcumin and quercetin, were tested for their sensitivity and activity towards calpain's proteolytic sequence and compared with the known calpain inhibitors via detailed molecular mechanics (MM), molecular dynamics (MD), and docking simulations. The MM and MD energy profiles (SJA6017 < AK275 < AK295 < PD151746 < quercetin < leupeptin < PD150606 < curcumin < ALLN < ALLM < MDL-28170 < calpeptin) and the docking analysis (AK275 < AK295 < PD151746 < ALLN < PD150606 < curcumin < leupeptin < quercetin < calpeptin < SJA6017 < MDL-28170 < ALLM) demonstrated that polyphenols conferred comparable calpain inhibition profiling. The modeling paradigm used in this study provides the first detailed account of corroboration of enzyme inhibition efficacy of calpain inhibitors and the respective calpain-calpain inhibitor molecular complexes' energetic landscape and in addition stimulates the polyphenol bioactive paradigm for post-SCI intervention with implications reaching to experimental in vitro, in cyto, and in vivo studies. PMID:25546626

  11. Increased μ-Calpain Activity in Blasts of Common B-Precursor Childhood Acute Lymphoblastic Leukemia Correlates with Their Lower Susceptibility to Apoptosis.

    Directory of Open Access Journals (Sweden)

    Anna Mikosik

    Full Text Available Childhood acute lymphoblastic leukemia (ALL blasts are characterized by inhibited apoptosis promoting fast disease progress. It is known that in chronic lymphocytic and acute myeloid leukemias the reduced apoptosis is strongly related with the activity of calpain-calpastatin system (CCS composed of cytoplasmic proteases--calpains--performing the modulatory proteolysis of key proteins involved in cell proliferation and apoptosis, and of their endogenous inhibitor--calpastatin. Here, the CCS protein abundance and activity was for the first time studied in childhood ALL blasts and in control bone marrow CD19+ B cells by semi-quantitative flow cytometry and western blotting of calpastatin fragments resulting from endogenous calpain activity. Significantly higher μ-calpain (CAPN1 gene transcription, protein amounts and activity (but not those of m-calpain, with calpastatin amount and transcription of its gene (CAST greatly varying were observed in CD19(+ ALL blasts compared to control cells. Significant inverse relation between the amount/activity of calpain and spontaneous apoptosis was noted. Patients older than 10 years (considered at higher risk displayed increased amounts and activities of blast calpain. Finally, treatment of blasts with the tripeptide calpain inhibitors II and IV significantly and in dose-dependent fashion increased the percentage of blasts entering apoptosis. Together, these findings make the CCS a potential new predictive tool and therapeutic target in childhood ALL.

  12. Expression of the gene for large subunit of m-calpain is elevated in skeletal muscle from Duchenne muscular dystrophy patients

    Indian Academy of Sciences (India)

    Tajamul Hussain; Harleen Mangath; C. Sundaram; M. P. J. S. Anandaraj

    2000-08-01

    Calpain is an intracellular nonlysosomal protease involved in essential regulatory or processing functions of the cell, mediated by physiological concentrations of Ca2+. However, in an environment of abnormal intracellular calcium, such as that seen in Duchenne muscular dystrophy (DMD), calpain is suggested to cause degeneration of muscle owing to enhanced activity. To test whether the reported increase in calpain activity in DMD results from de novo synthesis of the protease, we have assessed the quantitative changes in mRNA specific for m-calpain. mRNA isolated from DMD and control muscle was analysed by dot blot hybridization using a cDNA probe for the large subunit of m-calpain. Compared to control a four-fold increase in specific mRNAwas observed in dystrophic muscle. This enhanced expression of the m-calpain gene in dystrophic condition suggests that the reported increase in m-calpain activity results from de novo synthesis of protease and underlines the important role of m-calpain in DMD.

  13. A possible therapeutic potential of quercetin through inhibition of μ-calpain in hypoxia induced neuronal injury: a molecular dynamics simulation study.

    Science.gov (United States)

    Pandey, Anand Kumar; Shukla, Swet Chand; Bhattacharya, Pallab; Patnaik, Ranjana

    2016-08-01

    The neuroprotective property of quercetin is well reported against hypoxia and ischemia in past studies. This property of quercetin lies in its antioxidant property with blood-brain barrier permeability and anti-inflammatory capabilities. µ-Calpain, a calcium ion activated intracellular cysteine protease causes serious cellular insult, leading to cell death in various pathological conditions including hypoxia and ischemic stroke. Hence, it may be considered as a potential drug target for the treatment of hypoxia induced neuronal injury. As the inhibitory property of µ-calpain is yet to be explored in details, hence, in the present study, we investigated the interaction of quercetin with µ-calpain through a molecular dynamics simulation study as a tool through clarifying the molecular mechanism of such inhibition and determining the probable sites and modes of quercetin interaction with the µ-calpain catalytic domain. In addition, we also investigated the structure-activity relationship of quercetin with μ-calpain. Affinity binding of quercetin with µ-calpain had a value of -28.73 kJ/mol and a Ki value of 35.87 µM that may be a probable reason to lead to altered functioning of µ-calpain. Hence, quercetin was found to be an inhibitor of µ-calpain which might have a possible therapeutic role in hypoxic injury. PMID:27651771

  14. Calpains are involved in asexual and sexual development, cell wall integrity and pathogenicity of the rice blast fungus.

    Science.gov (United States)

    Liu, Xiao-Hong; Ning, Guo-Ao; Huang, Lu-Yao; Zhao, Ya-Hui; Dong, Bo; Lu, Jian-Ping; Lin, Fu-Cheng

    2016-01-01

    Calpains are ubiquitous and well-conserved proteins that belong to the calcium-dependent, non-lysosomal cysteine protease family. In this study, 8 putative calpains were identified using Pfam domain analysis and BlastP searches in M. oryzae. Three single gene deletion mutants (ΔMocapn7, ΔMocapn9 and ΔMocapn14) and two double gene deletion mutants (ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7) were obtained using the high-throughput gene knockout system. The calpain disruption mutants showed defects in colony characteristics, conidiation, sexual reproduction and cell wall integrity. The mycelia of the ΔMocapn7, ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7 mutants showed reduced pathogenicity on rice and barley. PMID:27502542

  15. Zilpaterol hydrochloride improves beef yield, changes palatability traits, and increases calpain-calpastatin gene expression in Nellore heifers.

    Science.gov (United States)

    Cônsolo, Nara Regina Brandão; Ferrari, Viviane Borba; Mesquita, Ligia Garcia; Goulart, Rodrigo Silva; Silva, Luis Felipe Prada E

    2016-11-01

    This research aimed to evaluate the effects of the beta-agonist zilpaterol hydrochloride (ZH) on carcass traits, subprimal yield, meat quality, palatability traits, and gene expression in Nellore heifers. Zilpaterol increased Longissimus lumborum area and did not change back fat thickness, meat color, and cooking loss. Heifers fed ZH had greater hindquarter weight and carcass percentage. Muscles from hindquarter were heavier for animals fed ZH. Forequarter (% of carcass) decreased and brisket did not change with ZH supplementation. There were no differences between treatments for steak aroma, beef flavor, and off-flavor. However, tenderness and juiciness were reduced by ZH, depending on postmortem aging. Zilpaterol increased Calpain-1, Calpain-2, and calpastatin mRNA expression, with no effect of day of slaughter or ZH×Day interaction. In conclusion, ZH supplementation improved hypertrophy, meat production, and debone yield in Nellore heifers, which led to decreased tenderness and to increased mRNA expression in the calpain-calpastatin system. PMID:27427783

  16. The calpain inhibitor MDL28170 induces the expression of apoptotic markers in Leishmania amazonensis promastigotes.

    Directory of Open Access Journals (Sweden)

    Fernanda A Marinho

    Full Text Available BACKGROUND: Human cutaneous leishmaniasis is caused by distinct species, including Leishmania amazonensis. Treatment of cutaneous leishmaniasis is far from satisfactory due to increases in drug resistance and relapses, and toxicity of compounds to the host. As a consequence for this situation, the development of new leishmanicidal drugs and the search of new targets in the parasite biology are important goals. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the mechanism of death pathway induced by the calpain inhibitor MDL28170 on Leishmania amazonensis promastigote forms. The combined use of different techniques was applied to contemplate this goal. MDL28170 treatment with IC50 (15 µM and two times the IC50 doses induced loss of parasite viability, as verified by resazurin assay, as well as depolarization of the mitochondrial membrane, which was quantified by JC-1 staining. Scanning and transmission electron microscopic images revealed drastic alterations on the parasite morphology, some of them resembling apoptotic-like death, including cell shrinking, surface membrane blebs and altered chromatin condensation pattern. The lipid rearrangement of the plasma membrane was detected by Annexin-V labeling. The inhibitor also induced a significant increase in the proportion of cells in the sub-G0/G1 phase, as quantified by propidium iodide staining, as well as genomic DNA fragmentation, detected by TUNEL assay. In cells treated with MDL28170 at two times the IC50 dose, it was also possible to observe an oligonucleossomal DNA fragmentation by agarose gel electrophoresis. CONCLUSIONS/SIGNIFICANCE: The data presented in the current study suggest that MDL28170 induces apoptotic marker expression in promastigotes of L. amazonensis. Altogether, the results described in the present work not only provide a rationale for further exploration of the mechanism of action of calpain inhibitors against trypanosomatids, but may also widen the

  17. Regulation of calpain activity in rat brain with altered Ca2+ homeostasis.

    Science.gov (United States)

    Averna, Monica; Stifanese, Roberto; De Tullio, Roberta; Passalacqua, Mario; Defranchi, Enrico; Salamino, Franca; Melloni, Edon; Pontremoli, Sandro

    2007-01-26

    Activation of calpain occurs as an early event in correlation with an increase in [Ca2+]i induced in rat brain upon treatment with a high salt diet for a prolonged period of time. The resulting sequential events have been monitored in the brain of normal and hypertensive rats of the Milan strain, diverging for a constitutive alteration in the level of [Ca2+]i found to be present in nerve cells of hypertensive animals. After 2 weeks of treatment, the levels of the plasma membrane Ca2+-ATPase and of native calpastatin are profoundly decreased. These degradative processes, more pronounced in the brain of hypertensive rats, are progressively and efficiently compensated in the brain of both rat strains by different incoming mechanisms. Along with calpastatin degradation, 15-kDa still-active inhibitory fragments are accumulated, capable of efficiently replacing the loss of native inhibitor molecules. A partial return to a more efficient control of Ca2+ homeostasis occurs in parallel, assured by an early increase in the expression of Ca2+-ATPase and of calpastatin, both producing, after 12 weeks of a high salt (sodium) diet, the restoration of almost original levels of the Ca2+ pump and of significant amounts of native inhibitor molecules. Thus, conservative calpastatin fragmentation, associated with an increased expression of Ca2+-ATPase and of the calpain natural inhibitor, has been demonstrated to occur in vivo in rat brain. This represents a sequential adaptive response capable of overcoming the effects of calpain activation induced by a moderate long term elevation of [Ca2+]i.

  18. A calcium- and calpain-dependent pathway determines the response to lenalidomide in myelodysplastic syndromes.

    Science.gov (United States)

    Fang, Jing; Liu, Xiaona; Bolanos, Lyndsey; Barker, Brenden; Rigolino, Carmela; Cortelezzi, Agostino; Oliva, Esther N; Cuzzola, Maria; Grimes, H Leighton; Fontanillo, Celia; Komurov, Kakajan; MacBeth, Kyle; Starczynowski, Daniel T

    2016-07-01

    Despite the high response rates of individuals with myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)) to treatment with lenalidomide (LEN) and the recent identification of cereblon (CRBN) as the molecular target of LEN, the cellular mechanism by which LEN eliminates MDS clones remains elusive. Here we performed an RNA interference screen to delineate gene regulatory networks that mediate LEN responsiveness in an MDS cell line, MDSL. We identified GPR68, which encodes a G-protein-coupled receptor that has been implicated in calcium metabolism, as the top candidate gene for modulating sensitivity to LEN. LEN induced GPR68 expression via IKAROS family zinc finger 1 (IKZF1), resulting in increased cytosolic calcium levels and activation of a calcium-dependent calpain, CAPN1, which were requisite steps for induction of apoptosis in MDS cells and in acute myeloid leukemia (AML) cells. In contrast, deletion of GPR68 or inhibition of calcium and calpain activation suppressed LEN-induced cytotoxicity. Moreover, expression of calpastatin (CAST), an endogenous CAPN1 inhibitor that is encoded by a gene (CAST) deleted in del(5q) MDS, correlated with LEN responsiveness in patients with del(5q) MDS. Depletion of CAST restored responsiveness of LEN-resistant non-del(5q) MDS cells and AML cells, providing an explanation for the superior responses of patients with del(5q) MDS to LEN treatment. Our study describes a cellular mechanism by which LEN, acting through CRBN and IKZF1, has cytotoxic effects in MDS and AML that depend on a calcium- and calpain-dependent pathway.

  19. Effect of exercise training on calpain systems in lean and obese Zucker rats

    OpenAIRE

    Hsieh, Yao-Yuan; Chang, Chi-Chen; Hsu, Kung-Hao; Tsai, Fuu-Jen; Chen, Chih-Ping; Tsai, Horng-Der

    2008-01-01

    Exercise training plays a major role in the improving physiology of diabetes. Herein we aimed to investigate the influence of exercise upon the calcium-dependent calpain-isoform expressions of lean or obese Zucker rats, a model of obesity and type II diabetes (NIDDM). Five-month-old rats were divided: (1) obese sedentary (OS, n=7); (2) obese exercise (OE, n=7); (3) lean sedentary (LS, n=7); (4) lean exercise (LE, n=7). After 2-month exercise (treadmill running), the body weight (BW) and expre...

  20. Skeletal Muscle-specific Calpain and Protein Degradation%骨骼肌特异性钙蛋白酶与蛋白质降解

    Institute of Scientific and Technical Information of China (English)

    张勇; 邓科

    2011-01-01

    骨骼肌特异性钙蛋白酶calpain-3是钙蛋白酶系统的一员,与肌细胞生成和细胞凋亡密切相关,同时也被认为参与了蛋白质降解过程.本文主要从骨骼肌特异性钙蛋白酶的结构功能和生理活性,并结合近年来国际上的研究成果来分析骨骼肌特异性钙蛋白酶在骨骼肌蛋白质降解中的作用.%Calpain-3, also named as skeletal muscle-specific calpain, is a member of the calpain system. Calpain-3 has been shown to be involved in myogenesis and apoptosis, and it also plays a role in protein degradation. This article mainly reviewed the structure, function and physiological activity of calpain-3, combined with recent international research advances, to analyze the roles of calpain-3 in skeletal muscle protein degradation. [Chinese Journal of Animal Nutrition, 2011,23 ( 4 ): 542-545

  1. Calcium Regulates the Activity and Structural Stability of Tpr, a Bacterial Calpain-like Peptidase.

    Science.gov (United States)

    Staniec, Dominika; Ksiazek, Miroslaw; Thøgersen, Ida B; Enghild, Jan J; Sroka, Aneta; Bryzek, Danuta; Bogyo, Matthew; Abrahamson, Magnus; Potempa, Jan

    2015-11-01

    Porphyromonas gingivalis is a peptide-fermenting asaccharolytic periodontal pathogen. Its genome contains several genes encoding cysteine peptidases other than gingipains. One of these genes (PG1055) encodes a protein called Tpr (thiol protease) that has sequence similarity to cysteine peptidases of the papain and calpain families. In this study we biochemically characterize Tpr. We found that the 55-kDa Tpr inactive zymogen proteolytically processes itself into active forms of 48, 37, and 33 kDa via sequential truncations at the N terminus. These processed molecular forms of Tpr are associated with the bacterial outer membrane where they are likely responsible for the generation of metabolic peptides required for survival of the pathogen. Both autoprocessing and activity were dependent on calcium concentrations >1 mm, consistent with the protein's activity within the intestinal and inflammatory milieus. Calcium also stabilized the Tpr structure and rendered the protein fully resistant to proteolytic degradation by gingipains. Together, our findings suggest that Tpr is an example of a bacterial calpain, a calcium-responsive peptidase that may generate substrates required for the peptide-fermenting metabolism of P. gingivalis. Aside from nutrient generation, Tpr may also be involved in evasion of host immune response through degradation of the antimicrobial peptide LL-37 and complement proteins C3, C4, and C5. Taken together, these results indicate that Tpr likely represents an important pathogenesis factor for P. gingivalis.

  2. Activation of mitochondrial calpain and increased cardiac injury: beyond AIF release.

    Science.gov (United States)

    Thompson, Jeremy; Hu, Ying; Lesnefsky, Edward J; Chen, Qun

    2016-02-01

    Calpain 1 (CPN1) is a ubiquitous cysteine protease that exists in both cytosol and cardiac mitochondria. Mitochondrial CPN1 (mit-CPN1) is located in the intermembrane space and matrix. Activation of mit-CPN1 within the intermembrane space increases cardiac injury by releasing apoptosis-inducing factor from mitochondria during ischemia-reperfusion (IR). We asked if activation of mit-CPN1 is involved in mitochondrial injury during IR. MDL-28170 (MDL) was used to inhibit CPN1 in buffer-perfused hearts following 25-min ischemia and 30-min reperfusion. MDL treatment decreased the release of lactate dehydrogenase into coronary effluent compared with untreated hearts, indicating that inhibition of CPN1 decreases cardiac injury. MDL also prevented the cleavage of spectrin (a substrate of CPN1) in cytosol during IR, supporting that MDL treatment decreased cytosolic calpain activation. In addition, MDL markedly improved calcium retention capacity compared with untreated heart, suggesting that MDL treatment decreases mitochondrial permeability transition pore opening. In addition, we found that IR led to decreased complex I activity, whereas inhibition of mit-CPN1 using MDL protected complex I. Pyruvate dehydrogenase content was decreased following IR. However, pyruvate dehydrogenase content was preserved in MDL-treated mitochondria. Taken together, MDL treatment decreased cardiac injury during IR by inhibiting both cytosolic and mit-CPN1. Activation of mit-CPN1 increases cardiac injury during IR by sensitizing mitochondrial permeability transition pore opening and impairing mitochondrial metabolism through damage of complex I. PMID:26637561

  3. Calpain 3 is a rapid-action, unidirectional proteolytic switch central to muscle remodeling.

    Directory of Open Access Journals (Sweden)

    Antoine de Morrée

    Full Text Available Calpain 3 (CAPN3 is a cysteine protease that when mutated causes Limb Girdle Muscular Dystrophy 2A. It is thereby the only described Calpain family member that genetically causes a disease. Due to its inherent instability little is known of its substrates or its mechanism of activity and pathogenicity. In this investigation we define a primary sequence motif underlying CAPN3 substrate cleavage. This motif can transform non-related proteins into substrates, and identifies >300 new putative CAPN3 targets. Bioinformatic analyses of these targets demonstrate a critical role in muscle cytoskeletal remodeling and identify novel CAPN3 functions. Among the new CAPN3 substrates are three E3 SUMO ligases of the Protein Inhibitor of Activated Stats (PIAS family. CAPN3 can cleave PIAS proteins and negatively regulates PIAS3 sumoylase activity. Consequently, SUMO2 is deregulated in patient muscle tissue. Our study thus uncovers unexpected crosstalk between CAPN3 proteolysis and protein sumoylation, with strong implications for muscle remodeling.

  4. Polymorphisms in calpastatin and mu-calpain genes are associated with beef iron content.

    Science.gov (United States)

    Casas, E; Duan, Q; Schneider, M J; Shackelford, S D; Wheeler, T L; Cundiff, L V; Reecy, J M

    2014-04-01

    The objective of this study was to assess the association of markers in the calpastatin and mu-calpain loci with iron in beef cattle muscle. The population consisted of 259 cross-bred steers from Beefmaster, Brangus, Bonsmara, Romosinuano, Hereford and Angus sires. Total iron and heme iron concentrations were measured. Markers in the calpastatin (referred to as CAST) and mu-calpain (referred to as CAPN4751) genes were used to assess their association with iron levels. The mean and standard error for iron and heme iron content in the population was 35.6 ± 1.3 μg and 27.1 ± 1.4 μg respectively. Significant associations (P < 0.01) of markers were observed for both iron and heme iron content. For CAST, animals with the CC genotype had higher levels of iron and heme iron in longissimus dorsi muscle. For CAPN4751, individuals with the TT genotype had higher concentrations of iron and heme iron than did animals with the CC and CT genotypes. Genotypes known to be associated with tougher meat were associated with higher levels of iron concentration. PMID:24303986

  5. The growth and tumor suppressors NORE1A and RASSF1A are targets for calpain-mediated proteolysis.

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    Sergey Kuznetsov

    Full Text Available BACKGROUND: NORE1A and RASSF1A are growth and tumour suppressors inactivated in a variety of cancers. Methylation of NORE1A and RASSF1A promoters is the predominant mechanism for downregulation of these proteins; however, other mechanisms are likely to exist. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a proteolysis of NORE1A and RASSF1A by calpains as alternative mechanism of their downregulation. Extracts of H358 cell line, a human bronchoalveolar carcinoma, and H460, a large cell carcinoma, were capable of proteolysis of NORE1A protein in the calpain-dependent manner. Likewise, RASSF1A tumor suppressor was proteolyzed by the H358 cell extract. Addition of calpain inhibitor to H358 and H460 cells growing in tissue culture resulted in re-expression of endogenous NORE1A. A survey of 10 human lung tumours revealed that three of them contain an activity capable of inducing NORE1A degradation. CONCLUSIONS/SIGNIFICANCE: Thus, degradation by calpains is a novel mechanism for downregulation of NORE1A and RASSF1A proteins and might be the mechanism allowing cancer cells to escape growth suppression.

  6. Propofol Ameliorates Calpain-induced Collapsin Response Mediator Protein-2 Proteolysis in Traumatic Brain Injury in Rats

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    Yun Yu

    2015-01-01

    Full Text Available Background: Collapsin response mediator protein-2 (CRMP2, a multifunctional cytosolic protein highly expressed in the brain, is degraded by calpain following traumatic brain injury (TBI, possibly inhibiting posttraumatic neurite regeneration. Lipid peroxidation (LP is involved in triggering postinjury CRMP2 proteolysis. We examined the hypothesis that propofol could attenuate LP, calpain-induced CRMP2 degradation, and brain injury after TBI. Methods: A unilateral moderate controlled cortical impact injury was induced in adult male Sprague-Dawley rats. The animals were randomly divided into seven groups: Sham control group, TBI group, TBI + propofol groups (including propofol 1 h, 2 h, and 4 h groups, TBI + U83836E group and TBI + fat emulsion group. The LP inhibitor U83836E was used as a control to identify that antioxidation partially accounts for the potential neuroprotective effects of propofol. The solvent of propofol, fat emulsion, was used as the vehicle control. Ipsilateral cortex tissues were harvested at 24 h post-TBI. Immunofluorescent staining, Western blot analysis, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling were used to evaluate LP, calpain activity, CRMP2 proteolysis and programmed cell death. The data were statistically analyzed using one-way analysis of variance and a paired t-test. Results: Propofol and U83836E significantly ameliorated the CRMP2 proteolysis. In addition, both propofol and U83836E significantly decreased the ratio of 145-kDa αII-spectrin breakdown products to intact 270-kDa spectrin, the 4-hydroxynonenal expression and programmed cell death in the pericontusional cortex at 24 h after TBI. There was no difference between the TBI group and the fat emulsion group. Conclusions: These results demonstrate that propofol postconditioning alleviates calpain-mediated CRMP2 proteolysis and provides neuroprotective effects following moderate TBI potentially by counteracting LP and reducing

  7. Glutamate protects against Ca(2+) paradox-induced injury and inhibits calpain activity in isolated rat hearts.

    Science.gov (United States)

    Zhang, Jian-Ying; Kong, Ling-Heng; Lai, Dong; Jin, Zhen-Xiao; Gu, Xiao-Ming; Zhou, Jing-Jun

    2016-10-01

    This study determined the effects of glutamate on the Ca(2+) paradoxical heart, which is a model for Ca(2+) overload-induced injury during myocardial ischaemia and reperfusion, and evaluated its effect on a known mediator of injury, calpain. An isolated rat heart was retrogradely perfused in a Langendorff apparatus. Ca(2+) paradox was elicited via perfusion with a Ca(2+) -free Krebs-Henseleit (KH) solution for 3 minutes followed by Ca(2+) -containing normal KH solution for 30 minutes. The Ca(2+) paradoxical heart exhibited almost no viable tissue on triphenyltetrazolium chloride staining and markedly increased LDH release, caspase-3 activity, cytosolic cytochrome c content, and apoptotic index. These hearts also displayed significantly increased LVEDP and a disappearance of LVDP. Glutamate (5 and 20 mmol/L) significantly alleviated Ca(2+) paradox-induced injury. In contrast, 20 mmol/L mannitol had no effect on Ca(2+) paradox. Ca(2+) paradox significantly increased the extent of the translocation of μ-calpain to the sarcolemmal membrane and the proteolysis of α-fodrin, which suggests calpain activation. Glutamate also blocked these effects. A non-selective inhibitor of glutamate transporters, dl-TBOA (10 μmol/L), had no effect on control hearts, but it reversed glutamate-induced cardioprotection and reduction in calpain activity. Glutamate treatment significantly increased intracellular glutamate content in the Ca(2+) paradoxical heart, which was also blocked by dl-TBOA. We conclude that glutamate protects the heart against Ca(2+) overload-induced injury via glutamate transporters, and the inhibition of calpain activity is involved in this process.

  8. Calpastatin overexpression reduces oxidative stress-induced mitochondrial impairment and cell death in human neuroblastoma SH-SY5Y cells by decreasing calpain and calcineurin activation, induction of mitochondrial fission and destruction of mitochondrial fusion.

    Science.gov (United States)

    Tangmansakulchai, Kulvadee; Abubakar, Zuroida; Kitiyanant, Narisorn; Suwanjang, Wilasinee; Leepiyasakulchai, Chaniya; Govitrapong, Piyarat; Chetsawang, Banthit

    2016-09-01

    Calpain is an intracellular Ca(2+)-dependent protease, and the activation of calpain has been implicated in neurodegenerative diseases. Calpain activity can be regulated by calpastatin, an endogenous specific calpain inhibitor. Several lines of evidence have demonstrated a potential role of calpastatin in preventing calpain-mediated pathogenesis. Additionally, several studies have revealed that calpain activation and mitochondrial damage are involved in the cell death process; however, recent evidence has not clearly indicated a neuroprotective mechanism of calpastatin against calpain-dependent mitochondrial impairment in the process of neuronal cell death. Therefore, the purpose of this study was to investigate the potential ability of calpastatin to inhibit calpain activation and mitochondrial impairment in oxidative stress-induced neuron degeneration. Calpastatin was stably overexpressed in human neuroblastoma SH-SY5Y cells. In non-calpastatin overexpressing SH-SY5Y cells, hydrogen peroxide significantly decreased cell viability, superoxide dismutase activity, mitochondrial membrane potential, ATP production and mitochondrial fusion protein (Opa1) levels in the mitochondrial fraction but increased reactive oxygen species formation, calpain and calcineurin activation, mitochondrial fission protein (Fis1 and Drp1) levels in the mitochondrial fraction and apoptotic cells. Nevertheless, these toxic effects were abolished in hydrogen peroxide-treated calpastatin-overexpressing SH-SY5Y cells. The results of the present study demonstrate the potential ability of calpastatin to diminish calpain and calcineurin activation and mitochondrial impairment in neurons that are affected by oxidative damage. PMID:27453331

  9. Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell–matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yan; Li, Zheng; He, Yan; Shang, Dandan; Pan, Jigang; Wang, Hongmei; Chen, Huamei; Zhu, Zhuxia [Department of Physiology/Cancer Research Group, Guiyang Medical University School of Basic Medicine, 9 Beijing Road, Guiyang 550004, Guizhou (China); Wan, Lei [Department of Pharmacology, Guiyang Medical University School of Basic Medicine, 9 Beijing Road, Guiyang 550004, Guizhou (China); Wang, Xudong, E-mail: xdwang@gmc.edu.cn [Department of Physiology/Cancer Research Group, Guiyang Medical University School of Basic Medicine, 9 Beijing Road, Guiyang 550004, Guizhou (China)

    2014-03-01

    Fulvestrant (ICI 182 780, ICI) has been used in treating patients with hormone-sensitive breast cancer, yet initial or acquired resistance to endocrine therapies frequently arises and, in particular, cancer recurs as metastasis. We demonstrate here that both 17-beta-estradiol (E2) and ICI enhance cell adhesion to matrigel in MCF-7 breast cancer cells, with increased autolysis of calpain 1 (large subunit) and proteolysis of focal adhesion kinase (FAK), indicating calpain activation. Additionally, either E2 or ICI induced down-regulation of estrogen receptor α without affecting G protein coupled estrogen receptor 30 (GPR30) expression. Interestingly, GPR30 agonist G1 triggered calpain 1 autolysis but not calpain 2, whereas ER agonist diethylstilbestrol caused no apparent calpain autolysis. Furthermore, the actions of E2 and ICI on calpain and cell adhesion were tremendously suppressed by G15, or knockdown of GPR30. E2 and ICI also induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 phosphorylation by U0126 profoundly impeded calpain activation triggered by estrogenic and antiestrogenic stimulations indicating implication of ERK1/2 in the GPR30-mediated action. Lastly, the E2- or ICI-induced cell adhesion was dramatically impaired by calpain-specific inhibitors, ALLN or calpeptin, suggesting requirement of calpain in the GPR30-associated action. These data show that enhanced cell adhesion by E2 and ICI occurs via a novel GPR30-ERK1/2-calpain pathway. Our results indicate that targeting the GPR30 signaling may be a potential strategy to reduce metastasis and improve the efficacy of antiestrogens in treatment of advanced breast cancer. - Highlights: • Estrogen and ICI augment adhesion to matrigel with calpain activation in MCF-7 cells. • GPR30 mediates cell–matrigel adhesion and calpain activation via ERK1/2. • Calpain is required in the cell–matrigel adhesion induced by E2 and ICI.

  10. Calpain Inhibitor Reduces Cancer-induced Bone Pain Possibly Through Inhibition of Osteoclastogenesis in Rat Cancer-induced Bone Pain Model

    Institute of Scientific and Technical Information of China (English)

    Jia-Ying Xu; Yu Jiang; Wei Liu; Yu-Guang Huang

    2015-01-01

    Background:Calpain,a calcium-dependent cysteine protease,has been demonstrated to regulate osteoclastogenesis,which is considered one of the major reasons for cancer-induced bone pain (CIBP).In the present study,calpain inhibitor was applied in a rat CIBP model to determine whether it could reduce CIBP through regulation of osteoclastogenesis activity.Methods:A rat CIBP model was established with intratibial injection of Walker 256 cells.Then,the efficacy of intraperitoneal administered calpain inhibitor Ⅲ (MDL28170,1 mg/kg) on mechanical withdrawal threshold (MWT) of bilateral hind paws was examined on postoperative days (PODs) 2,5,8,11,and 14.On POD 14,the calpain inhibitor's effect on tumor bone tartrate-resistant acid phosphatase (TRAP) stain and radiology was also carefully investigated.Results:Pain behavioral tests in rats showed that the calpain inhibitor effectively attenuated MWTs of both the surgical side and contralateral side hind paws on POD 5,8,and 11 (P < 0.05).TRAP-positive cell count of the surgical side bone was significantly decreased in the calpain inhibitor group compared with the vehicle group (P < 0.05).However,bone resorption and destruction measured by radiographs showed no difference between the two groups.Conclusions:Calpain inhibitor can effectively reduce CIBP of both the surgical side and nonsurgical side after tumor injection in a rat CIBP model.It may be due to the inhibition of receptor activator of nuclear factor-kappa B ligand-induced osteoclastogenesis.Whether a calpain inhibitor could be a novel therapeutic target to treat CIBP needs further investigation.

  11. Proteolysis of the human DNA polymerase delta smallest subunit p12 by μ-calpain in calcium-triggered apoptotic HeLa cells.

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    Xiaoting Fan

    Full Text Available Degradation of p12 subunit of human DNA polymerase delta (Pol δ that results in an interconversion between Pol δ4 and Pol δ3 forms plays a significant role in response to replication stress or genotoxic agents triggered DNA damage. Also, the p12 is readily degraded by human calpain in vitro. However, little has been done for the investigation of its degree of participation in any of the more common apoptosis. Here, we first report that the p12 subunit is a substrate of μ-calpain. In calcium-triggered apoptotic HeLa cells, the p12 is degraded at 12 hours post-induction (hpi, restored thereafter by 24 hpi, and then depleted again after 36 hpi in a time-dependent manner while the other three subunits are not affected. It suggests a dual function of Pol δ by its interconversion between Pol δ4 and Pol δ3 that is involved in a novel unknown apoptosis mechanism. The proteolysis of p12 could be efficiently blocked by both calpain inhibitor ALLN and proteasome inhibitor MG132. In vitro pull down and co-immunoprecipitation assays show that the μ-calpain binds to p12 through the interaction of μ-calpain with Pol δ other three subunits, not p12 itself, and PCNA, implying that the proteolysis of p12 by μ-calpain might be through a Pol δ4/PCNA complex. The p12 cleavage sites by μ-calpain are further determined as the location within a 16-amino acids peptide 28-43 by in vitro cleavage assays. Thus, the p12/Pol δ is a target as a nuclear substrate of μ-calpain in a calcium-triggered apoptosis and appears to be a potential marker in the study of the chemotherapy of cancer therapies.

  12. Restriction fragment length polymorphism in calpain (CAPN2 gene in crossbred cattle

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    Maria Aparecida Cassiano Lara

    2012-12-01

    Full Text Available With advances in molecular genetics have been possible to predict the genetic value of the animal, in particular its potential to transmit desired characters to their offspring, including characters difficult to evaluate or with low heritability, as is the case of the meat tenderization. It is known that Bos taurus indicus features differences in meat tenderization, being assigned this variability to their lowest proteolysis post-mortem, as result of high activity of calpastatin. This inhibitor decreases the activity of calpain, which are the enzymes responsible for the degradation of muscle fibers during the maturation of the meat. Moreover, there were previously observed differences in the frequencies of allele A of calpain among European breeds (Hereford, Aberdeen Angus and Holstein and Bos taurus indicus (Gir, Guzerá and Nelore. This variability has been related to tenderness of meat, as cattle with Bos taurus taurus origin have more tender meat than Bos taurus indicus, showing small values of shear force. One explanation is that the Capn2A product could confer greater proteolytic activity than the encoded by the allele Capn2B. If allele A is associated with tender meat, it will be possible the early identification of the animals that have the potential to produce meat with qualities that attend the needs of the consumer market, in order to add economic value to the final product of the animal production chain. For this reason, biochemical and genetic studies related to calpain and calpastatin systems have been considered promising for the clarification of the physiological changes that occur in muscle structure during the period post-mortem, whose results have contributed to the improvement of meat quality. The objectives of this study were to investigate the RFLP in calpain (Capn2 gene and its relation with meat tenderization in 252 crossbred (Bos taurus taurus x Bos taurus indicus. The analyses were carried through by PCR-RFLP technique

  13. In Silico Affinity Profiling of Neuroactive Polyphenols for Post-Traumatic Calpain Inactivation: A Molecular Docking and Atomistic Simulation Sensitivity Analysis

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    Pradeep Kumar

    2014-12-01

    Full Text Available Calcium-activated nonlysosomal neutral proteases, calpains, are believed to be early mediators of neuronal damage associated with neuron death and axonal degeneration after traumatic neural injuries. In this study, a library of biologically active small molecular weight calpain inhibitors was used for model validation and inhibition site recognition. Subsequently, two natural neuroactive polyphenols, curcumin and quercetin, were tested for their sensitivity and activity towards calpain’s proteolytic sequence and compared with the known calpain inhibitors via detailed molecular mechanics (MM, molecular dynamics (MD, and docking simulations. The MM and MD energy profiles (SJA6017 < AK275 < AK295 < PD151746 < quercetin < leupeptin < PD150606 < curcumin < ALLN < ALLM < MDL-28170 < calpeptin and the docking analysis (AK275 < AK295 < PD151746 < ALLN < PD150606 < curcumin < leupeptin < quercetin < calpeptin < SJA6017 < MDL-28170 < ALLM demonstrated that polyphenols conferred comparable calpain inhibition profiling. The modeling paradigm used in this study provides the first detailed account of corroboration of enzyme inhibition efficacy of calpain inhibitors and the respective calpain–calpain inhibitor molecular complexes’ energetic landscape and in addition stimulates the polyphenol bioactive paradigm for post-SCI intervention with implications reaching to experimental in vitro, in cyto, and in vivo studies.

  14. Identification of different domains of calpain and calpastatin from chicken blood and their role in post-mortem aging of meat during holding at refrigeration temperatures.

    Science.gov (United States)

    Biswas, A K; Tandon, S; Beura, C K

    2016-06-01

    The aim of this study was to develop a simple, specific and rapid analytical method for accurate identification of calpain and calpastatin from chicken blood and muscle samples. The method is based on liquid-liquid extraction technique followed by casein Zymography detection. The target compounds were extracted from blood and meat samples by tris buffer, and purified and separated on anion exchange chromatography. It has been observed that buffer (pH 6.7) containing 50 mM tris-base appears to be excellent extractant as activity of analytes was maximum for all samples. The concentrations of μ-, m-calpain and calpastatin detected in the extracts of blood, breast and thigh samples were 0.28-0.55, 1.91-2.05 and 1.38-1.52 Unit/g, respectively. For robustness, the analytical method was applied to determine the activity of calpains (μ and m) in eighty postmortem muscle samples. It has been observed that μ-calpain activity in breast and thigh muscles declined very rapidly at 48 h and 24 h, respectively while activity of m-calpain remained stable. Shear force values were also declined with the increase of post-mortem aging showing the presence of ample tenderness of breast and thigh muscles. Finally, it is concluded that the method standardized for the detection of calpain and calpastatin has the potential to be applied to identify post-mortem aging of chicken meat samples. PMID:26830594

  15. Effects of genetic variants for the bovine calpain gene on meat tenderness.

    Science.gov (United States)

    Chung, Hoyoung; Shin, Sungchul; Chung, Euiryong

    2014-05-01

    The objective of this study was to determine whether the genetic variants of CAPN1 developed in several cattle populations can be applied for Hanwoo, regarding genetic effects on meat traits. The traits were examined for 286 purebred Hanwoo steers with genotypes classified by restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP) analysis. The nucleotide positions of primers and previously identified genetic variants were based on sequences of the calpain 1 (CAPN1) gene with GenBank accession numbers (AF252504, AF248054, and AY639597). The analysis of genetic distribution estimated levels of minor allele frequencies ranged from 0.165 to 0.392, showing no significant departures from Hardy-Weinberg Equilibrium for all markers. Overall averages of heterozygosites (He) and polymorphic information contents (PICs) for all markers were calculated to 0.503 and 0.429, respectively, and the g.4558G>A marker showed the lowest He (0.425) and PIC (0.367). Animals from 29 months of age were slaughtered to measure Warner-Bratzler shear force (WBSF), cooking loss, water-holding capacity, pH, fat, and moisture. All the CAPN1 markers explained variations of WBSF, showing significant additive effects except g.5709G>A. A significant marginal mean difference in genotypes of g.6545C>T (P=0.046) was found in moisture with additive effects. From the result it may be possible to use three calpain markers (g.4558G>A, g.4685C>T, and g.6545C>T) classified by RFLP and SSCP analysis in marker assisted selection programs to improve WBSF as meat tenderness in Hanwoo.

  16. Calpain 3 Expression Pattern during Gastrocnemius Muscle Atrophy and Regeneration Following Sciatic Nerve Injury in Rats

    Directory of Open Access Journals (Sweden)

    Ronghua Wu

    2015-11-01

    Full Text Available Calpain 3 (CAPN3, also known as p94, is a skeletal muscle-specific member of the calpain family that is involved in muscular dystrophy; however, the roles of CAPN3 in muscular atrophy and regeneration are yet to be understood. In the present study, we attempted to explain the effect of CAPN3 in muscle atrophy by evaluating CAPN3 expression in rat gastrocnemius muscle following reversible sciatic nerve injury. After nerve injury, the wet weight ratio and cross sectional area (CSA of gastrocnemius muscle were decreased gradually from 1–14 days and then recovery from 14–28 days. The active form of CAPN3 (~62 kDa protein decreased slightly on day 3 and then increased from day 7 to 14 before a decrease from day 14 to 28. The result of linear correlation analysis showed that expression of the active CAPN3 protein level was negatively correlated with muscle wet weight ratio. CAPN3 knockdown by short interfering RNA (siRNA injection improved muscle recovery on days 7 and 14 after injury as compared to that observed with control siRNA treatment. Depletion of CAPN3 gene expression could promote myoblast differentiation in L6 cells. Based on these findings, we conclude that the expression pattern of the active CAPN3 protein is linked to muscle atrophy and regeneration following denervation: its upregulation during early stages may promote satellite cell renewal by inhibiting differentiation, whereas in later stages, CAPN3 expression may be downregulated to stimulate myogenic differentiation and enhance recovery. These results provide a novel mechanistic insight into the role of CAPN3 protein in muscle regeneration after peripheral nerve injury.

  17. Effect of nutrient restriction and re-feeding on calpain family genes in skeletal muscle of channel catfish (Ictalurus punctatus.

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    Elena Preziosa

    Full Text Available BACKGROUND: Calpains, a superfamily of intracellular calcium-dependent cysteine proteases, are involved in the cytoskeletal remodeling and wasting of skeletal muscle. Calpains are generated as inactive proenzymes which are activated by N-terminal autolysis induced by calcium-ions. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we characterized the full-length cDNA sequences of three calpain genes, clpn1, clpn2, and clpn3 in channel catfish, and assessed the effect of nutrient restriction and subsequent re-feeding on the expression of these genes in skeletal muscle. The clpn1 cDNA sequence encodes a protein of 704 amino acids, Clpn2 of 696 amino acids, and Clpn3 of 741 amino acids. Phylogenetic analysis of deduced amino acid sequences indicate that catfish Clpn1 and Clpn2 share a sequence similarity of 61%; catfish Clpn1 and Clpn3 of 48%, and Clpn2 and Clpn3 of only 45%. The domain structure architectures of all three calpain genes in channel catfish are similar to those of other vertebrates, further supported by strong bootstrap values during phylogenetic analyses. Starvation of channel catfish (average weight, 15-20 g for 35 days influenced the expression of clpn1 (2.3-fold decrease, P<0.05, clpn2 (1.3-fold increase, P<0.05, and clpn3 (13.0-fold decrease, P<0.05, whereas the subsequent refeeding did not change the expression of these genes as measured by quantitative real-time PCR analysis. Calpain catalytic activity in channel catfish skeletal muscle showed significant differences only during the starvation period, with a 1.2- and 1.4- fold increase (P<0.01 after 17 and 35 days of starvation, respectively. CONCLUSION/SIGNIFICANCE: We have assessed that fasting and refeeding may provide a suitable experimental model to provide us insight into the role of calpains during fish muscle atrophy and how they respond to changes in nutrient supply.

  18. The calpain, caspase 12, caspase 3 cascade leading to apoptosis is altered in F508del-CFTR expressing cells.

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    Mathieu Kerbiriou

    Full Text Available In cystic fibrosis (CF, the most frequent mutant variant of the cystic fibrosis transmembrane conductance regulator (CFTR, F508del-CFTR protein, is misfolded and retained in the endoplasmic reticulum (ER. We previously showed that the unfolded protein response (UPR may be triggered in CF. Since prolonged UPR activation leads to apoptosis via the calcium-calpain-caspase-12-caspase-3 cascade and because apoptosis is altered in CF, our aim was to compare the ER stress-induced apoptosis pathway between wild type (Wt and F508del-CFTR expressing cells. Here we show that the calcium-calpain-caspase-12-caspase-3 cascade is altered in F508del-CFTR expressing cells. We propose that this alteration is involved in the altered apoptosis triggering observed in CF.

  19. The Calpain Inhibitor A-705253 Attenuates Alcohol-Seeking and Relapse with Low Side-Effect Profile.

    Science.gov (United States)

    Vengeliene, Valentina; Moeller, Achim; Meinhardt, Marcus W; Beardsley, Patrick M; Sommer, Wolfgang H; Spanagel, Rainer; Bespalov, Anton

    2016-03-01

    Preclinical studies revealed contribution of N-methyl-D-aspartate receptors (NMDARs) to a variety of neuropsychiatric diseases including alcoholism, but development of NMDAR antagonists for therapeutic use has been a challenge, in part due to severe side effects. One of the key intracellular events resulting from stimulation of NMDAR is activation of calpains-calcium-dependent cysteine proteases. Here we studied whether inhibition of calpains would produce therapeutic-like effects of NMDAR antagonists but without their NMDAR-mediated side-effect profile. The calpain inhibitor A-705253 (3-10 mg/kg) was tested in a model of cue-induced reinstatement of alcohol-seeking behavior in post-dependent Wistar rats and in an alcohol deprivation effect (ADE) model in long-term alcohol drinking Wistar rats, two behavioral models for alcohol-seeking and relapse, respectively. We also tested the effect of A-705253 on the saccharine deprivation effect (SDE) as a selectivity measure. Acute treatment with A-705253 dose-dependently reduced cue-induced reinstatement of alcohol-seeking behavior. Repeated administration of A-705253 caused significant reductions of relapse-like excessive alcohol intake during the post-abstinence drinking days, an effect that persisted during two more successive drug-free drinking weeks, which was selective for the ADE as the SDE was unaffected. However, A-705253 did not produce psychostimulant, cognition impairing (delayed-matching-to-position), or psychotomimetic effects (specifically, phencyclidine discriminative stimulus effects). Taken together, these results demonstrate the involvement of calpains in alcohol-seeking and relapse and present a rationale for a novel pharmacological intervention that may reduce craving and relapse with minimal side effects in alcohol-dependent patients. PMID:26216521

  20. Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

    OpenAIRE

    Pasupathi Sundaramoorthy; Jae Jun Sim; Yeong-Su Jang; Siddhartha Kumar Mishra; Keun-Yeong Jeong; Poonam Mander; Oh Byung Chul; Won-Sik Shim; Seung Hyun Oh; Ky-Youb Nam; Hwan Mook Kim

    2015-01-01

    Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca2+) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca2+ bound lactate (CaLa), its downstrea...

  1. Calpain-controlled detachment of major glycoproteins from the cytoskeleton regulates adhesive properties of activated phosphatidylserine-positive platelets.

    Science.gov (United States)

    Artemenko, Elena O; Yakimenko, Alena O; Pichugin, Alexey V; Ataullakhanov, Fazly I; Panteleev, Mikhail A

    2016-02-15

    In resting platelets, adhesive membrane glycoproteins are attached to the cytoskeleton. On strong activation, phosphatidylserine(PS)-positive and -negative platelet subpopulations are formed. Platelet activation is accompanied by cytoskeletal rearrangement, although the glycoprotein attachment status in these two subpopulations is not clear. We developed a new, flow cytometry-based, single-cell approach to investigate attachment of membrane glycoproteins to the cytoskeleton in cell subpopulations. In PS-negative platelets, adhesive glycoproteins integrin αIIbβ3, glycoprotein Ib and, as shown for the first time, P-selectin were associated with the cytoskeleton. In contrast, this attachment was disrupted in PS-positive platelets; it was retained to some extent only in the small convex regions or 'caps'. It correlated with the degradation of talin and filamin observed only in PS-positive platelets. Calpain inhibitors essentially prevented the disruption of membrane glycoprotein attachment in PS-positive platelets, as well as talin and filamin degradation. With the suggestion that detachment of glycoproteins from the cytoskeleton may affect platelet adhesive properties, we investigated the ability of PS-positive platelets to resist shear-induced breakaway from the immobilized fibrinogen. Shear rates of 500/s caused PS-positive platelet breakaway, but their adhesion stability increased more than 10-fold after pretreatment of the platelets with calpain inhibitor. In contrast, the ability of PS-positive platelets to adhere to immobilized von Willebrand's factor at 100/s was low, but this was not affected by the preincubation of platelets with a calpain inhibitor. Our data suggest that calpain-controlled detachment of membrane glycoproteins is a new mechanism that is responsible for the loss of ability of the procoagulant platelets to resist detachment from thrombi by high shear stress.

  2. Alterations in the expression of atrial calpains in electrical and structural remodeling during aging and atrial fibrillation.

    Science.gov (United States)

    Xu, Guo-Jun; Gan, Tian-Yi; Tang, Bao-Peng; Chen, Zu-Heng; Mahemuti, Ailiman; Jiang, Tao; Song, Jian-Guo; Guo, Xia; Li, Yao-Dong; Zhou, Xian-Hui; Zhang, Yu; Li, Jin-Xin

    2013-11-01

    The aim of this study was to investigate the correlation between the change in the expression of atrial calpains and electrical, molecular and structural remodeling during aging and atrial fibrillation (AF). Adult and aged canines in sinus rhythm (SR) and with persistent AF (induced by rapid atrial pacing) were investigated. A whole-cell patch clamp was used to measure the L-type Ca2+ current (ICa-L) in cells in the left atrium. The mRNA and protein expression of the L-type calcium channel alc subunit (LVDCCa1c) and calpains were measured by quantitative (q)PCR and western blot analysis. Histopathological and ultrastructural changes were analyzed via light and electron microscopy. The quantity of apoptotic myocytes was determined by a terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assay. In SR groups, atrial cells of the aged canines exhibited a longer action potential (AP) duration to 90% repolarization (APD90), lower AP plateau potential and peak ICa-L current densities (Pcontrol group, the mRNA and protein expression levels of LVDCCa1c were decreased in the aged groups; however, the mRNA and protein expression of calpain 1 was increased in the adult and the aged groups with AF (Patrial tissue exhibited abnormal histopathological and ultrastructural changes, such as accelerated fibrosis and apoptosis with aging and in AF. Age-related alterations in atrial tissues were attributed to the increased expression of calpain 1. The general pathophysiological alterations in normal aged atria may therefore produce a substrate that is conducive to AF. PMID:24043247

  3. Production and processing studies on calpain-system gene markers for beef tenderness: consumer assessments of eating quality.

    Science.gov (United States)

    Robinson, D L; Cafe, L M; McIntyre, B L; Geesink, G H; Barendse, W; Pethick, D W; Thompson, J M; Polkinghorne, R; Greenwood, P L

    2012-08-01

    We investigated the effects of calpain-system genetic markers on consumer beef quality ratings, including interactions of marker effects with hormonal growth promotant (HGP) use and tenderstretch hanging. Brahman cattle in New South Wales (NSW; n = 164) and Western Australia (WA; n = 141) were selected at weaning from commercial and research herds to achieve balance and divergence in calpastatin (CAST) and calpain 3 (CAPN3) gene marker status. Genotypes for μ-calpain (CAPN1-4751 and CAPN1-316) were also determined. Angus cattle (49 in NSW, 17 in WA) with favorable CAST and CAPN3 alleles, balanced for CAPN1-316 status, were also studied. Half the cattle at each site had HGP (Revalor-H, containing 200 mg trenbolone acetate and 20 mg 17β-estradiol) implants during grain finishing. One side of each carcass was suspended from the Achilles tendon (AT) and the other from the pelvis [tenderstretch (TS)]. Meat Standards Australia consumer panels scored 7-d aged striploin steaks from both AT and TS sides, and 7-d aged rump and oyster blade steaks from the AT side of each carcass. Two favorable CAST alleles increased tenderness ratings of AT-striploin, TS-striploin, rump, and oyster blade steaks by, respectively, 6.1, 4.2, 4.2, and 3.1 units, and overall liking by 4.7, 2.8, 2.9, 3.7 (all P Brahman steaks from the same location with the same marker alleles had similar scores. In contrast, NSW Angus striploin steaks scored about 15 units greater for tenderness and overall liking (P < 0.001) than cattle with the same marker alleles at the other 3 location × breed combinations, which had generally similar scores. Therefore, calpain-system gene markers have beneficial effects on eating quality, consistent with our previous findings for objective meat quality.

  4. Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain.

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    Kepa B Uribe

    Full Text Available Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active "soluble AC". The calpain-mediated ACT processing allows trafficking of the "soluble AC" domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP "pools", which would play different roles in the cell pathophysiology.

  5. Calpain inhibitor attenuates ER stress-induced apoptosis in injured spinal cord after bone mesenchymal stem cells transplantation.

    Science.gov (United States)

    Wang, Chao; Shi, Dongling; Song, Xinghui; Chen, Yingying; Wang, Linlin; Zhang, Xiaoming

    2016-07-01

    Bone marrow mesenchymal stem cells (BMSCs) therapy for tissue repair is limited by low survival of cells transplanted in the recipient sites after spinal cord injury (SCI). Here, we investigated the effects of a calpain inhibitor (MDL28170) on BMSCs survival by a rat model of spinal cord injury in vitro and in vivo. Conditioned medium from hypoxia injured VSC4.1 motor neurons (Hypoxia-CM) were collected to mimic the micro-environment of injured spinal cord. Tunicamycin was also applied to induce endoplasmic reticulum (ER) stress in BMSCs. The CCK-8 assay, LDH leakage assay and flow cytometer assay demonstrated that MDL28170 could enhance BMSCs survival in response to Hypoxia-CM and tunicamycin. Moreover, MDL28170 significantly enhanced GFP-positive BMSCs survival in vivo after transplantation into the contused spinal cord of SCI rats. The protective effects of MDL28170 on BMSCs survival may inhibit the activation of calpain and the downstream ER stress-induced apoptosis. The present results suggested for the first time that MDL28170 with BMSCs transplant helped to rescue cells in injured spinal cord by modulating the ER stress-induced apoptosis. The calpain inhibitor, MDL28170 may have the promising new strategies for promoting the survival of transplanted BMSCs on cell-based regenerative medicine. PMID:27137651

  6. Calpain inhibitor attenuates ER stress-induced apoptosis in injured spinal cord after bone mesenchymal stem cells transplantation.

    Science.gov (United States)

    Wang, Chao; Shi, Dongling; Song, Xinghui; Chen, Yingying; Wang, Linlin; Zhang, Xiaoming

    2016-07-01

    Bone marrow mesenchymal stem cells (BMSCs) therapy for tissue repair is limited by low survival of cells transplanted in the recipient sites after spinal cord injury (SCI). Here, we investigated the effects of a calpain inhibitor (MDL28170) on BMSCs survival by a rat model of spinal cord injury in vitro and in vivo. Conditioned medium from hypoxia injured VSC4.1 motor neurons (Hypoxia-CM) were collected to mimic the micro-environment of injured spinal cord. Tunicamycin was also applied to induce endoplasmic reticulum (ER) stress in BMSCs. The CCK-8 assay, LDH leakage assay and flow cytometer assay demonstrated that MDL28170 could enhance BMSCs survival in response to Hypoxia-CM and tunicamycin. Moreover, MDL28170 significantly enhanced GFP-positive BMSCs survival in vivo after transplantation into the contused spinal cord of SCI rats. The protective effects of MDL28170 on BMSCs survival may inhibit the activation of calpain and the downstream ER stress-induced apoptosis. The present results suggested for the first time that MDL28170 with BMSCs transplant helped to rescue cells in injured spinal cord by modulating the ER stress-induced apoptosis. The calpain inhibitor, MDL28170 may have the promising new strategies for promoting the survival of transplanted BMSCs on cell-based regenerative medicine.

  7. Propofol Ameliorates Calpain-induced Collapsin Response Mediator Protein-2 Proteolysis in Traumatic Brain Injury in Rats

    Institute of Scientific and Technical Information of China (English)

    Yun Yu; Min-Yu Jian; Yun-Zhen Wang; Ru-Quan Han

    2015-01-01

    Background:Collapsin response mediator protein-2 (CRMP2),a multifunctional cytosolic protein highly expressed in the brain,is degraded by calpain following traumatic brain injury (TBI),possibly inhibiting posttraumatic neurite regeneration.Lipid peroxidation (LP) is involved in triggering postinjury CRMP2 proteolysis.We examined the hypothesis that propofol could attenuate LP,calpain-induced CRMP2 degradation,and brain injury after TBI.Methods:A unilateral moderate controlled cortical impact injury was induced in adult male Sprague-Dawley rats.The animals were randomly divided into seven groups:Sham control group,TBI group,TBI + propofol groups (including propofol 1 h,2 h,and 4 h groups),TBI + U83836E group and TBI + fat emulsion group.The LP inhibitor U83836E was used as a control to identify that antioxidation partially accounts for the potential neuroprotective effects of propofol.The solvent of propofol,fat emulsion,was used as the vehicle control.Ipsilateral cortex tissues were harvested at 24 h post-TBI.Immunofluorescent staining,Western blot analysis,and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling were used to evaluate LP,calpain activity,CRMP2 proteolysis and programmed cell death.The data were statistically analyzed using one-way analysis of variance and a paired t-test.Results:Propofol and U83836E significantly ameliorated the CRMP2 proteolysis.In addition,both propofol and U83836E significantly decreased the ratio of 145-kDa αⅡ-spectrin breakdown products to intact 270-kDa spectrin,the 4-hydroxynonenal expression and programmed cell death in the pericontusional cortex at 24 h after TBI.There was no difference between the TBI group and the fat emulsion group.Conclusions:These results demonstrate that propofol postconditioning alleviates calpain-mediated CRMP2 proteolysis and provides neuroprotective effects following moderate TBI potentially by counteracting LP and reducing calpain activation.

  8. Assessment of calpain and caspase systems activities during ageing of two bovine muscles by degradation patterns of αII spectrin and PARP-1.

    Science.gov (United States)

    Saccà, Elena; Pizzutti, Nicoletta; Corazzin, Mirco; Lippe, Giovanna; Piasentier, Edi

    2016-03-01

    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 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. PMID:26950517

  9. Neuroprotective effect of synthetic chalcone derivatives as competitive dual inhibitors against μ-calpain and cathepsin B through the downregulation of tau phosphorylation and insoluble Aβ peptide formation.

    Science.gov (United States)

    Jeon, Kyung-Hwa; Lee, Eunyoung; Jun, Kyu-Yeon; Eom, Ji-Eun; Kwak, Soo Yeon; Na, Younghwa; Kwon, Youngjoo

    2016-10-01

    A series of chalcone derivatives were synthesized and evaluated for their μ-calpain and cathepsin B inhibitory activities. Among the tested chalcone derivatives, two compounds, 7 and 11, showed potent inhibitory activities against μ-calpain and cathepsin B and were selected for further evaluation. Compounds 7 and 11 showed enzyme inhibitory activities at the cellular level and displayed neuroprotective effects against oxidative stress-induced apoptosis in SH-SY5Y cells, a human neuroblastoma cell line. Moreover, compounds 7 and 11 reduced p25 formation, tau phosphorylation and insoluble Aβ peptide formation. Enzyme kinetic experiments and docking studies revealed that compounds 7 and 11 competitively inhibited both μ-calpain and cathepsin B enzymes. PMID:27318120

  10. The Relation Between Calpain System and Muscle Tenderness%钙蛋白酶系统与肌肉嫩度的关系

    Institute of Scientific and Technical Information of China (English)

    赵红艳

    2008-01-01

    钙蛋白酶系统主要由钙蛋白酶(μ-calpain,m-calpain)及钙蛋白酶抑制蛋白(calpastatin)组成,calpain是存在于细胞质中的依赖于ca2+的中性蛋白酶,calpastatin是钙蛋白酶的内源抑制蛋白.本文综述了钙蛋白酶系统各种酶的结构、作用、活性调节机能及其与肉质嫩度的关系.

  11. Production and processing studies on calpain-system gene markers for beef tenderness: consumer assessments of eating quality.

    Science.gov (United States)

    Robinson, D L; Cafe, L M; McIntyre, B L; Geesink, G H; Barendse, W; Pethick, D W; Thompson, J M; Polkinghorne, R; Greenwood, P L

    2012-08-01

    We investigated the effects of calpain-system genetic markers on consumer beef quality ratings, including interactions of marker effects with hormonal growth promotant (HGP) use and tenderstretch hanging. Brahman cattle in New South Wales (NSW; n = 164) and Western Australia (WA; n = 141) were selected at weaning from commercial and research herds to achieve balance and divergence in calpastatin (CAST) and calpain 3 (CAPN3) gene marker status. Genotypes for μ-calpain (CAPN1-4751 and CAPN1-316) were also determined. Angus cattle (49 in NSW, 17 in WA) with favorable CAST and CAPN3 alleles, balanced for CAPN1-316 status, were also studied. Half the cattle at each site had HGP (Revalor-H, containing 200 mg trenbolone acetate and 20 mg 17β-estradiol) implants during grain finishing. One side of each carcass was suspended from the Achilles tendon (AT) and the other from the pelvis [tenderstretch (TS)]. Meat Standards Australia consumer panels scored 7-d aged striploin steaks from both AT and TS sides, and 7-d aged rump and oyster blade steaks from the AT side of each carcass. Two favorable CAST alleles increased tenderness ratings of AT-striploin, TS-striploin, rump, and oyster blade steaks by, respectively, 6.1, 4.2, 4.2, and 3.1 units, and overall liking by 4.7, 2.8, 2.9, 3.7 (all P Brahman steaks from the same location with the same marker alleles had similar scores. In contrast, NSW Angus striploin steaks scored about 15 units greater for tenderness and overall liking (P quality, consistent with our previous findings for objective meat quality. PMID:22367069

  12. Modulation of intracellular calcium levels by calcium lactate affects colon cancer cell motility through calcium-dependent calpain.

    Directory of Open Access Journals (Sweden)

    Pasupathi Sundaramoorthy

    Full Text Available Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca2+ supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca2+ bound lactate (CaLa, its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca2+ (iCa2+ levels for a prolonged period of time. Ca2+ influx induced cleavage of FAK into an N-terminal FAK (FERM domain in a dose-dependent manner. Phosphorylated FAK (p-FAK was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca2+ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca2+ supplementation to patient undergoing treatment for metastatic cancer.

  13. mdm Muscular Dystrophy: Interactions with Calpain 3 and a Novel Functional Role for Titin’s N2A Domain

    OpenAIRE

    Huebsch, Kimberly A.; Kudryashova, Elena; WOOLEY, CHRISTINE M.; SHER, ROGER B.; Seburn, Kevin L.; Spencer, Melissa J.; Cox, Gregory A.

    2005-01-01

    Human tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J) are caused by mutations in the giant sarcomeric protein titin (TTN) adjacent to a binding site for the muscle-specific protease calpain 3 (CAPN3). Muscular dystrophy with myositis (mdm) is a recessive mouse mutation with severe and progressive muscular degeneration caused by a deletion in the N2A domain of titin (TTN-N2AΔ83), disrupting a putative binding site for CAPN3. To determine whether the muscular dyst...

  14. Calcium paradox induces apoptosis in the isolated perfused Rana ridibunda heart: involvement of p38-MAPK and calpain.

    Science.gov (United States)

    Aggeli, Ioanna-Katerina; Zacharias, Triantafyllos; Papapavlou, Georgia; Gaitanaki, Catherine; Beis, Isidoros

    2013-12-01

    "Calcium paradox" as a term describes the deleterious effects conferred to a heart perfused with a calcium-free solution followed by repletion, including loss of mechanical activity and sarcomere disruption. Given that the signaling mechanisms triggered by calcium paradox remain elusive, in the present study, we tried to investigate them in the isolated perfused heart from Rana ridibunda. Calcium paradox was found to markedly activate members of the MAPKs (p43-ERK, JNKs, p38-MAPK). In addition to lactate dehydrogenase (LDH) release in the perfusate (indicative of necrosis), we also confirmed the occurrence of apoptosis by using the TUNEL assay and identifying poly(ADP-ribose) polymerase (PARP) fragmentation and upregulated Bax expression. Furthermore, using MDL28170 (a selective calpain inhibitor), a role for this protease was revealed. In addition, various divalent cations were shown to exert a protective effect against the calcium paradox. Interestingly, SB203580, a p38-MAPK inhibitor, alleviated calcium-paradox-conferred apoptosis. This result indicates that p38-MAPK plays a pro-apoptotic role, contributing to the resulting myocardial dysfunction and cell death. To our knowledge, this is the first time that the calcium paradox has been shown to induce apoptosis in amphibians, with p38-MAPK and calpain playing significant roles.

  15. Cav1.4 L-Type Calcium Channels Contribute to Calpain Activation in Degenerating Photoreceptors of rd1 Mice.

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    Christian Schön

    Full Text Available Retinitis pigmentosa is an inherited blinding disorder characterized by progressive degeneration and loss of photoreceptors. The exact mechanism of degeneration and cell death of photoreceptors is not known, but is thought to involve disturbed Ca2+-signaling. Ca2+ can enter the photoreceptor cell via outer segment cyclic nucleotide-gated (CNG channels or synaptic Cav1.4 L-type voltage-gated calcium channels (VGCC. Previously, we have shown that genetic ablation of the Cngb1 gene encoding the B subunit of the rod CNG channel delays the fast progressing degeneration in the rd1 mutant mouse model of retinitis pigmentosa. In this study, we crossbred rd1 mice with the Cacna1f-deficient mouse lacking the Cav1.4 α1 subunit of the L-type VGCC. Longitudinal in vivo examinations of photoreceptor layer thickness by optical coherence tomography revealed a significant, but not sustained delay of retinal degeneration in Cacna1f x rd1 double mutant mice compared to rd1 mice. This was accompanied by a reduction of TUNEL positive cells in the early phase of rod degeneration. Remarkably, Cacna1f x rd1 double mutant mice displayed a strong decrease in the activation of the Ca2+-dependent protease calpain during photoreceptor loss. Our results show that genetic deletion of the synaptic Cav1.4 L-type VGCCs impairs calpain activation and leads to a short-term preservation of photoreceptors in the rd1 mouse.

  16. Calpastatin and µ-calpain differ in their control of genotype specific residual variance of beef tenderness in Angus and MARC III steers

    Science.gov (United States)

    Genotype variant effects of calpastatin (CAST) and µ-calpain (CAPN1) on mean beef tenderness have been widely characterized. We have tested whether these genetic variants also control residual (non-genetic) variation, and subsequently total phenotypic variation, of tenderness. Observation of rare ...

  17. Human U87 astrocytoma cell invasion induced by interaction of βig-h3 with integrin α5β1 involves calpain-2.

    Directory of Open Access Journals (Sweden)

    Jie Ma

    Full Text Available It is known that βig-h3 is involved in the invasive process of many types of tumors, but its mechanism in glioma cells has not been fully clarified. Using immunofluorescent double-staining and confocal imaging analysis, and co-immunoprecipitation assays, we found that βig-h3 co-localized with integrin α5β1 in U87 cells. We sought to elucidate the function of this interaction by performing cell invasion assays and gelatin zymography experiments. We found that siRNA knockdowns of βig-h3 and calpain-2 impaired cell invasion and MMP secretion. Moreover, βig-h3, integrins and calpain-2 are known to be regulated by Ca(2+, and they are also involved in tumor cell invasion. Therefore, we further investigated if calpain-2 was relevant to βig-h3-integrin α5β1 interaction to affect U87 cell invasion. Our data showed that βig-h3 co-localized with integrin α5β1 to enhance the invasion of U87 cells, and that calpain-2, is involved in this process, acting as a downstream molecule.

  18. Genetic disruption of calpain correlates with loss of membrane blebbing and differential expression of RhoGDI-1, cofilin and tropomyosin

    DEFF Research Database (Denmark)

    Larsen, Anna Karina; Lametsch, Rene; Elce, John S.;

    2008-01-01

    blebbing was significantly reduced in calpain-knockout cells, and genetic rescue fully restored the wild-type phenotype in knockout cells. Proteomic comparison of wild-type and knockout cells identified decreased levels of RhoGDI-1 (Rho GDP-dissociation inhibitor) and cofilin 1, and increased levels...

  19. Effect of sire on mu- and m-calpain activity and rate of tenderization as indicated by myofibril fragmentation indices of steaks from Brahman cattle.

    Science.gov (United States)

    Riley, D G; Chase, C C; Pringle, T D; West, R L; Johnson, D D; Olson, T A; Hammond, A C; Coleman, S W

    2003-10-01

    The objectives of this study were to assess the influence of sire on mu- and m-calpain activities, to evaluate the relationships of activities of these enzymes to other traits related to beef palatability, and to assess the influence of sire on the rate of tenderization (as measured by myofibril fragmentation index [MFI]) in Brahman longissimus muscle. Brahman calves (n = 87), sired by nine bulls, were born, weaned, fed, and slaughtered in central Florida. Traits evaluated were mu- and m-calpain activities and MFI after 1, 7, 14, and 21 d of aging. Other traits were analyzed to determine their associations with mu- and m-calpain activity and MFI, including calpastatin activity, percentage of raw and cooked lipids, Warner-Bratzler shear force (WBSF) values after 7, 14, and 21 d of aging, and sensory panel rating of tenderness, juiciness, and connective tissue amount after 14 d of aging. Data were analyzed using a model with sire, sex, year, and slaughter group (calves of the same sex slaughtered on the same date) as fixed effects, and adjusted to a constant adjusted 12th-rib fat thickness. Sire affected mu-calpain activity (P carcass sorting program represents an alternative consideration for tenderization improvement programs.

  20. 钙蛋白酶与心血管疾病的关系%Roles of calpains in cardiovascular system diseases

    Institute of Scientific and Technical Information of China (English)

    吴金兰; 万福生

    2011-01-01

    钙蛋白酶(calpain)是一种依赖 Ca2+激活的蛋白水解酶,属于半胱氨酸蛋白水解酶超家族成员.钙蛋白酶广泛分布于心血管系统,可被Ca2+澈活,产生多种生物学效应,如降解心肌收缩蛋白、促进细胞凋亡、参与心血管重构等.近年,钙蛋白酶与心肌缺血再灌注损伤、血栓、房颤、动脉粥样硬化等心血管疾病的关系正受到越来越多的关注.%Calpain is a Ca2+-activated protease, which belongs to the super family of homocystein protease. Upon activation, calpains can cleave myocardial contractile protein, promote myocardial apoptosis, be involved in cardiovascular remodeling,and so on. The relationship between calpains and cardiovascular system diseases such as ischemia reperfusion injury, thrombosis,atrial fibrillation, atherosclerosis, etc, has received more and more attention in recent years.

  1. Effect of sire on mu- and m-calpain activity and rate of tenderization as indicated by myofibril fragmentation indices of steaks from Brahman cattle.

    Science.gov (United States)

    Riley, D G; Chase, C C; Pringle, T D; West, R L; Johnson, D D; Olson, T A; Hammond, A C; Coleman, S W

    2003-10-01

    The objectives of this study were to assess the influence of sire on mu- and m-calpain activities, to evaluate the relationships of activities of these enzymes to other traits related to beef palatability, and to assess the influence of sire on the rate of tenderization (as measured by myofibril fragmentation index [MFI]) in Brahman longissimus muscle. Brahman calves (n = 87), sired by nine bulls, were born, weaned, fed, and slaughtered in central Florida. Traits evaluated were mu- and m-calpain activities and MFI after 1, 7, 14, and 21 d of aging. Other traits were analyzed to determine their associations with mu- and m-calpain activity and MFI, including calpastatin activity, percentage of raw and cooked lipids, Warner-Bratzler shear force (WBSF) values after 7, 14, and 21 d of aging, and sensory panel rating of tenderness, juiciness, and connective tissue amount after 14 d of aging. Data were analyzed using a model with sire, sex, year, and slaughter group (calves of the same sex slaughtered on the same date) as fixed effects, and adjusted to a constant adjusted 12th-rib fat thickness. Sire affected mu-calpain activity (P carcass sorting program represents an alternative consideration for tenderization improvement programs. PMID:14552370

  2. Effects of calpastatin and micro-calpain markers in beef cattle on tenderness traits.

    Science.gov (United States)

    Casas, E; White, S N; Wheeler, T L; Shackelford, S D; Koohmaraie, M; Riley, D G; Chase, C C; Johnson, D D; Smith, T P L

    2006-03-01

    The objective of this study was to assess the association of single nucleotide polymorphisms (SNP) developed at the calpastatin (CAST) and mu-calpain (CAPN1) genes with meat tenderness and palatability traits in populations with diverse genetic backgrounds. Three populations were used in the study. One population consisted of Bos taurus that included crossbred animals derived from Hereford, Angus, Red Angus, Limousin, Charolais, Gelbvieh, and Simmental (GPE7; n = 539). Another population consisted of Bos taurus with Bos indicus influence, including crossbred animals from Hereford, Angus, Brangus, Beefmaster, Bonsmara, and Romosinuano (GPE8; n = 580). The third population was Bos indicus and consisted of purebred Brahman (STARS; n = 444). Traits evaluated were meat tenderness measured as Warner-Bratzler shear force (WBSF; kg) at 14 d postmortem, and traits evaluated by trained sensory panels that included tenderness score, juiciness, and flavor intensity. A SNP at the CAST gene had a significant (P < 0.003) effect on WBSF and tenderness score in the GPE7 and GPE8 populations. Animals inheriting the TT genotype at CAST had meat that was more tender than those inheriting the CC genotype. The marker at the CAPN1 gene was significant (P < 0.03) for tenderness score in GPE7 and GPE8. Animals inheriting the CC genotype at CAPN1 had meat that was more tender than those inheriting the TT genotype. Markers at the CAST and CAPN1 genes were associated with flavor intensity in the GPE8 population. Animals inheriting the CC genotype at CAST and the TT genotype at CAPN1 produced steaks with an intense flavor when compared with the other genotypes. An interaction between CAST and CAPN1 was detected (P < 0.05) for WBSF on GPE8. The statistical significance of the interaction is questionable because of the limited number of observations in some cells. Markers developed at the CAST and CAPN1 genes are suitable for use in identifying animals with the genetic potential to produce meat

  3. Similar to spironolactone, oxymatrine is protective in aldosterone-induced cardiomyocyte injury via inhibition of calpain and apoptosis-inducing factor signaling.

    Directory of Open Access Journals (Sweden)

    Ting-Ting Xiao

    Full Text Available Accumulating evidence indicates that oxymatrine (OMT possesses variously pharmacological properties, especially on the cardiovascular system. We previously demonstrated that activated calpain/apoptosis-inducing factor (AIF-mediated pathway was the key molecular mechanism in aldosterone (ALD induces cardiomyocytes apoptosis. In the present study, we extended the experimentation by investigating the effect of OMT on cardiomyocytes exposed to ALD, as compared to spironolactone (Spiro, a classical ALD receptor antagonist. Cardiomyocytes were pre-incubated with OMT, Spiro or vehicle for 1 h, and then, cardiomyocytes were exposed to ALD 24 h. The cell injury was evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and lactate dehydrogenase (LDH leakage ratio. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay, annexin V/PI staining, and relative caspase-3 activity assay. Furthermore, expression of pro-apoptotic proteins including truncated Bid (tBid, calpain and AIF were evaluated by western blot analysis. ALD stimulation increased cardiomyocytes apoptosis, caspase-3 activity and protein expression of calpain, tBid and AIF in the cytosol (p<0.05. Pre-incubated with cardiomyocytes injury and increased caspase-3 activity were significantly attenuated (p<0.05. Furthermore, OMT suppressed ALD-induced high expression of calpain and AIF. And these effects of OMT could be comparable to Spiro. These findings indicated that OMT might be a potential cardioprotective-agent against excessive ALD-induced cardiotoxicity, at least in part, mediated through inhibition of calpain/AIF signaling.

  4. Production and processing studies on calpain-system gene markers for tenderness in Brahman cattle: 2. Objective meat quality.

    Science.gov (United States)

    Cafe, L M; McIntyre, B L; Robinson, D L; Geesink, G H; Barendse, W; Pethick, D W; Thompson, J M; Greenwood, P L

    2010-09-01

    Effects and interactions of calpain-system tenderness gene markers on objective meat quality traits of Brahman (Bos indicus) cattle were quantified within 2 concurrent experiments at different locations. Cattle were selected for study from commercial and research herds at weaning based on their genotype for calpastatin (CAST) and calpain 3 (CAPN3) gene markers for beef tenderness. Gene marker status for mu-calpain (CAPN1-4751 and CAPN1-316) was also determined for inclusion in statistical analyses. Eighty-two heifer and 82 castrated male cattle with 0 or 2 favorable alleles for CAST and CAPN3 were studied in New South Wales (NSW), and 143 castrated male cattle with 0, 1, or 2 favorable alleles for CAST and CAPN3 were studied in Western Australia (WA). The cattle were backgrounded for 6 to 8 mo and grain-fed for 117 d (NSW) or 80 d (WA) before slaughter. One-half the cattle in each experiment were implanted with a hormonal growth promotant during feedlotting. One side of each carcass was suspended from the Achilles tendon (AT) and the other from the pelvis (tenderstretch). The M. longissimus lumborum from both sides and the M. semitendinosus from the AT side were collected; then samples of each were aged at 1 degrees C for 1 or 7 d. Favorable alleles for one or more markers reduced shear force, with little effect on other meat quality traits. The size of effects of individual markers varied with site, muscle, method of carcass suspension, and aging period. Individual marker effects were additive as evident in cattle with 4 favorable alleles for CAST and CAPN3 markers, which had shear force reductions of 12.2 N (P 0.05) of interactions between the gene markers, or between the hormonal growth promotant and gene markers for any meat quality traits. This study provides further evidence that selection based on the CAST or CAPN3 gene markers improves meat tenderness in Brahman cattle, with little if any detrimental effects on other meat quality traits. The CAPN1-4751 gene

  5. Production and processing studies on calpain-system gene markers for tenderness in Brahman cattle: 2. Objective meat quality.

    Science.gov (United States)

    Cafe, L M; McIntyre, B L; Robinson, D L; Geesink, G H; Barendse, W; Pethick, D W; Thompson, J M; Greenwood, P L

    2010-09-01

    Effects and interactions of calpain-system tenderness gene markers on objective meat quality traits of Brahman (Bos indicus) cattle were quantified within 2 concurrent experiments at different locations. Cattle were selected for study from commercial and research herds at weaning based on their genotype for calpastatin (CAST) and calpain 3 (CAPN3) gene markers for beef tenderness. Gene marker status for mu-calpain (CAPN1-4751 and CAPN1-316) was also determined for inclusion in statistical analyses. Eighty-two heifer and 82 castrated male cattle with 0 or 2 favorable alleles for CAST and CAPN3 were studied in New South Wales (NSW), and 143 castrated male cattle with 0, 1, or 2 favorable alleles for CAST and CAPN3 were studied in Western Australia (WA). The cattle were backgrounded for 6 to 8 mo and grain-fed for 117 d (NSW) or 80 d (WA) before slaughter. One-half the cattle in each experiment were implanted with a hormonal growth promotant during feedlotting. One side of each carcass was suspended from the Achilles tendon (AT) and the other from the pelvis (tenderstretch). The M. longissimus lumborum from both sides and the M. semitendinosus from the AT side were collected; then samples of each were aged at 1 degrees C for 1 or 7 d. Favorable alleles for one or more markers reduced shear force, with little effect on other meat quality traits. The size of effects of individual markers varied with site, muscle, method of carcass suspension, and aging period. Individual marker effects were additive as evident in cattle with 4 favorable alleles for CAST and CAPN3 markers, which had shear force reductions of 12.2 N (P 0.05) of interactions between the gene markers, or between the hormonal growth promotant and gene markers for any meat quality traits. This study provides further evidence that selection based on the CAST or CAPN3 gene markers improves meat tenderness in Brahman cattle, with little if any detrimental effects on other meat quality traits. The CAPN1-4751 gene

  6. Effect of two dietary concentrate levels on tenderness, calpain and calpastatin activities, and carcass merit in Waguli and Brahman steers.

    Science.gov (United States)

    Ibrahim, R M; Goll, D E; Marchello, J A; Duff, G C; Thompson, V F; Mares, S W; Ahmad, H A

    2008-06-01

    The objective of this study was to compare carcass characteristics of a newly introduced breed, the Waguli (Wagyu x Tuli), with the carcass characteristics of the Brahman breed. Brahman cattle are used extensively in the Southwest of the United States because of their tolerance to adverse environmental conditions. However, Brahman carcasses are discounted according to the height of their humps because of meat tenderness issues. The Waguli was developed in an attempt to obtain a breed that retained the heat tolerance of the Brahman but had meat quality attributes similar to the Wagyu. Twenty-four animals were used. Six steers from each breed were fed a 94% concentrate diet and 6 steers from each breed were fed an 86% concentrate diet. Eight steers, 2 from each group, were harvested after 128 d, after 142 d, and after 156 d on feed. Waguli steers had larger LM, greater backfat thickness, greater marbling scores, and greater quality grades than the Brahman steers (P meat, and these traits are also present in the Waguli. The Waguli had significantly lower Warner-Bratzler shear force values than the Brahman steers after 7 and 10 d of postmortem aging (P Brahman had increased to acceptable levels. Toughness of the Brahman has been associated with high levels of calpastatin in Brahman muscle, and the Waguli LM had significantly less calpastatin activity (P = 0.02) at 0 h postmortem than the Brahman LM. At 0-h postmortem, the total LM calpain activity did not differ between the Brahman and Waguli (P = 0.57). Neither diet nor days on feed had any significant effect on the 0-h postmortem calpain or at 0-h postmortem calpastatin activity, nor an effect on Warner-Bratzler shear-force values. In conclusion, LM muscle from the Waguli steers had a high degree of marbling, lower shear force values, and low calpastatin activity, all of which are related to more tender meat. PMID:18310491

  7. Effect of two dietary concentrate levels on tenderness, calpain and calpastatin activities, and carcass merit in Waguli and Brahman steers.

    Science.gov (United States)

    Ibrahim, R M; Goll, D E; Marchello, J A; Duff, G C; Thompson, V F; Mares, S W; Ahmad, H A

    2008-06-01

    The objective of this study was to compare carcass characteristics of a newly introduced breed, the Waguli (Wagyu x Tuli), with the carcass characteristics of the Brahman breed. Brahman cattle are used extensively in the Southwest of the United States because of their tolerance to adverse environmental conditions. However, Brahman carcasses are discounted according to the height of their humps because of meat tenderness issues. The Waguli was developed in an attempt to obtain a breed that retained the heat tolerance of the Brahman but had meat quality attributes similar to the Wagyu. Twenty-four animals were used. Six steers from each breed were fed a 94% concentrate diet and 6 steers from each breed were fed an 86% concentrate diet. Eight steers, 2 from each group, were harvested after 128 d, after 142 d, and after 156 d on feed. Waguli steers had larger LM, greater backfat thickness, greater marbling scores, and greater quality grades than the Brahman steers (P Brahman steers after 7 and 10 d of postmortem aging (P Brahman had increased to acceptable levels. Toughness of the Brahman has been associated with high levels of calpastatin in Brahman muscle, and the Waguli LM had significantly less calpastatin activity (P = 0.02) at 0 h postmortem than the Brahman LM. At 0-h postmortem, the total LM calpain activity did not differ between the Brahman and Waguli (P = 0.57). Neither diet nor days on feed had any significant effect on the 0-h postmortem calpain or at 0-h postmortem calpastatin activity, nor an effect on Warner-Bratzler shear-force values. In conclusion, LM muscle from the Waguli steers had a high degree of marbling, lower shear force values, and low calpastatin activity, all of which are related to more tender meat.

  8. Isoform-specific function of calpains in cell adhesion disruption: studies in postlactational mammary gland and breast cancer.

    Science.gov (United States)

    Rodríguez-Fernández, Lucía; Ferrer-Vicens, Iván; García, Concha; Oltra, Sara S; Zaragozá, Rosa; Viña, Juan R; García-Trevijano, Elena R

    2016-09-15

    Cleavage of adhesion proteins is the first step for physiological clearance of undesired cells during postlactational regression of the mammary gland, but also for cell migration in pathological states such as breast cancer. The intracellular Ca(2+)-dependent proteases, calpains (CAPNs), are known to cleave adhesion proteins. The isoform-specific function of CAPN1 and CAPN2 was explored and compared in two models of cell adhesion disruption: mice mammary gland during weaning-induced involution and breast cancer cell lines according to tumor subtype classification. In both models, E-cadherin, β-catenin, p-120, and talin-1 were cleaved as assessed by western blot analysis. Both CAPNs were able to cleave adhesion proteins from lactating mammary gland in vitro Nevertheless, CAPN2 was the only isoform found to co-localize with E-cadherin in cell junctions at the peak of lactation. CAPN2/E-cadherin in vivo interaction, analyzed by proximity ligation assay, was dramatically increased during involution. Calpain inhibitor administration prevented the cytosolic accumulation of truncated E-cadherin cleaved by CAPN2. Conversely, in breast cancer cells, CAPN2 was restricted to the nuclear compartment. The isoform-specific expression of CAPNs and CAPN activity was dependent on the breast cancer subtype. However, CAPN1 and CAPN2 knockdown cells showed that cleavage of adhesion proteins and cell migration was mediated by CAPN1, independently of the breast cancer cell line used. Data presented here suggest that the subcellular distribution of CAPN1 and CAPN2 is a major issue in target-substrate recognition; therefore, it determines the isoform-specific role of CAPNs during disruption of cell adhesion in either a physiological or a pathological context. PMID:27402795

  9. Inhibitors of cysteine cathepsin and calpain do not prevent ultraviolet-B-induced apoptosis in human keratinocytes and HeLa cells

    DEFF Research Database (Denmark)

    Bang, Bo; Baadsgaard, Ole; Skov, Lone;

    2004-01-01

    Caspases, members of the cysteine protease family, execute UVB-induced apoptosis in several cell lines and keratinocytes. Several researchers investigating UVB-induced apoptosis have demonstrated a dose-dependent protective effect of the synthetic peptide caspase inhibitor zVAD-fmk. However, z......VAD-fmk displays a dose-dependent protective effect against UVB-induced apoptosis, even at doses higher than those required to block all known proapoptotic caspases. In addition, it is known that zVAD-fmk also inhibits other cysteine proteases including cathepsins and calpains, and these proteases have recently...... been demonstrated to play a role in the execution of programmed cell death induced by other stimuli, e.g. TNF-alpha. The purpose of the present study was therefore to investigate whether inhibitors of cysteine cathepsins and calpains could prevent UVB-induced apoptosis in HeLa cells and keratinocytes...

  10. 宰后牦牛肉成熟过程中钙激活酶与嫩度指标的相关性分析%Correlation Analysis between Calpains and Tenderness Indexes during Postmortem Aging of Yak Meat

    Institute of Scientific and Technical Information of China (English)

    师希雄; 余群力; 党欣

    2013-01-01

    Longissimus dorsi muscles of ten yaks from the south part of Gansu province were tested for myofibril fragmentation index (MFI),shear force,muscle fiber diameter and the activities of calpains (μ-calpain,m-calpain and calpastatin) during 8 d of postmortem aging.Furthermore,the calpain activities were analyzed for correlation with MFI,shear force and muscle fiber diameter.The results showed that each calpain activity was positively correlated with shear force and muscle fiber diameter,but negatively correlated with MFI; a significant correlation with MFI was observed for μ-calpain and calpastatin (P<0.05).Therefore,the changes in calpain activity may cause in the increase in MFI,weaken myofibrils and tenderize meat andμ-calpain seems to mainly contribute to tenderizing yak meat.%以10头甘南牦牛为研究对象,对宰后8d成熟期间肌原纤维小片化指数、剪切力、肌纤维直径、μ-钙蛋白酶(μ-calpain)、m-钙蛋白酶(m-calpain)、钙蛋白酶抑素(calpastatin)的活力进行了测定,同时研究了3种酶活力与肌原纤维小片化指数、剪切力、肌纤维直径3个嫩度指标之间的相关性.结果表明:μ-calpain、m-calpain、calpastatin 3种酶与剪切力值及肌纤维直径均呈正相关;3种酶与肌原纤维小片化指数呈负相关,其中μ-calpain与calpastatin呈显著负相关(P<0.05).因此,钙激活酶活力的变化可能导致了肌原纤维小片化指数的增加,肌原纤维的弱化和肉的嫩化,μ-calpain可能是牦牛肉嫩化的主要贡献者.

  11. Truncation and Activation of Dual Specificity Tyrosine Phosphorylation-regulated Kinase 1A by Calpain I: A MOLECULAR MECHANISM LINKED TO TAU PATHOLOGY IN ALZHEIMER DISEASE.

    Science.gov (United States)

    Jin, Nana; Yin, Xiaomin; Gu, Jianlan; Zhang, Xinhua; Shi, Jianhua; Qian, Wei; Ji, Yuhua; Cao, Maohong; Gu, Xiaosong; Ding, Fei; Iqbal, Khalid; Gong, Cheng-Xin; Liu, Fei

    2015-06-12

    Hyperphosphorylation and dysregulation of exon 10 splicing of Tau are pivotally involved in pathogenesis of Alzheimer disease (AD) and/or other tauopathies. Alternative splicing of Tau exon 10, which encodes the second microtubule-binding repeat, generates Tau isoforms containing three and four microtubule-binding repeats, termed 3R-Taus and 4R-Taus, respectively. Dual specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1A) lies at the Down syndrome critical region of chromosome 21. Overexpression of this kinase may contribute to the early Tau pathology in Down syndrome via phosphorylation of Tau and dysregulation of Tau exon 10. Here, we report that Dyrk1A was truncated at the C terminus and was associated with overactivation of calpain I in AD brain. Calpain I proteolyzed Dyrk1A in vitro first at the C terminus and further at the N terminus and enhanced its kinase activity toward Tau via increased Vmax but not Km. C-terminal truncation of Dyrk1A resulted in stronger activity than its full-length protein in promotion of exon 10 exclusion and phosphorylation of Tau. Dyrk1A was truncated in kainic acid-induced excitotoxic mouse brains and coincided with an increase in 3R-Tau expression and phosphorylation of Tau via calpain activation. Moreover, truncation of Dyrk1A was correlated with an increase in the ratio of 3R-Tau/4R-Tau and Tau hyperphosphorylation in AD brain. Collectively, these findings suggest that truncation/activation of Dyrk1A by Ca(2+)/calpain I might contribute to Tau pathology via promotion of exon 10 exclusion and hyperphosphorylation of Tau in AD brain. PMID:25918155

  12. Chronic administration of a leupeptin-derived calpain inhibitor fails to ameliorate severe muscle pathology in a canine model of Duchenne muscular dystrophy

    Directory of Open Access Journals (Sweden)

    Martin K Childers

    2012-01-01

    Full Text Available Calpains likely play a role in the pathogenesis of Duchenne muscular dystrophy (DMD. Accordingly, calpain inhibition may provide therapeutic benefit to DMD patients. In the present study, we sought to measure benefit from administration of a novel calpain inhibitor, C101, in a canine muscular dystrophy model. Specifically, we tested the hypothesis that treatment with C101 mitigates progressive weakness and severe muscle pathology observed in young dogs with golden retriever muscular dystrophy (GRMD. Young (6 week-old GRMD dogs were treated daily with either C101 (17mg/kg twice daily oral dose, n=9 or placebo (vehicle only, n=7 for 8 weeks. A battery of functional tests, including tibiotarsal joint angle, muscle/fat composition, and pelvic limb muscle strength were performed at baseline and every two weeks during the 8-week study. Results indicate that C101-treated GRMD dogs maintained strength in their cranial pelvic limb muscles (tibiotarsal flexors while placebo-treated dogs progressively lost strength. However, concomitant improvement was not observed in posterior pelvic limb muscles (tibiotarsal extensors. C101 treatment did not mitigate force drop following repeated eccentric contractions and no improvement was seen in the development of joint contractures, lean muscle mass or muscle histopathology. Taken together, these data do not support the hypothesis that treatment with C101 mitigates progressive weakness or ameliorates severe muscle pathology observed in young dogs with GRMD.

  13. FRET-FLIM investigation of PSD95-NMDA receptor interaction in dendritic spines; control by calpain, CaMKII and Src family kinase.

    Directory of Open Access Journals (Sweden)

    Kim Doré

    Full Text Available Little is known about the changes in protein interactions inside synapses during synaptic remodeling, as their live monitoring in spines has been limited. We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction. NMDAR stimulation caused a transient decrease in FRET between the NMDAR and PSD95 in spines of young and mature neurons. The activity of both CaMKII and calpain were essential for this effect in both developmental stages. Meanwhile, inhibition of Src family kinase (SFK had opposing impacts on this decrease in FRET in young versus mature neurons. Our data suggest concerted roles for CaMKII, SFK and calpain activity in regulating activity-dependent separation of PSD95 from GluN2A or GluN2B. Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.

  14. Involvement of calpain/p35-p25/Cdk5/NMDAR signaling pathway in glutamate-induced neurotoxicity in cultured rat retinal neurons.

    Directory of Open Access Journals (Sweden)

    Yanying Miao

    Full Text Available We investigated possible involvement of a calpain/p35-p25/cyclin-dependent kinase 5 (Cdk5 signaling pathway in modifying NMDA receptors (NMDARs in glutamate-induced injury of cultured rat retinal neurons. Glutamate treatment decreased cell viability and induced cell apoptosis, which was accompanied by an increase in Cdk5 and p-Cdk5(T15 protein levels. The Cdk5 inhibitor roscovitine rescued the cell viability and inhibited the cell apoptosis. In addition, the protein levels of both calpain 2 and calpain-specific alpha-spectrin breakdown products (SBDPs, which are both Ca(2+-dependent, were elevated in glutamate-induced cell injury. The protein levels of Cdk5, p-Cdk5(T15, calpain 2 and SBDPs tended to decline with glutamate treatments of more than 9 h. Furthermore, the elevation of SBDPs was attenuated by either D-APV, a NMDAR antagonist, or CNQX, a non-NMDAR antagonist, but was hardly changed by the inhibitors of intracellular calcium stores dantrolene and xestospongin. Moreover, the Cdk5 co-activator p35 was significantly up-regulated, whereas its cleaved product p25 expression showed a transient increase. Glutamate treatment for less than 9 h also considerably enhanced the ratio of the Cdk5-phosphorylated NMDAR subunit NR2A at Ser1232 site (p-NR2A(S1232 and NR2A (p-NR2A(S1232/NR2A, and caused a translocation of p-NR2A(S1232 from the cytosol to the plasma membrane. The enhanced p-NR2A(S1232 was inhibited by roscovitine, but augmented by over-expression of Cdk5. Calcium imaging experiments further showed that intracellular Ca(2+ concentrations ([Ca(2+](i of retinal cells were steadily increased following glutamate treatments of 2 h, 6 h and 9 h. All these results suggest that the activation of the calpain/p35-p25/Cdk5 signaling pathway may contribute to glutamate neurotoxicity in the retina by up-regulating p-NR2A(S1232 expression.

  15. Real-time CARS imaging reveals a calpain-dependent pathway for paranodal myelin retraction during high-frequency stimulation.

    Directory of Open Access Journals (Sweden)

    Terry B Huff

    Full Text Available High-frequency electrical stimulation is becoming a promising therapy for neurological disorders, however the response of the central nervous system to stimulation remains poorly understood. The current work investigates the response of myelin to electrical stimulation by laser-scanning coherent anti-Stokes Raman scattering (CARS imaging of myelin in live spinal tissues in real time. Paranodal myelin retraction at the nodes of Ranvier was observed during 200 Hz electrical stimulation. Retraction was seen to begin minutes after the onset of stimulation and continue for up to 10 min after stimulation was ceased, but was found to reverse after a 2 h recovery period. The myelin retraction resulted in exposure of Kv 1.2 potassium channels visualized by immunofluorescence. Accordingly, treating the stimulated tissue with a potassium channel blocker, 4-aminopyridine, led to the appearance of a shoulder peak in the compound action potential curve. Label-free CARS imaging of myelin coupled with multiphoton fluorescence imaging of immuno-labeled proteins at the nodes of Ranvier revealed that high-frequency stimulation induced paranodal myelin retraction via pathologic calcium influx into axons, calpain activation, and cytoskeleton degradation through spectrin break-down.

  16. Phosphorylation of Def Regulates Nucleolar p53 Turnover and Cell Cycle Progression through Def Recruitment of Calpain3

    Science.gov (United States)

    Tao, Ting; Shi, Hui; Lo, Li Jan; Wang, Yingchun; Chen, Jun; Peng, Jinrong

    2016-01-01

    Digestive organ expansion factor (Def) is a nucleolar protein that plays dual functions: it serves as a component of the ribosomal small subunit processome for the biogenesis of ribosomes and also mediates p53 degradation through the cysteine proteinase calpain-3 (CAPN3). However, nothing is known about the exact relationship between Def and CAPN3 or the regulation of the Def function. In this report, we show that CAPN3 degrades p53 and its mutant proteins p53A138V, p53M237I, p53R248W, and p53R273P but not the p53R175H mutant protein. Importantly, we show that Def directly interacts with CAPN3 in the nucleoli and determines the nucleolar localisation of CAPN3, which is a prerequisite for the degradation of p53 in the nucleolus. Furthermore, we find that Def is modified by phosphorylation at five serine residues: S50, S58, S62, S87, and S92. We further show that simultaneous phosphorylations at S87 and S92 facilitate the nucleolar localisation of Capn3 that is not only essential for the degradation of p53 but is also important for regulating cell cycle progression. Hence, we propose that the Def-CAPN3 pathway serves as a nucleolar checkpoint for cell proliferation by selective inactivation of cell cycle-related substrates during organogenesis. PMID:27657329

  17. Identification and association of the single nucleotide polymorphisms in calpain3 (CAPN3 gene with carcass traits in chickens

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    Du Hua-Rui

    2009-03-01

    Full Text Available Abstract Background The aim of this study is to screen single nucleotide polymorphisms (SNP of chicken Calpain3 (CAPN3 gene and to analyze the potential association between CAPN3 gene polymorphisms and carcass traits in chickens. We screened CAPN3 single nucleotide polymorphisms (SNP in 307 meat-type quality chicken from 5 commercial pure lines (S01, S02, S03, S05, and D99 and 4 native breeds from Guangdong Province (Huiyang Huxu chicken and Qingyuan Ma chicken and Sichuan Province (Caoke chicken and Shandi Black-bone chicken, China. Results Two SNPs (11818T>A and 12814T>G were detected by single strand conformation polymorphism (SSCP method and were verified by DNA sequencing. Association analysis showed that the 12814T>G genotypes were significantly associated with body weight (BW, carcass weight (CW, breast muscle weight (BMW, and leg muscle weight (LMW. Haplotypes constructed on the two SNPs (H1, TG; H2, TT; H3, AG; and H4, AT were associated with BW, CW (P P Conclusion We speculated that the CAPN3 gene was a major gene affecting chicken muscle growth and carcass traits or it was linked with the major gene(s. Diplotypes H1H2 and H2H2 might be advantageous for carcass traits.

  18. M(E)CANISMES MOL(E)CULAIRES IMPLIQU(E)S DANS L'ALT(E)RATION DU PH(E)NOTYPE DES CELLULES (E)PITHELIAL(E)S TUBULAIRES PAR LES CALPA(I)NES EXTRACELLULAIRES

    Institute of Scientific and Technical Information of China (English)

    ZHANG Wen-hui; Joelle Perez; Laurent Baud

    2008-01-01

    Objectif Rechercher les mécanismes moléculaires par lesquels des calpaines extracellulaires af-fectent l'adhérence et la mobilité des cellules épithéliales HK-2 dérivées du tubule proximal humain. Méthodes Western blot pour détecter le clivage des chaines α des intégrines; dosage radioimmunologique pour mesurer l' AMP cyclique intracellulaire; fluorescence-activated cell sorting (FACS) pour tester l' apoptose cellulaire. La morpholo-gie des cellules HK-2 a été observée et photographiée. Résultats (1) L ' exposition des cellules HK-2 à la calpaine μ n'a pas entrainé de clivage des chaines o3 et αV des intégrines; (2)l' exposition des cellules HK-2 à la calpaine μ entrainait une augmentation progressive de l' accumulation intracellulaire d' AMP cyclique (P<0.05) qui était associée une résistance cellulaire à l'apoptose(P <0. 05 ) ; (3)l'addition d'un inhibiteur pharmacologique de la protéine kinase A(PKA) prévenait totalement les modifications d'adhérence et de mobilité cellulaires induites par calpaine μ Conclusion Les calpaines externalisées peuvent modifier l' adherence et la mobilité cellulaires via un mécanisme qui implique l'accumulation d' AMP cyclique et l' activation de la PKA. Par ces mécanismes, les calpaines externalisées pourraient jouer un role dans l'induction de la réparation au cours de l'insuffucance rénale aiguё.

  19. Calpain inhibition reduces amplitude and accelerates decay of the late sodium current in ventricular myocytes from dogs with chronic heart failure.

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    Albertas Undrovinas

    Full Text Available Calpain is an intracellular Ca²⁺-activated protease that is involved in numerous Ca²⁺ dependent regulation of protein function in many cell types. This paper tests a hypothesis that calpains are involved in Ca²⁺-dependent increase of the late sodium current (INaL in failing heart. Chronic heart failure (HF was induced in 2 dogs by multiple coronary artery embolization. Using a conventional patch-clamp technique, the whole-cell INaL was recorded in enzymatically isolated ventricular cardiomyocytes (VCMs in which INaL was activated by the presence of a higher (1 μM intracellular [Ca²⁺] in the patch pipette. Cell suspensions were exposed to a cell- permeant calpain inhibitor MDL-28170 for 1-2 h before INaL recordings. The numerical excitation-contraction coupling (ECC model was used to evaluate electrophysiological effects of calpain inhibition in silico. MDL caused acceleration of INaL decay evaluated by the two-exponential fit (τ₁ = 42±3.0 ms τ₂ = 435±27 ms, n = 6, in MDL vs. τ₁ = 52±2.1 ms τ₂ = 605±26 control no vehicle, n = 11, and vs. τ₁ = 52±2.8 ms τ₂ = 583±37 ms n = 7, control with vehicle, P<0.05 ANOVA. MDL significantly reduced INaL density recorded at -30 mV (0.488±0.03, n = 12, in control no vehicle, 0.4502±0.0210, n = 9 in vehicle vs. 0.166±0.05pA/pF, n = 5, in MDL. Our measurements of current-voltage relationships demonstrated that the INaL density was decreased by MDL in a wide range of potentials, including that for the action potential plateau. At the same time the membrane potential dependency of the steady-state activation and inactivation remained unchanged in the MDL-treated VCMs. Our ECC model predicted that calpain inhibition greatly improves myocyte function by reducing the action potential duration and intracellular diastolic Ca²⁺ accumulation in the pulse train.Calpain inhibition reverses INaL changes in failing dog ventricular

  20. Targeting the nNOS/peroxynitrite/calpain system to confer neuroprotection and aid functional recovery in a mouse model of TBI.

    Science.gov (United States)

    Khan, Mushfiquddin; Dhammu, Tajinder S; Matsuda, Fumiyo; Annamalai, Balasubramaniam; Dhindsa, Tejbir Singh; Singh, Inderjit; Singh, Avtar K

    2016-01-01

    Traumatic brain injury (TBI) derails nitric oxide (NO)-based anti-inflammatory and anti-excitotoxicity mechanisms. NO is consumed by superoxide to form peroxynitrite, leading to decreased NO bioavailability for S-nitrosoglutathione (GSNO) synthesis and regulation of neuroprotective pathways. Neuronal peroxynitrite is implicated in neuronal loss and functional deficits following TBI. Using a contusion mouse model of TBI, we investigated mechanisms for the opposed roles of GSNO versus peroxynitrite for neuroprotection and functional recovery. TBI was induced by controlled cortical impact (CCI) in adult male mice. GSNO treatment at 2h after CCI decreased the expression levels of phospho neuronal nitric oxide synthase (pnNOS), alpha II spectrin degraded products, and 3-NT, while also decreasing the activities of nNOS and calpains. Treatment of TBI with FeTPPS, a peroxynitrite scavenger, had effects similar to GSNO treatment. GSNO treatment of TBI also reduced neuronal degeneration and improved neurobehavioral function in a two-week TBI study. In a cell free system, SIN-1 (a peroxynitrite donor and 3-nitrotyrosinating agent) increased whereas GSNO (an S-nitrosylating agent) decreased calpain activity, and these activities were reversed by, respectively, FeTPPS and mercuric chloride, a cysteine-NO bond cleaving agent. These data indicate that peroxynitrite-mediated activation and GSNO-mediated inhibition of the deleterious nNOS/calpain system play critical roles in the pathobiology of neuronal protection and functional recovery in TBI disease. Given GSNO׳s safety record in other diseases, its neuroprotective efficacy and promotion of functional recovery in this TBI study make low-dose GSNO a potential candidate for preclinical evaluation. PMID:26596859

  1. Pharmacological inhibition of caspase and calpain proteases: a novel strategy to enhance the homing responses of cord blood HSPCs during expansion.

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    V M Sangeetha

    Full Text Available BACKGROUND: Expansion of hematopoietic stem/progenitor cells (HSPCs is a well-known strategy employed to facilitate the transplantation outcome. We have previously shown that the prevention of apoptosis by the inhibition of cysteine proteases, caspase and calpain played an important role in the expansion and engraftment of cord blood (CB derived HSPCs. We hypothesize that these protease inhibitors might have maneuvered the adhesive and migratory properties of the cells rendering them to be retained in the bone marrow for sustained engraftment. The current study was aimed to investigate the mechanism of the homing responses of CB cells during expansion. METHODOLOGY/PRINCIPAL FINDINGS: CB derived CD34(+ cells were expanded using a combination of growth factors with and without Caspase inhibitor -zVADfmk or Calpain 1 inhibitor- zLLYfmk. The cells were analyzed for the expression of homing-related molecules. In vitro adhesive/migratory interactions and actin polymerization dynamics of HSPCs were assessed. In vivo homing assays were carried out in NOD/SCID mice to corroborate these observations. We observed that the presence of zVADfmk or zLLYfmk (inhibitors caused the functional up regulation of CXCR4, integrins, and adhesion molecules, reflecting in a higher migration and adhesive interactions in vitro. The enhanced actin polymerization and the RhoGTPase protein expression complemented these observations. Furthermore, in vivo experiments showed a significantly enhanced homing to the bone marrow of NOD/SCID mice. CONCLUSION/SIGNIFICANCE: Our present study reveals another novel aspect of the regulation of caspase and calpain proteases in the biology of HSPCs. The priming of the homing responses of the inhibitor-cultured HSPCs compared to the cytokine-graft suggests that the modulation of these proteases may help in overcoming the major homing defects prevalent in the expansion cultures thereby facilitating the manipulation of cells for transplant

  2. Production and processing studies on calpain-system gene markers for tenderness in Brahman cattle: 1. Growth, efficiency, temperament, and carcass characteristics.

    Science.gov (United States)

    Cafe, L M; McIntyre, B L; Robinson, D L; Geesink, G H; Barendse, W; Greenwood, P L

    2010-09-01

    Experiments were conducted concurrently at 2 locations to quantify effects and interactions of calpain-system tenderness gene markers on growth, efficiency, temperament, and carcass traits of Brahman cattle. Cattle were selected at weaning from commercial and research herds based on their genotype for commercially available calpastatin (CAST) and calpain 3 (CAPN3) gene markers for beef tenderness. Genotypes for mu-calpain gene markers (CAPN1-4751 and CAPN1-316) were also determined and included in statistical analyses. The New South Wales (NSW) herd was composed of 82 heifers and 82 castrated male cattle with 0 or 2 favorable alleles for CAST and CAPN3. The Western Australia (WA) herd was composed of 173 castrated male cattle with 0, 1, or 2 favorable alleles for CAST and CAPN3. One-half of the cattle at each site were implanted with a hormonal growth promotant (HGP: Revalor-H) during grain finishing. Cattle were backgrounded at pasture for 6 to 8 mo and grain-fed for 117 d (NSW) or 80 d (WA) before slaughter. Individually, or in combination with each other and with CAPN1-4751 status, CAST and CAPN3 status had no significant (all P > 0.05) effects on BW, growth, feed efficiency, or temperament traits. The only significant effect of CAST or CAPN3 on carcass characteristics was a small increase in rib fat with increasing number of favorable CAST alleles (P = 0.042) in the WA herd. There were no significant interactions (all P > 0.05) between the markers, or between the markers and sex or HGP treatment apart from CAST x HGP for area of the M. longissimus lumborum (P = 0.024) in the NSW experiment. Favorable CAST or CAPN3 alleles appear unlikely to have detrimental effects on growth, efficiency, temperament, or carcass characteristics of Brahman cattle; however, some effects evident for CAPN1 status indicate the need for further production studies on effects of these markers. Overall, the findings of the present study indicate that calpain-system gene markers are

  3. Production and processing studies on calpain-system gene markers for tenderness in Brahman cattle: 1. Growth, efficiency, temperament, and carcass characteristics.

    Science.gov (United States)

    Cafe, L M; McIntyre, B L; Robinson, D L; Geesink, G H; Barendse, W; Greenwood, P L

    2010-09-01

    Experiments were conducted concurrently at 2 locations to quantify effects and interactions of calpain-system tenderness gene markers on growth, efficiency, temperament, and carcass traits of Brahman cattle. Cattle were selected at weaning from commercial and research herds based on their genotype for commercially available calpastatin (CAST) and calpain 3 (CAPN3) gene markers for beef tenderness. Genotypes for mu-calpain gene markers (CAPN1-4751 and CAPN1-316) were also determined and included in statistical analyses. The New South Wales (NSW) herd was composed of 82 heifers and 82 castrated male cattle with 0 or 2 favorable alleles for CAST and CAPN3. The Western Australia (WA) herd was composed of 173 castrated male cattle with 0, 1, or 2 favorable alleles for CAST and CAPN3. One-half of the cattle at each site were implanted with a hormonal growth promotant (HGP: Revalor-H) during grain finishing. Cattle were backgrounded at pasture for 6 to 8 mo and grain-fed for 117 d (NSW) or 80 d (WA) before slaughter. Individually, or in combination with each other and with CAPN1-4751 status, CAST and CAPN3 status had no significant (all P > 0.05) effects on BW, growth, feed efficiency, or temperament traits. The only significant effect of CAST or CAPN3 on carcass characteristics was a small increase in rib fat with increasing number of favorable CAST alleles (P = 0.042) in the WA herd. There were no significant interactions (all P > 0.05) between the markers, or between the markers and sex or HGP treatment apart from CAST x HGP for area of the M. longissimus lumborum (P = 0.024) in the NSW experiment. Favorable CAST or CAPN3 alleles appear unlikely to have detrimental effects on growth, efficiency, temperament, or carcass characteristics of Brahman cattle; however, some effects evident for CAPN1 status indicate the need for further production studies on effects of these markers. Overall, the findings of the present study indicate that calpain-system gene markers are

  4. Sialoglycosylation of RBC in visceral leishmaniasis leads to enhanced oxidative stress, calpain-induced fragmentation of spectrin and hemolysis.

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    Sajal Samanta

    Full Text Available Visceral leishmaniasis (VL caused by the intracellular parasite Leishmania donovani accounts for an estimated 12 million cases of human infection. It is almost always associated with anemia, which severely complicates the disease course. However, the pathological processes leading to anemia in VL have thus far not been adequately characterized to date. In studying the glycosylation patterns of peripheral blood cells we found that the red blood cells (RBC of VL patients (RBC(VL express eight 9-O-acetylated sialoglycoproteins (9-O-AcSGPs that are not detected in the RBC of healthy individuals (RBC(N. At the same time, the patients had high titers of anti-9-O-AcSGP IgG antibodies in their sera. These two conditions appear to be linked and related to the anemic state of the patients, as exposure of RBC(VL but not RBC(N to anti-9-O-AcSGPs antibodies purified from patient sera triggered a series of responses. These included calcium influx via the P/Q-type but not L-type channels, activation of calpain I, proteolysis of spectrin, enhanced oxidative stress, lipid peroxidation, externalization of phosphatidyl serine with enhanced erythrophagocytosis, enhanced membrane fragility and, finally, hemolysis. Taken together, this study suggests that the enhanced hemolysis is linked to an impairment of membrane integrity in RBC(VL which is mediated by ligand-specific interaction of surface 9-O-AcSGPs. This affords a potential explanation for the structural and functional features of RBC(VL which are involved in the hemolysis related to the anemia which develops in VL patients.

  5. Variation at the Calpain 3 gene is associated with meat tenderness in zebu and composite breeds of cattle

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    Bunch Rowan J

    2008-07-01

    Full Text Available Abstract Background Quantitative Trait Loci (QTL affecting meat tenderness have been reported on Bovine chromosome 10. Here we examine variation at the Calpain 3 (CAPN3 gene in cattle, a gene located within the confidence interval of the QTL, and which is a positional candidate gene based on the biochemical activity of the protein. Results We identified single nucleotide polymorphisms (SNP in the genomic sequence of the CAPN3 gene and tested three of these in a sample of 2189 cattle. Of the three SNP genotyped, the CAPN3:c.1538+225G>T had the largest significant additive effect, with an allele substitution effect in the Brahman of α = -0.144 kg, SE = 0.060, P = 0.016, and the polymorphism explained 1.7% of the residual phenotypic variance in that sample of the breed. Significant haplotype substitution effects were found for all three breeds, the Brahman, the Belmont Red, and the Santa Gertrudis. For the common haplotype, the haplotype substitution effect in the Brahman was α = 0.169 kg, SE = 0.056, P = 0.003. The effect of this gene was compared to Calpastatin in the same sample. The SNP show negligible frequencies in taurine breeds and low to moderate minor allele frequencies in zebu or composite animals. Conclusion These associations confirm the location of a QTL for meat tenderness in this region of bovine chromosome 10. SNP in or near this gene may be responsible for part of the overall difference between taurine and zebu breeds in meat tenderness, and the greater variability in meat tenderness found in zebu and composite breeds. The evidence provided so far suggests that none of these tested SNP are causative mutations.

  6. Systemic and cerebral vascular endothelial growth factor levels increase in murine cerebral malaria along with increased calpain and caspase activity and can be reduced by erythropoietin treatment

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    Casper eHempel

    2014-06-01

    Full Text Available The pathogenesis of cerebral malaria includes compromised microvascular perfusion, increased inflammation, cytoadhesion and endothelial activation. These events cause blood-brain barrier disruption and neuropathology and can be associated with the vascular endothelial growth factor (VEGF signalling pathway. We studied this pathway in mice infected with Plasmodium berghei ANKA causing murine cerebral malaria with or without the use of erythropoietin as adjunct therapy. ELISA and western blotting was used for quantification of VEGF and relevant proteins in brain and plasma. Cerebral malaria increased levels of VEGF in brain and plasma and decreased plasma levels of soluble VEGF receptor 2. Erythropoietin treatment normalised VEGF receptor 2 levels and reduced brain VEGF levels. Hypoxia-inducible factor (HIF-1α was significantly upregulated whereas cerebral HIF-2α and erythropoietin levels remained unchanged. Furthermore, we noticed increased caspase-3 and calpain activity in terminally ill mice, as measured by protease-specific cleavage of α-spectrin and p35. In conclusion, we detected increased cerebral and systemic VEGF as well as HIF-1α, which in the brain were reduced to normal in erythropoietin-treated mice. Also caspase and calpain activity was reduced markedly in erythropoietin-treated mice.

  7. Interaction between Calpain 5, Peroxisome proliferator-activated receptor-gamma and Peroxisome proliferator-activated receptor-delta genes: a polygenic approach to obesity

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    Ruiz Agustín

    2008-07-01

    Full Text Available Abstract Context Obesity is a multifactorial disorder, that is, a disease determined by the combined effect of genes and environment. In this context, polygenic approaches are needed. Objective To investigate the possibility of the existence of a crosstalk between the CALPAIN 10 homologue CALPAIN 5 and nuclear receptors of the peroxisome proliferator-activated receptors family. Design Cross-sectional, genetic association study and gene-gene interaction analysis. Subjects The study sample comprise 1953 individuals, 725 obese (defined as body mass index ≥ 30 and 1228 non obese subjects. Results In the monogenic analysis, only the peroxisome proliferator-activated receptor delta (PPARD gene was associated with obesity (OR = 1.43 [1.04–1.97], p = 0.027. In addition, we have found a significant interaction between CAPN5 and PPARD genes (p = 0.038 that reduces the risk for obesity in a 55%. Conclusion Our results suggest that CAPN5 and PPARD gene products may also interact in vivo.

  8. Calpain2调节自噬相关基因ATG7的表达在非酒精性脂肪性肝病中的作用%Role of regulation of autophagy related gene 7 by Calpain 2 in non-alcoholic fatty liver disease in rats

    Institute of Scientific and Technical Information of China (English)

    陈洁; 熊吉; 陈潇迪; 牟歌; 王军; 樊丽琳; 陈东风

    2011-01-01

    Objective To investigate the expression and significance of autophagy related gene 7 ( ATG7 ) and Calpain 2 in nonalcoholic fatty liver disease ( NAFLD ). Methods In vivo model of NAFLD was established in SD rats by high fat diet, while the rats fed with normal food were set as control group. The rats were killed at 4, 8, 12 and 16 weeks after feeding. Blood samples were collected to check serum aspartate transaminase (AST) , alanine aminotransferase (ALT) , and free fatty acid(FFA). Steatosis of liver tissues were observed by HE staining. The expression of Calpain 2 and ATG7 was detected by real-time PCR and Western blotting respectively for mRNA and protein levels. Results HE staining implicated that the degree of hepatic steatosis was increased with the time of high fat diet feeding. Compared with the control group, the serum contents of ALT, AST, and FFA in NAFLD rats were increased with different degree, and significantly increased at 16th week (165.95 ±7. 24 U/L, 249. 52 ±4. 20 U/L, 0. 83 ±0. 05 mmol/L, respectively P < · 0. 01). The relative expression of Calpain 2 at mRNA level was increased after high fat diet feeding and reached its peak at the 16th week, and was 9. 83 ±0. 85 fold higher as compared with the control group (P <0. 01). While the relative expression of ATG7 began to decrease at the 4th week (0. 82 ±0. 02) , and reached its lowest level at the 16th week (0. 20 ±0. 03, P <0. 01) when compared with the control group. As with mRNA level, the protein level of Calpain 2 began to increase at the 4th week (2. 32 ± 0.45 ) , and was 9. 87 ± 1. 20 fold higher as the control group (P <0. 01). While the expression of ATG7 at protein level was decreased with progress of steatosis, and was significantly reduced at 16th week (0.18 ±0.05, P<0.01). Conclusion The up-regulation of Calpain 2 inhibits the expression of ATG7, which further attenuates the cell protection through autophagy, and then induces injury of hepatocyte in NAFLD. Autophagy may

  9. Long-term application of diethylstilbestrol upregulates expressions of μ- and m-calpains in pituitary intermediate lobe of female Wistar rats

    Institute of Scientific and Technical Information of China (English)

    Weijiang Zhao; Fang Yuan; Guilin Li; Zhongfang Shi; Yun Cui; Yazhuo Zhang; Zhongcheng Wang

    2007-01-01

    BACKGROUND: During formation of prolactin neoplasia, how cells and its structure in adenohypophysis affect prolactin cells should be further studied. Intermediate lobe can be regarded as a driving region to release prolactin (PRL) and may promote formation of prolactin neoplasia in pituitary anterior lobe. OBJECTIVE: To observe the effect of diethylstilbestrol (DES) on the expressions of μ and m-calpains in pituitary intermediate lobe of female Wistar rats. DESIGN: Observational contrast animal study. SETTING: Beijing Neurosurgical Institute.MATERIALS: A total of 21 female Wistar rats, 3 weeks old weighing 70 - 80 g were housed with free access to tap water and standard pellet food. They were kept in a CL-grade condition, at (24±1) ℃ and a humidity of (55±5)%, and with a 12 hours day-night cycle. Caprine anti-μ- and m-calpains antibodies were provided by Santa Cruz Biotechnology, CA, USA; rabbit-anti-PRL antibodies by Dako, Denmark; rabbit-anti-ACTH antibody by Boster Company, Wuhan.METHODS: The experiment was carried out in Pathophysiological Department and Animal Laboratory, Beijing Neurosurgical Institute from August 2006 to January 2007. ①Rats were randomly divided into groups with 7 in each group, including vehicle control group, in which rats were injected intraperitoneally with sun-flower seed oil (1 Ml/kg, twice a week) for 16 weeks; DES group, where animals were administered with DES (5 mg/kg, twice a week) for 16 weeks; DES + vehicle control group, in which DES was administered for 12 weeks at the same dose with those in DES group, and then was discontinued and replaced by sun-flower seed oil (1 Ml/kg, twice a week) for the following 4 weeks. ②At 16 weeks later, pituitary tissue was dealt with HE staining and PRL immunohistochemical examination to observe evoke of tumor; meanwhile, immunohistochemical examination was used to observe expression of PRL of pituitary anterior lobe, expressions ofμ- and m-calpains of pituitary intermediate lobe and

  10. Association between myocardial calpain activation and apoptosis in lipopolysaccharide-induced septic mouse model%钙激活中性蛋白酶在脓毒症小鼠心肌半胱氨酸蛋白酶-3活化中的作用及其机制

    Institute of Scientific and Technical Information of China (English)

    李小平; 李浪; 陈瑞珍; 刘唐威; 伍伟锋; 申锷; 杨英珍; 陈灏珠

    2010-01-01

    目的 探讨钙激活中性蛋白酶(calpain)在脓毒症小鼠心肌半胱氨酸蛋白酶-3(caspase-3)活化中的作用及其机制.方法 (1)体内实验:腹腔注射脂多糖(LPS,4 mg/kg)建立脓毒症小鼠模型.Western blot检测心肌组织中calpain、caspase-3活性和calpain-1、calpain-2、calpain特异性抑制蛋白calpastatin水平以及凋亡相关蛋白Bcl-2、Bid水平及剪切片段,TUNEL法检测心肌细胞凋亡情况,Langendorff灌注装置评价小鼠心脏的收缩和舒张功能.(2)体外实验:成年大鼠心肌细胞给予LPS(1μg/ml)处理4 h,或同时予以calpain抑制剂calpain inhibitor-Ⅲ(10 μmol/L)干预后,检测心肌细胞calpain和caspase-3活性,Bcl-2、Bid蛋白水平以及心肌细胞凋亡情况.结果 (1)体内实验:在脓毒症小鼠心肌组织中,calpain活性增高2.7倍,caspase-3活性增高1.8倍,给予calpain-inhibitor-Ⅲ或PD150606,均可抑制caspase-3活性的增高.脓毒症小鼠心肌组织calpain-1、calpain-2、calpastatin以及Bcl-2、Bid蛋白水平未见改变,亦未检测到Bcl-2、Bid剪切片段.Calpain inhibitor-Ⅲ则可使脓毒症小鼠心室最快压力上升速率和心室最快压力下降速率分别增加34.5%和34.6%,从而改善脓毒症小鼠心功能障碍.(2)体外实验:LPS可诱导成年大鼠心肌细胞calpain和caspase-3活性增高,给予calpain inhibitor-Ⅲ则可抑制caspase-3活性的增高,心肌细胞Bcl-2、Bid蛋白水平未见改变.体内外实验均未发现LPS可诱导心肌细胞凋亡的增加.结论 脓毒症小鼠心肌calpain活性增高,可活化心肌caspase-3,但未导致心肌细胞凋亡,其机制与凋亡蛋白Bcl-2和Bid无关.%Objective In septic mice, myocardial calpain was activated and induced caspase-3 activation, the association between calpain activation and apoptosis was explored in this experiment. Methods In in vivo model, adult C57 mice were injected with lipopolysaccharide (LPS, 4rg/kg, i. p. ) to induce sepsis. Myocardial calpain and

  11. Systemic and cerebral vascular endothelial growth factor levels increase in murine cerebral malaria along with increased Calpain and caspase activity and can be reduced by erythropoietin treatment

    DEFF Research Database (Denmark)

    Hempel, Casper; Hoyer, Nils; Kildemoes, Anna;

    2014-01-01

    The pathogenesis of cerebral malaria (CM) includes compromised microvascular perfusion, increased inflammation, cytoadhesion, and endothelial activation. These events cause blood-brain barrier disruption and neuropathology and associations with the vascular endothelial growth factor (VEGF...... increased levels of VEGF in brain and plasma and decreased plasma levels of soluble VEGF receptor 2. EPO treatment normalized VEGF receptor 2 levels and reduced brain VEGF levels. Hypoxia-inducible factor (HIF)-1α was significantly upregulated whereas cerebral HIF-2α and EPO levels remained unchanged....... Furthermore, we noticed increased caspase-3 and calpain activity in terminally ill mice, as measured by protease-specific cleavage of α-spectrin and p35. In conclusion, we detected increased cerebral and systemic VEGF as well as HIF-1α, which in the brain were reduced to normal in EPO-treated mice. Also...

  12. Association of polymorphisms in calpain 1, (mu/I) large subunit, calpastatin, and cathepsin D genes with meat quality traits in double-muscled Piemontese cattle.

    Science.gov (United States)

    Ribeca, Cinzia; Bonfatti, Valentina; Cecchinato, Alessio; Albera, Andrea; Maretto, Fabio; Gallo, Luigi; Carnier, Paolo

    2013-04-01

    Five single-nucleotide polymorphisms (SNPs) located in the calpain 1, (mu/I) large subunit (CAPN1), calpastatin (CAST), and cathepsin D (CTSD) genes were analyzed in a large sample of Piemontese cattle. The aim of this study was to evaluate allele and genotype frequencies of these SNPs and to investigate associations of CAPN1, CAST, and CTSD gene variants with meat quality traits. Minor allele frequencies ranged from 30 to 48%. The presence of the A allele at CAPN530 increased yellowness and drip loss. The CAST282 G allele was associated with an increased drip loss compared to the C allele, and the CAST2959 A allele decreased redness compared to the G allele.

  13. Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation and cell differentiation

    DEFF Research Database (Denmark)

    Grossi, Alberto; Lawson, Moira Ann

    Abstract Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation and cell differentiation. A. Grossi, M. A. Lawson; Department of Food Science, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark The process of muscle...... development and growth is a complex sequence of events whereby muscle cells respond to a number of stimuli in order to form organised muscle tissue. Increase in muscle mass is greatly influenced by the rate of skeletal muscle protein synthesis and degradation, processes that can be altered by mechanical...... forces. Stretch- or load-induced signaling is now beginning to be understood as a factor which affects the mass and phenotype of muscles as well as the expression of a number of proteins within muscle cells. Use of magnetic field to produce mechanical forces to stimulate cell populations has been well...

  14. Calpain3 is expressed in a proteolitically active form in papillomavirus-associated urothelial tumors of the urinary bladder in cattle.

    Directory of Open Access Journals (Sweden)

    Sante Roperto

    Full Text Available 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

  15. Abnormal activation of calpain and protein kinase Cα promotes a constitutive release of matrix metalloproteinase 9 in peripheral blood mononuclear cells from cystic fibrosis patients.

    Science.gov (United States)

    Averna, Monica; Bavestrello, Margherita; Cresta, Federico; Pedrazzi, Marco; De Tullio, Roberta; Minicucci, Laura; Sparatore, Bianca; Salamino, Franca; Pontremoli, Sandro; Melloni, Edon

    2016-08-15

    Matrix metalloproteinase 9 (MMP9) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies. We here report that peripheral blood mononuclear cells (PBMCs), isolated from cystic fibrosis (CF) patients homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR), express constitutively and release at high rate MMP9 due to the alteration in their intracellular Ca(2+) homeostasis. This spontaneous and sustained MMP9 secretion may contribute to the accumulation of this protease in fluids of CF patients. Conversely, in PBMCs isolated from healthy donors, expression and secretion of MMP9 are undetectable but can be evoked, after 12 h of culture, by paracrine stimulation which also promotes an increase in [Ca(2+)]i. We also demonstrate that in both CF and control PBMCs the Ca(2+)-dependent MMP9 secretion is mediated by the concomitant activation of calpain and protein kinase Cα (PKCα), and that MMP9 expression involves extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation. Our results are supported by the fact that either the inhibition of Ca(2+) entry or chelation of [Ca(2+)]i as well as the inhibition of single components of the signaling pathway or the restoration of CFTR activity all promote the reduction of MMP9 secretion. PMID:27349634

  16. 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside confers neuroprotection in cell and animal models of ischemic stroke through calpain1/PKA/CREB-mediated induction of neuronal glucose transporter 3

    International Nuclear Information System (INIS)

    Salidroside is proven to be a neuroprotective agent of natural origin, and its analog, 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside (named SalA-4 g), has been synthesized in our lab. In this study, we showed that SalA-4 g promoted neuronal survival and inhibited neuronal apoptosis in primary hippocampal neurons exposed to oxygen and glucose deprivation (OGD) and in rats subjected to ischemia by transient middle cerebral artery occlusion (MCAO), respectively, and that SalA-4 g was more neuroprotective than salidroside. We further found that SalA-4 g elevated glucose uptake in OGD-injured primary hippocampal neurons and increased the expression and recruitment of glucose transporter 3 (GLUT3) in ischemic brain. Signaling analysis revealed that SalA-4 g triggered the phosphorylation of CREB, and increased the expression of PKA RII in primary hippocampal neurons exposed to OGD injury, while inhibition of PKA/CREB by H-89 alleviated the elevation in glucose uptake and GLUT3 expression, and blocked the protective effects of SalA-4 g. Moreover, SalA-4 g was noted to inhibit intracellular Ca2+ influx and calpain1 activation in OGD-injured primary hippocampal neurons. Our results suggest that SalA-4 g neuroprotection might be mediated by increased glucose uptake and elevated GLUT3 expression through calpain1/PKA/CREB pathway. - Highlights: • A salidroside (Sal) analog (SalA-4 g) is prepared to be more neuroprotective than Sal. • SalA-4 g protected hippocampal neurons from oxygen and glucose deprivation insult. • SalA-4 g reduced ischemic injury after transient middle cerebral artery occlusion in rats. • Neuroprotection of SalA-4 g was mediated by GLUT3 level via calpain/PKA/CREB pathway

  17. 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside confers neuroprotection in cell and animal models of ischemic stroke through calpain1/PKA/CREB-mediated induction of neuronal glucose transporter 3

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Shu; Cheng, Qiong; Li, Lu; Liu, Mei; Yang, Yumin; Ding, Fei, E-mail: dingfei@ntu.edu.cn

    2014-06-15

    Salidroside is proven to be a neuroprotective agent of natural origin, and its analog, 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside (named SalA-4 g), has been synthesized in our lab. In this study, we showed that SalA-4 g promoted neuronal survival and inhibited neuronal apoptosis in primary hippocampal neurons exposed to oxygen and glucose deprivation (OGD) and in rats subjected to ischemia by transient middle cerebral artery occlusion (MCAO), respectively, and that SalA-4 g was more neuroprotective than salidroside. We further found that SalA-4 g elevated glucose uptake in OGD-injured primary hippocampal neurons and increased the expression and recruitment of glucose transporter 3 (GLUT3) in ischemic brain. Signaling analysis revealed that SalA-4 g triggered the phosphorylation of CREB, and increased the expression of PKA RII in primary hippocampal neurons exposed to OGD injury, while inhibition of PKA/CREB by H-89 alleviated the elevation in glucose uptake and GLUT3 expression, and blocked the protective effects of SalA-4 g. Moreover, SalA-4 g was noted to inhibit intracellular Ca{sup 2+} influx and calpain1 activation in OGD-injured primary hippocampal neurons. Our results suggest that SalA-4 g neuroprotection might be mediated by increased glucose uptake and elevated GLUT3 expression through calpain1/PKA/CREB pathway. - Highlights: • A salidroside (Sal) analog (SalA-4 g) is prepared to be more neuroprotective than Sal. • SalA-4 g protected hippocampal neurons from oxygen and glucose deprivation insult. • SalA-4 g reduced ischemic injury after transient middle cerebral artery occlusion in rats. • Neuroprotection of SalA-4 g was mediated by GLUT3 level via calpain/PKA/CREB pathway.

  18. 脊髓缺血再灌注损伤后μ-CalpainmRNA及蛋白的表达%Expressions ofμ-Calpain mRNA and protein after spinal cord ischemia-reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    卜国云; 杜区成; 吴叶; 邓树才; 朱加亮; 商卫林

    2014-01-01

    Objective To explore the expressions and signiifcance ofμ-Calpain after spinal cord ischemia-reperfusion injury. Methods An adult Sprague-Dawley ( SD ) rat model of spinal cord ischemia-reperfusion injury was established. Quantitative real-time lfuroscent polymerase chin reaction ( PCR ) and Western-blot technique were used to detect the expressions of mRNA and protein ofμ-Calpain at 2 h, 6 h, 12 h, 24 h, 48 h and 72 h after the model was established. The degradation ofα-II specrin of the speciifc substrate of ofμ-Calpain was detected by Western-blot technique, and the results were compared with that of the control group. Results The expressions ofμ-Calpain mRNA of the injured spinal cord began to increase at 2 h after the model was established, but there were no statistically significant differences. The expressions were obviously increased at 12 h, and there were statistically significant differences ( P<0.05 ). The peak was reached at 48 h after the model was established ( P<0.001 ). The expressions ofμ-Calpain mRNA remained at a higher level at 72 h when compared with that of the control group, and there were statistically signiifcant differences ( P<0.05 ). The expressions ofμ-Calpain protein of the injured spinal cord began to increase at 2 h after the model was established, and the peak was reached at 48 h ( P<0.001 ). The expressions ofμ-Calpain protein remained at a higher level at 72 h after the model was established when compared with that of the control group, and there were statistically signiifcant differences ( P<0.05 ). Theα-II spectrin began to degenerate at 2 h after the model was established, but there were no statistically signiifcant differences. There were still someα-II spectrin remains at 72 h. Conclusions The expressions ofμ-Calpain mRNA and protein are increased after the spinal cord ischemia-reperfusion injury model is established, and meanwhile theα-II specrin of its speciifc substrate begins to degenerate. Theμ-Calpain is

  19. Arsenic Exposure and Calpain-10 Polymorphisms Impair the Function of Pancreatic Beta-Cells in Humans: A Pilot Study of Risk Factors for T2DM

    Science.gov (United States)

    Díaz-Villaseñor, Andrea; Cruz, Laura; Cebrián, Arturo; Hernández-Ramírez, Raúl U.; Hiriart, Marcia; García-Vargas, Gonzálo; Bassol, Susana; Sordo, Monserrat; Gandolfi, A. Jay; Klimecki, Walter T.; López-Carillo, Lizbeth; Cebrián, Mariano E.; Ostrosky-Wegman, Patricia

    2013-01-01

    The incidence of type 2 diabetes mellitus (T2DM) is increasing worldwide and diverse environmental and genetic risk factors are well recognized. Single nucleotide polymorphisms (SNPs) in the calpain-10 gene (CAPN-10), which encodes a protein involved in the secretion and action of insulin, and chronic exposure to inorganic arsenic (iAs) through drinking water have been independently associated with an increase in the risk for T2DM. In the present work we evaluated if CAPN-10 SNPs and iAs exposure jointly contribute to the outcome of T2DM. Insulin secretion (beta-cell function) and insulin sensitivity were evaluated indirectly through validated indexes (HOMA2) in subjects with and without T2DM who have been exposed to a gradient of iAs in their drinking water in northern Mexico. The results were analyzed taking into account the presence of the risk factor SNPs SNP-43 and -44 in CAPN-10. Subjects with T2DM had significantly lower beta-cell function and insulin sensitivity. An inverse association was found between beta-cell function and iAs exposure, the association being more pronounced in subjects with T2DM. Subjects without T2DM who were carriers of the at-risk genotype SNP-43 or -44, also had significantly lower beta-cell function. The association of SNP-43 with beta-cell function was dependent on iAs exposure, age, gender and BMI, whereas the association with SNP-44 was independent of all of these factors. Chronic exposure to iAs seems to be a risk factor for T2DM in humans through the reduction of beta-cell function, with an enhanced effect seen in the presence of the at-risk genotype of SNP-43 in CAPN-10. Carriers of CAPN-10 SNP-44 have also shown reduced beta-cell function. PMID:23349674

  20. Variations in the calpain-10 gene are associated with the risk of type 2 diabetes and hypertension in northern Han Chinese population

    Institute of Scientific and Technical Information of China (English)

    CHEN Shu-feng; LU Xiang-feng; YAN Wei-li; HUANG Jian-feng; GU Dong-feng

    2007-01-01

    Background Calpain-10(CAPN10)has been identified as a susceptibility gene in type 2 diabetes mellitus (T2DM) and insulin resistance.The present study aimed to identify the effects of genetic variations in the CAPN10 gene on the development of type 2 diabetes and hypertension in northern Han Chinese population.Methods We performed a case-control study and genotyped single nucleotide polymorphism(SNP)-44,-43,-19 and -63 of CAPN10 gene in 1046 subjects from the northern China,including 493 patients with T2DM and hypertension and 553 age-and gender-matched normal healthy controls.Results Univariate analysis showed that the four polymorphisms were not independently associated with T2DM and hypertension.However,the frequency distributions of SNP-44 allele C(allele 2)(17.89% vs 9.80%,P=0.0016)and genotype CC(22)(4.21% vs 1.01%,P=0.0059)in obese patients(body mass index≥30 kg/m2)were different from those in non-obese patients.Logistic regression analyses revealed that carriers of lhe 1112/1221 diplotype had a significantly lower odds ratio for diabetes and hypertension(OR=0.399,95% CI,0.196-0.814,P=0.0115).The 1112/1121 diplotype associated with significantly increased risk of type 2 diabetes in Mexican-American was not associated with the increased risk in Chinese.Conclusion These results suggested that CAPN10 gene variations might play roles in the risk of diabetes and hypertension in northern Han Chinese population.

  1. Arsenic exposure and calpain-10 polymorphisms impair the function of pancreatic beta-cells in humans: a pilot study of risk factors for T2DM.

    Directory of Open Access Journals (Sweden)

    Andrea Díaz-Villaseñor

    Full Text Available The incidence of type 2 diabetes mellitus (T2DM is increasing worldwide and diverse environmental and genetic risk factors are well recognized. Single nucleotide polymorphisms (SNPs in the calpain-10 gene (CAPN-10, which encodes a protein involved in the secretion and action of insulin, and chronic exposure to inorganic arsenic (iAs through drinking water have been independently associated with an increase in the risk for T2DM. In the present work we evaluated if CAPN-10 SNPs and iAs exposure jointly contribute to the outcome of T2DM. Insulin secretion (beta-cell function and insulin sensitivity were evaluated indirectly through validated indexes (HOMA2 in subjects with and without T2DM who have been exposed to a gradient of iAs in their drinking water in northern Mexico. The results were analyzed taking into account the presence of the risk factor SNPs SNP-43 and -44 in CAPN-10. Subjects with T2DM had significantly lower beta-cell function and insulin sensitivity. An inverse association was found between beta-cell function and iAs exposure, the association being more pronounced in subjects with T2DM. Subjects without T2DM who were carriers of the at-risk genotype SNP-43 or -44, also had significantly lower beta-cell function. The association of SNP-43 with beta-cell function was dependent on iAs exposure, age, gender and BMI, whereas the association with SNP-44 was independent of all of these factors. Chronic exposure to iAs seems to be a risk factor for T2DM in humans through the reduction of beta-cell function, with an enhanced effect seen in the presence of the at-risk genotype of SNP-43 in CAPN-10. Carriers of CAPN-10 SNP-44 have also shown reduced beta-cell function.

  2. 先天性肌性斜颈钙蛋白酶-1、泛素和20S蛋白酶体表达%Expressions of calpain-1, ubiquitin and 20S proteasome in congenital muscular torticollis

    Institute of Scientific and Technical Information of China (English)

    陈焕雄; 唐盛平; 王帅印; 江贤萍; 曹娟; 付桂兵; 孙客

    2012-01-01

    Objective To detect the expressions of calpain-1,ubiquitin and 20S proteasome in sternocleidomastoid muscle (SCM) of the patients with congenital muscular torticollis (CMT).Methods CMT group consisted of 40 patients aged from 4 months to 16 years old who were randomly chosen from 188 CMT patients.According to their age,these patients were divided into 4 groups with 10 in each:group 1 included patients aged from 4 to 6 months old; group 2 was 7 to 12 months old;group 3 was 1 to 3 years old; and group 4 was 4 to 16 years old.Five specimens collected from adductor muscle,including 1 from the patient with cerebral palsy and the other 4 from development dysplasia of the hip,were taken as control group.All resected surgical specimens vere processed for H&.E staining,Masson and immunohistochemical staining for calpain 1,ubiquitin and 20S proteasome.Results Atrophic muscle fibers were noted in all 40 CMT patients.Adipose hyperplasia was noted in 29patients with CMT.The percentage of strongly calpain-1-positive fibers in CMT group was significantly higher than that of controls (40.7%±13.8% vs.0.52%± 0.54%,P<0.001 ),and was positively correlated with the level of fibrosis (r=0.750,P<0.001 ).The percentage of strongly ubiquitin and 20S proteasome positive fibers in CMT group was significantly higher than that of controls (43.7%±14.7%,32.4%±12.5% vs.1.1%±0.63%,0.62%±0.40%,P<0.001,respectively),and was positively correlated with the level of fibrosis (r=0.758,P<0.001 ; r=0.571,P<0.001,respectively).The level of calpain-1 was positively correlated with ubiquitin (r=0.956,P<0.001 ).The level of ubiquitin was positive correlated with 20S proteasome (r=0.786,P<0.001 ).Conclusions Muscle atrophy and adipose hyperplasia are the basic pathological changes in CMT.The calpain and ubiquitin-proteasome dependent proteolytic pathway may play a role in muscle atrophy of CMT.%目的 检测先天性肌性斜颈(CMT)病变组织中钙蛋白酶1

  3. 洛伐他汀通过抑制Calpain和CDK5的过度激活减轻NMDA的毒性损害%Lovastatin attenuates Calpain and CDK5 over-activation induced by NMDA

    Institute of Scientific and Technical Information of China (English)

    马涛; 许著一; 姚晴宇; 孔岳南

    2012-01-01

    Objective To observe the effect of lovastatin on the excitotoxicity induced by NMDA in cortical neurons in rats and to investigate the underlying mechanism. Methods Cortical neurons prepared from E17 rats were assigned into 4 groups:NMDA group (addition of 100 μmol/L of NMDA for 15 minutes),LOV group (pretreatment of the neurons for 3 days with 500 nmol/L of LOV),LOV+NMDA group (pretreatment of the neurons for 3 days with 500 nmol/L of LOV and addition of 100 μmol/L of NMDA for 15 minutes) and untreated group (addition of isodose solvent).Cell viability was evaluated with the trypan blue dye exclusion test,the morphology and number of neurons were assessed with MAP-2 immunofluorescence staining,and the level of protein was measured with Western blotting assay. Results Trypan blue staining demonstrated that the pretreatment with 500 nmol/L of lovastatin for 3 days significantly protected the neurons against the excitotoxicity induced by NMDA (P<0.05 vs NMDA).Immunofluorescence staining demonstrated the number of MAP-2 positive neurons decreased and the surviving neurons showed a loss of MAP-2 positive dendrites after NMDA treatment (P<0.05 vs untreated),which were not observed after lovastatin pretreatment (P<0.05 vs NMDA).Excitotoxicity was mediated in part by the Calpain over-activation and the subsequent protein truncation events on Calpain substrate, CDK5 co-activator P35 to P25 cleavage. Lovastatin pretreatment remarkably suppressed Calpain over-activation and the conversion from P35 to P25 in response to NMDA exposure as detected by Western blotting analysis (P<0.05 vs NMDA). Conclusions Lovastatin significantly attenuates the excitotoxicity induced by NMDA. The neuroprotection of lovastatin may be mediated by blocking the Calpain and CDK5 over-activation.%目的 探讨洛伐他汀(LOV)对N-甲基-D-天门冬氨酸(NMDA)诱导的大鼠皮质神经元兴奋性毒性损害的神经保护作用及可能机制. 方法 原代培养的大鼠皮质神经

  4. μ-Calpain, calpastatin, and growth hormone receptor genetic effects on preweaning performance, carcass quality traits, and residual variance of tenderness in Angus cattle selected to increase minor haplotype and allele frequencies.

    Science.gov (United States)

    Tait, R G; Shackelford, S D; Wheeler, T L; King, D A; Casas, E; Thallman, R M; Smith, T P L; Bennett, G L

    2014-02-01

    Genetic marker effects and interactions are estimated with poor precision when minor marker allele frequencies are low. An Angus population was subjected to marker assisted selection for multiple years to increase divergent haplotype and minor marker allele frequencies to 1) estimate effect size and mode of inheritance for previously reported SNP on targeted beef carcass quality traits; 2) estimate effects of previously reported SNP on nontarget performance traits; and 3) evaluate tenderness SNP specific residual variance models compared to a single residual variance model for tenderness. Divergent haplotypes within µ-calpain (CAPN1), and SNP within calpastatin (CAST) and growth hormone receptor (GHR) were successfully selected to increase their frequencies. Traits evaluated were birth BW, weaning BW, final BW, fat thickness, LM area, USDA marbling score, yield grade, slice shear force (SSF), and visible and near infrared predicted slice shear force. Both CAPN1 and CAST exhibited additive (P grade (P meat yield and less trimmable fat. There were no significant effects (P ≥ 0.23) for GHR on any of the traits evaluated in this study. Furthermore, CAST specific residual variance models were found to fit significantly better (P quality. PMID:24398843

  5. Distribution and relative contents of m-calpain,cytoskeleton proteins α-Ⅱ spectrin and NF200 in retina after corneal penetrating injury%角膜穿通伤后视网膜钙蛋白酶、细胞骨架蛋白α-Ⅱ spectrin和NF200的分布及其相对含量

    Institute of Scientific and Technical Information of China (English)

    黄燕; 吴滢; 宗海洋; 肖寿华; 张志坚

    2011-01-01

    目的 观察角膜穿通伤后钙蛋白酶( m-calpain)、血影蛋白(α-Ⅱspectrin)及神经丝蛋白-200(neurofilament protein200,NF200)在视网膜中的分布特征,检测骨架蛋白降解产物相对含量的动态变化,初步探讨角膜穿通伤后视网膜继发性损伤的病理机制.方法清洁级成年雌性SD大鼠50只,随机分为正常对照组和角膜穿通伤后6h、24h、48 h、72 h组,每组各10只.于眼球颠侧角膜缘用无菌注射器刺穿角膜制作角膜穿通伤模型.各组5只大鼠制作眼球切片,用相应抗体免疫荧光染色,观察m-calpain、α-Ⅱspectrin和NF200在视网膜中的分布特征;同时用免疫印迹法检测各 组另外5只大鼠眼球壁组织中上述蛋白相对含量的动态变化.结果 正常对照组m-calpain主要分布于视网膜节细胞,组织中蛋白相对含量较低;角膜穿通伤组组织中阳性细胞数量增多,蛋白相对含量亦增高,于24h达高峰,各组相比差异均有统计学意义(均为P<0.05).正常对照组小Ⅱspcctrin和NF200主要分布于各种视细胞突起内,细胞界限和层次较清楚;角膜穿通伤组免疫荧光呈弥散分布,细胞界限和层次模糊不清;组织中α-Ⅱspectrln和NF200降解产物相对含量随m-capain含量增高而增高,于24h逮最大量,各组相比差异均有统计学意义(均为P<O.05).结论 角膜穿通伤可刺激视网膜细胞高表达m-calpain,后者对视网膜细胞骨架蛋白的降解是造成视网膜继发性损伤的重要因素.%Objective To investigate the mechanisms of the second injury of ret' ina after corneal penetrating injury by observing distribution of m-calpain, cytoskeleton proteins a- II spectrin and neuronlament protein-200 (NF200) and the dynamic changes of relative contents of degradation products of retinal nerve cell cytoskeleton proteins. Methods The 50 clear adult female rats were randomly divided into normal control group and corneal penetrating injury groups, which

  6. Role of calpain in spinal dorsal horn in development of paw inflammatory pain in rats%脊髓背角卡配因在大鼠足底炎性痛形成中的作用

    Institute of Scientific and Technical Information of China (English)

    王静捷; 陈广俊; 陈雯; 杜金; 罗爱伦; 黄宇光

    2011-01-01

    目的 探讨脊髓背角卡配因在大鼠足底炎性痛形成中的作用.方法 雄性SD大鼠48只,6周龄,体重160~200 g,采用随机数字表法,将其随机分为3组:正常对照组(C组,n=8)、PBS组(n=16)和酵母多糖诱发足底炎性痛组(Z组,n=24).Z组于大鼠左侧后足足底皮下注射酵母多糖1.25 mg,制备酵母多糖诱发足底炎性痛模型,PBS组给予等容量PBS 100μl.分别于给药前(T0)、给药后30 min(T1)、1 h(T2)、2 h(T3)、4 h(T4)、8 h(T5)、24 h(T6)和48 h(T7)时测定左侧后足机械刺激缩足阈值(MWT)、热缩足反应潜伏期(PWTL)和左侧后足足底最大厚度.PBS组于T4时处死8只大鼠,Z组分别于T4、T6和T7时各处死8只大鼠,取左侧脊髓L4~6节段,采用Western blot法测定脊髓背角spectrin αⅡ降解产物、IκBα、环氧化酶-2(COX-2)的表达和NF-κB活性.结果 与C组比较,Z组MWT降低,PWTL缩短,足底最大厚度增厚,脊髓背角spectrin αⅡ降解产物和COX-2的表达上调,IκBα表达下调,NF-κB活性升高(P<0.05或0.01),PBS组上述指标差异无统计学意义(P>0.05).结论 脊髓背角卡配因活化参与了大鼠足底炎性痛的形成,其机制与激活NF-κB,上调COX-2表达有关.%Objective To investigate the role of calpain in the spinal dorsal horn in development of paw inflammatory pain in rats.Methods Forty-eight male SD rats,aged 6 weeks,weighing 160-200 g,were randomly divided into three groups:normal control group(group C,n =8),PBS group( n =16),zymosan-induced paw inflammatory pain group (group Z,n =24).Inflammatory pain was induced by injection of zymosan 1.25 mg into the plantar surface of left hindpaw.Group PBS received the equal volume of PBS 100 μl.The mechanical paw withdrawal threshold (MWT),paw withdrawal thermal latency (PWTL) and maximum thickness of the plantar surface of left hindpaw were measured before (T0 ) and at 30 min,1,2,4,8,24 and 48 h(T1-7 ) after zymosan or PBS injection.Eight rats were sacrificed at T4 in

  7. Changes of BB Isoenzyme of Creatine Kinase, CaATPase and Calpain in Experimental Autoimmune Encephalomyelitis Mouse Brain and Spinal Cord%实验性自身免疫性脑脊髓炎小鼠脑组织和脊髓中脑型肌酸激酶、钙泵和钙中性蛋白酶的变化

    Institute of Scientific and Technical Information of China (English)

    王沛; 郑荣远; 林福虹; 王赵伟; 厉芳; 张正学

    2011-01-01

    Aim: To investigate the changes ofBB isoenzyme of creatine kinase(CK-BB), CaATPase and calpain in experimental autoimmune encephalomyelitis(EAE) mouse brain and spinal cord. Methods: C57BL/6 mice were induced into the models of EAE with multiple sclerosis by myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptides. Behavioral changes of the EAE mice were observed and recorded. With HE staining, LFB myelin staining, the changes of the central nervous tissues, CK-BB, CaATPase and calpain activity were assayed in the peak incidence by using microplate reader and spectrophotometer (19 days after immunization). Results: Compared with the control group, the results of the EAE group were as follows :① Mean daily clinical scores and cumulative scores were mcreased(P<0.01).② HE staining: Central inflammatory cell infiltration became obvious(P<0.05).③ LFB Clinical Analysis of 15 Cases with Spontaneous Intracranial Hypotension HeadacheKEY WORDS spontaneous intracranial hypotension; headache; secondary headacheABSTRACT Aim: To explore the clinical features of spontaneous intracranial hypotension(SIH) headache.Methods: Clinical data of 15 cases of SIH headache were retrospectively analyzed. Results: 12 0f 15 caseswere acute onset, 9 were female. The ages of onset were from 28 t0 56 years. 93.33% cases had posturalheadache, with the common concomitant symptoms of nausea and vomit. The average cerebrospinal fluidpressure was (41.2 + 30.85)mmH20, which was higher in male than in female (P<0.05). Radionuclidecisternography and imaging were normal. All cases were cured after conservative treatment. Conclusion:Typical postural headache and cerebrospinal fluid pressure less than 60 mmH.O were the main features in SIHheadache, which were with favorable prognosis.%目的:观察实验性自身免疫性脑脊髓炎(EAE)小鼠模型脑组织和脊髓中脑型肌酸激酶(CK-BB)、钙泵(CaATPase) 和钙中性蛋白酶(calpain)的变化.方法:C57BL/6

  8. CALPAIN AND MARCKS PROTEIN REGULATION OF AIRWAY MUCIN SECRETION

    OpenAIRE

    Lampe, W. Randall; Park, Joungjoa; Fang, Shijing; Crews, Anne L; Adler, Kenneth B.

    2012-01-01

    Hypersecretion of mucin plays an important role in the pathophysiology of many inflammatory airway diseases, including asthma, chronic bronchitis, and cystic fibrosis. Myristoylated alanine-rich C-kinase substrate (MARCKS) protein has been shown to play an important role in regulation of airway mucin secretion, as peptides analogous to the amino (N)-terminus of MARCKS attenuate mucin secretion by airway epithelium in vitro and in vivo. Here, we investigated a potential role for the protease C...

  9. SwissProt search result: AK064381 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064381 002-108-E01 (O08529) Calpain-2 catalytic subunit (EC 3.4.22.53) (Calpain-2... large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) (80 kDa M-calpain subunit) (CALP80) CAN2_MOUSE 5e-53 ...

  10. UniProt search blastx result: AK287903 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287903 J065211G19 P17655|CAN2_HUMAN Calpain-2 catalytic subunit precursor (EC 3.4.22.53) (Calpain...-2 large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain...) (Calpain large polypeptide L2) - Homo sapiens (Human) 0 ...

  11. SwissProt search result: AK072218 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072218 J013167O21 (P17655) Calpain-2 catalytic subunit (EC 3.4.22.53) (Calpain-2 ...large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) (Calpain large polypeptide L2) CAN2_HUMAN 6e-40 ...

  12. UniProt search blastx result: AK287903 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287903 J065211G19 O08529|CAN2_MOUSE Calpain-2 catalytic subunit precursor (EC 3.4.22.53) (Calpain...-2 large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain...) (80 kDa M-calpain subunit) (CALP80) - Mus musculus (Mouse) 0 ...

  13. SwissProt search result: AK064381 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064381 002-108-E01 (P17655) Calpain-2 catalytic subunit (EC 3.4.22.53) (Calpain-2... large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) (Calpain large polypeptide L2) CAN2_HUMAN 2e-53 ...

  14. SwissProt search result: AK072218 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072218 J013167O21 (O08529) Calpain-2 catalytic subunit (EC 3.4.22.53) (Calpain-2 ...large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) (80 kDa M-calpain subunit) (CALP80) CAN2_MOUSE 1e-38 ...

  15. NCYM promotes calpain-mediated Myc-nick production in human MYCN-amplified neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Shoji, Wataru [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Department of Pediatric Surgery, Graduate School of Medicine, Tohoku University, Sendai 980-8574 (Japan); Suenaga, Yusuke, E-mail: ysuenaga@chiba-cc.jp [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Cancer Genome Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Kaneko, Yoshiki; Islam, S.M. Rafiqul; Alagu, Jennifer [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Yokoi, Sana [Cancer Genome Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan); Nio, Masaki [Department of Pediatric Surgery, Graduate School of Medicine, Tohoku University, Sendai 980-8574 (Japan); Nakagawara, Akira, E-mail: nakagawara-a@koseikan.jp [Division of Biochemistry and Innovative Cancer Therapeutics and Children' s Cancer Research Center, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, Chiba 260-8717 (Japan)

    2015-06-05

    NCYM is a cis-antisense gene of MYCN and is amplified in human neuroblastomas. High NCYM expression is associated with poor prognoses, and the NCYM protein stabilizes MYCN to promote proliferation of neuroblastoma cells. However, the molecular mechanisms of NCYM in the regulation of cell survival have remained poorly characterized. Here we show that NCYM promotes cleavage of MYCN to produce the anti-apoptotic protein, Myc-nick, both in vitro and in vivo. NCYM and Myc-nick were induced at G2/M phase, and NCYM knockdown induced apoptotic cell death accompanied by Myc-nick downregulation. These results reveal a novel function of NCYM as a regulator of Myc-nick production in human neuroblastomas. - Highlights: • NCYM promotes cleavages of MYC and MYCN to produce Myc-nick in vitro. • NCYM increases Myc-nick production in MYCN-amplified neuroblastoma cells. • NCYM knockdown decreases Myc-nick production and induces apoptosis at G2/M phase.

  16. NCYM promotes calpain-mediated Myc-nick production in human MYCN-amplified neuroblastoma cells

    International Nuclear Information System (INIS)

    NCYM is a cis-antisense gene of MYCN and is amplified in human neuroblastomas. High NCYM expression is associated with poor prognoses, and the NCYM protein stabilizes MYCN to promote proliferation of neuroblastoma cells. However, the molecular mechanisms of NCYM in the regulation of cell survival have remained poorly characterized. Here we show that NCYM promotes cleavage of MYCN to produce the anti-apoptotic protein, Myc-nick, both in vitro and in vivo. NCYM and Myc-nick were induced at G2/M phase, and NCYM knockdown induced apoptotic cell death accompanied by Myc-nick downregulation. These results reveal a novel function of NCYM as a regulator of Myc-nick production in human neuroblastomas. - Highlights: • NCYM promotes cleavages of MYC and MYCN to produce Myc-nick in vitro. • NCYM increases Myc-nick production in MYCN-amplified neuroblastoma cells. • NCYM knockdown decreases Myc-nick production and induces apoptosis at G2/M phase

  17. Association of calpain 10 gene UCSNP-43 polymorphism (rs3792267) with polycystic ovarian syndrome

    OpenAIRE

    Sujatha Thathapudi; Jayashankar Erukkambattu; Qurratulain Hasan; Uma Addepally; Vijayalakshmi Kodati

    2015-01-01

    Background: The principle features of polycystic ovarian syndrome (PCOS) are insulin resistance (IR), hyperandrogenism (HA), obesity (Ob), oligo/anovulation and polycystic ovaries (PCO). PCOS is known to be associated with increased risk of type-2 diabetes mellitus (T2DM) and genes related to T2DM may also play a role in PCOS pathogenesis. Our aim is to study the association of CAPN-10 gene UCSNP-43 (rs3792267) polymorphism with PCOS. Methods: Case-control study, involved 204 women with P...

  18. Calcitriol enhances fat synthesis factors and calpain activity in co-cultured cells.

    Science.gov (United States)

    Choi, Hyuck; Myung, Kyuho

    2014-08-01

    We have conducted an in vitro experiment to determine whether calcitriol can act as a fat synthesizer and/or meat tenderizer when skeletal muscle cells, adipose tissue, and macrophages are co-cultured. When co-cultured, pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) expression increased, whereas decreased anti-inflammatory cytokine (IL-10 and IL-15) expression decreased in both C2C12 and 3T3-L1 cells. Calcitriol increased reactive oxygen species (ROS) production in the media. While adiponectin gene expression decreased, leptin, resistin, CCAAT-enhancer-binding protein-beta (C/EBP-β), and peroxisome proliferator-activated receptor gamma (PPAR-γ) gene expression was significantly (P meat tenderizer, in meat-producing animals. PMID:24687633

  19. Dexamethasone enhances necrosis-like neuronal death in ischemic rat hippocampus involving μ-calpain activation

    DEFF Research Database (Denmark)

    Müller, Georg Johannes; Hasseldam, Henrik; Rasmussen, Rune Skovgaard;

    2014-01-01

    Transient forebrain ischemia (TFI) leads to hippocampal CA1 pyramidal cell death which is aggravated by glucocorticoids (GC). It is unknown how GC affect apoptosis and necrosis in cerebral ischemia. We therefore investigated the co-localization of activated caspase-3 (casp-3) with apoptosis...

  20. SwissProt search result: AK064381 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064381 002-108-E01 (P06814) Calpain-2 catalytic subunit (EC 3.4.22.53) (Calpain-2... large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) (Fragment) CAN2_RABIT 6e-16 ...

  1. SwissProt search result: AK072218 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072218 J013167O21 (P06814) Calpain-2 catalytic subunit (EC 3.4.22.53) (Calpain-2 ...large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) (Fragment) CAN2_RABIT 2e-16 ...

  2. SwissProt search result: AK072218 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072218 J013167O21 (Q92178) Calpain-2 catalytic subunit (EC 3.4.22.53) (Calpain-2 ...large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) CAN2_CHICK 3e-38 ...

  3. UniProt search blastx result: AK287903 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287903 J065211G19 Q92178|CAN2_CHICK Calpain-2 catalytic subunit precursor (EC 3.4.22.53) (Calpain...-2 large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) - Gallus gallus (Chicken) 0 ...

  4. SwissProt search result: AK103409 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103409 J033128E16 (Q92178) Calpain-2 catalytic subunit (EC 3.4.22.53) (Calpain-2 ...large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) CAN2_CHICK 3e-11 ...

  5. SwissProt search result: AK059278 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059278 001-025-C08 (Q92178) Calpain-2 catalytic subunit (EC 3.4.22.53) (Calpain-2... large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) CAN2_CHICK 1e-11 ...

  6. SwissProt search result: AK072218 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072218 J013167O21 (Q9GLG1) Calpain-2 catalytic subunit (EC 3.4.22.53) (Calpain-2 ...large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) CAN2_MACFA 8e-40 ...

  7. SwissProt search result: AK064381 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064381 002-108-E01 (Q07009) Calpain-2 catalytic subunit (EC 3.4.22.53) (Calpain-2... large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) CAN2_RAT 9e-52 ...

  8. Arabidopsis CDS blastp result: AK121261 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK121261 J023104H13 At1g55350.4 calpain-type cysteine protease family identical to calpain...-like protein GI:20268660 from [Arabidopsis thaliana]; contains Pfam profiles: PF00648 Calpain family... cysteine protease, PF01067 Calpain large subunit,domain III; identical to cDNA calpain-like protein GI:20268659 0.0 ...

  9. SwissProt search result: AK064381 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064381 002-108-E01 (Q92178) Calpain-2 catalytic subunit (EC 3.4.22.53) (Calpain-2... large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) CAN2_CHICK 1e-51 ...

  10. SwissProt search result: AK064381 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064381 002-108-E01 (Q9GLG1) Calpain-2 catalytic subunit (EC 3.4.22.53) (Calpain-2... large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) CAN2_MACFA 3e-53 ...

  11. SwissProt search result: AK072218 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072218 J013167O21 (Q07009) Calpain-2 catalytic subunit (EC 3.4.22.53) (Calpain-2 ...large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) CAN2_RAT 4e-38 ...

  12. UniProt search blastx result: AK287903 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287903 J065211G19 P43367|CAN2_PIG Calpain-2 catalytic subunit (EC 3.4.22.53) (Calpain...-2 large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain) (Fragment) - Sus scrofa (Pig) 0 ...

  13. UniProt search blastx result: AK287903 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287903 J065211G19 Q9GLG1|CAN2_MACFA Calpain-2 catalytic subunit precursor (EC 3.4.22.53) (Calpain...-2 large subunit) (Calcium-activated neutral proteinase 2) (CANP 2) (Calpain M-type) (M-calpain) (Millimolar-calpain

  14. Arabidopsis CDS blastp result: AK072218 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072218 J013167O21 At1g55350.4 calpain-type cysteine protease family identical to calpain...-like protein GI:20268660 from [Arabidopsis thaliana]; contains Pfam profiles: PF00648 Calpain family... cysteine protease, PF01067 Calpain large subunit,domain III; identical to cDNA calpain-like protein GI:20268659 1e-150 ...

  15. Calpain inhibition prevents pacing-induced cellular remodeling in a HL-1 myocyte model for atrial fibrillation

    NARCIS (Netherlands)

    Brundel, BJJM; Kampinga, HH; Henning, RH

    2004-01-01

    Objective: Atrial fibrillation (AF) is a progressive disease. Previously, clinical and animal experimental studies in AF revealed a variety of myocyte remodeling processes including L-type Ca(2+) channel reduction and structural changes, which finally result in electrical remodeling and contractile

  16. Activity-Dependent Calpain Activation Plays a Critical Role in Synaptic Facilitation and Post-Tetanic Potentiation

    Science.gov (United States)

    Khoutorsky, Arkady; Spira, Micha E.

    2009-01-01

    Synaptic facilitation and post-tetanic potentiation (PTP) are believed to necessitate active regeneration of the release machinery and supply of synaptic vesicles to a ready-releasable site. The prevailing hypothesis assumes that synapsins play pivotal roles in these processes. Using a cholinergic synapse formed between cultured "Aplysia" neurons…

  17. Role of the Calpain in Meat Tenderness%钙激活酶对肉嫩度的影响

    Institute of Scientific and Technical Information of China (English)

    黄镭; 熊汉国

    2010-01-01

    宰后肌肉嫩度的变化是在多种酶的协同作用下完成的,其中一个重要部分是钙激活酶.本文主要论述了钙激活酶的分子结构特征,影响酶活性的因素,以及对肌肉嫩化的作用机理等方面的内容.

  18. Effect of ageing and μ-calpain markers on meat quality from Brangus steers finished on pasture.

    Science.gov (United States)

    Mazzucco, Juliana Papaleo; Melucci, Lilia M; Villarreal, Edgardo L; Mezzadra, Carlos A; Soria, Liliana; Corva, Pablo; Motter, Mariana M; Schor, Alejandro; Miquel, María C

    2010-11-01

    Brangus steers (n=247) finished on pasture were used to evaluate the effects of post-mortem ageing and polymorphism CAPN1 316 and CAPN1 4751 markers on meat tenderness and objective colour measurements (CIEL*a*b*) of m. Longissimus dorsi. Ageing meat for 7 days decreased shear force (SF) by 13.7% and improved a* (8.4%) and b* (10%) compared to ageing for 1 day. No difference between 7 and 14 days of ageing was found for SF, a* and b*. However, L* increased markedly with ageing. Fitting both markers simultaneously, CAPN1 316 showed association with SF and L* and CAPN1 4751 with a* and b*. Fitting the markers individually, CAPN1 4751 affected all traits and CAPN1 316 showed association with SF and L*. Post-mortem ageing and the use of markers represent two independent and alternative tools that could be used for improving quality of meat from Brangus cattle. PMID:20709460

  19. SwissProt search result: AK099458 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK099458 J013022O12 (P06815) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) (Fragment) CAN1_RABIT 2e-11 ...

  20. SwissProt search result: AK059278 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059278 001-025-C08 (P07384) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1... large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_HUMAN 2e-12 ...

  1. SwissProt search result: AK059278 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059278 001-025-C08 (P35750) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1... large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_PIG 2e-12 ...

  2. SwissProt search result: AK103409 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103409 J033128E16 (O35350) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_MOUSE 2e-11 ...

  3. SwissProt search result: AK064381 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064381 002-108-E01 (P97571) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1... large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_RAT 1e-53 ...

  4. SwissProt search result: AK064381 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064381 002-108-E01 (P07384) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1... large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_HUMAN 2e-52 ...

  5. SwissProt search result: AK103409 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103409 J033128E16 (Q9GLG2) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_MACFA 2e-12 ...

  6. SwissProt search result: AK072218 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072218 J013167O21 (P07384) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_HUMAN 1e-39 ...

  7. SwissProt search result: AK103409 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103409 J033128E16 (P06815) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) (Fragment) CAN1_RABIT 8e-12 ...

  8. SwissProt search result: AK072218 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072218 J013167O21 (O35350) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_MOUSE 9e-41 ...

  9. NCBI nr-aa BLAST: CBRC-PABE-11-0002 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PABE-11-0002 ref|NP_001013789.1| calpain 11 [Mus musculus] gb|AAT27434.1| calpain... 11 [Mus musculus] gb|AAI48638.1| Calpain 11 [synthetic construct] gb|AAI53196.1| Calpain 11 [synthetic construct] NP_001013789.1 2.9 28% ...

  10. SwissProt search result: AK065151 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK065151 J013002B09 (P06815) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) (Fragment) CAN1_RABIT 2e-11 ...

  11. SwissProt search result: AK103409 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103409 J033128E16 (P07384) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_HUMAN 2e-12 ...

  12. SwissProt search result: AK064381 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064381 002-108-E01 (O35350) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1... large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_MOUSE 2e-53 ...

  13. SwissProt search result: AK064381 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064381 002-108-E01 (Q9GLG2) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1... large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_MACFA 1e-52 ...

  14. SwissProt search result: AK059278 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059278 001-025-C08 (Q9GLG2) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1... large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_MACFA 2e-12 ...

  15. SwissProt search result: AK103409 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103409 J033128E16 (Q27970) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_BOVIN 2e-11 ...

  16. SwissProt search result: AK064381 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064381 002-108-E01 (P35750) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1... large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_PIG 7e-52 ...

  17. SwissProt search result: AK072218 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072218 J013167O21 (Q27970) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_BOVIN 2e-39 ...

  18. SwissProt search result: AK072218 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072218 J013167O21 (P97571) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_RAT 7e-41 ...

  19. SwissProt search result: AK065151 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK065151 J013002B09 (P35750) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_PIG 4e-11 ...

  20. SwissProt search result: AK059278 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059278 001-025-C08 (Q27970) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1... large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_BOVIN 2e-11 ...

  1. SwissProt search result: AK072218 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072218 J013167O21 (P35750) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_PIG 4e-39 ...

  2. SwissProt search result: AK103409 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103409 J033128E16 (P97571) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_RAT 2e-11 ...

  3. SwissProt search result: AK059278 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059278 001-025-C08 (P97571) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1... large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_RAT 2e-11 ...

  4. SwissProt search result: AK103409 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103409 J033128E16 (P35750) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_PIG 2e-12 ...

  5. SwissProt search result: AK099458 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK099458 J013022O12 (P35750) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_PIG 4e-11 ...

  6. SwissProt search result: AK059278 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059278 001-025-C08 (O35350) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1... large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_MOUSE 2e-11 ...

  7. SwissProt search result: AK059278 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059278 001-025-C08 (P06815) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1... large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) (Fragment) CAN1_RABIT 6e-12 ...

  8. SwissProt search result: AK072218 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072218 J013167O21 (Q9GLG2) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1 ...large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_MACFA 1e-39 ...

  9. SwissProt search result: AK064381 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064381 002-108-E01 (Q27970) Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain-1... large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) (Calpain mu-type) (muCANP) (Micromolar-calpain) CAN1_BOVIN 5e-52 ...

  10. Arabidopsis CDS blastp result: AK064381 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064381 002-108-E01 At1g55350.4 calpain-type cysteine protease family identical to calpain...-like protein GI:20268660 from [Arabidopsis thaliana]; contains Pfam profiles: PF00648 Calpain famil...y cysteine protease, PF01067 Calpain large subunit,domain III; identical to cDNA calpain-like protein GI:20268659 0.0 ...

  11. Characterization of the role of full-length CRMP3 and its calpain-cleaved product in inhibiting microtubule polymerization and neurite outgrowth

    International Nuclear Information System (INIS)

    Collapsin response mediator proteins (CRMPs) are key modulators of cytoskeletons during neurite outgrowth in response to chemorepulsive guidance molecules. However, their roles in adult injured neurons are not well understood. We previously demonstrated that CRMP3 underwent calcium-dependent N-terminal protein cleavage during excitotoxicity-induced neurite retraction and neuronal death. Here, we report findings that the full-length CRMP3 inhibits tubulin polymerization and neurite outgrowth in cultured mature cerebellar granule neurons, while the N-terminal truncated CRMP3 underwent nuclear translocation and caused a significant nuclear condensation. The N-terminal truncated CRMP3 underwent nuclear translocation through nuclear pores. Nuclear protein pull-down assay and mass spectrometry analysis showed that the N-terminal truncated CRMP3 was associated with nuclear vimentin. In fact, nuclear-localized CRMP3 co-localized with vimentin during glutamate-induced excitotoxicity. However, the association between the truncated CRMP3 and vimentin was not critical for nuclear condensation and neurite outgrowth since over-expression of truncated CRMP3 in vimentin null neurons did not alleviate nuclear condensation and neurite outgrowth inhibition. Together, these studies showed CRMP3's role in attenuating neurite outgrowth possibility through inhibiting microtubule polymerization, and also revealed its novel association with vimentin during nuclear condensation prior to neuronal death.

  12. Characterization of the role of full-length CRMP3 and its calpain-cleaved product in inhibiting microtubule polymerization and neurite outgrowth

    Energy Technology Data Exchange (ETDEWEB)

    Aylsworth, Amy [Experimental NeuroTherapeutics Laboratory, Institute for Biological Sciences, National Research Council Canada, 1200 Montreal Road, Bldg M54, Ottawa, Ontario, Canada K1A 0R6 (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario (Canada); Jiang, Susan X.; Desbois, Angele [Experimental NeuroTherapeutics Laboratory, Institute for Biological Sciences, National Research Council Canada, 1200 Montreal Road, Bldg M54, Ottawa, Ontario, Canada K1A 0R6 (Canada); Hou, Sheng T., E-mail: Sheng.hou@nrc-cnrc.gc.ca [Experimental NeuroTherapeutics Laboratory, Institute for Biological Sciences, National Research Council Canada, 1200 Montreal Road, Bldg M54, Ottawa, Ontario, Canada K1A 0R6 (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario (Canada)

    2009-10-01

    Collapsin response mediator proteins (CRMPs) are key modulators of cytoskeletons during neurite outgrowth in response to chemorepulsive guidance molecules. However, their roles in adult injured neurons are not well understood. We previously demonstrated that CRMP3 underwent calcium-dependent N-terminal protein cleavage during excitotoxicity-induced neurite retraction and neuronal death. Here, we report findings that the full-length CRMP3 inhibits tubulin polymerization and neurite outgrowth in cultured mature cerebellar granule neurons, while the N-terminal truncated CRMP3 underwent nuclear translocation and caused a significant nuclear condensation. The N-terminal truncated CRMP3 underwent nuclear translocation through nuclear pores. Nuclear protein pull-down assay and mass spectrometry analysis showed that the N-terminal truncated CRMP3 was associated with nuclear vimentin. In fact, nuclear-localized CRMP3 co-localized with vimentin during glutamate-induced excitotoxicity. However, the association between the truncated CRMP3 and vimentin was not critical for nuclear condensation and neurite outgrowth since over-expression of truncated CRMP3 in vimentin null neurons did not alleviate nuclear condensation and neurite outgrowth inhibition. Together, these studies showed CRMP3's role in attenuating neurite outgrowth possibility through inhibiting microtubule polymerization, and also revealed its novel association with vimentin during nuclear condensation prior to neuronal death.

  13. Calpain Activity and Toll-Like Receptor 4 Expression in Platelet Regulate Haemostatic Situation in Patients Undergoing Cardiac Surgery and Coagulation in Mice

    Directory of Open Access Journals (Sweden)

    Jui-Chi Tsai

    2014-01-01

    Full Text Available Human platelets express Toll-like receptors (TLR 4. However, the mechanism by which TLR4 directly affects platelet aggregation and blood coagulation remains to be explored. Therefore, in this study, we evaluated the platelet TLR4 expression in patients who underwent CABG surgery; we explored the correlation between platelet TLR4 expression and the early outcomes in hospital of patients. Additionally, C57BL/6 and C57BL/6-TlrLPS−/− mice were used to explore the roles of platelet TLR4 in coagulation by platelet aggregometry and rotation thromboelastometry. In conclusion, our results highlight the important roles of TLR4 in blood coagulation and platelet function. Of clinical relevance, we also explored novel roles for platelet TLR4 that are associated with early outcomes in cardiac surgery.

  14. Combined calpain-induced downregulation of TrkB-FL and TrkB-T1 upregulation causes neuronal death in excitotoxicity and ischemia

    OpenAIRE

    Díaz-Guerra, Margarita; Vidaurre, Oscar G.; Gascón, Sergio

    2012-01-01

    Electronic response to "Excitotoxicity downregulates TrkB.FL signaling and upregulates the neuroprotective truncated TrkB receptors in cultured hippocampal and striatal neurons" Gomes, et al., 32(13): 4610-4622; doi: 10.1523/JNEUROSCI.0374-12.2012

  15. The role of extracellular and intracellular proteolytic systems in aneurysms of the ascending aorta.

    Science.gov (United States)

    Werner, Isabella; Schack, Stephanie; Richter, Manfred; Stock, Ulrich A; Ahmad, Ali El-Sayed; Moritz, Anton; Beiras-Fernandez, Andres

    2016-05-01

    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. PMID:26582478

  16. 异氟烷通过激活Calpain影响大鼠海马神经干细胞的增殖与分化%Isoflurane Affects Proliferation and Differentiation of Neural Stem Cells by Activating Calpain

    Institute of Scientific and Technical Information of China (English)

    张建芳; 陈欣; 王伟; 方茜; 罗爱林; 周碧云

    2015-01-01

    目的 探讨异氟烷影响神经干细胞(NSCs)增殖与分化的机制.方法 原代培养SD新生大鼠(出生1d内)海马NSCs,取传至第2代细胞,将其随机分为4组(n=5):空白对照组(CON),给予含有21%O2和5%CO2的混合气体;异氟烷组(ISO),给予3.4%异氟烷,干预6 h;Calpeptin组(CP),给予10μmol/L Calpeptin和含有21%O2和5%CO2的混合气体,干预6h;异氟烷+Calpeptin组(ISO+ CP),给予10 μmol/L Calpeptin和3.4%异氟烷,干预6h.BrdU检测NSCs增殖,Western blot检测GFAP、Tuj-1、αⅡ-spectrin及其裂解产物SBDP145的蛋白水平.结果 与CON组相比,ISO组在干预后0、12、24 h时BrdU+细胞数目明显减少,说明异氟烷抑制了NSCs增殖并有后遗效应;ISO组SBDP145表达明显上调,说明异氟烷可上调Calpain活性.掺入Calpain抑制剂Calpeptin后,与ISO组相比,ISO+ CP组SB-DP145表达量明显下降,表明Calpeptin可拮抗异氟烷致Calpain活性增强的作用;ISO+ CP组BrdU+细胞数目增多,Calpeptin减弱了异氟烷抑制NSCs增殖的作用.与CON组相比,ISO组GFAP表达水平上调,Tuj-1水平无明显改变;CP组GFAP水平无明显改变,而Tuj-1表达上调.与ISO组相比,ISO+ CP组GFAP水平降低,Tuj-1水平无明显改变,表明异氟烷促进NSCs分化为胶质细胞,而Calpeptin可拮抗异氟烷的这种作用.结论 异氟烷通过激活Calpain信号通路抑制NSCs的自我更新并促进NSCs分化为胶质细胞.

  17. The effects of Capn1 gene inactivation on skeletal muscle growth, development, and atrophy, and the compensatory role of other proteolytic systems

    Science.gov (United States)

    Myofibrillar protein turnover is a key component of muscle growth and degeneration, requiring proteolytic enzymes to degrade the skeletal muscle proteins. The objective of this study was to investigate the role of the calpain proteolytic system in muscle growth development using µ-calpain knockout (...

  18. Mechanical Signal Transduction in Countermeasures to Muscle Atrophy

    Science.gov (United States)

    Tidball, James G.; Chu, Amy (Technical Monitor)

    2002-01-01

    We have shown that modifications in muscle use result in changes in the expression and activity of calpains and nitric oxide synthase (NOS). Although muscle unloading for 10 days produced no change in the concentrations of calpain 1 or 2 and no change in calpain activation, muscle reloading produced a 90% increase in calpain 2 concentration. We developed an in vitro model to test our hypothesis that nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin. Talin was selected because it is a well-characterized calpain substrate and it is codistributed with calpain in muscle cells. We found that intermittant loading during hindlimb suspension that is sufficient to prevent muscle mass loss that occurs during muscle unloading is also sufficient to prevent the decrease in NOS expression that normally occurs during hindlimb unloading. These findings indicate that therapeutics directed toward regulating the calpain/calpastatin system may be beneficial in preventing muscle mass loss in muscle injury, unloading and disease.

  19. Evaluation of Limb-Girdle Muscular Dystrophy

    Science.gov (United States)

    2014-03-06

    Becker Muscular Dystrophy; Limb-Girdle Muscular Dystrophy, Type 2A (Calpain-3 Deficiency); Limb-Girdle Muscular Dystrophy, Type 2B (Miyoshi Myopathy, Dysferlin Deficiency); Limb-Girdle Muscular Dystrophy, Type 2I (FKRP-deficiency)

  20. Calpastatin is regulated by protein never in mitosis gene A interacting-1 (PIN1) in endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Tongzheng, E-mail: liu.tongzheng@mayo.edu [Division of Oncology Research, Department of Oncology, Mayo Clinic, Rochester, MN 55905 (United States); Schneider, Ryan A., E-mail: schneiderr@findlay.edu [College of Pharmacy, The University of Findlay, Findlay, OH 45840 (United States); Hoyt, Dale G., E-mail: hoyt.27@osu.edu [The Dorothy M. Davis Heart and Lung Research Institute, and the Division of Pharmacology, College of Pharmacy, The Ohio State University, 500 West Twelfth Avenue, Columbus, OH 43210 (United States)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer Depletion of PIN1 increases inhibitory effect of calpastatin against calpain in endothelial cells. Black-Right-Pointing-Pointer PIN1 associates with calpastatin. Black-Right-Pointing-Pointer PIN1, but not mutants, reduces the inhibitory activity of calpastatin in vitro. Black-Right-Pointing-Pointer Depletion of calpastatin shows that it is required for PIN1 depletion to reduce calpain activity. -- Abstract: The peptidyl-proline isomerase, protein never in mitosis gene A interacting-1 (PIN1) binds and isomerizes proteins phosphorylated on serine/threonine before a proline. It was previously found that depletion of PIN1 greatly increased induction of cyclooxygenase-2 and inducible nitric oxide synthase by lowering calpain activity in murine aortic endothelial cells (MAEC). Here we investigated the effect of PIN1 on the endogenous inhibitor of heterodimeric {mu}- and m-calpains, calpastatin. MAEC were transduced with small hairpin (sh) RNA to knock down PIN1 (KD) or an inactive Control shRNA. Cells were also treated with non-targeted double stranded small inhibitory RNA (siRNA) or siRNA designed to deplete calpastatin. Despite reducing calpain activity, PIN1 KD did not significantly affect the expression of {mu}- and m-calpains, or calpastatin, compared to Control shRNA. Instead, depletion of PIN1 increased the inhibitory activity of calpastatin. Calpastatin co-immunoprecipitated with endogenous PIN1 and was pulled down with glutathione-S-transferase (GST)-PIN1 fusion protein. Adding GST-PIN1 to KD cell extracts lacking PIN1 reduced calpastatin inhibitory activity. Substrate binding and catalytic domain mutants of PIN1 failed to do so. These results suggest that protein interaction and the proline isomerase functions of PIN1 are required for it to inhibit calpastatin. Furthermore, depletion of calpastatin raised calpain activity and reduced calpain inhibitory activity to similar levels in KD and Control MAEC, indicating that

  1. PEST sequences in the malaria parasite Plasmodium falciparum: a genomic study

    Directory of Open Access Journals (Sweden)

    Bell Angus

    2003-06-01

    Full Text Available Abstract Background Inhibitors of the protease calpain are known to have selectively toxic effects on Plasmodium falciparum. The enzyme has a natural inhibitor calpastatin and in eukaryotes is responsible for turnover of proteins containing short sequences enriched in certain amino acids (PEST sequences. The genome of P. falciparum was searched for this protease, its natural inhibitor and putative substrates. Methods The publicly available P. falciparum genome was found to have too many errors to permit reliable analysis. An earlier annotation of chromosome 2 was instead examined. PEST scores were determined for all annotated proteins. The published genome was searched for calpain and calpastatin homologs. Results Typical PEST sequences were found in 13% of the proteins on chromosome 2, including a surprising number of cell-surface proteins. The annotated calpain gene has a non-biological "intron" that appears to have been created to avoid an unrecognized frameshift. Only the catalytic domain has significant similarity with the vertebrate calpains. No calpastatin homologs were found in the published annotation. Conclusion A calpain gene is present in the genome and many putative substrates of this enzyme have been found. Calpastatin homologs may be found once the re-annotation is completed. Given the selective toxicity of calpain inhibitors, this enzyme may be worth exploring further as a potential drug target.

  2. 中国肾移植患者钙蛋白酶10基因多态性与移植后糖尿病的相关性研究%Calpain10 gene polymorphisms are associated with posttransplantation diabetes mellitus in Chinese renal allograft recipients

    Institute of Scientific and Technical Information of China (English)

    余爱荣; 范星; 刘慧明; 辛华雯

    2012-01-01

    目的:探讨中国肾移植患者钙蛋白酶10(CAPN10)基因多态性与移植后糖尿病(PTDM)的相关性.方法:采用等位基因特异性PCR、限制性片断长度多态性PCR (PCR-RFLP)分别检测了97例PTDM患者(PTDM组)和301例未发生PTDM的肾移植患者(对照组)的CAPN10基因SNP-19、SNP-43、SNP-63的基因型,采用lo-gistic回归分析该基因多态性与PTDM的相关性.结果:PTDM组患者SNP-19的11+12基因型频率和SNP-43的GG基因型频率明显高于对照组(P<0.05).用性别、移植时年龄、体重和BMI进行校正后,SNP-19的11基因型和12基因型携带者移植术后发生PTDM的风险分别是22基因型的1.502倍(OR=1.502,95% CI:1.016~2.347,P=0.048)和1.764倍(O)R=1.764,95%CI:1.055~2.947,P=0.030),SNP-43的GG基因型携带者移植术后发生PTDM的风险是AA和GA基因型患者的2.19倍(OR=2.190,95%CI:1.047~~3.473,P=0.044),SNP-63与PTDM的发生无明显相关性(P>0.05).结论:CAPN10基因SNP-19的1等位基因和SNP-43的GG基因型是肾移植后发生PTDM的独立危险因素.%AIM: To investigate the association between the CalpainlO gene polymorphism and the risk of PTDM in Chinese renal allograft recipients. METHODS; Three single nucleotide polymorphisms (CAPN10 gene SNP-19, SNP-43, SNP-63) were genotyped in the cohort, which consisted of 97 renal allograft recipients with PTDM (PTDM group) and 301 renal allograft recipients without PTDM ( control group). The genotypes of polymorphisms were performed by allele specific polymerase chain reaction ( ASPCR ) , PCR-restriction fragment length polymorphism (PCR-RFLP). Logistic regression test was used to identify risk factors for PTDM development and calculate the odds ratio, RESULTS: The 11 and 12 genotypes of SNP-19 and the GG genotypes of SNP-43 were more common in patients with PTDM than those without PTDM (F<0. 05). After adjustments for age, sex, body weight and BMI, the effect of genotype remained significant (11 vs 22, OR = 1.502, 95%CI: 1.016-2.347, P = 0.048; 12 vs 22, OR=1. 764, 95%CI: 1.055 - 2.947, P = 0. 030) in SNP-19 and the patients carrying gen otype GG had higher risk comparison with carriers with genotype AA or GA(OR = 2. 190, 95% CI: 1.047 - 3.473, P = 0. 044) in SNP-43. But SNP-63 was not association with PTDM. CONCLUSION; The 1-allele in SNP-19 and GG genotype in SNP-43 of CAPN10 gene are the independent risk factors of PTDM in Chinese renal allograft recipients.

  3. Multiple mutations in a specific gene in a small geographic area: A common phenomenon

    Energy Technology Data Exchange (ETDEWEB)

    Zlotogora, J.; Bach, G.; Gieselmann, V.

    1996-01-01

    We read with interest the article from Allamand et al., which demonstrates in a genetic isolate the presence of at least six different haplotypes in the limb-girdle muscular dystrophy type 2A chromosome. Several hypotheses were proposed by the authors to explain this finding, but, after the identification of calpain, the gene involved in the disorder, multiple mutations were proved to be at the origin of this observation. The authors proposed that both the presence of multiple distinct calpain mutations within the Reunion Island pedigrees and the relatively low frequency of the disease in the isolate may be explained by a digenic inheritance of the disorder. Their hypothesis postulates that, although calpain mutations may be frequent in all populations, the disease manifestations are controlled by another frequently mutated nuclear or mitochondrial gene in the Reunion isolate. 8 refs.

  4. A heterozygous 21-bp deletion in CAPN3 causes dominantly inherited limb girdle muscular dystrophy

    DEFF Research Database (Denmark)

    Vissing, John; Barresi, Rita; Witting, Nanna;

    2016-01-01

    Limb girdle muscular dystrophy type 2A is the most common limb girdle muscular dystrophy form worldwide. Although strict recessive inheritance is assumed, patients carrying a single mutation in the calpain 3 gene (CAPN3) are reported. Such findings are commonly attributed to incomplete mutation...... creatine kinase or myoglobin. Muscle weakness was generally milder than observed in limb girdle muscular dystrophy type 2A, but affected the same muscle groups (proximal leg, lumbar paraspinal and medial gastrocnemius muscles). In some cases, the weakness was severely disabling. The 21-bp deletion did...... affecting the calpain 3 homodimer. This renders patients deficient in calpain 3 as in limb girdle muscular dystrophy type 2A, albeit in a milder form in most cases. Based on findings in 10 families, our study indicates that a dominantly inherited pattern of calpainopathy exists, and should be considered...

  5. Degradation of the transcription factors NF-κB, STAT3, and STAT5 is involved in Entamoeba histolytica-induced cell death in Caco-2 colonic epithelial cells.

    Science.gov (United States)

    Kim, Kyeong Ah; Min, Arim; Lee, Young Ah; Shin, Myeong Heon

    2014-10-01

    Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-κB (p65) in Caco-2 cells. However, IκB, an inhibitor of NF-κB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-κB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-κB and STATs in colonic epithelial cells, which ultimately accelerates cell death.

  6. Effects of Sunphenon and Polyphenon 60 on proteolytic pathways, inflammatory cytokines and myogenic markers in H22-treated C2C12 cells

    Indian Academy of Sciences (India)

    Allur Subramaniyan Sivakumar; Inho Hwang

    2015-03-01

    The effect of Sunphenon and Polyphenon 60 in oxidative stress response, myogenic regulatory factors, inflammatory cytokines, apoptotic and proteolytic pathways on H2O2-induced myotube atrophy was addressed. Cellular responses of H2O2-induced C2C12cells were examined, including mRNA expression of myogenic regulatory factors, such as MyoD and myogenin, inflammatory pathways, such as TNF- and NF-kB, as well as proteolytic enzymes, such as -calpain and m-calpain. The pre-treatment of Sunphenon (50 g/mL)/Polyphenon 60 (50 g/mL) on H2O2-treated C2C12 cells significantly down-regulated the mRNA expression of myogenin and MyoD when compared to those treated with H2O2-induced alone. Additionally, the mRNA expression of -calpain and m-calpain were significantly ( < 0.05) increased in H2O2-treated C2C12 cells, whereas pre-treatment with Sunphenon/Polyphenon significantly down-regulated the above genes, namely -calpain and m-calpain. Furthermore, the mRNA expression of TNF- and NF-kB were significantly increased in H2O2-treated C2C12 cells, while pre-treatment with Sunphenon (50 g/mL)/Polyphenon 60 (50 g/mL) significantly ( < 0.05) down-regulated it when compared to the untreated control group. Subsequent analysis of DNA degeneration and caspase activation revealed that Sunphenon (50 g/mL)/Polyphenon 60 (50 g/mL) inhibited activation of caspase-3 and showed an inhibitory effect on DNA degradation. From this result, we know that, in stress conditions, -calpain may be involved in the muscle atrophy through the suppression of myogenin and MyoD. Moreover, Sunphenon may regulate the skeletal muscle genes/promote skeletal muscle recovery by the up-regulation of myogenin and MyoD and suppression of -calpain and inflammatory pathways and may regulate the apoptosis pathways. Our findings suggest that dietary supplementation of Sunphenon might reduce inflammatory events in muscle-associated diseases, such as myotube atrophy.

  7. Prion pathogenesis is faithfully reproduced in cerebellar organotypic slice cultures.

    Directory of Open Access Journals (Sweden)

    Jeppe Falsig

    Full Text Available Prions cause neurodegeneration in vivo, yet prion-infected cultured cells do not show cytotoxicity. This has hampered mechanistic studies of prion-induced neurodegeneration. Here we report that prion-infected cultured organotypic cerebellar slices (COCS experienced progressive spongiform neurodegeneration closely reproducing prion disease, with three different prion strains giving rise to three distinct patterns of prion protein deposition. Neurodegeneration did not occur when PrP was genetically removed from neurons, and a comprehensive pharmacological screen indicated that neurodegeneration was abrogated by compounds known to antagonize prion replication. Prion infection of COCS and mice led to enhanced fodrin cleavage, suggesting the involvement of calpains or caspases in pathogenesis. Accordingly, neurotoxicity and fodrin cleavage were prevented by calpain inhibitors but not by caspase inhibitors, whereas prion replication proceeded unimpeded. Hence calpain inhibition can uncouple prion replication from its neurotoxic sequelae. These data validate COCS as a powerful model system that faithfully reproduces most morphological hallmarks of prion infections. The exquisite accessibility of COCS to pharmacological manipulations was instrumental in recognizing the role of calpains in neurotoxicity, and significantly extends the collection of tools necessary for rigorously dissecting prion pathogenesis.

  8. Relationships among calpastatin single nucleotide polymorphisms, calpastatin expression and tenderness in pork longissimus

    Science.gov (United States)

    Genome scans in the pig have identified a region on chromosome 2 (SSC2) associated with tenderness. Calpastatin is a likely positional candidate gene in this region because of its inhibitory role in the calpain system that is involved in postmortem tenderization. Novel single nucleotide polymorphism...

  9. Regulation of NKG2D-ligand cell surface expression by intracellular calcium after HDAC-inhibitor treatment

    DEFF Research Database (Denmark)

    Jensen, Helle; Hagemann-Jensen, Michael Henrik; Lauridsen, Felicia Kathrine Bratt;

    2013-01-01

    surface expression of ULBP2, but not MICA/B, was sensitive to treatment calmidazolium and trifluoperazine, two agents known to block calcium signaling. siRNA-mediated knock-down of the calcium-regulated proteins calmodulin or calpain did however not affect NKG2D-ligand cell surface expression on Jurkat T...

  10. [Activity of Ca(2+)-dependent neutral proteinases in rat organs under cobalt and mercury chloride injection].

    Science.gov (United States)

    Kaliman, P A; Samokhin, A A; Samokhina, L M

    2003-01-01

    The activity of Ca(2+)-dependent neutral proteinases in rats under cobalt and mercury chloride injection was investigated. The calpains activity increase in the lungs, heart, liver and kidneys was revealed after 2 h cobalt chloride action. The mercury chloride gives a reliable increase of calcium-dependent neutral proteinases only in the kidneys. PMID:14574747

  11. Relation of postmortem protease activity to tenderness in buffalo meat and Brahman beef

    Directory of Open Access Journals (Sweden)

    M. Hirabayashi

    2010-02-01

    Full Text Available We previously showed that meat from crossbred water buffalo had significantly higher tenderness than beef from crossbred Brahman cattle of the same age, gender, and diet. Extensive studies on meat tenderness have indicated that proteases degrade muscle fibre proteins during postmortem storage, leading to weakening of the myofibrillar structure and an increase in tenderness. Thus, we investigated the difference in protease activity immediately postmortem, in order to explain the difference in tenderness between buffalo meat and beef. Five female crossbred water-buffalo (Philippine Carabao x Bulgarian Murrah and five female crossbred cattle (Brahman x Philippine Native were slaughtered at 30 months of age, and Longissimus thoracis muscle was sampled immediately post-slaughter. Protease activity at different pH levels and the effect of various inhibitors on protease activity were examined. Results showed that buffalo meat had significantly higher protease activity compared to beef, and calpain inhibitor 1 was the most effective inhibitor. As calpain inhibitor 1 is a specific inhibitor of calpain 1 and 2, the results suggest that higher calpain activity in buffalo meat was responsible for the higher tenderness of buffalo meat compared to Brahman beef.

  12. Calpastatin inhibits motor neuron death and increases survival of hSOD1(G93A) mice.

    Science.gov (United States)

    Rao, Mala V; Campbell, Jabbar; Palaniappan, Arti; Kumar, Asok; Nixon, Ralph A

    2016-04-01

    Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease with a poorly understood cause and no effective treatment. Given that calpains mediate neurodegeneration in other pathological states and are abnormally activated in ALS, we investigated the possible ameliorative effects of inhibiting calpain over-activation in hSOD1(G93A) transgenic (Tg) mice in vivo by neuron-specific over-expression of calpastatin (CAST), the highly selective endogenous inhibitor of calpains. Our data indicate that over-expression of CAST in hSOD1(G93A) mice, which lowered calpain activation to levels comparable to wild-type mice, inhibited the abnormal breakdown of cytoskeletal proteins (spectrin, MAP2 and neurofilaments), and ameliorated motor axon loss. Disease onset in hSOD1(G93A) /CAST mice compared to littermate hSOD1(G93A) mice is delayed, which accounts for their longer time of survival. We also find that neuronal over-expression of CAST in hSOD1(G93A) transgenic mice inhibited production of putative neurotoxic caspase-cleaved tau and activation of Cdk5, which have been implicated in neurodegeneration in ALS models, and also reduced the formation of SOD1 oligomers. Our data indicate that inhibition of calpain with CAST is neuroprotective in an ALS mouse model. CAST (encoding calpastatin) inhibits hyperactivated calpain to prevent motor neuron disease operating through a cascade of events as indicated in the schematic, with relevance to amyotrophic lateral sclerosis (ALS). We propose that over-expression of CAST in motor neurons of hSOD1(G93A) mice inhibits activation of CDK5, breakdown of cytoskeletal proteins (NFs, MAP2 and Tau) and regulatory molecules (Cam Kinase IV, Calcineurin A), and disease-causing proteins (TDP-43, α-Synuclein and Huntingtin) to prevent neuronal loss and delay neurological deficits. In our experiments, CAST could also inhibit cleavage of Bid, Bax, AIF to prevent mitochondrial, ER and lysosome-mediated cell death mechanisms. Similarly

  13. Calpastatin overexpression prevents progression of S-1,2-dichlorovinyl-L-cysteine (DCVC)-initiated acute renal injury and renal failure (ARF) in diabetes

    International Nuclear Information System (INIS)

    Previously we have shown that 90% of streptozotocin (STZ)-induced type-1 diabetic (DB) mice survive from acute renal failure (ARF) and death induced by a normally LD9 dose (75 mg/kg, i.p.) of the nephrotoxicant S-1,2-dichlorovinyl-L-cysteine (DCVC). This remarkable protection is due to a combination of slower progression of DCVC-initiated renal injury and increased compensatory nephrogenic tissue repair in the DB kidneys. BRDU immunohistochemistry revealed that the DB condition led to 4-fold higher number of proximal tubular cells (PTC) entering S-phase of cell cycle. In the present study, we tested the hypothesis that DB-induced augmentation of PTC into S-phase is accompanied by overexpression of the calpain-inhibitor calpastatin, which endogenously prevents the progression of DCVC-initiated renal injury mediated by the calpain escaping out of damaged PTCs. Immunohistochemical detection of renal calpain and its activity in the urine, over a time course after treatment with the LD9 dose of DCVC, indicated progressive increase in leakage of calpain into the extracellular spaces of the injured PTCs of the non-diabetic (NDB) kidneys as compared to the DB kidneys. Calpastatin expression was minimally detected in the NDB kidneys, using immunohistochemistry, over the time course. On the other hand, consistently higher number of tubules in the DB kidney showed calpastatin expression over the time course. The lower leakage of calpain in the DB kidneys was commensurate with constitutively higher expression of calpastatin in the S-phase-laden PTCs of these mice. To test the protective role of newly divided/dividing PTCs, DB mice were given the anti-mitotic agent colchicine (CLC) (2 mg/kg and 1.5 mg/kg, i.p., on days 8 and 10 after STZ injection) prior to challenge with a LD9 dose of DCVC, which led to 100% mortality by 48 h. Mortality was due to rapid progression of DCVC-initiated renal injury, suggesting that newly divided/dividing cells are instrumental in mitigating the

  14. Our energy-Ca2+ signaling deficits hypothesis and its explanatory potential for key features of Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    Ming eChen

    2014-12-01

    Full Text Available Alzheimer’s disease (AD has not been explained by any current theories, so new hypotheses are urgently needed. We proposed that energy and Ca2+ signaling deficits are perhaps the earliest modifiable defects in brain aging underlying memory decline and tau deposits (by means of inactivating Ca2+-dependent protease calpain. Consistent with this hypothesis, we now notice that at least eight other known calpain substrates have also been reported to accumulate in aging and AD. Thus, protein accumulation or aggregation is not an accidental or random event, but occurs naturally and selectively to a peculiar family of proteins, corroborating the proposed changes of calpain. Why are only calpain substrates accumulated and how can they stay for decades in the brain without being attacked by many other non-specific proteases there? We believe that these long-lasting puzzles can be explained by calpain’s unique properties, especially its unusual specificity and exclusivity in substrate recognition, which can protect the substrates from other proteases’ attacks after calpain inactivation. Interestingly, the energy-Ca2+ deficits model, in essence, may also explain tau phosphorylation (by calcineurin inactivation and the formation of amyloid plaques. Our studies suggest that α-secretase is an energy-/Ca2+-dual dependent protease and is also the primary determinant for Aβ levels. Finally we discuss why β- and γ-secretases, the current enthusiastic study focuses, are unlikely to be responsible for Aβ genesis or be positively identified by biological laws. Overall, the study suggests that our hypothesis can coherently explain several basic AD features, thus pointing to a new strategy for AD prevention.

  15. Effects of cerebrolysin on motor-neuron-like NSC-34 cells

    International Nuclear Information System (INIS)

    Although the peripheral nervous system is capable of regeneration, this capability is limited. As a potential means of augmenting nerve regeneration, the effects of cerebrolysin (CL) – a proteolytic peptide fraction – were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypic spinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changing media and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferation using MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated. Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypic cultures, the effect of CL on motor neuron survival and neurite sprouting was tested by immunohistochemistry. CL had a temporary anti-proliferative but initially neuroprotective effect on OGD-stressed NSC-34 cells. High-dosed or repeatedly applied CL was deleterious for cell survival. CL amplified neurite reconstruction to limited extent, affected calpain-1 protein expression and influenced calpain-mediated spectrin cleavage as a function of Src expression. In organotypic spinal cord slice cultures, CL was not able to support motor neuron survival/neurite sprouting. Moreover, it hampered astroglia and microglia activities. The data suggest that CL may have only isolated positive effects on injured spinal motor neurons. High-dosed or accumulated CL seemed to have adverse effects in treatment of spinal cord injury. Further experiments are required to optimize the conditions for a safe clinical administration of CL in spinal cord injuries. - Highlights: • Cerebrolysin (CL) is anti-proliferative but initially neuroprotective in OGD-stressed NSC-34 cells. • CL amplified neurite reconstruction of NSC-34 cells. • CL affected calpain-1 expression and calpain-mediated spectrin cleavage as function of Src expression. • In organotypic spinal cord cultures, CL hampered motor neuron survival and

  16. Rescuing axons from degeneration does not affect retinal ganglion cell death

    Directory of Open Access Journals (Sweden)

    S. de Lima

    2016-01-01

    Full Text Available After a traumatic injury to the central nervous system, the distal stumps of axons undergo Wallerian degeneration (WD, an event that comprises cytoskeleton and myelin breakdown, astrocytic gliosis, and overexpression of proteins that inhibit axonal regrowth. By contrast, injured neuronal cell bodies show features characteristic of attempts to initiate the regenerative process of elongating their axons. The main molecular event that leads to WD is an increase in the intracellular calcium concentration, which activates calpains, calcium-dependent proteases that degrade cytoskeleton proteins. The aim of our study was to investigate whether preventing axonal degeneration would impact the survival of retinal ganglion cells (RGCs after crushing the optic nerve. We observed that male Wistar rats (weighing 200-400 g; n=18 treated with an exogenous calpain inhibitor (20 mM administered via direct application of the inhibitor embedded within the copolymer resin Evlax immediately following optic nerve crush showed a delay in the onset of WD. This delayed onset was characterized by a decrease in the number of degenerated fibers (P<0.05 and an increase in the number of preserved fibers (P<0.05 4 days after injury. Additionally, most preserved fibers showed a normal G-ratio. These results indicated that calpain inhibition prevented the degeneration of optic nerve fibers, rescuing axons from the process of axonal degeneration. However, analysis of retinal ganglion cell survival demonstrated no difference between the calpain inhibitor- and vehicle-treated groups, suggesting that although the calpain inhibitor prevented axonal degeneration, it had no effect on RGC survival after optic nerve damage.

  17. Release of full-length PrPC from cultured neurons following neurotoxic challenges

    Directory of Open Access Journals (Sweden)

    Kevin K W Wang

    2012-10-01

    Full Text Available The susceptibility of the normal cellular prion protein isoform, PrPC, to proteolytic digestion has been well documented. In addition, a link between PrPC and the cytosolic protease, calpain, has been reported although the specifics of the interaction remain unclear. We performed in vitro and in cell-based studies to examine this relationship. We observed that human recombinant PrP (HrPrP was readily cleaved by calpain-1 and -2, and we have identified and defined the targeted cleavage sites. In contrast, HrPrP was resistant to caspase-3 digestion. Unexpectedly, when brain lysates from PrPC-expressing mice were treated with calpain, no appreciable loss of the intact PrPC, nor the appearance of PrPC breakdown products (BDPs were observed, even though alpha II-spectrin was converted to its signature calpain-induced BDPs. In addition, when rat cerebrocortical neuronal cultures (RtCNC were subjected to the two neurotoxins at subacute levels, maitotoxin (MTX and N-methyl-D-aspartate (NMDA, PrPC BDPs were also not detectable. However, a novel finding from these cell-based studies is that apparently full-length, mature PrPC is released into culture media from RtCNC challenged with subacute doses of MTX and NMDA. Calpain inhibitor SNJ-1945 and caspase inhibitor IDN-6556 did not attenuate the release of PrPC. Similarly, the lysosomal protease inhibitor, NH4Cl, and the proteasome inhibitor, lactacystin, did not significantly alter the integrity of PrPC or its release from the RtCNC. In conclusion, rat neuronal PrPC is not a significant target for proteolytic modifications during MTX and NMDA neurotoxic challenges. However, the robust neurotoxin-mediated release of full-length PrPC into the cell culture media suggests an unidentified neuroprotective mechanism for PrPC.

  18. Effects of cerebrolysin on motor-neuron-like NSC-34 cells

    Energy Technology Data Exchange (ETDEWEB)

    Keilhoff, Gerburg, E-mail: Gerburg.keilhoff@med.ovgu.de [Institute of Biochemistry and Cell Biology, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg (Germany); Lucas, Benjamin; Pinkernelle, Josephine; Steiner, Michael [Institute of Biochemistry and Cell Biology, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg (Germany); Fansa, Hisham [Department of Plastic, Reconstructive and Aesthetic Surgery, Hand Surgery, Klinikum Bielefeld, Teutoburger Str. 50, D-33604 Bielefeld (Germany)

    2014-10-01

    Although the peripheral nervous system is capable of regeneration, this capability is limited. As a potential means of augmenting nerve regeneration, the effects of cerebrolysin (CL) – a proteolytic peptide fraction – were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypic spinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changing media and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferation using MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated. Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypic cultures, the effect of CL on motor neuron survival and neurite sprouting was tested by immunohistochemistry. CL had a temporary anti-proliferative but initially neuroprotective effect on OGD-stressed NSC-34 cells. High-dosed or repeatedly applied CL was deleterious for cell survival. CL amplified neurite reconstruction to limited extent, affected calpain-1 protein expression and influenced calpain-mediated spectrin cleavage as a function of Src expression. In organotypic spinal cord slice cultures, CL was not able to support motor neuron survival/neurite sprouting. Moreover, it hampered astroglia and microglia activities. The data suggest that CL may have only isolated positive effects on injured spinal motor neurons. High-dosed or accumulated CL seemed to have adverse effects in treatment of spinal cord injury. Further experiments are required to optimize the conditions for a safe clinical administration of CL in spinal cord injuries. - Highlights: • Cerebrolysin (CL) is anti-proliferative but initially neuroprotective in OGD-stressed NSC-34 cells. • CL amplified neurite reconstruction of NSC-34 cells. • CL affected calpain-1 expression and calpain-mediated spectrin cleavage as function of Src expression. • In organotypic spinal cord cultures, CL hampered motor neuron survival and

  19. Neuroprotective effects of a novel single compound 1-methoxyoctadecan-1-ol isolated from Uncaria sinensis in primary cortical neurons and a photothrombotic ischemia model.

    Directory of Open Access Journals (Sweden)

    Ji Yeon Jang

    Full Text Available We identified a novel neuroprotective compound, 1-methoxyoctadecan-1-ol, from Uncaria sinensis (Oliv. Havil and investigated its effects and mechanisms in primary cortical neurons and in a photothrombotic ischemic model. In primary rat cortical neurons against glutamate-induced neurotoxicity, pretreatment with 1-methoxyoctadecan-1-ol resulted in significantly reduced neuronal death in a dose-dependent manner. In addition, treatment with 1-methoxyoctadecan-1-ol resulted in decreased neuronal apoptotic death, as assessed by nuclear morphological approaches. To clarify the neuroprotective mechanism of 1-methoxyoctadecan-1-ol, we explored the downstream signaling pathways of N-methyl-D-aspartate receptor (NMDAR with calpain activation. Treatment with glutamate leads to early activation of NMDAR, which in turn leads to calpain-mediated cleavage of striatal-enriched protein tyrosine phosphatase (STEP and subsequent activation of p38 mitogen activated protein kinase (MAPK. However, pretreatment with 1-methoxyoctadecan-1-ol resulted in significantly attenuated activation of GluN2B-NMDAR and a decrease in calpain-mediated STEP cleavage, leading to subsequent attenuation of p38 MAPK activation. We confirmed the critical role of p38 MAPK in neuroprotective effects of 1-methoxyoctadecan-1-ol using specific inhibitor SB203580. In the photothrombotic ischemic injury in mice, treatment with 1-methoxyoctadecan-1-ol resulted in significantly reduced infarct volume, edema size, and improved neurological function. 1-methoxyoctadecan-1-ol effectively prevents cerebral ischemic damage through down-regulation of calpain-mediated STEP cleavage and activation of p38 MAPK. These results suggest that 1-methoxyoctadecan-1-ol showed neuroprotective effects through down-regulation of calpain-mediated STEP cleavage with activation of GluN2B-NMDAR, and subsequent alleviation of p38 MAPK activation. In addition, 1-methoxyoctadecan-1-ol might be a useful therapeutic agent for

  20. Neuroprotective effects of a novel single compound 1-methoxyoctadecan-1-ol isolated from Uncaria sinensis in primary cortical neurons and a photothrombotic ischemia model.

    Science.gov (United States)

    Jang, Ji Yeon; Choi, Young Whan; Kim, Ha Neui; Kim, Yu Ri; Hong, Jin Woo; Bae, Dong Won; Park, Se Jin; Shin, Hwa Kyoung; Choi, Byung Tae

    2014-01-01

    We identified a novel neuroprotective compound, 1-methoxyoctadecan-1-ol, from Uncaria sinensis (Oliv.) Havil and investigated its effects and mechanisms in primary cortical neurons and in a photothrombotic ischemic model. In primary rat cortical neurons against glutamate-induced neurotoxicity, pretreatment with 1-methoxyoctadecan-1-ol resulted in significantly reduced neuronal death in a dose-dependent manner. In addition, treatment with 1-methoxyoctadecan-1-ol resulted in decreased neuronal apoptotic death, as assessed by nuclear morphological approaches. To clarify the neuroprotective mechanism of 1-methoxyoctadecan-1-ol, we explored the downstream signaling pathways of N-methyl-D-aspartate receptor (NMDAR) with calpain activation. Treatment with glutamate leads to early activation of NMDAR, which in turn leads to calpain-mediated cleavage of striatal-enriched protein tyrosine phosphatase (STEP) and subsequent activation of p38 mitogen activated protein kinase (MAPK). However, pretreatment with 1-methoxyoctadecan-1-ol resulted in significantly attenuated activation of GluN2B-NMDAR and a decrease in calpain-mediated STEP cleavage, leading to subsequent attenuation of p38 MAPK activation. We confirmed the critical role of p38 MAPK in neuroprotective effects of 1-methoxyoctadecan-1-ol using specific inhibitor SB203580. In the photothrombotic ischemic injury in mice, treatment with 1-methoxyoctadecan-1-ol resulted in significantly reduced infarct volume, edema size, and improved neurological function. 1-methoxyoctadecan-1-ol effectively prevents cerebral ischemic damage through down-regulation of calpain-mediated STEP cleavage and activation of p38 MAPK. These results suggest that 1-methoxyoctadecan-1-ol showed neuroprotective effects through down-regulation of calpain-mediated STEP cleavage with activation of GluN2B-NMDAR, and subsequent alleviation of p38 MAPK activation. In addition, 1-methoxyoctadecan-1-ol might be a useful therapeutic agent for brain disorder

  1. Amiloride attenuates lipopolysaccharide-accelerated atherosclerosis via inhibition of NHE1-dependent endothelial cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Gui-mei CUI; Yu-xi ZHAO; Na-na ZHANG; Zeng-shan LIU; Wan-chun SUN; Qi-sheng PENG

    2013-01-01

    Aim: To investigate the effects of the potassium-sparing diuretic amiloride on endothelial cell apoptosis during lipopolysaccharide (LPS)-accelerated atherosclerosis.Methods: Human umbilical vein endothelial cells (HUVECs) were exposed to LPS (100 ng/mL) in the presence of drugs tested.The activity of Na+/H+ exchanger 1 (NHE1) and calpain,intracellular free Ca2+ level ([Ca2+]i),as well as the expression of apoptosis-related proteins in the cells were measured.For in vivo study,ApoE-deficient (ApoE-/-) mice were fed high-fat diets with 0.5% (w/w) amiloride for 4 weeks and LPS (10 μg/mouse) infusion into caudal veins.Afterwards,atherosclerotic lesions,NHE1 activity and Bcl-2 expression in the aortic tissues were evaluated.Results: LPS treatment increased NHE1 activity and [Ca2+]i in HUVECs in a time-dependent manner,which was associated with increased activity of the Ca2+-dependent protease calpain.Amiloride (1-10 μmol/L) significantly suppressed LPS-induced increases in NHE1 activity,[Ca2+]i.and calpain activity.In the presence of the Ca2+ chelator BAPTA (0.5 mmol/L),LPS-induced increase of calpain activity was also abolished.In LPS-treated HUVECs,the expression of Bcl-2 protein was significantly decreased without altering its mRNA level.In the presence of amiloride (10 μmol/L) or the calpain inhibitor ZLLal (50 μmol/L),the down-regulation of Bcl-2 protein by LPS was blocked.LPS treatment did not alter the expression of Bax and Bak proteins in HUVECs.In the presence of amiloride,BAPTA or ZLLal,LPS-induced HUVEC apoptosis was significantly attenuated.In ApoE-/-mice,administration of amiloride significantly suppressed LPS-accelerated atherosclerosis and LPS-induced increase of NHE1 activity,and reversed LPS-induced down-regulation of Bcl-2 expression.Conclusion: LPS stimulates NHE1 activity,increases [Ca2+]i,and activates calpain,which leads to endothelial cell apoptosis related to decreased Bcl-2 expression.Amiloride inhibits NHE1 activity,thus attenuates LPS

  2. A heterozygous 21-bp deletion in CAPN3 causes dominantly inherited limb girdle muscular dystrophy.

    Science.gov (United States)

    Vissing, John; Barresi, Rita; Witting, Nanna; Van Ghelue, Marijke; Gammelgaard, Lise; Bindoff, Laurence A; Straub, Volker; Lochmüller, Hanns; Hudson, Judith; Wahl, Christoph M; Arnardottir, Snjolaug; Dahlbom, Kathe; Jonsrud, Christoffer; Duno, Morten

    2016-08-01

    Limb girdle muscular dystrophy type 2A is the most common limb girdle muscular dystrophy form worldwide. Although strict recessive inheritance is assumed, patients carrying a single mutation in the calpain 3 gene (CAPN3) are reported. Such findings are commonly attributed to incomplete mutation screening. In this investigation, we report 37 individuals (age range: 21-85 years, 21 females and 16 males) from 10 families in whom only one mutation in CAPN3 could be identified; a 21-bp, in-frame deletion (c.643_663del21). This mutation co-segregated with evidence of muscle disease and autosomal dominant transmission in several generations. Evidence of muscle disease was indicated by muscle pain, muscle weakness and wasting, significant fat replacement of muscles on imaging, myopathic changes on muscle biopsy and loss of calpain 3 protein on western blotting. Thirty-one of 34 patients had elevated creatine kinase or myoglobin. Muscle weakness was generally milder than observed in limb girdle muscular dystrophy type 2A, but affected the same muscle groups (proximal leg, lumbar paraspinal and medial gastrocnemius muscles). In some cases, the weakness was severely disabling. The 21-bp deletion did not affect mRNA maturation. Calpain 3 expression in muscle, assessed by western blot, was below 15% of normal levels in the nine mutation carriers in whom this could be tested. Haplotype analysis in four families from three different countries suggests that the 21-bp deletion is a founder mutation. This study provides strong evidence that heterozygosity for the c.643_663del21 deletion in CAPN3 results in a dominantly inherited muscle disease. The normal expression of mutated mRNA and the severe loss of calpain 3 on western blotting, suggest a dominant negative effect with a loss-of-function mechanism affecting the calpain 3 homodimer. This renders patients deficient in calpain 3 as in limb girdle muscular dystrophy type 2A, albeit in a milder form in most cases. Based on findings

  3. The Hypertrophic Marchigiana: physical and biochemical parameters for meat quality evaluation

    Directory of Open Access Journals (Sweden)

    F. M. Sarti

    2010-04-01

    Full Text Available The aim of this study was to evaluate the meat quality of double muscled Marchigiana young bulls characterized by different genotypes for the hypertrophy: normal and mutated (heterozygous. Calpain and calpastatin activities were determined to verify the state of aging meat on a sample of Longissimus thoracis muscle (XIII thoracic rib taken at slaughtering (0h and after 24 hours (24h. After 14 days of aging, another sample of muscle was taken to evaluate physical and chemical parameters of meat quality. The results showed a better meat quality of mutated animals respect normal animals. Another interesting result was the correlation between the biochemical parameters and some physical parameters, such as WBS (Warner Bratzler Shear Force, CL (Cooking loss. These results showed the relationship between the proteolytic activity of calpain system and meat tenderness.

  4. Cannabinoid treatment renders neurons less vulnerable than oligodendrocytes in experimental autoimmune encephalomyelitis

    DEFF Research Database (Denmark)

    Hasseldam, Henrik; Johansen, Flemming Fryd

    2011-01-01

    effects. EAE was induced using MOG(1-125) in Dark Agouti rats and treatment was initiated at symptom debut and continued until first relapse culminated. The central nervous system (CNS) cell death including caspase and calpain activation, axonal degeneration and demyelination as well as a wide range......ABSTRACT Using the rat model Experimental Autoimmune Encephalomyelitis (EAE), we have investigated the cytokinetical and cellular events of axonal degeneration and demyelination following treatment with 5 mg/kg/24h R(+)WIN55,212-2 or 10 mg/kg/24h R(+)WIN55,212-2, which have immunosuppressive...... of immunological parameters were quantified. We found a significant reduction in axonal degeneration associated with reduced calpain 1 following treatment with 5 mg/kg/24h R(+)WIN55,212-2. Treatment with 10 mg/kg/24h resulted furthermore in an improved clinical performance and a reduction in inflammatory activity...

  5. Methylanthraquinone from Hedyotis diffusa WILLD induces Ca(2+)-mediated apoptosis in human breast cancer cells.

    Science.gov (United States)

    Liu, Zheng; Liu, Ming; Liu, Miao; Li, Jianchun

    2010-02-01

    Methylanthraquinone from Hedyotis diffusa WILLD exhibited potent anticancer activity in many kinds of cancer cells. However, the exact mechanism and signaling pathway involved in methylanthraquinone-induced apoptosis have not been fully elucidated. Therefore, we explored the mechanisms of methylanthraquinone-mediated apoptosis in MCF-7 human breast cancer cells. When MCF-7 cells were co-incubated with methylanthraquinone, the percentage of apoptotic cell and S phase of cell cycle was markedly increased. In addition, a rise in intracellular calcium levels, phosphorylation of JNK and activation of calpain were found in MCF-7 cells after exposure to methylanthraquinone. With the methylanthraquinone-mediated reduction of mitochondrial membrane potential, cytochrome c was released from mitochondria to cytosol. Moreover, methylanthraquinone strongly induced cleavage of caspase-4, caspase-9 and caspase-7 in MCF-7 cells. These results suggested that methylanthraquinone from Hedyotis diffusa WILLD induced MCF-7 cells apoptosis via Ca(2+)/calpain/caspase-4 pathway. PMID:19686834

  6. Matrix Metalloproteinases are Modifiers of Huntingtin Proteolysis and Toxicity in Huntington’s Disease

    OpenAIRE

    Miller, John P.; Holcomb, Jennifer; Al-Ramahi, Ismael; de Haro, Maria; Gafni, Juliette; Zhang, Ningzhe; Kim, Eugene; Sanhueza, Mario; Torcassi, Cameron; Kwak, Seung; Botas, Juan; Hughes, Robert E.; Ellerby, Lisa M.

    2010-01-01

    Proteolytic cleavage of huntingtin (Htt) is known to be a key event in the pathogenesis of Huntington’s disease (HD). Our understanding of proteolytic processing of Htt has thus far focused on the cysteine protease families of caspases and calpains. Identifying critical protease families involved in Htt proteolysis and toxicity using an unbiased approach has not been reported. To accomplish this, we designed a high-throughput western blot-based screen to examine the generation of the smallest...

  7. Diospyrin derivative, an anticancer quinonoid, regulates apoptosis at endoplasmic reticulum as well as mitochondria by modulating cytosolic calcium in human breast carcinoma cells

    International Nuclear Information System (INIS)

    Highlights: ► Diospyrin diethylether (D7) caused oxidative stress-dependent activation of PC-PLC. ► Activated PC-PLC induced a sustained-release of Ca2+ from endoplasmic reticulum. ► The elevated cytosolic Ca+2 led to the calpain-caspase12 dependent apoptosis. ► D7-Induced Ca+2 also found to accentuate the mitochondrial pathway of apoptosis. -- Abstract: Diospyrin diethylether (D7), a bisnaphthoquinonoid derivative, exhibited an oxidative stress-dependent apoptosis in several human cancer cells and tumor models. The present study was aimed at evaluation of the increase in cytosolic calcium [Ca2+]c leading to the apoptotic cell death triggered by D7 in MCF7 human breast carcinoma cells. A phosphotidylcholine-specific phospholipase C (PC-PLC) inhibitor, viz. U73122, and an antioxidant, viz. N-acetylcysteine, could significantly prevent the D7-induced rise in [Ca2+]c and PC-PLC activity. Using an endoplasmic reticulum (ER)-Ca2+ mobilizer (thapsigargin) and an ER-IP3R antagonist (heparin), results revealed ER as a major source of [Ca2+]c which led to the activation of calpain and caspase12, and cleavage of fodrin. These effects including apoptosis were significantly inhibited by the pretreatment of Bapta-AM (a cell permeable Ca2+-specific chelator), or calpeptin (a calpain inhibitor). Furthermore, D7-induced [Ca2+]c was found to alter mitochondrial membrane potential and induce cytochrome c release, which was inhibited by either Bapta-AM or ruthenium red (an inhibitor of mitochondrial Ca2+ uniporter). Thus, these results provided a deeper insight into the D7-induced redox signaling which eventually integrated the calcium-dependent calpain/caspase12 activation and mitochondrial alterations to accentuate the induction of apoptotic cell death.

  8. Delivering minocycline into brain endothelial cells with liposome-based technology

    OpenAIRE

    Xing, Changhong; Levchenko, Tatyana; Guo, Shuzhen; Stins, Monique; Torchilin, Vladimir P.; Eng H Lo

    2012-01-01

    Minocycline has been proposed as a way to blunt neurovascular injury from matrix metalloproteinases (MMPs) during stroke. However, recent clinical trials suggest that high levels of minocycline may have deleterious side-effects. Here, we showed that very high minocycline concentrations damage endothelial cells via calpain/caspase pathways. To alleviate this potential cytotoxicity, we encapsulated minocycline in liposomes. Low concentrations of minocycline could not reduce tumor necrosis facto...

  9. A single nucleotide polymorphism in CAPN1 associated with marbling score in Korean cattle

    OpenAIRE

    Kim Ji; Han Chang; Lee Hae; Namgoong Sohg; Bae Joon; Kim Lyoung; Park Byung; Yoon Du-Hak; Cheong Hyun; Cheong Il-Cheong; Shin Hyoung

    2008-01-01

    Abstract Background Marbling score (MS) is the major quantitative trait that affects carcass quality in beef cattle. In this study, we examined the association between genetic polymorphisms of the micromolar calcium-activated neutral protease gene (micro-calpain, CAPN1) and carcass traits in Korean cattle (also known as Hanwoo). Results By direct DNA sequencing in 24 unrelated Korean cattle, we identified 39 sequence variants within exons and their flanking regions in CAPN1. Among them, 12 co...

  10. Gene expression profiling in limb-girdle muscular dystrophy 2A.

    Directory of Open Access Journals (Sweden)

    Amets Sáenz

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

  11. Genetic polymorphisms related to meat traits in purebred and crossbred Nelore cattle Polimorfismos genéticos relacionados às características da carne em bovinos Nelore puros e cruzados

    OpenAIRE

    Rogério Abdallah Curi; Marina Rufino Salinas Fortes; Luis Artur Loyola Chardulo; Antonio Carlos Silveira; Mário de Beni Arrigoni; Cyntia Ludovico Martins; Mayra Elena Ortiz D' Avila Assumpção; Henrique Nunes de Oliveira

    2009-01-01

    The objective of this work was to estimate the allelic and genotypic frequencies of CAST/XmnI, a calpastatin gene polymorphism, and CAPN530, a calpain 1 large subunit gene polymorphism, in different beef genetic groups (Nelore and Nelore x Bos taurus), and to investigate associations between these polymorphisms and carcass and meat traits. Three hundred animals - comprising 114 Nelore, 67 Angus x Nelore, 44 Rubia Gallega x Nelore, 41 Canchim, 19 Brangus three-way cross and 15 Braunvieh three-...

  12. Defects in the CAPN1 Gene Result in Alterations in Cerebellar Development and Cerebellar Ataxia in Mice and Humans

    Directory of Open Access Journals (Sweden)

    Yubin Wang

    2016-06-01

    Full Text Available A CAPN1 missense mutation in Parson Russell Terrier dogs is associated with spinocerebellar ataxia. We now report that homozygous or heterozygous CAPN1-null mutations in humans result in cerebellar ataxia and limb spasticity in four independent pedigrees. Calpain-1 knockout (KO mice also exhibit a mild form of ataxia due to abnormal cerebellar development, including enhanced neuronal apoptosis, decreased number of cerebellar granule cells, and altered synaptic transmission. Enhanced apoptosis is due to absence of calpain-1-mediated cleavage of PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1, which results in inhibition of the Akt pro-survival pathway in developing granule cells. Injection of neonatal mice with the indirect Akt activator, bisperoxovanadium, or crossing calpain-1 KO mice with PHLPP1 KO mice prevented increased postnatal cerebellar granule cell apoptosis and restored granule cell density and motor coordination in adult mice. Thus, mutations in CAPN1 are an additional cause of ataxia in mammals, including humans.

  13. Defects in the CAPN1 gene result in alterations in cerebellar development and in cerebellar ataxia in mice and humans

    Science.gov (United States)

    Wang, Yubin; Hersheson, Joshua; Lopez, Dulce; Hamad, Monia Ben; Liu, Yan; Lee, Ka-Hung; Pinto, Vanessa; Seinfeld, Jeff; Wiethoff, Sarah; Sun, Jiandong; Amouri, Rim; Hentati, Faycal; Baudry, Neema; Tran, Jennifer; Singleton, Andrew B; Coutelier, Marie; Brice, Alexis; Stevanin, Giovanni; Durr, Alexandra; Bi, Xiaoning; Houlden, Henry; Baudry, Michel

    2016-01-01

    SUMMARY A CAPN1 missense mutation in Parson Russell Terrier dogs is associated with spinocerebellar ataxia. We now report that homozygous CAPN1 null mutations in humans result in cerebellar ataxia and limb spasticity in four independent pedigrees. Calpain-1 knock-out (KO) mice also exhibit a mild form of ataxia due to abnormal cerebellar development, including enhanced neuronal apoptosis, decreased number of cerebellar granule cells, and altered synaptic transmission. Enhanced apoptosis is due to absence of calpain-1 mediated cleavage of PH domain and Leucine rich repeat Protein Phosphatase 1 (PHLPP1), which results in inhibition of the Akt pro-survival pathway in developing granule cells. Injection of neonatal mice with the indirect Akt activator, bisperoxovanadium, or crossing calpain-1 KO mice with PHLPP1 KO mice prevented increased postnatal cerebellar granule cell apoptosis, and restored granule cell density and motor coordination in adult mice. Thus, mutations in CAPN1 are an additional cause of ataxia in mammals, including humans. PMID:27320912

  14. Postmortem degradation of skeletal muscle proteins: a novel approach to determine the time since death.

    Science.gov (United States)

    Pittner, Stefan; Monticelli, Fabio C; Pfisterer, Alexander; Zissler, Angela; Sänger, Alexandra M; Stoiber, Walter; Steinbacher, Peter

    2016-03-01

    Estimating the time since death is a very important aspect in forensic sciences which is pursued by a variety of methods. The most precise method to determine the postmortem interval (PMI) is the temperature method which is based on the decrease of the body core temperature from 37 °C. However, this method is only useful in the early postmortem phase (~0-36 h). The aim of the present work is to develop an accurate method for PMI determination beyond this present limit. For this purpose, we used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and casein zymography to analyze the time course of degradation of selected proteins and calpain activity in porcine biceps femoris muscle until 240 h postmortem (hpm). Our results demonstrate that titin, nebulin, desmin, cardiac troponin T, and SERCA1 degraded in a regular and predictable fashion in all samples investigated. Similarly, both the native calpain 1 and calpain 2 bands disintegrate into two bands subsequently. This degradation behavior identifies muscular proteins and enzymes as promising substrates for future molecular-based PMI determination technologies. PMID:26041514

  15. Dysfunction of the β2-spectrin-based pathway in human heart failure.

    Science.gov (United States)

    Smith, Sakima A; Hughes, Langston D; Kline, Crystal F; Kempton, Amber N; Dorn, Lisa E; Curran, Jerry; Makara, Michael; Webb, Tyler R; Wright, Patrick; Voigt, Niels; Binkley, Philip F; Janssen, Paul M L; Kilic, Ahmet; Carnes, Cynthia A; Dobrev, Dobromir; Rasband, Matthew N; Hund, Thomas J; Mohler, Peter J

    2016-06-01

    β2-Spectrin is critical for integrating membrane and cytoskeletal domains in excitable and nonexcitable cells. The role of β2-spectrin for vertebrate function is illustrated by dysfunction of β2-spectrin-based pathways in disease. Recently, defects in β2-spectrin association with protein partner ankyrin-B were identified in congenital forms of human arrhythmia. However, the role of β2-spectrin in common forms of acquired heart failure and arrhythmia is unknown. We report that β2-spectrin protein levels are significantly altered in human cardiovascular disease as well as in large and small animal cardiovascular disease models. Specifically, β2-spectrin levels were decreased in atrial samples of patients with atrial fibrillation compared with tissue from patients in sinus rhythm. Furthermore, compared with left ventricular samples from nonfailing hearts, β2-spectrin levels were significantly decreased in left ventricle of ischemic- and nonischemic heart failure patients. Left ventricle samples of canine and murine heart failure models confirm reduced β2-spectrin protein levels. Mechanistically, we identify that β2-spectrin levels are tightly regulated by posttranslational mechanisms, namely Ca(2+)- and calpain-dependent proteases. Furthermore, consistent with this data, we observed Ca(2+)- and calpain-dependent loss of β2-spectrin downstream effector proteins, including ankyrin-B in heart. In summary, our findings illustrate that β2-spectrin and downstream molecules are regulated in multiple forms of cardiovascular disease via Ca(2+)- and calpain-dependent proteolysis. PMID:27106045

  16. Myoglobin inhibition of most protease activities measured with fluorescent substrates is an artifact!

    Science.gov (United States)

    Volle, B; Dutaud, D; Ouali, A

    1999-05-01

    Myoglobin has been suggested to be a potential inhibitor of endogenous muscle proteases as different as cathepsin B, cathepsin L, cathepsin H and calpains all being supposed to be important in post-mortem muscle. The present work aimed at verifying the ability of myoglobin and its prosthetic group, hemin, to inhibit a series of endopeptidases including papain, cathepsin B, trypsin, calpains as well as two activities of the 20S proteasome. The conclusion of the present work was that inhibition of proteolytic activities of endopeptidases by myoglobin is an artifact. This was based on the following evidences: (1) a similar extent of inhibition was observed for all proteases tested whether myoglobin or hemin were added before starting the reaction or after having stopped it; (2) a quenching of the probes fluorescence by myoglobin and hemin; (3) no inhibition of calpains were found when assayed with non labeled casein as substrate and the activity expressed as the increase in the absorbency at 280 nm of the TCA soluble protein fragments.(1). PMID:22062146

  17. Activation of Cyclin-Dependent Kinase 5 Is a Consequence of Cell Death

    Directory of Open Access Journals (Sweden)

    Yixia Ye

    2009-01-01

    Full Text Available Cyclin-dependent kinase 5 (Cdk5 is similar to other Cdks but is activated during cell differentiation and cell death rather than cell division. Since activation of Cdk5 has been reported in many situations leading to cell death, we attempted to determine if it was required for any form of cell death. We found that Cdk5 is activated during apoptotic deaths and that the activation can be detected even when the cells continue to secondary necrosis. This activation can occur in the absence of Bim, calpain, or neutral cathepsins. The kinase is typically activated by p25, derived from p35 by calpain-mediated cleavage, but inhibition of calpain does not affect cell death or the activation of Cdk5. Likewise, RNAi-forced suppression of the synthesis of Cdk5 does not affect the incidence or kinetics of cell death. We conclude that Cdk5 is activated as a consequence of metabolic changes that are common to many forms of cell death. Thus its activation suggests processes during cell death that will be interesting or important to understand, but activation of Cdk5 is not necessary for cells to die.

  18. A single fraction from Uncaria sinensis exerts neuroprotective effects against glutamate-induced neurotoxicity in primary cultured cortical neurons.

    Science.gov (United States)

    Kim, Ha Neui; Jang, Ji Yeon; Choi, Byung Tae

    2015-06-01

    We identified a neuroprotective single fraction among 62 ones of hexane extract from Uncaria sinensis (JGH43IA) and investigated its effects and mechanisms in primary cortical neurons. Pretreatment with JGH43IA showed a significantly increase cell viability in a dose-dependent manner with a decrease in the lactate dehydrogenase release. When we performed morphological assay and flow cytometry to determination of the type of cell death, pretreatment with JGH43IA showed a significant reduction of glutamate-induced apoptotic cell death. Then we explored the downstream signaling pathways of N-methyl-D-aspartate receptor (NMDAR) with calpain activation to elucidate possible pathways of neuroprotection by JGH43IA. Pretreatment with JGH43IA exhibited a significant attenuation of NMDAR GluN2B subunit activation and a decrease in active form of calpain 1 leading to subsequent cleavage of striatal-enriched protein tyrosine phosphatase (STEP). In addition, pretreatment with JGH43IA showed a marked increase of cAMP responsive element binding protein. These results suggest that JGH43IA may have neuroprotective effects through down-regulation of NMDAR GluN2B subunit and calpain 1 activation, and subsequent alleviation of STEP cleavage. This single fraction from U. sinensis might be a useful therapeutic agent for brain disorder associated with glutamate injury. PMID:26140220

  19. Kihi-to, a herbal traditional medicine, improves Abeta(25–35-induced memory impairment and losses of neurites and synapses

    Directory of Open Access Journals (Sweden)

    Joyashiki Eri

    2008-08-01

    Full Text Available Abstract Background We previously hypothesized that achievement of recovery of brain function after the injury requires the reconstruction of neuronal networks, including neurite regeneration and synapse reformation. Kihi-to is composed of twelve crude drugs, some of which have already been shown to possess neurite extension properties in our previous studies. The effect of Kihi-to on memory deficit has not been examined. Thus, the goal of the present study is to determine the in vivo and in vitro effects of Kihi-to on memory, neurite growth and synapse reconstruction. Methods Effects of Kihi-to, a traditional Japanese-Chinese traditional medicine, on memory deficits and losses of neurites and synapses were examined using Alzheimer's disease model mice. Improvements of Aβ(25–35-induced neuritic atrophy by Kihi-to and the mechanism were investigated in cultured cortical neurons. Results Administration of Kihi-to for consecutive 3 days resulted in marked improvements of Aβ(25–35-induced impairments in memory acquisition, memory retention, and object recognition memory in mice. Immunohistochemical comparisons suggested that Kihi-to attenuated neuritic, synaptic and myelin losses in the cerebral cortex, hippocampus and striatum. Kihi-to also attenuated the calpain increase in the cerebral cortex and hippocampus. When Kihi-to was added to cells 4 days after Aβ(25–35 treatment, axonal and dendritic outgrowths in cultured cortical neurons were restored as demonstrated by extended lengths of phosphorylated neurofilament-H (P-NF-H and microtubule-associated protein (MAP2-positive neurites. Aβ(25–35-induced cell death in cortical culture was also markedly inhibited by Kihi-to. Since NF-H, MAP2 and myelin basic protein (MBP are substrates of calpain, and calpain is known to be involved in Aβ-induced axonal atrophy, expression levels of calpain and calpastatin were measured. Treatment with Kihi-to inhibited the Aβ(25–35-evoked increase in

  20. Limb-girdle muscular dystrophy: an immunohistochemical diagnostic approach Distrofias musculares de cinturas: uma abordagem diagnóstica imuno-histoquímica

    Directory of Open Access Journals (Sweden)

    Enio Alberto Comerlato

    2005-06-01

    Full Text Available The limb-girdle muscle dystrophy (LGMD represents a heterogeneous group of muscular diseases with dominant and recessive inheritance, individualized by gene mutation. A group of 56 patients, 32 males and 24 females, with suggestive LGMD diagnosis were submitted to clinical evaluation, serum muscle enzymes, electromyography, muscle biopsy, and the immunoidentification (ID of sarcoglycans (SG alpha, beta, gamma and delta, dysferlin and western blot for calpain-3. All the patients had normal ID for dystrophin (rod domain, carboxyl and amine terminal. The alpha-SG was normal in 42 patients, beta-SG in 28, beta-SG in 45, delta-SG in 32, dysferlin in 37 and calpain-3 in 9. There was a reduction in the alpha-SG in 7 patients, beta-SG in 4, gamma-SG in 2, and delta-SG in 8. There was deficiency of alpha-SG in 7 patients, beta-SG in 6, gamma-SG in 9, delta-SG in 5, dysferlin in 8, and calpain-3 in 5. The patients were grouped according the ID as sarcoglycans deficiency 18 cases, dysferlin deficiency 8 cases and calpain-3 deficiency 5 cases. Only the sarcoglycans deficiency group showed calf hypertrophy. The dysferlin deficiency group was more frequent in females and the onset was later than sarcoglycan and calpain-3 deficiency groups. The calpain-3 deficiency group occurred only in males and showed an earlier onset and weaker muscular strength.As distrofias musculares de cinturas (DMC representam grupo heterogêneo de doenças musculares com heranças autossômicas dominante ou recessivas, caracterizadas geneticamente por mutações gênicas específicas. Cinqüenta e seis pacientes, 32 masculinos e 24 femininos, com diagnóstico sugestivo de DMC, foram submetidos a avaliação clínica, dosagem séricas das enzimas musculares, eletromiografia, biópsia muscular e imunoidentificação (ID das proteínas sarcoglicanas (SG alfa, beta, gama e delta, disferlina e calpaína-3. A ID da distrofina (domínio rod e terminais carboxila e amino era normal em todos

  1. Effects of cerebrolysin on motor-neuron-like NSC-34 cells.

    Science.gov (United States)

    Keilhoff, Gerburg; Lucas, Benjamin; Pinkernelle, Josephine; Steiner, Michael; Fansa, Hisham

    2014-10-01

    Although the peripheral nervous system is capable of regeneration, this capability is limited. As a potential means of augmenting nerve regeneration, the effects of cerebrolysin (CL)--a proteolytic peptide fraction--were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypic spinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changing media and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferation using MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated. Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypic cultures, the effect of CL on motor neuron survival and neurite sprouting was tested by immunohistochemistry. CL had a temporary anti-proliferative but initially neuroprotective effect on OGD-stressed NSC-34 cells. High-dosed or repeatedly applied CL was deleterious for cell survival. CL amplified neurite reconstruction to limited extent, affected calpain-1 protein expression and influenced calpain-mediated spectrin cleavage as a function of Src expression. In organotypic spinal cord slice cultures, CL was not able to support motor neuron survival/neurite sprouting. Moreover, it hampered astroglia and microglia activities. The data suggest that CL may have only isolated positive effects on injured spinal motor neurons. High-dosed or accumulated CL seemed to have adverse effects in treatment of spinal cord injury. Further experiments are required to optimize the conditions for a safe clinical administration of CL in spinal cord injuries. PMID:24997385

  2. Detoxification and protein quality control markers in the mussel Mytilus edulis (Linnaeus) exposed to crude oil: Salinity-induced modulation

    Science.gov (United States)

    Lysenko, Liudmila; Sukhovskaya, Irina; Borvinskaya, Ekaterina; Krupnova, Marina; Kantserova, Nadezda; Bakhmet, Igor'; Nemova, Nina

    2015-12-01

    Marine and coastal ecosystems are influenced by oil from chronic contamination or sporadic oil spills. An oil spill was simulated in an aquarium-based experiment designed to reproduce interactions of crude oil with inert environmental components, particularly adhesion on shore gravel and dissolution in sea water. Total experimental oil concentrations were in the range of comparable hydrocarbon concentrations following an oil spill. Furthermore, the possible interaction of a chemical (anthropogenic) stressor, such as oil PAHs, and a "natural" stressor like desalination, was simulated. In order to assess the biological effects of crude oil contamination and desalination (each individually and in combination) on the blue mussel Mytilus edulis L., biochemical responses were estimated including: detoxification capacity by glutathione-S-transferase (GST) activity, reduced glutathione (GSH) level, and protein quality control by autophagy-related proteases cathepsin B (CatB), cathepsin D (CatD), and calcium-dependent calpain-like proteases. Oil treatment stimulated defense system response in the mussels with primary effects on GST and protease-mediated reactions such as the activation of CatB, CatD, and calpains. Most of biomarkers responded to oil in a dose- and time-dependent manner. Additional environmental stress, such as desalination, promoted the oil-induced activation of GST and CatD while resulting in a delay or impairement of the defense response to oil by GSH and proteases CatB and calpains. Thus, biomarker data shows that combined effects of oil compounds and desalination can be realized in both a synergistic and an antagonistic manner. The evaluated interaction between oil pollution effects and sub-optimal salinity on M. edulis indicates the potential risk of maladaptation to the biota of estuaries.

  3. Retinoids and glucocorticoids have opposite effects on actin cytoskeleton rearrangement in hippocampal HT22 cells.

    Science.gov (United States)

    Hélène, Roumes; Julie, Brossaud; Aloïs, Lemelletier; Marie-Pierre, Moisan; Véronique, Pallet; Anabelle, Redonnet; Jean-Benoît, Corcuff

    2016-02-01

    A chronic excess of glucocorticoids elicits deleterious effects in the hippocampus. Conversely, retinoic acid plays a major role in aging brain plasticity. As synaptic plasticity depends on mechanisms related to cell morphology, we investigated the involvement of retinoic acid and glucocorticoids in the remodelling of the HT22 neurons actin cytoskeleton. Cells exhibited a significantly more elongated shape with retinoic acid and a rounder shape with dexamethasone; retinoic acid reversed the effects of dexamethasone. Actin expression and abundance were unchanged by retinoic acid or dexamethasone but F-actin organization was dramatically modified. Indeed, retinoic acid and dexamethasone increased (70 ± 7% and 176 ± 5%) cortical actin while retinoic acid suppressed the effect of dexamethasone (90 ± 6%). Retinoic acid decreased (-22 ± 9%) and dexamethasone increased (134 ± 16%) actin stress fibres. Retinoic acid also suppressed the effect of dexamethasone (-21 ± 7%). Spectrin is a key protein in the actin network remodelling. Its abundance was decreased by retinoic acid and increased by dexamethasone (-21 ± 11% and 52 ± 10%). However, retinoic acid did not modify the effect of dexamethasone (48 ± 7%). Calpain activity on spectrin was increased by retinoic acid and decreased by dexamethasone (26 ± 14% and -57 ± 5%); retinoic acid mildly but significantly modified the effect of dexamethasone (-44 ± 7%). The calpain inhibitor calpeptin suppressed the effects of retinoic acid and dexamethasone on cell shape and actin stress fibres remodelling but did not modify the effects on cortical actin. Retinoic acid and dexamethasone have a dramatic but mainly opposite effect on actin cytoskeleton remodelling. These effects originate, at least partly, from calpain activity. PMID:26748244

  4. 大鼠角膜穿通伤后局部应用 IL-10抑制视网膜炎症和抗神经溃变的研究%Topical application of IL-10 inhibits inflammation and reduces neural degeneration of the retina after corneal penetrating injury in rats

    Institute of Scientific and Technical Information of China (English)

    黄燕; 周月鹏; 肖寿华; 张志坚

    2014-01-01

    目的:观察角膜穿通伤后局部应用 IL-10对视网膜炎症反应的抑制和对神经溃变的保护作用。方法:将清洁级成年雌性 SD 大鼠50只,随机分为对照组(8只)、损伤组和治疗组,后两组(每组21只)再分为损伤1 d、2 d 和3 d 组(每小组7只)。损伤组用无菌注射器刺穿眼球颞侧角膜缘角膜,制作角膜穿通伤模型。治疗组在角膜穿通伤后用 IL-10溶液滴眼。各组2只大鼠用于制作眼球切片,免疫荧光染色观察角膜穿通伤后炎症因子 IL-1β、IL-6和 TNF-α以及神经丝蛋白-200(neurofilament protein-200,NF-200)、血影蛋白(α-Ⅱspectrin)及钙蛋白酶2(m-calpain)在视网膜中的分布特征;另5只大鼠用于提取视网膜蛋白,采用免疫印迹法检测各组大鼠于角膜穿通伤24,48,72 h 后,视网膜组织内 NF-200和血影蛋白降解产物以及活性钙蛋白酶2相对含量的动态变化。结果:对照组视网膜结构层次清楚, IL-1β、IL-6、TNF-α和 m-calpain 免疫荧光很弱;血影蛋白和 NF-200免疫荧光染色可清晰显示阳性神经纤维;角膜穿通伤后,视网膜结构层次模糊,TNF-α、IL-1β,IL-6和 m-calpain 免疫荧光增强,主要分布在内层;NF-200和血影蛋白免疫荧光染色显示阳性神经纤维明显减少;IL-10治疗后,TNF-α、IL-1β,IL-6和 m-calpain 免疫荧光减弱,血影蛋白和 NF-200阳性神经纤维明显多于损伤组。免疫印迹检测结果表明,损伤组视网膜组织内大分子的 NF-200和血影蛋白含量逐渐减少,其小分子的降解产物以及小分子的活性 m-calpain 逐渐增加;在 IL-10治疗组,NF-200和血影蛋白的降解产物以及活性 m-calpain 的增加程度明显减轻。结论:角膜穿通伤可刺激视网膜细胞高表达炎症因子 IL-1β,IL-6和TNF-α,导致组织的炎症损伤,同时激活 m-calpain,后者降解视网膜细胞骨架蛋白 NF

  5. Calcium-activated tenderization of strip loin, top sirloin, and top round steaks in diverse genotypes of cattle.

    Science.gov (United States)

    Pringle, T D; Harrelson, J M; West, R L; Williams, S E; Johnson, D D

    1999-12-01

    Steers of known percentage Brahman (B) and Angus (A) breeding (100% A, n = 6; F1 B x A, n = 6; and 100% B, n = 6) were used to determine the effect of calcium chloride injection on the calpain proteinase system and meat tenderness. The steers were slaughtered in six replications (at either 9 or 14 mm of backfat, determined ultrasonically), with each breed type represented. Calpains and calpastatin activities were measured on fresh, prerigor longissimus muscle samples. Carcass data were collected after a 24-h chill, and the short loin (IMPS #180), top sirloin (IMPS #184), and top round (IMPS #168) were removed from both sides of each carcass. The cuts from the right side were then injected at 5% (wt/wt) with CaCl2 solution (2.2%). Longissimus muscle calpain and calpastatin activities were also measured at 48 h postmortem from the injected and control sides of each carcass. Warner-Bratzler shear force was measured on steaks from the three subprimals aged 1, 2, 5, 15, or 31 d. Marbling scores and USDA quality grades were higher (Pcarcasses. Calpastatin activity was higher (P.05) across breed type. Calcium injection improved strip loin and top sirloin steak tenderness, but it did not affect top round steak tenderness. Collectively, these data show that CaC12 injection can be used to improve meat tenderness, with similar responses shown in cattle containing 0, 50, and 100% B inheritance. However, even with CaCl2 injection, B steaks are less tender than their A and F1 B x A counterparts.

  6. Role of platelet plasma membrane Ca2+-ATPase in health and disease

    Institute of Scientific and Technical Information of China (English)

    William; L; Dean

    2010-01-01

    Platelets have essential roles in both health and disease. Normal platelet function is required for hemostasis.Inhibition of platelet function in disease or by pharmacological treatment results in bleeding disorders.On the other hand,hyperactive platelets lead to heart attack and stroke.Calcium is a major second messenger in platelet activation,and elevated intracellular calcium leads to hyperactive platelets.Elevated platelet calcium has been documented in hypertension and diabetes;both conditions increase the likelihood of heart attack and stroke. Thus,proper regulation of calcium metabolism in the platelet is extremely important.Plasma membrane Ca2+-ATPase(PMCA)is a major player in platelet calcium metabolism since it provides the only significant route for calcium efflux.In keeping with the important role of calcium in platelet function,PMCA is a highly regulated transporter.In human platelets,PMCA is activated by Ca2+/calmodulin,by cAMP-dependent phosphorylation and by calpain-dependent removal of the inhibitory peptide.It is inhibited by tyrosine phosphorylation and calpain-dependent proteolysis.In addition,the cellular location of PMCA is regulated by a PDZ-domain-dependent interaction with the cytoskeleton during platelet activation.Rapid regulation by phosphorylation results in changes in the rate of platelet activation,whereas calpain-dependent proteolysis and interaction with the cytoskeleton appears to regulate later events such as clot retraction.In hypertension and diabetes,PMCA expression is upregulated while activity is decreased, presumably due to tyrosine phosphorylation.Clearly,a more complete understanding of PMCA function in human platelets could result in the identification of new ways to control platelet function in disease states.

  7. Orai1 mediates exacerbated Ca(2+ entry in dystrophic skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Xiaoli Zhao

    Full Text Available There is substantial evidence indicating that disruption of Ca(2+ homeostasis and activation of cytosolic proteases play a key role in the pathogenesis and progression of Duchenne Muscular Dystrophy (DMD. However, the exact nature of the Ca(2+ deregulation and the Ca(2+ signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca(2+ entry (SOCE for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca(2+ storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca(2+ storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca(2+ homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca(2+ store contributes to the disrupted Ca(2+ homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of

  8. Autophagic signaling and proteolytic enzyme activity in cardiac and skeletal muscle of spontaneously hypertensive rats following chronic aerobic exercise.

    Directory of Open Access Journals (Sweden)

    Elliott M McMillan

    Full Text Available Hypertension is a cardiovascular disease associated with deleterious effects in skeletal and cardiac muscle. Autophagy is a degradative process essential to muscle health. Acute exercise can alter autophagic signaling. Therefore, we aimed to characterize the effects of chronic endurance exercise on autophagy in skeletal and cardiac muscle of normotensive and hypertensive rats. Male Wistar Kyoto (WKY and spontaneously hypertensive rats (SHR were assigned to a sedentary condition or 6 weeks of treadmill running. White gastrocnemius (WG of hypertensive rats had higher (p<0.05 caspase-3 and proteasome activity, as well as elevated calpain activity. In addition, skeletal muscle of hypertensive animals had elevated (p<0.05 ATG7 and LC3I protein, LAMP2 mRNA, and cathepsin activity, indicative of enhanced autophagic signaling. Interestingly, chronic exercise training increased (p<0.05 Beclin-1, LC3, and p62 mRNA as well as proteasome activity, but reduced (p<0.05 Beclin-1 and ATG7 protein, as well as decreased (p<0.05 caspase-3, calpain, and cathepsin activity. Left ventricle (LV of hypertensive rats had reduced (p<0.05 AMPKα and LC3II protein, as well as elevated (p<0.05 p-AKT, p-p70S6K, LC3I and p62 protein, which collectively suggest reduced autophagic signaling. Exercise training had little effect on autophagy-related signaling factors in LV; however, exercise training increased (p<0.05 proteasome activity but reduced (p<0.05 caspase-3 and calpain activity. Our results suggest that autophagic signaling is altered in skeletal and cardiac muscle of hypertensive animals. Regular aerobic exercise can effectively alter the proteolytic environment in both cardiac and skeletal muscle, as well as influence several autophagy-related factors in skeletal muscle of normotensive and hypertensive rats.

  9. Calcium-activated tenderization of strip loin, top sirloin, and top round steaks in diverse genotypes of cattle.

    Science.gov (United States)

    Pringle, T D; Harrelson, J M; West, R L; Williams, S E; Johnson, D D

    1999-12-01

    Steers of known percentage Brahman (B) and Angus (A) breeding (100% A, n = 6; F1 B x A, n = 6; and 100% B, n = 6) were used to determine the effect of calcium chloride injection on the calpain proteinase system and meat tenderness. The steers were slaughtered in six replications (at either 9 or 14 mm of backfat, determined ultrasonically), with each breed type represented. Calpains and calpastatin activities were measured on fresh, prerigor longissimus muscle samples. Carcass data were collected after a 24-h chill, and the short loin (IMPS #180), top sirloin (IMPS #184), and top round (IMPS #168) were removed from both sides of each carcass. The cuts from the right side were then injected at 5% (wt/wt) with CaCl2 solution (2.2%). Longissimus muscle calpain and calpastatin activities were also measured at 48 h postmortem from the injected and control sides of each carcass. Warner-Bratzler shear force was measured on steaks from the three subprimals aged 1, 2, 5, 15, or 31 d. Marbling scores and USDA quality grades were higher (P.05) across breed type. Calcium injection improved strip loin and top sirloin steak tenderness, but it did not affect top round steak tenderness. Collectively, these data show that CaC12 injection can be used to improve meat tenderness, with similar responses shown in cattle containing 0, 50, and 100% B inheritance. However, even with CaCl2 injection, B steaks are less tender than their A and F1 B x A counterparts.

  10. Retinoids and glucocorticoids have opposite effects on actin cytoskeleton rearrangement in hippocampal HT22 cells.

    Science.gov (United States)

    Hélène, Roumes; Julie, Brossaud; Aloïs, Lemelletier; Marie-Pierre, Moisan; Véronique, Pallet; Anabelle, Redonnet; Jean-Benoît, Corcuff

    2016-02-01

    A chronic excess of glucocorticoids elicits deleterious effects in the hippocampus. Conversely, retinoic acid plays a major role in aging brain plasticity. As synaptic plasticity depends on mechanisms related to cell morphology, we investigated the involvement of retinoic acid and glucocorticoids in the remodelling of the HT22 neurons actin cytoskeleton. Cells exhibited a significantly more elongated shape with retinoic acid and a rounder shape with dexamethasone; retinoic acid reversed the effects of dexamethasone. Actin expression and abundance were unchanged by retinoic acid or dexamethasone but F-actin organization was dramatically modified. Indeed, retinoic acid and dexamethasone increased (70 ± 7% and 176 ± 5%) cortical actin while retinoic acid suppressed the effect of dexamethasone (90 ± 6%). Retinoic acid decreased (-22 ± 9%) and dexamethasone increased (134 ± 16%) actin stress fibres. Retinoic acid also suppressed the effect of dexamethasone (-21 ± 7%). Spectrin is a key protein in the actin network remodelling. Its abundance was decreased by retinoic acid and increased by dexamethasone (-21 ± 11% and 52 ± 10%). However, retinoic acid did not modify the effect of dexamethasone (48 ± 7%). Calpain activity on spectrin was increased by retinoic acid and decreased by dexamethasone (26 ± 14% and -57 ± 5%); retinoic acid mildly but significantly modified the effect of dexamethasone (-44 ± 7%). The calpain inhibitor calpeptin suppressed the effects of retinoic acid and dexamethasone on cell shape and actin stress fibres remodelling but did not modify the effects on cortical actin. Retinoic acid and dexamethasone have a dramatic but mainly opposite effect on actin cytoskeleton remodelling. These effects originate, at least partly, from calpain activity.

  11. Targeting microparticle biogenesis: a novel approach to the circumvention of cancer multidrug resistance.

    Science.gov (United States)

    Roseblade, Ariane; Luk, Frederick; Ung, Alison; Bebawy, Mary

    2015-01-01

    Microparticles (MPs) are released from most eukaryotic cells after the vesiculation of the plasma membrane and serve as vectors of long and short-range signaling. MPs derived from multidrug resistant (MDR) cancer cells carry molecular components of the donor cell such as nucleic acids and proteins, and can alter the activity of drug-sensitive recipient cells through the transfer of their cargo. Given the substantial role of MPs in the acquisition and dissemination of MDR, we propose that the inhibition of MP release provides a novel therapeutic approach. This study characterises the effect of a panel of molecules known to act on MP-biosynthetic pathways. We demonstrate a differential effect by these molecules on MP inhibition that appear dependent on the release of intracellular calcium stores following activation with the calcium ionophore A23187. Calpain inhibitor, PD-150606; a selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK), Y-27632; and the vitamin B5 derivative pantethine, inhibited MP release only upon prior activation with A23187. Calpain inhibitor II showed significant inhibition in the absence of cell activation, whereas the vitamin B5 derivatives cystamine dihydrochloride and cysteamine hydrochloride showed no effect on MP inhibition under either condition. In contrast the classical pharmacological inhibitor of MDR, the calcium channel blocker Verapamil, showed an increase in MP formation on resting cells. These results suggest a potential role for calcium in the mechanism of action for PD-150606, Y-27632 and pantethine. These molecules, together with calpain inhibitor II have shown promise as modulators of MP release and warrant consideration as potential candidates for the development of an alternative therapeutic strategy for the prevention of MP-mediated MDR in cancer. PMID:25714701

  12. Effects of cerebrolysin on motor-neuron-like NSC-34 cells.

    Science.gov (United States)

    Keilhoff, Gerburg; Lucas, Benjamin; Pinkernelle, Josephine; Steiner, Michael; Fansa, Hisham

    2014-10-01

    Although the peripheral nervous system is capable of regeneration, this capability is limited. As a potential means of augmenting nerve regeneration, the effects of cerebrolysin (CL)--a proteolytic peptide fraction--were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypic spinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changing media and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferation using MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated. Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypic cultures, the effect of CL on motor neuron survival and neurite sprouting was tested by immunohistochemistry. CL had a temporary anti-proliferative but initially neuroprotective effect on OGD-stressed NSC-34 cells. High-dosed or repeatedly applied CL was deleterious for cell survival. CL amplified neurite reconstruction to limited extent, affected calpain-1 protein expression and influenced calpain-mediated spectrin cleavage as a function of Src expression. In organotypic spinal cord slice cultures, CL was not able to support motor neuron survival/neurite sprouting. Moreover, it hampered astroglia and microglia activities. The data suggest that CL may have only isolated positive effects on injured spinal motor neurons. High-dosed or accumulated CL seemed to have adverse effects in treatment of spinal cord injury. Further experiments are required to optimize the conditions for a safe clinical administration of CL in spinal cord injuries.

  13. Involvement of Different networks in mammary gland involution after the pregnancy/lactation cycle: Implications in breast cancer.

    Science.gov (United States)

    Zaragozá, Rosa; García-Trevijano, Elena R; Lluch, Ana; Ribas, Gloria; Viña, Juan R

    2015-04-01

    Early pregnancy is associated with a reduction in a woman's lifetime risk for breast cancer. However, different studies have demonstrated an increase in breast cancer risk in the years immediately following pregnancy. Early and long-term risk is even higher if the mother age is above 35 years at the time of first parity. The proinflammatory microenvironment within the mammary gland after pregnancy renders an "ideal niche" for oncogenic events. Signaling pathways involved in programmed cell death and tissue remodeling during involution are also activated in breast cancer. Herein, the major signaling pathways involved in mammary gland involution, signal transducer and activator of transcription (STAT3), nuclear factor-kappa B (NF-κB), transforming growth factor beta (TGFβ), and retinoid acid receptors (RARs)/retinoid X receptors (RXRs), are reviewed as part of the complex network of signaling pathways that crosstalk in a contextual-dependent manner. These factors, also involved in breast cancer development, are important regulatory nodes for signaling amplification after weaning. Indeed, during involution, p65/p300 target genes such as MMP9, Capn1, and Capn2 are upregulated. Elevated expression and activities of these proteases in breast cancer have been extensively documented. The role of these proteases during mammary gland involution is further discussed. MMPs, calpains, and cathepsins exert their effect by modification of the extracellular matrix and intracellular proteins. Calpains, activated in the mammary gland during involution, cleave several proteins located in cell membrane, lysosomes, mitochondria, and nuclei favoring cell death. Besides, during this period, Capn1 is most probably involved in the modulation of preadipocyte differentiation through chromatin remodeling. Calpains can be implicated in cell anchoring loss, providing a proper microenvironment for tumor growth. A better understanding of the role of any of these proteases in tumorigenesis may

  14. Diospyrin derivative, an anticancer quinonoid, regulates apoptosis at endoplasmic reticulum as well as mitochondria by modulating cytosolic calcium in human breast carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Binod [Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032 (India); Radiation and Cancer Biology Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Kumar, Amit [Radiation and Cancer Biology Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Ghosh, Subhalakshmi [Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032 (India); Pandey, Badri N., E-mail: bnp@barc.gov.in [Radiation and Cancer Biology Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Mishra, Kaushala P. [Radiation and Cancer Biology Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Hazra, Banasri, E-mail: banasrihazra@yahoo.co.in [Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032 (India)

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Diospyrin diethylether (D7) caused oxidative stress-dependent activation of PC-PLC. Black-Right-Pointing-Pointer Activated PC-PLC induced a sustained-release of Ca{sup 2+} from endoplasmic reticulum. Black-Right-Pointing-Pointer The elevated cytosolic Ca{sup +2} led to the calpain-caspase12 dependent apoptosis. Black-Right-Pointing-Pointer D7-Induced Ca{sup +2} also found to accentuate the mitochondrial pathway of apoptosis. -- Abstract: Diospyrin diethylether (D7), a bisnaphthoquinonoid derivative, exhibited an oxidative stress-dependent apoptosis in several human cancer cells and tumor models. The present study was aimed at evaluation of the increase in cytosolic calcium [Ca{sup 2+}]{sub c} leading to the apoptotic cell death triggered by D7 in MCF7 human breast carcinoma cells. A phosphotidylcholine-specific phospholipase C (PC-PLC) inhibitor, viz. U73122, and an antioxidant, viz. N-acetylcysteine, could significantly prevent the D7-induced rise in [Ca{sup 2+}]{sub c} and PC-PLC activity. Using an endoplasmic reticulum (ER)-Ca{sup 2+} mobilizer (thapsigargin) and an ER-IP3R antagonist (heparin), results revealed ER as a major source of [Ca{sup 2+}]{sub c} which led to the activation of calpain and caspase12, and cleavage of fodrin. These effects including apoptosis were significantly inhibited by the pretreatment of Bapta-AM (a cell permeable Ca{sup 2+}-specific chelator), or calpeptin (a calpain inhibitor). Furthermore, D7-induced [Ca{sup 2+}]{sub c} was found to alter mitochondrial membrane potential and induce cytochrome c release, which was inhibited by either Bapta-AM or ruthenium red (an inhibitor of mitochondrial Ca{sup 2+} uniporter). Thus, these results provided a deeper insight into the D7-induced redox signaling which eventually integrated the calcium-dependent calpain/caspase12 activation and mitochondrial alterations to accentuate the induction of apoptotic cell death.

  15. Apoptotic Versus Angiogenic Factors in Gastric and Colorectal Cancers

    Directory of Open Access Journals (Sweden)

    Enas A Hamed

    2012-04-01

    Conclusions. Gastric-colon malignancy patients exhibited decreased apoptosis, as evident by an increase in antiapoptotic indices, i.e. sFas and bcl-2, and increased angiogenic activity, as evident by enhanced proteolytic activity of cathepsin-D and calpain I and II. These parameters were higher in gastric than colorectal cancers reflecting aggressive behavior of the earlier. Thus, decreased apoptosis and enhanced angiogenesis give growth priority in gastric-colon cancers, and the angiogenic factors and #8217; blockage may delay the tumor and #8217;s spread. [Arch Clin Exp Surg 2012; 1(2.000: 71-84

  16. Candidate gene effects on beef quality

    OpenAIRE

    Ekerljung, Marie

    2012-01-01

    The contribution of five candidate genes to the variation in meat tenderness, pH, colour, marbling and water holding capacity (WHC) was analysed in muscle samples from 243 young bulls of Angus, Charolais, Hereford, Limousin, or Simmental breed, raised in Swedish commercial herds. The animals were genotyped for single nucleotide polymorphisms (SNPs) in the genes encoding calpain 1 (CAPN1:c.947G>C), calpastatin, (CAST:c.155C>T), diacylglycerol O-acyltransferase 1 (DGAT1), leptin (UASMS2C>T) a...

  17. Retinoic Acid Induces Apoptosis of Prostate Cancer DU145 Cells through Cdk5 Overactivation

    OpenAIRE

    Mei-Chih Chen; Chih-Yang Huang; Shih-Lan Hsu; Eugene Lin; Chien-Te Ku; Ho Lin; Chuan-Mu Chen

    2012-01-01

    Retinoic acid (RA) has been believed to be an anticancer drug for a long history. However, the molecular mechanisms of RA actions on cancer cells remain diverse. In this study, the dose-dependent inhibition of RA on DU145 cell proliferation was identified. Interestingly, RA treatment triggered p35 cleavage (p25 formation) and Cdk5 overactivation, and all could be blocked by Calpain inhibitor, Calpeptin (CP). Subsequently, RA-triggered DU145 apoptosis detected by sub-G1 phase accumulation and ...

  18. Type 2 diabetes mellitus: phylogenetic motifs for predicting protein functional sites

    Indian Academy of Sciences (India)

    Ashok Sharma; Tanuja Rastogi; Meenakshi Bhartiya; A K Shasany; S P S Khanuja

    2007-08-01

    Diabetes mellitus, commonly referred to as diabetes, is a medical condition associated with abnormally high levels of glucose (or sugar) in the blood. Keeping this view, we demonstrate the phylogenetic motifs (PMs) identification in type 2 diabetes mellitus very likely corresponding to protein functional sites. In this article, we have identified PMs for all the candidate genes for type 2 diabetes mellitus. Glycine 310 remains conserved for glucokinase and potassium channel KCNJ11. Isoleucine 137 was conserved for insulin receptor and regulatory subunit of a phosphorylating enzyme. Whereas residues valine, leucine, methionine were highly conserved for insulin receptor. Occurrence of proline was very high for calpain 10 gene and glucose transporter

  19. Studies on changes of atrial function and its mechanism in a cannie atrial fibrillation model%心房颤动犬心房功能改变特点及机制的研究

    Institute of Scientific and Technical Information of China (English)

    马骁; 张薇; 杨贵荣; 钟明; 黎莉; 张运

    2007-01-01

    目的 应用声学定量技术评价心房颤动(房颤)犬左心房功能改变,探讨钙激活蛋白酶系统与左房功能重构的关系.方法 17只杂种犬随机分为房颤组(11只)和对照组(6只),通过心房快速起搏8周建立犬房颤模型,于起搏前后应用声学定量技术记录左心房容量-时间曲线,并计算相关指标.采用Western blot技术检测左心房肌钙依赖性中性蛋白酶(calpain)及其抑制剂钙蛋白酶抑制蛋白(calpastatin)的蛋白表达量.结果 与对照组比较,房颤组左房储存器容积无明显改变;管道容积显著增加(P<0.01);左房射血分数、峰值心房排空率(PAER)显著降低(P <0.01);左房心室收缩末期容积、快速排空末期容积、心室舒张末期容积、心房排空容积增加(P<0.01).房颤组calpain Ⅰ、calpain Ⅱ蛋白表达明显高于对照组(P<0.05),calpastatin蛋白表达明显低于对照组(P <0.05).calpain Ⅰ蛋白表达水平与左房PAER、左房排空分数、左室射血分数呈显著负相关(P<0.05);calpastatin蛋白表达水平与左心房PAER、左房排空分数呈显著正相关(P <0.05).结论 房颤时心房发生功能重构,表现为助力泵功能减低,管道功能增强,calpain系统激活可能是房颤时心房功能重构的重要分子机制.

  20. cDNA analyses of CAPN3 enhance mutation detection and reveal a low prevalence of LGMD2A patients in Denmark

    DEFF Research Database (Denmark)

    Duno, M.; Sveen, M.L.; Schwartz, M.;

    2008-01-01

    Calpainopathy or limb-girdle muscular dystrophy type 2A (LGMD2A) is generally recognized as the most prevalent form of recessive LGMD and is caused by mutations in the CAPN3 gene. Out of a cohort of 119 patients fulfilling clinical criteria for LGMD2, referred to our neuromuscular clinic, 46 were......, only a single heterozygous mutation could be identified both at the genomic level and on full-length CAPN3 cDNA. All three patients exhibited a highly abnormal western blot for calpain-3 and clinical characteristics of LGMD2A. Only three of the genetically confirmed LGMD2A patients were of Danish...

  1. The association of CAPN1 316 marker genotypes with growth and meat quality traits of steers finished on pasture

    OpenAIRE

    María C. Miquel; Edgardo Villarreal; Carlos Mezzadra; Lilia Melucci; Liliana Soria; Pablo Corva; Alejandro Schor

    2009-01-01

    The objective of this paper was to determine the association of a SNP in the μ-calpain gene at position 316 with growth and quality of meat traits of steers grown on pasture. Fifty-nine Brangus and 20 Angus steers were genotyped for CAPN1 316. Warner Bratzler shear force was measured in l. lumborum samples after a 7-day aging period. A multivariate analysis of variance was performed, including shear force (WBSF), final weight (FW), average daily gain (ADG), backfat thickness (BFT), average mo...

  2. Synthesis and extended activity of triazole-containing macrocyclic protease inhibitors

    DEFF Research Database (Denmark)

    Pehere, A.D.; Pietsch, M.; Gütschow, M.;

    2013-01-01

    Peptide-derived protease inhibitors are an important class of compounds with the potential to treat a wide range of diseases. Herein, we describe the synthesis of a series of triazole- containing macrocyclic protease inhibitors pre-organized into a b-strand conformation and an evaluation...... of their activity against a panel of proteases. Acyclic azidoalkyne-based aldehydes are also evaluated for comparison. The macrocyclic peptidomimetics showed considerable activity towards calpain II, cathepsin L and S, and the 20S proteasome chymotrypsin-like activity. Some of the first examples of highly potent...

  3. Deleting both PHLPP1 and CANP1 rescues impairments in long-term potentiation and learning in both single knockout mice.

    Science.gov (United States)

    Liu, Yan; Sun, Jiandong; Wang, Yubin; Lopez, Dulce; Tran, Jennifer; Bi, Xiaoning; Baudry, Michel

    2016-08-01

    Calpain-1 (CANP1) has been shown to play a critical role in synaptic plasticity and learning and memory, as its deletion in mice results in impairment in theta-burst stimulation- (TBS) induced LTP and various forms of learning and memory. Likewise, PHLPP1 (aka SCOP) has also been found to participate in learning and memory, as PHLPP1 overexpression impairs hippocampus-dependent learning. We previously showed that TBS-induced LTP was associated with calpain-1 mediated truncation of PHLPP1.To better understand the roles of these 2 genes in synaptic plasticity and learning and memory, we generated a double knockout (DKO) mouse by crossing the parent strains. Surprisingly, DKO mice exhibit normal TBS-induced LTP, and the learning impairments in fear conditioning and novel object or novel location recognition were absent in the DKO mice. Moreover, TBS-induced ERK activation in field CA1 of hippocampal slices, which is impaired in both single deletion mice, was restored in the DKO mice. These results further strengthen the roles of both CANP1 and PHLPP1 in synaptic plasticity and learning and memory, and illustrate the complexities of the interactions between multiple pathways participating in synaptic plasticity. PMID:27421891

  4. Suppression of myofibrillar proteolysis in chick skeletal muscles by alpha-ketoisocaproate.

    Science.gov (United States)

    Nakashima, K; Yakabe, Y; Ishida, A; Yamazaki, M; Abe, H

    2007-09-01

    We previously reported that L-leucine suppresses myofibrillar proteolysis in chick skeletal muscles. In the current study, we compared the effects of L- and D-enantiomers of leucine on myofibrillar proteolysis in skeletal muscle of chicks. We also assessed whether leucine itself or its metabolite, alpha-ketoisocaproate (alpha-KIC), mediates the effects of leucine. Food-deprived (24 h) chicks were orally administered 225 mg/100 g body weight L-leucine, D-leucine or alpha-KIC and were sacrificed after 2 h. L-Leucine administration had an obvious inhibitory effect on myofibrillar proteolysis (plasma N(tau)-methylhistidine concentration) in chicks while D-leucine and alpha-KIC were much more effective. We also examined the expression of the proteolytic-related genes (ubiquitin, proteasome, m-calpain and cathepsin B) by real-time PCR of cDNA in chick skeletal muscles. Ubiquitin mRNA expression was decreased by D-leucine and alpha-KIC but not L-leucine. Proteasome and m-calpain mRNA expressions as well as cathepsin B mRNA expression were likewise decreased by L-leucine, D-leucine and alpha-KIC. These results indicate that D-leucine and alpha-KIC suppress proteolytic-related genes, resulting in an decrease in myofibrillar proteolysis while L-leucine is much less effective in skeletal muscle of chicks, may be explain by conversion of D-leucine to alpha-KIC. PMID:16998714

  5. Selective inhibition of caspases in skeletal muscle reverses the apoptotic synaptic degeneration in slow-channel myasthenic syndrome.

    Science.gov (United States)

    Zhu, Haipeng; Pytel, Peter; Gomez, Christopher M

    2014-01-01

    Slow-channel syndrome (SCS) is a congenital myasthenic disorder caused by point mutations in subunits of skeletal muscle acetylcholine receptor leading to Ca(2+) overload and degeneration of the postsynaptic membrane, nuclei and mitochondria of the neuromuscular junction (NMJ). In both SCS muscle biopsies and transgenic mouse models for SCS (mSCS), the endplate regions are shrunken, and there is evidence of DNA damage in the subsynaptic region. Activated caspase-9, -3 and -7 are intensely co-localized at the NMJ, and the Ca(2+)-activated protease, calpain, and the atypical cyclin-dependent kinase (Cdk5) are overactivated in mSCS. Thus, the true mediator(s) of the disease process is not clear. Here, we demonstrate that selective inhibition of effector caspases, caspase-3 and -7, or initiator caspase, caspase-9, in limb muscle in vivo by localized expression of recombinant inhibitor proteins dramatically decreases subsynaptic DNA damage, increases endplate area and improves ultrastructural abnormalities in SCS transgenic mice. Calpain and Cdk5 are not affected by this treatment. On the other hand, inhibition of Cdk5 by expression of a dominant-negative form of Cdk5 has no effect on the degeneration. Together with previous studies, these results indicate that focal activation of caspase activity at the NMJ is the principal pathological process responsible for the synaptic apoptosis in SCS. Thus, treatments that reduce muscle caspase activity are likely to be of benefit for SCS patients.

  6. Pretreatment with Pancaspase Inhibitor (Z-VAD-FMK Delays but Does Not Prevent Intraperitoneal Heat-Killed Group B Streptococcus-Induced Preterm Delivery in a Pregnant Mouse Model

    Directory of Open Access Journals (Sweden)

    Ozlem Equils

    2009-01-01

    Full Text Available Caspases and apoptosis are thought to play a role in infection-associated preterm-delivery. We have shown that in vitro treatment with pancaspase inhibitor Z-VAD-FMK protects trophoblasts from microbial antigen-induced apoptosis. Objective. To examine whether in vivo administration of Z-VAD-FMK would prevent infection-induced preterm-delivery. Methods. We injected 14.5 day-pregnant-mice with heat-killed group B streptococcus (HK-GBS. Apoptosis within placentas and membranes was assessed by TUNEL staining. Calpain expression and caspase-3 activation were assessed by immunohistochemistry. Preterm-delivery was defined as expulsion of a fetus within 48 hours after injection. Results. Intrauterine (i.u. or intraperitoneal (i.p. HK-GBS injection led to preterm-delivery and induced apoptosis in placentas and membranes at 14 hours. The expression of calpain, a caspase-independent inducer of apoptosis, was increased in placenta. Treatment with the specific caspase inhibitor Z-VAD-FMK (i.p. prior to HK-GBS (i.p. delayed but did not prevent preterm-delivery. Conclusion. Caspase-dependent apoptosis appears to play a role in the timing but not the occurrence of GBS-induced preterm delivery in the mouse.

  7. Magnesium homeostasis in colon carcinoma LoVo cells sensitive or resistant to doxorubicin.

    Science.gov (United States)

    Castiglioni, Sara; Cazzaniga, Alessandra; Trapani, Valentina; Cappadone, Concettina; Farruggia, Giovanna; Merolle, Lucia; Wolf, Federica I; Iotti, Stefano; Maier, Jeanette A M

    2015-11-13

    Neoplastic cells accumulate magnesium, an event which provides selective advantages and is frequently associated with TRPM7 overexpression. Little is known about magnesium homeostasis in drug-resistant cancer cells. Therefore, we used the colon cancer LoVo cell model and compared doxorubicin-resistant to sensitive cells. In resistant cells the concentration of total magnesium is higher while its influx capacity is lower than in sensitive cells. Accordingly, resistant cells express lower amounts of the TRPM6 and 7, both involved in magnesium transport. While decreased TRPM6 levels are due to transcriptional regulation, post-transcriptional events are involved in reducing the amounts of TRPM7. Indeed, the calpain inhibitor calpeptin markedly increases the levels of TRPM7 in resistant cells. In doxorubicin-sensitive cells, silencing TRPM7 shifts the phenotype to one more similar to resistant cells, since in these cells silencing TRPM7 significantly decreases the influx of magnesium, increases its intracellular concentration and increases resistance to doxorubicin. On the other hand, calpain inhibition upregulates TRPM7, decreases intracellular magnesium and enhances the sensitivity to doxorubicin of resistant LoVo cells. We conclude that in LoVo cells drug resistance is associated with alteration of magnesium homeostasis through modulation of TRPM7. Our data suggest that TRPM7 expression may be an additional undisclosed player in chemoresistance.

  8. Targeted proteolysis of plectin isoform 1a accounts for hemidesmosome dysfunction in mice mimicking the dominant skin blistering disease EBS-Ogna.

    Directory of Open Access Journals (Sweden)

    Gernot Walko

    2011-12-01

    Full Text Available Autosomal recessive mutations in the cytolinker protein plectin account for the multisystem disorders epidermolysis bullosa simplex (EBS associated with muscular dystrophy (EBS-MD, pyloric atresia (EBS-PA, and congenital myasthenia (EBS-CMS. In contrast, a dominant missense mutation leads to the disease EBS-Ogna, manifesting exclusively as skin fragility. We have exploited this trait to study the molecular basis of hemidesmosome failure in EBS-Ogna and to reveal the contribution of plectin to hemidesmosome homeostasis. We generated EBS-Ogna knock-in mice mimicking the human phenotype and show that blistering reflects insufficient protein levels of the hemidesmosome-associated plectin isoform 1a. We found that plectin 1a, in contrast to plectin 1c, the major isoform expressed in epidermal keratinocytes, is proteolytically degraded, supporting the notion that degradation of hemidesmosome-anchored plectin is spatially controlled. Using recombinant proteins, we show that the mutation renders plectin's 190-nm-long coiled-coil rod domain more vulnerable to cleavage by calpains and other proteases activated in the epidermis but not in skeletal muscle. Accordingly, treatment of cultured EBS-Ogna keratinocytes as well as of EBS-Ogna mouse skin with calpain inhibitors resulted in increased plectin 1a protein expression levels. Moreover, we report that plectin's rod domain forms dimeric structures that can further associate laterally into remarkably stable (paracrystalline polymers. We propose focal self-association of plectin molecules as a novel mechanism contributing to hemidesmosome homeostasis and stabilization.

  9. Integrin β1A Upregulates p27 Protein Amount at the Post-translational Level in Human Hepatocellular Carcinoma Cell Line SMMC-7721

    Institute of Scientific and Technical Information of China (English)

    Yi FU; Li-Ying WANG; Yu-Long LIANG; Jia-Wei JIN; Zheng-Yu FANG; Xi-Liang ZHA

    2006-01-01

    Integrins mediate many fundamental cellular processes by binding to components of the extracellular matrix. We showed previously that integrin β1A could inhibit cell proliferation. Integrin β1A stimulated the promoter activity of p21cip1 and enhanced its transcription in SMMC-7721 cells. In this study,we demonstrated that integrin β1A upregulated p27kip1 at the post-translational level in SMMC-7721 cells. Our results showed that integrin β1A increased the p27 protein amount, both in cytoplasm and nucleus, but did not affect the p27m RNA amount. Cycloheximide treatment experiment revealed that the half-life of p27 protein was prolonged in integrin β1A overexpressing cells, indicating that integrin β1A inhibited the degradation of p27 protein. Our data also provided evidence that both the proteasome and calpain were involved in the degradation of p27 protein in SMMC-7721 cells. Integrin β1A decreased the Skp2 expression and repressed the activity of calpain during G1 phase in SMMC-7721 cells. Taken together, these results indicated that integrin β1A might upregulate the protein amount of p27 through repressing Skp2-dependent proteasome degradation and calpainmediated proteolysis in SMMC-7721 cells.

  10. Evolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories

    Science.gov (United States)

    Nakayama, S.; Moncrief, N. D.; Kretsinger, R. H.

    1992-01-01

    In the first report in this series we described the relationships and evolution of 152 individual proteins of the EF-hand subfamilies. Here we add 66 additional proteins and define eight (CDC, TPNV, CLNB, LPS, DGK, 1F8, VIS, TCBP) new subfamilies and seven (CAL, SQUD, CDPK, EFH5, TPP, LAV, CRGP) new unique proteins, which we assume represent new subfamilies. The main focus of this study is the classification of individual EF-hand domains. Five subfamilies--calmodulin, troponin C, essential light chain, regulatory light chain, CDC31/caltractin--and three uniques--call, squidulin, and calcium-dependent protein kinase--are congruent in that all evolved from a common four-domain precursor. In contrast calpain and sarcoplasmic calcium-binding protein (SARC) each evolved from its own one-domain precursor. The remaining 19 subfamilies and uniques appear to have evolved by translocation and splicing of genes encoding the EF-hand domains that were precursors to the congruent eight and to calpain and to SARC. The rates of evolution of the EF-hand domains are slower following formation of the subfamilies and establishment of their functions. Subfamilies are not readily classified by patterns of calcium coordination, interdomain linker stability, and glycine and proline distribution. There are many homoplasies indicating that similar variants of the EF-hand evolved by independent pathways.

  11. Evaluation of biochemical parameters and genetic markers for association with meat tenderness in South African feedlot cattle.

    Science.gov (United States)

    Frylinck, L; van Wyk, G L; Smith, T P L; Strydom, P E; van Marle-Köster, E; Webb, E C; Koohmaraie, M; Smith, M F

    2009-12-01

    A large proportion of South African feedlot cattle are crossbreds of Brahman (BrX, Bos indicus), and Simmental (SiX, Bos taurus). A sample of 20 grain fed bulls from each of these crossbreeds was used to compare meat quality with that of the small frame indigenous Nguni (NgX, Sanga) by evaluating a variety of biochemical and genetic parameters previously shown to be associated with meat tenderness. Shear force values were generally high (5.6kg average at 14days post mortem), with SiX animals higher than BrX or NgX (P=0.051) despite higher calpastatin:calpain ratio in BrX (P<0.05). Calpain activity and cold shortening were both correlated with tenderness for all classes. The sample size was too small to accurately estimate genotypic effects of previously published markers in the CAST and CAPN1 genes, but the allele frequencies suggest that only modest progress would be possible in these South African crossbreds using these markers. PMID:20416642

  12. Report of limb girdle muscular dystrophy type 2a in 6 Iranian patients, one with a novel deletion in CAPN3 gene.

    Science.gov (United States)

    Fadaee, Mahsa; Kariminejad, Ariana; Fattahi, Zohreh; Nafissi, Shahriar; Godarzi, Hamed Reza; Beheshtian, Maryam; Vazehan, Raheleh; Akbari, Mohammad Reza; Kahrizi, Kimia; Najmabadi, Hossein

    2016-01-01

    Calpain3 is a calcium-dependent intracellular protease involved in an autosomal recessive form of muscular dystrophy known as limb-girdle muscular dystrophy type 2A. Many pathogenic mutations have been identified in calpain3, encoded by the CAPN3 gene, which leads to weakness of the pelvic and shoulder girdle muscles. In the present study, whole exome sequencing was performed on six unrelated Iranian families who presented with progressive muscle weakness, with a strong suspicion of Calpainopathies. Genetic analysis of CAPN3 gene revealed five causative variants which had not been reported in the Iranian population before including a novel 6 bp deletion (c.795_800delCATTGA) and four previously reported mutations (c.1939G > T, c.2243G > A, c.2257delGinsAA, and c.2380 + 2T > G). Our findings indicate that exome sequencing can be a very effective and affordable method to diagnose heterogeneous muscular dystrophies, especially in consanguineous populations such as Iran. PMID:27020652

  13. Fragmented inositol 1,4,5-trisphosphate receptors retain tetrameric architecture and form functional Ca2+ release channels.

    Science.gov (United States)

    Alzayady, Kamil J; Chandrasekhar, Rahul; Yule, David I

    2013-04-19

    Inositol 1,4,5-trisphosphate receptor isoforms are a family of ubiquitously expressed ligand-gated channels encoded by three individual genes. The proteins are localized to membranes of intracellular Ca(2+) stores and play pivotal roles in Ca(2+) homeostasis. Previous studies have demonstrated that IP3R1 is cleaved by the intracellular proteases calpain and caspase both in vivo and in vitro. However, the resultant cleavage products are poorly defined, and the functional consequences of these proteolytic events are not fully understood. We demonstrate that IP3R1 is cleaved during staurosporine-induced apoptosis, yielding N-terminal fragments encompassing the ligand-binding domain and the majority of the central modulatory domain together with a C-terminal fragment containing the channel domain and cytosolic tail. Notably, these fragments remain associated with the membrane after initiation of apoptotic cleavage. Furthermore, when recombinant IP3R1 fragments, corresponding to those predicted to be generated by caspase or calpain cleavage, are stably coexpressed in cells, they physically associate and form functional channels. These data provide novel insights regarding the regulation of IP3R1 during proteolysis and provide direct evidence that polypeptide continuity is not required for IP3R activation and Ca(2+) release.

  14. Apoptosis-related genes change their expression with age and hearing loss in the mouse cochlea.

    Science.gov (United States)

    Tadros, Sherif F; D'Souza, Mary; Zhu, Xiaoxia; Frisina, Robert D

    2008-11-01

    To understand possible causative roles of apoptosis gene regulation in age-related hearing loss (presbycusis), apoptotic gene expression patterns in the CBA mouse cochlea of four different age and hearing loss groups were compared, using GeneChip and real-time (qPCR) microarrays. GeneChip transcriptional expression patterns of 318 apoptosis-related genes were analyzed. Thirty eight probes (35 genes) showed significant differences in expression. The significant gene families include Caspases, B-cell leukemia/lymphoma2 family, P53, Calpains, Mitogen activated protein kinase family, Jun oncogene, Nuclear factor of kappa light chain gene enhancer in B-cells inhibitor-related and tumor necrosis factor-related genes. The GeneChip results of 31 genes were validated using the new TaqMan Low Density Array (TLDA). Eight genes showed highly correlated results with the GeneChip data. These genes are: activating transcription factor3, B-cell leukemia/lymphoma2, Bcl2-like1, caspase4 apoptosis-related cysteine protease 4, Calpain2, dual specificity phosphatase9, tumor necrosis factor receptor superfamily member12a, and Tumor necrosis factor superfamily member13b, suggesting they may play critical roles in inner ear aging.

  15. Molecular fingerprint of high fat diet induced urinary bladder metabolic dysfunction in a rat model.

    Directory of Open Access Journals (Sweden)

    Andreas Oberbach

    Full Text Available AIMS/HYPOTHESIS: Diabetic voiding dysfunction has been reported in epidemiological dimension of individuals with diabetes mellitus. Animal models might provide new insights into the molecular mechanisms of this dysfunction to facilitate early diagnosis and to identify new drug targets for therapeutic interventions. METHODS: Thirty male Sprague-Dawley rats received either chow or high-fat diet for eleven weeks. Proteomic alterations were comparatively monitored in both groups to discover a molecular fingerprinting of the urinary bladder remodelling/dysfunction. Results were validated by ELISA, Western blotting and immunohistology. RESULTS: In the proteome analysis 383 proteins were identified and canonical pathway analysis revealed a significant up-regulation of acute phase reaction, hypoxia, glycolysis, β-oxidation, and proteins related to mitochondrial dysfunction in high-fat diet rats. In contrast, calcium signalling, cytoskeletal proteins, calpain, 14-3-3η and eNOS signalling were down-regulated in this group. Interestingly, we found increased ubiquitin proteasome activity in the high-fat diet group that might explain the significant down-regulation of eNOS, 14-3-3η and calpain. CONCLUSIONS/INTERPRETATION: Thus, high-fat diet is sufficient to induce significant remodelling of the urinary bladder and alterations of the molecular fingerprint. Our findings give new insights into obesity related bladder dysfunction and identified proteins that may indicate novel pathophysiological mechanisms and therefore constitute new drug targets.

  16. Examining the Neural and Astroglial Protective Effects of Cellular Prion Protein Expression and Cell Death Protease Inhibition in Mouse Cerebrocortical Mixed Cultures.

    Science.gov (United States)

    Wang, Kevin K W; Yang, Zhihui; Chiu, Allen; Lin, Fan; Rubenstein, Richard

    2016-09-01

    Overexpression of cellular prion protein, PrP(C), has cytoprotective effects against neuronal injuries. Inhibition of cell death-associated proteases such as necrosis-linked calpain and apoptosis-linked caspase are also neuroprotective. Here, we systematically studied how PrP(C) expression levels and cell death protease inhibition affect cytotoxic challenges to both neuronal and glial cells in mouse cerebrocortical mixed cultures (CCM). Primary CCM derived from three mouse lines expressing no (PrP(C) knockout mice (PrPKO)), normal (wild-type (wt)), or high (tga20) levels of PrP(C) were subjected to necrotic challenge (calcium ionophore A23187) and apoptotic challenge (staurosporine (STS)). CCM which originated from tga20 mice provided the most robust neuron-astroglia protective effects against necrotic and early apoptotic cell death (lactate dehydrogenase (LDH) release) at 6 h but subsequently lost its cytoprotective effects. In contrast, PrPKO-derived cultures displayed elevated A23187- and STS-induced cell death at 24 h. Calpain inhibitor SNJ-1945 protected against A23187 challenge at 6 h in CCM from all three mouse lines but protected only against A23187 and STS treatments by 24 h in the PrPKO line. In parallel, caspase inhibitor Z-D-DCB protected against pro-apoptotic STS challenge at 6 and 24 h. Furthermore, we also examined αII-spectrin breakdown products (primarily from neurons) and glial fibrillary acidic protein (GFAP) breakdown products (from astroglia) as cytoskeletal proteolytic biomarkers. Overall, it appeared that both neurons and astroglial cells were less vulnerable to proteolytic attack during A23187 and STS challenges in tga20-derived cultures but more vulnerable in PrPKO-derived cultures. In addition, calpain and caspase inhibitors provide further protection against respective protease attacks on these neuronal and glial cytoskeletal proteins in CCM regardless of mouse-line origin. Lastly, some synergistic cytoprotective effects between Pr

  17. 内源性蛋白酶对宰后肌肉嫩化机制研究进展%Advances in Research on Postmortem Tenderization Mechanism of Endogenous Proteolytic Enzymes in Muscle

    Institute of Scientific and Technical Information of China (English)

    黄明; 黄峰; 黄继超; 徐宝才; 周光宏; 徐幸莲

    2011-01-01

    The mechanism of postmortem tenderization of muscle has been the focus of meat science for years. It is generally accepted that improvement of meat tenderness during postmortem aging mainly results from limited degradation of myofibrillar proteins by endogenous proteolytic enzymes. Calpains are widely considered to be a major contributor, but not only ones to postmortem improvement of meat tenderness, which is the result of multi-enzymatic interaction. The roles of lysosomal cathepsins, proteasomes and calpains during postmortem tenderization of meat were briefly reviewed in the paper. The characteristics of apoptosis, activating pathways and caspases were introduced, meanwhile the potential contribution of caspases to postmortem tenderization was also discussed. In the last, the possibilities of interactions between caspases and calpains and contribution of both proteases together to meat tenderization were also analyzed in the review.%肌肉的宰后嫩化机制一直是国内外肉品科学研究的一个热点,目前认为肌肉宰后嫩度的改善主要归因于内源性水解酶作用所引起的肌原纤维蛋白的有限降解.钙激活酶虽然是宰后嫩度改善的一个主要贡献者,但不是惟一的,而是多种内源酶类协同作用的结果.本文综述了溶酶体组织蛋白酶、蛋白酶体、钙激活酶在宰后嫩化过程的作用;介绍了细胞凋亡的特点、通路及凋亡酶,并对细胞凋亡酶对宰后嫩度改善的潜在贡献进行了讨论;最后分析了细胞凋亡酶和钙激活酶在宰后成熟过程中的交互作用及共同贡献于肌肉宰后嫩化的可能性.

  18. A post-transcriptional mechanism regulates calpastatin expression in bovine skeletal muscle.

    Science.gov (United States)

    Nattrass, G S; Cafe, L M; McIntyre, B L; Gardner, G E; McGilchrist, P; Robinson, D L; Wang, Y H; Pethick, D W; Greenwood, P L

    2014-02-01

    The objective of this study was to investigate whether single nucleotide polymorphisms (SNP) in the calpain 1 (CAPN1), calpain 3 (CAPN3) and calpastatin (CAST) genes, which have been shown to be associated with shear force and tenderness differences in the skeletal muscle of cattle, contribute to phenotypic variation in muscle tenderness by modulating the transcriptional activity of their respective gene. The mRNA expression of the calpain and CAST genes was assessed in the longissimus lumborum muscle (LLM) of cattle from two herds located in distinct production zones on the east (New South Wales, NSW) and west (Western Australia, WA) of Australia. The cattle in the herds were mainly Brahman cattle (Bos indicus) with smaller populations of Angus cattle (Bos taurus). There were 191 steers in the WA herd and 107 steers and 106 heifers in the NSW herd. These herds were established by choosing cattle from the diverse population which had different single nucleotide polymorphism (SNP) genotypes at the CAPN1, CAPN3 and CAST loci. Using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), the transcriptional activities of the CAPN1 and the CAST genes, but not the CAPN3 gene, were found to differ between favorable, positively associated with tenderness, and unfavorable, negatively associated with tenderness, allelic variants of these genes. These findings suggest that the muscle shear force and consumer taste panel differences in tenderness explained by the CAPN1 and CAST gene markers are a consequence of alterations in their mRNA levels, which may ultimately influence the protein activity of these genes, thereby altering the rate and(or) the extent of postmortem proteolysis in skeletal muscle. Of particular importance were the significantly lower type II and type III CAST 5' splice variant mRNA levels that were detected in the LLM muscle of Brahman and Angus cattle with 2 favourable alleles of the CAST:c.2832A > G polymorphism. Moreover, a reduction in

  19. Fluoxetine is neuroprotective in slow-channel congenital myasthenic syndrome.

    Science.gov (United States)

    Zhu, Haipeng; Grajales-Reyes, Gary E; Alicea-Vázquez, Vivianette; Grajales-Reyes, Jose G; Robinson, KaReisha; Pytel, Peter; Báez-Pagán, Carlos A; Lasalde-Dominicci, Jose A; Gomez, Christopher M

    2015-08-01

    The slow-channel congenital myasthenic syndrome (SCS) is an inherited neurodegenerative disease that caused mutations in the acetylcholine receptor (AChR) affecting neuromuscular transmission. Leaky AChRs lead to Ca(2+) overload and degeneration of the neuromuscular junction (NMJ) attributed to activation of cysteine proteases and apoptotic changes of synaptic nuclei. Here we use transgenic mouse models expressing two different mutations found in SCS to demonstrate that inhibition of prolonged opening of mutant AChRs using fluoxetine not only improves motor performance and neuromuscular transmission but also prevents Ca(2+) overload, the activation of cysteine proteases, calpain, caspase-3 and 9 at endplates, and as a consequence, reduces subsynaptic DNA damage at endplates, suggesting a long term benefit to therapy. These studies suggest that prolonged treatment of SCS patients with open ion channel blockers that preferentially block mutant AChRs is neuroprotective.

  20. Delivering minocycline into brain endothelial cells with liposome-based technology

    Science.gov (United States)

    Xing, Changhong; Levchenko, Tatyana; Guo, Shuzhen; Stins, Monique; Torchilin, Vladimir P; Lo, Eng H

    2012-01-01

    Minocycline has been proposed as a way to blunt neurovascular injury from matrix metalloproteinases (MMPs) during stroke. However, recent clinical trials suggest that high levels of minocycline may have deleterious side-effects. Here, we showed that very high minocycline concentrations damage endothelial cells via calpain/caspase pathways. To alleviate this potential cytotoxicity, we encapsulated minocycline in liposomes. Low concentrations of minocycline could not reduce tumor necrosis factor α (TNFα)-induced MMP-9 release from endothelial cells. But low concentrations of minocycline-loaded liposomes significantly reduced TNFα-induced MMP-9 release. This study provides proof-of-concept that liposomes may be used to deliver lower levels of minocycline for targeting MMPs in cerebral endothelium. PMID:22491155

  1. Variation in meat quality characteristics between Sanga (Bos taurus africanus) and Sanga-derived cattle breeds and between Sanga and Brahman (Bos indicus).

    Science.gov (United States)

    Strydom, P E; Frylinck, L; Smith, M F

    2011-03-01

    Cattle breeds indigenous to Africa (Sanga) compare favourably to Bos indicus breeds with regard to adaptation to harsh environments. This study compared the meat quality of three Sanga breeds (Nguni, Tuli and Drakensberger), a Sanga-related breed (Bonsmara) and a B. indicus breed (Brahman) and supported these results with biochemical and histological measurements on the M. longissimus lumborum. Twelve young grain-fed steers of each breed were slaughtered and carcasses were electrically stimulated. All Sanga (and related) breeds, with the exception of the Tuli, had lower Warner-Bratzler shear force (SF) values at 2 and 21 days post mortem compared with the BR (P meat than BR, mainly due to favourable calpain-to-calpastatin ratios. Small differences in colour, drip loss and cooking properties were found among breeds (P < 0.05). PMID:22445415

  2. 老化红细胞变形能力与膜钙依赖中性蛋白酶活性的关系

    Institute of Scientific and Technical Information of China (English)

    刘成玉; 林荣军; 万希琴; 谭润鸾

    1999-01-01

    @@ 研究表明,老化红细胞变形能力明显降低,且其降低与血红蛋白浓度增高及膜弹性降低有关,而与红细胞膜钙依赖中性蛋白酶(Calpain)活性的关系尚不清楚.为此,我们检测42例健康人老化红细胞及年轻红细胞变形能力、Calpain和Ca2+-ATP酶活性及膜收缩蛋白(spectrin,SP)相对含量的变化,以探讨老化细胞变形能力降低与Calpain活性的关系.

  3. Rare disease clinical trials: Power in numbers.

    Science.gov (United States)

    Wicklund, Matthew P

    2016-08-01

    The limb-girdle muscular dystrophies (LGMDs) encompass a collection of genetic muscle diseases with proximal-predominant weakness of the limbs. Thirty-two of these disorders are named via the common nomenclature, including 8 autosomal-dominant (LGMD1A-H) and 24 autosomal-recessive (LGMD2A-X) disorders.(1) In addition, numerous other genetic muscle diseases, including Bethlem myopathy, dystrophinopathies, ryanodine receptor-associated myopathies, and many more, may clinically present with similar proximal-predominant weakness.(2) Therefore, current genetic testing panels targeting neuromuscular weakness frequently encompass >75 genes. These disorders are quite rare, each with minimum prevalence estimates of 0.01-0.60 cases per 100,000 persons.(3) LGMD2A (attributable to mutations in the gene for calpain-3) and LGMD2B (attributable to mutations in the gene for dysferlin) consistently are the 2 most prevalent LGMD subtypes in a variety of ethnic cohorts. PMID:27540592

  4. Single nucleotide polymorphisms in Brahman steers and their association with carcass and tenderness traits.

    Science.gov (United States)

    Smith, T; Thomas, M G; Bidner, T D; Paschal, J C; Franke, D E

    2009-01-01

    Data from purebred Brahman steers (N = 467) were used to study the association of single nucleotide polymorphisms (SNP) with carcass traits and measures of tenderness. Fall weaned calves were grazed and fed in a subtropical environment and then harvested for processing in a commercial facility. Carcass data were recorded 24 h postmortem. Muscle samples and primal ribs were obtained to measure calpastatin activity and shear force. DNA was used to determine genotypes of thyroglobulin (TG5), calpastatin (CAST) and mu-calpain (CAPN 316 and CAPN 4751) SNP. Minor allele frequencies for CAST, CAPN 316 and CAPN 4751 were 0.342, 0.031, and 0.051, respectively. CAST genotypes were associated with calpastatin enzyme activity (P carcass traits.

  5. Regulation of neuronal axon specification by glia-neuron gap junctions in C. elegans

    Science.gov (United States)

    Meng, Lingfeng; Zhang, Albert; Jin, Yishi; Yan, Dong

    2016-01-01

    Axon specification is a critical step in neuronal development, and the function of glial cells in this process is not fully understood. Here, we show that C. elegans GLR glial cells regulate axon specification of their nearby GABAergic RME neurons through GLR-RME gap junctions. Disruption of GLR-RME gap junctions causes misaccumulation of axonal markers in non-axonal neurites of RME neurons and converts microtubules in those neurites to form an axon-like assembly. We further uncover that GLR-RME gap junctions regulate RME axon specification through activation of the CDK-5 pathway in a calcium-dependent manner, involving a calpain clp-4. Therefore, our study reveals the function of glia-neuron gap junctions in neuronal axon specification and shows that calcium originated from glial cells can regulate neuronal intracellular pathways through gap junctions. DOI: http://dx.doi.org/10.7554/eLife.19510.001 PMID:27767956

  6. Mechanistic studies on a sequential PDT protocol

    Science.gov (United States)

    Kessel, David

    2016-03-01

    A low (~LD15) PDT dose resulting in selective lysosomal photodamage can markedly promote photokilling by subsequent photodamage targeted to mitochondria. Experimental data are consistent with the proposal that cleavage of the autophagyassociated protein ATG5 to a pro-apoptotic fragment is responsible for this effect. This process is known to be dependent on the proteolytic activity of calpain. We have proposed that Ca2+ released from photodamaged lysosomes is the trigger for ATG5 cleavage. We can now document the conversion of ATG5 to the truncated form after lysosomal photodamage. Photofrin, a photosensitizer that targets both mitochondria and lysosomes, can be used for either phase of the sequential PDT process. The ability of Photofrin to target both loci may explain the well-documented efficacy of this agent.

  7. A single nucleotide polymorphism in CAPN1 associated with marbling score in Korean cattle

    Directory of Open Access Journals (Sweden)

    Kim Ji

    2008-04-01

    Full Text Available Abstract Background Marbling score (MS is the major quantitative trait that affects carcass quality in beef cattle. In this study, we examined the association between genetic polymorphisms of the micromolar calcium-activated neutral protease gene (micro-calpain, CAPN1 and carcass traits in Korean cattle (also known as Hanwoo. Results By direct DNA sequencing in 24 unrelated Korean cattle, we identified 39 sequence variants within exons and their flanking regions in CAPN1. Among them, 12 common polymorphic sites were selected for genotyping in the beef cattle (n = 421. Statistical analysis revealed that a polymorphism in the 3'UTR (c.2151*479C>T showed significant association with MS (Pcor. = 0.02. Conclusion Our findings suggest that polymorphisms in CAPN1 might be one of the important genetic factors involved in carcass quality in beef cattle, although it could be false positive association.

  8. Caspase Family Proteases and Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Ting-Jun FAN; Li-Hui HAN; Ri-Shan CONG; Jin LIANG

    2005-01-01

    Apoptosis, or programmed cell death, is an essential physiological process that plays a critical role in development and tissue homeostasis. The progress of apoptosis is regulated in an orderly way by a series of signal cascades under certain circumstances. The caspase-cascade system plays vital roles in the induction, transduction and amplification of intracellular apoptotic signals. Caspases, closely associated with apoptosis, are aspartate-specific cysteine proteases and members of the interleukin-1β-converting enzyme family. The activation and function of caspases, involved in the delicate caspase-cascade system, are regulated by various kinds of molecules, such as the inhibitor of apoptosis protein, Bcl-2 family proteins, calpain,and Ca2+. Based on the latest research, the members of the caspase family, caspase-cascade system and caspase-regulating molecules involved in apoptosis are reviewed.

  9. Calcium-dependent proteasome activation is required for axonal neurofilament degradation.

    Science.gov (United States)

    Park, Joo Youn; Jang, So Young; Shin, Yoon Kyung; Suh, Duk Joon; Park, Hwan Tae

    2013-12-25

    Even though many studies have identified roles of proteasomes in axonal degeneration, the molecular mechanisms by which axonal injury regulates proteasome activity are still unclear. In the present study, we found evidence indicating that extracellular calcium influx is an upstream regulator of proteasome activity during axonal degeneration in injured peripheral nerves. In degenerating axons, the increase in proteasome activity and the degradation of ubiquitinated proteins were significantly suppressed by extracellular calcium chelation. In addition, electron microscopic findings revealed selective inhibition of neurofilament degradation, but not microtubule depolymerization or mitochondrial swelling, by the inhibition of calpain and proteasomes. Taken together, our findings suggest that calcium increase and subsequent proteasome activation are an essential initiator of neurofilament degradation in Wallerian degeneration. PMID:25206662

  10. Incorporation of noncanonical amino acids into Rosetta and use in computational protein-peptide interface design.

    Directory of Open Access Journals (Sweden)

    P Douglas Renfrew

    Full Text Available Noncanonical amino acids (NCAAs can be used in a variety of protein design contexts. For example, they can be used in place of the canonical amino acids (CAAs to improve the biophysical properties of peptides that target protein interfaces. We describe the incorporation of 114 NCAAs into the protein-modeling suite Rosetta. We describe our methods for building backbone dependent rotamer libraries and the parameterization and construction of a scoring function that can be used to score NCAA containing peptides and proteins. We validate these additions to Rosetta and our NCAA-rotamer libraries by showing that we can improve the binding of a calpastatin derived peptides to calpain-1 by substituting NCAAs for native amino acids using Rosetta. Rosetta (executables and source, auxiliary scripts and code, and documentation can be found at (http://www.rosettacommons.org/.

  11. Field populations of native Indian honey bees from pesticide intensive agricultural landscape show signs of impaired olfaction

    Science.gov (United States)

    Chakrabarti, Priyadarshini; Rana, Santanu; Bandopadhyay, Sreejata; Naik, Dattatraya G.; Sarkar, Sagartirtha; Basu, Parthiba

    2015-07-01

    Little information is available regarding the adverse effects of pesticides on natural honey bee populations. This study highlights the detrimental effects of pesticides on honey bee olfaction through behavioural studies, scanning electron microscopic imaging of antennal sensillae and confocal microscopic studies of honey bee brains for calcium ions on Apis cerana, a native Indian honey bee species. There was a significant decrease in proboscis extension response and biologically active free calcium ions and adverse changes in antennal sensillae in pesticide exposed field honey bee populations compared to morphometrically similar honey bees sampled from low/no pesticide sites. Controlled laboratory experiments corroborated these findings. This study reports for the first time the changes in antennal sensillae, expression of Calpain 1(an important calcium binding protein) and resting state free calcium in brains of honey bees exposed to pesticide stress.

  12. Single nucleotide polymorphisms in Brahman steers and their association with carcass and tenderness traits.

    Science.gov (United States)

    Smith, T; Thomas, M G; Bidner, T D; Paschal, J C; Franke, D E

    2009-01-20

    Data from purebred Brahman steers (N = 467) were used to study the association of single nucleotide polymorphisms (SNP) with carcass traits and measures of tenderness. Fall weaned calves were grazed and fed in a subtropical environment and then harvested for processing in a commercial facility. Carcass data were recorded 24 h postmortem. Muscle samples and primal ribs were obtained to measure calpastatin activity and shear force. DNA was used to determine genotypes of thyroglobulin (TG5), calpastatin (CAST) and mu-calpain (CAPN 316 and CAPN 4751) SNP. Minor allele frequencies for CAST, CAPN 316 and CAPN 4751 were 0.342, 0.031, and 0.051, respectively. CAST genotypes were associated with calpastatin enzyme activity (P carcass traits.

  13. Neuroprotective Effect of Calpeptin on Acrylamide-Induced Neuropathy in Rats.

    Science.gov (United States)

    Wei, Xiaomin; Yan, Fengfeng; E, Meng; Zhang, Cuili; Li, Guozhen; Yang, Xiwei; Zhang, Fengmei; Wang, Shue; Yu, Sufang

    2015-11-01

    Acrylamide (ACR) is a vinyl monomer with established human neurotoxic effects, which is characterized by the accumulation of neurofilaments (NFs) in the distal swellings of large axons in peripheral and central nervous systems. However, the mechanisms of neurotoxicity remain unclear. The objective is to investigate the neuroprotective effect of calpeptin (CP) on ACR-induced neuropathy and its mechanism. Female adult Wistar rats were randomly divided into four groups (control, CP, ACR, and ACR + CP group). Control group received 0.9 % saline, ACR and ACR + CP groups received 30 mg/kg ACR by intraperitoneal injection. In addition, CP and ACR + CP groups also received 200 µg/kg CP. Gait analysis and hind limb splay were measured weekly to analyze neurobehavioral changes. The calpain activity and the changes of NFs protein levels in spinal cord are determined. Compared with control group, body weight of rats in ACR group decreased by 11.3 % (P < 0.01), while in ACR + CP group body weight increased significantly by 8.3 % (P < 0.01) compared with ACR group by the end of the 4th week; gait score of rats in both ACR and ACR + CP groups increased significantly by 167 % and 100 % (P < 0.01) compared with control group, while it decreased significantly by 25.1 % (P < 0.01) in ACR + CP group compared with ACR group; the distance of hind limb splay in both ACR and ACR + CP groups increased by 76.7 % and 49.5 % (P < 0.01) compared with control group, while it decreased by 15.4 % (P < 0.01) in ACR + CP group compared with ACR group; calpain activity of spinal cord at ACR and ACR + CP groups increased significantly by 14.9 % and 10.0 % (P < 0.01) compared with control group, while it decreased 4.2 % (P < 0.01) in ACR + CP group compared with ACR group; compared with control group, the levels of light NF (NF-L), medium NF (NF-M) and heavy NF (NF-H) subunits increased by 81.2 %, 263.6 % and 22.6 % (P < 0.01) in the supernatant of ACR group in spinal cord tissue and increased by 28

  14. Calcium-dependent proteasome activation is required for axonal neurofilament degradation

    Institute of Scientific and Technical Information of China (English)

    Joo Youn Park; So Young Jang; Yoon Kyung Shin; Duk Joon Suh; Hwan Tae Park

    2013-01-01

    Even though many studies have identified roles of proteasomes in axonal degeneration, the mo-lecular mechanisms by which axonal injury regulates proteasome activity are stil unclear. In the present study, we found evidence indicating that extracellular calcium influx is an upstream regula-tor of proteasome activity during axonal degeneration in injured peripheral nerves. In degenerating axons, the increase in proteasome activity and the degradation of ubiquitinated proteins were sig-nificantly suppressed by extracellular calcium chelation. In addition, electron microscopic findings revealed selective inhibition of neurofilament degradation, but not microtubule depolymerization or mitochondrial swel ing, by the inhibition of calpain and proteasomes. Taken together, our findings suggest that calcium increase and subsequent proteasome activation are an essential initiator of neurofilament degradation in Wal erian degeneration.

  15. Association of single nucleotide polymorphisms in CAPN1, CAST and MB genes with meat color of Brahman and crossbreed cattle.

    Science.gov (United States)

    Castro, Susan; Ríos, Marcela; Ortiz, Yurany; Manrique, Carlos; Jiménez, Ariel; Ariza, Fernando

    2016-07-01

    The objective of this research was to determine the association of SNPs in the candidate genes Calpain (CAPN1), Calpastatin (CAST) and Myoglobin (MB) with colorimetric parameters (L *, a *, b *, C *, hue) in a F1 population (n = 164) obtained from crossing Bos taurus × Bos indicus and Bos indicus × Bos indicus. SNPs were analyzed using PCR-RFLP and SSCP. Colorimetric measurements were performed in the muscles Longissimus thoracis et lumborum (LTL) and Semitendinosus (ST) at 7, 14 and 21 days postmortem applying the methodology CIE L* a* b*. The CAST gene showed a significant effect on the b* and hue* parameters in both muscles. MB gene showed significant association with all colorimetric parameters in both LTL and ST muscles, except with b* parameter. The CAPN1 gene did not show any significant association. These results suggest an important role of genetics in meat color variation for cattle raised under the tropic conditions.

  16. Long term anoxia in rainbow trout investigated by 2-DE and MS/MS

    DEFF Research Database (Denmark)

    Wulff, Tune; Jessen, Flemming; Roepstorff, P.;

    2008-01-01

    Twenty-four hours of N-2 induced anoxia induced global perturbations on protein expression in rainbow trout hypodermal fibroblasts cell line. Anoxia was obtained by depleting the medium of O-2 by flushing with N-2, and protein changes were studied by 2-DE coupled with MS providing quantitative...... measurements of a large number of proteins in one single study. The anoxic insult changed the level of 33 protein spots: 22 of these were up-regulated compared to the control situation and 11 were down-regulated. Using MS/MS sequencing 19 of the 33 protein spots that changed were identified, corresponding...... to a success rate of more than 50%. The identified proteins included two proteins involved in energy metabolism namely phosphoglycerate mutase and isocitrate dehydrogenase. In addition we observed the up-regulation of a cluster of proteins that contribute to cytoskeleton function. These are calpain, EB1...

  17. Association of single nucleotide polymorphisms in CAPN1, CAST and MB genes with meat color of Brahman and crossbreed cattle.

    Science.gov (United States)

    Castro, Susan; Ríos, Marcela; Ortiz, Yurany; Manrique, Carlos; Jiménez, Ariel; Ariza, Fernando

    2016-07-01

    The objective of this research was to determine the association of SNPs in the candidate genes Calpain (CAPN1), Calpastatin (CAST) and Myoglobin (MB) with colorimetric parameters (L *, a *, b *, C *, hue) in a F1 population (n = 164) obtained from crossing Bos taurus × Bos indicus and Bos indicus × Bos indicus. SNPs were analyzed using PCR-RFLP and SSCP. Colorimetric measurements were performed in the muscles Longissimus thoracis et lumborum (LTL) and Semitendinosus (ST) at 7, 14 and 21 days postmortem applying the methodology CIE L* a* b*. The CAST gene showed a significant effect on the b* and hue* parameters in both muscles. MB gene showed significant association with all colorimetric parameters in both LTL and ST muscles, except with b* parameter. The CAPN1 gene did not show any significant association. These results suggest an important role of genetics in meat color variation for cattle raised under the tropic conditions. PMID:26946475

  18. Mechanisms and pathophysiological significance of eryptosis, the suicidal erythrocyte death.

    Science.gov (United States)

    Lang, Elisabeth; Lang, Florian

    2015-03-01

    Eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling, is stimulated by Ca(2+) entry through Ca(2+)-permeable, PGE2-activated cation channels, by ceramide, caspases, calpain, complement, hyperosmotic shock, energy depletion, oxidative stress, and deranged activity of several kinases (e.g. AMPK, GK, PAK2, CK1α, JAK3, PKC, p38-MAPK). Eryptosis is triggered by intoxication, malignancy, hepatic failure, diabetes, chronic renal insufficiency, hemolytic uremic syndrome, dehydration, phosphate depletion, fever, sepsis, mycoplasma infection, malaria, iron deficiency, sickle cell anemia, thalassemia, glucose 6-phosphate dehydrogenase deficiency, and Wilson's disease. Eryptosis may precede and protect against hemolysis but by the same token result in anemia and deranged microcirculation. PMID:25636585

  19. Reduced amyloidogenic processing of the amyloid beta-protein precursor by the small-molecule Differentiation Inducing Factor-1.

    Science.gov (United States)

    Myre, Michael A; Washicosky, Kevin; Moir, Robert D; Tesco, Giuseppina; Tanzi, Rudolph E; Wasco, Wilma

    2009-04-01

    The detection of cell cycle proteins in Alzheimer's disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Abeta properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid beta-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Abeta40 and Abeta42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Abeta42 to Abeta40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Abeta. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a gamma-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668. PMID:19154786

  20. Reduced amyloidogenic processing of the amyloid β-protein precursor by the small-molecule Differentiation Inducing Factor-1

    Science.gov (United States)

    Myre, Michael A.; Washicosky, Kevin; Moir, Robert D.; Tesco, Giuseppina; Tanzi, Rudolph E.; Wasco, Wilma

    2013-01-01

    The detection of cell cycle proteins in Alzheimer’s disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Aβ properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid β-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Aβ40 and Aβ42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Aβ42 to Aβ40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Aβ. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a γ-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668. PMID:19154786

  1. Poly (ADP-ribose polymerase plays an important role in intermittent hypoxia-induced cell death in rat cerebellar granule cells

    Directory of Open Access Journals (Sweden)

    Chiu Sheng-Chun

    2012-03-01

    Full Text Available Abstract Background Episodic cessation of airflow during sleep in patients with sleep apnea syndrome results in intermittent hypoxia (IH. Our aim was to investigate the effects of IH on cerebellar granule cells and to identify the mechanism of IH-induced cell death. Methods Cerebellar granule cells were freshly prepared from neonatal Sprague-Dawley rats. IH was created by culturing the cerebellar granule cells in the incubators with oscillating O2 concentration at 20% and 5% every 30 min for 1-4 days. The results of this study are based on image analysis using a confocal microscope and associated software. Cellular oxidative stress increased with increase in IH. In addition, the occurrence of cell death (apoptosis and necrosis increased as the duration of IH increased, but decreased in the presence of an iron chelator (phenanthroline or poly (ADP-ribose polymerase (PARP inhibitors [3-aminobenzamide (3-AB and DPQ]. The fluorescence of caspase-3 remained the same regardless of the duration of IH, and Western blots did not detect activation of caspase-3. However, IH increased the ratio of apoptosis-inducing factor (AIF translocation to the nucleus, while PARP inhibitors (3-AB reduced this ratio. Results According to our findings, IH increased oxidative stress and subsequently leading to cell death. This effect was at least partially mediated by PARP activation, resulting in ATP depletion, calpain activation leading to AIF translocation to the nucleus. Conclusions We suggest that IH induces cell death in rat primary cerebellar granule cells by stimulating oxidative stress PARP-mediated calpain and AIF activation.

  2. Higher levels of phosphorylated Y1472 on GluN2B subunits in the frontal cortex of aged mice are associated with good spatial reference memory, but not cognitive flexibility.

    Science.gov (United States)

    Zamzow, Daniel R; Elias, Val; Acosta, Varinia A; Escobedo, Emily; Magnusson, Kathy R

    2016-06-01

    The N-methyl-D-aspartate receptor (NMDAr) is particularly vulnerable to aging. The GluN2B subunit of the NMDAr, compared to other NMDAr subunits, suffers the greatest losses of expression in the aging brain, especially in the frontal cortex. While expression levels of GluN2B mRNA and protein in the aged brain are well documented, there has been little investigation into age-related posttranslational modifications of the subunit. In this study, we explored some of the mechanisms that may promote differences in the NMDAr complex in the frontal cortex of aged animals. Two ages of mice, 3 and 24 months, were behaviorally tested in the Morris water maze. The frontal cortex and hippocampus from each mouse were subjected to differential centrifugation followed by solubilization in Triton X-100. Proteins from Triton-insoluble membranes, Triton-soluble membranes, and intracellular membranes/cytosol were examined by Western blot. Higher levels of GluN2B tyrosine 1472 phosphorylation in frontal cortex synaptic fractions of old mice were associated with better reference learning but poorer cognitive flexibility. Levels of GluN2B phosphotyrosine 1336 remained steady, but there were greater levels of the calpain-induced 115 kDa GluN2B cleavage product on extrasynaptic membranes in these old good learners. There was an age-related increase in calpain activity, but it was not associated with better learning. These data highlight a unique aging change for aged mice with good spatial learning that might be detrimental to cognitive flexibility. This study also suggests that higher levels of truncated GluN2B on extrasynaptic membranes are not deleterious to spatial memory in aged mice. PMID:27094400

  3. Regulation of the transcriptional coactivator FHL2 licenses activation of the androgen receptor in castrate-resistant prostate cancer.

    Science.gov (United States)

    McGrath, Meagan J; Binge, Lauren C; Sriratana, Absorn; Wang, Hong; Robinson, Paul A; Pook, David; Fedele, Clare G; Brown, Susan; Dyson, Jennifer M; Cottle, Denny L; Cowling, Belinda S; Niranjan, Birunthi; Risbridger, Gail P; Mitchell, Christina A

    2013-08-15

    It is now clear that progression from localized prostate cancer to incurable castrate-resistant prostate cancer (CRPC) is driven by continued androgen receptor (AR), signaling independently of androgen. Thus, there remains a strong rationale to suppress AR activity as the single most important therapeutic goal in CRPC treatment. Although the expression of ligand-independent AR splice variants confers resistance to AR-targeted therapy and progression to lethal castrate-resistant cancer, the molecular regulators of AR activity in CRPC remain unclear, in particular those pathways that potentiate the function of mutant AR in CRPC. Here, we identify FHL2 as a novel coactivator of ligand-independent AR variants that are important in CRPC. We show that the nuclear localization of FHL2 and coactivation of the AR is driven by calpain cleavage of the cytoskeletal protein filamin, a pathway that shows differential activation in prostate epithelial versus prostate cancer cell lines. We further identify a novel FHL2-AR-filamin transcription complex, revealing how deregulation of this axis promotes the constitutive, ligand-independent activation of AR variants, which are present in CRPC. Critically, the calpain-cleaved filamin fragment and FHL2 are present in the nucleus only in CRPC and not benign prostate tissue or localized prostate cancer. Thus, our work provides mechanistic insight into the enhanced AR activation, most notably of the recently identified AR variants, including AR-V7 that drives CRPC progression. Furthermore, our results identify the first disease-specific mechanism for deregulation of FHL2 nuclear localization during cancer progression. These results offer general import beyond prostate cancer, given that nuclear FHL2 is characteristic of other human cancers where oncogenic transcription factors that drive disease are activated like the AR in prostate cancer.

  4. Roles of integrin β3 cytoplasmic tail in bidirectional signal transduction in a trans-dominant inhibition model.

    Science.gov (United States)

    Huang, Jiansong; Zhou, Yulan; Su, Xiaoyu; Lyu, Yuanjing; Tao, Lanlan; Shi, Xiaofeng; Liu, Ping; Long, Zhangbiao; Ruan, Zheng; Xiao, Bing; Xi, Wenda; Zhou, Quansheng; Mao, Jianhua; Xi, Xiaodong

    2016-09-01

    We evaluated the roles of calpain cleavage-related mutations of the integrin β3 cytoplasmic tail in integrin αIIbβ3 bidirectional signaling using a trans-dominant inhibition model. Chimeric Tac-β3 proteins (i.e., Tac-β3, Tac-β3Δ741, Tac-β3Δ747, Tac-β3Δ754, Tac-β3Δ759, and Tac-β3ΔNITY) consisting of the extracellular and transmembrane domains of human IL-2 receptor (Tac) and the human integrin β3 cytoplasmic domain were stably expressed in the 123 CHO cells harboring human glycoprotein Ib-IX and wild-type integrin αIIbβ3. The different cells were assayed for stable adhesion and spreading on immobilized fibrinogen, and for binding soluble fibrinogen representing outside-in and inside-out signaling events, respectively. The chimeric protein Tac-β3 inhibited, and Tac-β3ΔNITY partially attenuated stable adhesion and spreading. Tac-β3, Tac-β3Δ759, Tac-β3ΔNITY, and Tac-β3Δ754, but not Tac-β3Δ747 or Tac-β3Δ741, impaired the soluble fibrinogen binding. Results indicated that the bidirectional signaling was significantly inhibited by Tac-β3 and Tac-β3ΔNITY, albeit to a much lesser extent. Moreover, only inside-out signaling was impaired in the 123/Tac-β3Δ759 and 123/Tac-β3Δ754 cells in contrast to an intact bidirectional signaling in the 123/Tac-β3Δ747 and 123/Tac-β3Δ741 cells. In conclusion, the calpain cleavage of integrin β3 resulted in the regulatory effects on signaling by interrupting its interaction with cytoplasmic proteins rather than altering its conformation, and may thus regulate platelet function.

  5. Role of Copper and Cholesterol Association in the Neurodegenerative Process

    Directory of Open Access Journals (Sweden)

    Nathalie Arnal

    2013-01-01

    Full Text Available Age is one of the main factors involved in the development of neurological illnesses, in particular, Alzheimer, and it is widely held that the rapid aging of the world population is accompanied by a rise in the prevalence and incidence of Alzheimer disease. However, evidence from recent decades indicates that Cu and Cho overload are emerging causative factors in neurodegeneration, a hypothesis that has been partially investigated in experimental models. The link between these two variables and the onset of Alzheimer disease has opened up interesting new possibilities requiring more in-depth analysis. The aim of the present study was therefore to investigate the effect of the association of Cu + Cho (CuCho as a possible synergistic factor in the development of an Alzheimer-like pathology in Wistar rats. We measured total- and nonceruloplasmin-bound Cu and Cho (free and sterified contents in plasma and brain zones (cortex and hippocampus, markers of oxidative stress damage, inflammation, and programmed cell death (caspase-3 and calpain isoforms. The ratio beta-amyloid (1-42/(1-40 was determined in plasma and brain as neurodegenerative biomarker. An evaluation of visuospatial memory (Barnes maze test was also performed. The results demonstrate the establishment of a prooxidative and proinflammatory environment after CuCho treatment, hallmarked by increased TBARS, protein carbonyls, and nitrite plus nitrate levels in plasma and brain zones (cortex and hippocampus with a consequent increase in the activity of calpains and no significant changes in caspase-3. A simultaneous increase in the plasma Aβ1-42/Aβ1-40 ratio was found. Furthermore, a slight but noticeable change in visuospatial memory was observed in rats treated with CuCho. We conclude that our model could reflect an initial stage of neurodegeneration in which Cu and Cho interact with one another to exacerbate neurological damage.

  6. Neuroprotection by sodium ferulate against glutamate-induced apoptosis is mediated by ERK and P13 kinase pathways

    Institute of Scientific and Technical Information of China (English)

    Ying JIN; En-zhi YAN; Ying FAN; Xiao-li GUO; Yan-jie ZHAO; Zhi-hong ZONG; Zhuo LIU

    2007-01-01

    Aim: To investigate whether sodium ferulate (SF) can protect cortical neurons from glutamate-induced neurotoxicity and the mechanisms responsible for this protection. Methods: Cultured cortical neurons were incubated with 50 μmol/L glutamate for either 30 min or 24 h, with or without pre-incubation with SF (100, 200, and 500 μmol/L, respectively). LY294002, wortmannin, PD98059, and U0126 were added respectively to the cells 1 h prior to SF treatment. After incubation with glutamate for 24 h, neuronal apoptosis was quantified by scoring the per- centage of ceils with apoptotic nuclear morphology after Hoechst 33258 staining. After incubation with glutamate for either 30 min or 24 h, cellular extracts were prepared for Western blotting of active caspase-3, poly (ADP-ribose) polymerase (PARP), μ-calpain, Bcl-2, phospho-Akt, phosphorylated ribosomal protein S6 pro- tein kinase (p70S6K), phospho-mitogen-activated protein kinase kinase (MEK1/2) and phosphorylated extracellular signal-regulated kinase (ERK) 1/2. Results: SF reduced glutamate-evoked apoptotic morphology, active caspase-3 protein expression, and PARP cleavage and inhibited the glutamate-induced upregulation of the μ-calpain protein level. The inhibition of the phosphatidylinositol 3-kinase (PI3K) and the MEK/ERK1/2 pathways partly abrogated the protective effect ot SF against glutamate-induced neuronal apoptosis. SF prevented the glutamate-induced decrease in the activity of the PI3K/Akt/p70S6K and the MEK/ERK1/2 pathways. Moreover, incubation of cortical neurons with SF for 30 min inhibited the reduction of the Bcl-2 expression induced by glutamate. Conclusion: The results indicate that PI3K/Akt/p70S6K and the MEK/ERK signaling pathways play important roles in the protective effect of SF against glutamate toxicity in cortical neurons.

  7. Environmental neurotoxic challenge of conditional alpha-synuclein transgenic mice predicts a dopaminergic olfactory-striatal interplay in early PD.

    Science.gov (United States)

    Nuber, Silke; Tadros, Daniel; Fields, Jerel; Overk, Cassia Rose; Ettle, Benjamin; Kosberg, Kori; Mante, Michael; Rockenstein, Edward; Trejo, Margarita; Masliah, Eliezer

    2014-04-01

    The olfactory bulb (OB) is one of the first brain regions in Parkinson's disease (PD) to contain alpha-synuclein (α-syn) inclusions, possibly associated with nonmotor symptoms. Mechanisms underlying olfactory synucleinopathy, its contribution to progressive aggregation pathology and nigrostriatal dopaminergic loss observed at later stages, remain unclear. A second hit, such as environmental toxins, is suggestive for α-syn aggregation in olfactory neurons, potentially triggering disease progression. To address the possible pathogenic role of olfactory α-syn accumulation in early PD, we exposed mice with site-specific and inducible overexpression of familial PD-linked mutant α-syn in OB neurons to a low dose of the herbicide paraquat. Here, we found that olfactory α-syn per se elicited structural and behavioral abnormalities, characteristic of an early time point in models with widespread α-syn expression, including hyperactivity and increased striatal dopaminergic marker. Suppression of α-syn reversed the dopaminergic phenotype. In contrast, paraquat treatment synergistically induced degeneration of olfactory dopaminergic cells and opposed the higher reactive phenotype. Neither neurodegeneration nor behavioral abnormalities were detected in paraquat-treated mice with suppressed α-syn expression. By increasing calpain activity, paraquat induced a pathological cascade leading to inhibition of autophagy clearance and accumulation of calpain-cleaved truncated and insoluble α-syn, recapitulating biochemical and structural changes in human PD. Thus our results underscore the primary role of proteolytic failure in aggregation pathology. In addition, we provide novel evidence that olfactory dopaminergic neurons display an increased vulnerability toward neurotoxins in dependence to presence of human α-syn, possibly mediating an olfactory-striatal dopaminergic network dysfunction in mouse models and early PD. PMID:24509835

  8. The role of heat shock protein 70 in the protective effect of YC-1 on β-amyloid-induced toxicity in differentiated PC12 cells.

    Directory of Open Access Journals (Sweden)

    Yung-Chieh Tsai

    Full Text Available Neurodegenerative brain disorders such as Alzheimer's disease (AD have been well investigated. However, significant methods for the treatment of the progression of AD are unavailable currently. Heat shock protein 70 (Hsp70 plays important roles in neural protection from stress by assisting cellular protein folding. In this study, we investigated the effect and the molecular mechanism of YC-1, an activator of guanylyl cyclase (GC, on Aβ25-35-induced cytotoxicity in differentiated PC12 cells. The results of this study showed that Aβ25-35 (10 µM significantly increased p25 protein production in a pattern that was consistent with the increase in μ-calpain expression. Moreover, Aβ25-35 significantly increased tau hyperphosphorylation and induced differentiated PC12 cell death. YC-1 (0.5-10 µM prevented the cell death induced by Aβ25-35. In addition, YC-1 (1, 10 µM significantly blocked Aβ25-35-induced μ-calpain expression and decreased the formation of p25 and tau hyperphosphorylation. Moreover, YC-1 (5-20 µM alone or combined with Aβ25-35 (10 µM significantly increased the expression of Hsp70 in differentiated PC12 cells. The neuroprotective effect of YC-1 was significantly attenuated by an Hsp70 inhibitor (quercetin, 50 µM or in PC12 cells transfected with an Hsp70 small interfering RNA. However, pretreatment of cells with the GC inhibitor ODQ (10 µM did not affect the neuroprotective effect of YC-1 against Aβ25-35 in differentiated PC12 cells. These results suggest that the neuroprotective effect of YC-1 against Aβ25-35-induced toxicity is mainly mediated by the induction of Hsp70. Thus, YC-1 is a potential agent against AD.

  9. Analysis of proteolytic processes and enzymatic activities in the generation of huntingtin n-terminal fragments in an HEK293 cell model.

    Directory of Open Access Journals (Sweden)

    Andrew T N Tebbenkamp

    Full Text Available BACKGROUND: N-terminal fragments of mutant huntingtin (htt that terminate between residues 90-115, termed cleavage product A or 1 (cp-A/1, form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD. These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments. RESULTS: Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like, were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400-600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115-124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1. CONCLUSIONS: Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.

  10. Inhibition of PKC-Induced COX-2 and IL-8 Expression in Human Breast Cancer Cells by Glucosamine.

    Science.gov (United States)

    Chou, Wan-Yu; Chuang, Kun-Han; Sun, David; Lee, Yu-Hsiu; Kao, Pu-Hong; Lin, Yen-Yu; Wang, Hsei-Wei; Wu, Yuh-Lin

    2015-09-01

    Breast cancer is a common cancer leading to many deaths among females. Cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) are two highly expressed inflammatory mediators to be induced by the protein kinase C (PKC) signaling via various inflammatory stimuli and both contribute significantly to cancer metastasis/progression. Glucosamine has been shown to act as an anti-inflammation molecule. The aim of this study was to clarify the role and acting mechanism of glucosamine during the PKC-regulation of COX-2/IL-8 expression and the associated impact on breast cancer. In MCF-7 breast cancer cells, glucosamine effectively suppresses the PKC induction of COX-2 and IL-8 promoter activity, mRNA and protein levels, as well as the production of prostaglandin E(2) (PGE(2)) and IL-8. Glucosamine is able to promote COX-2 protein degradation in a calpain-dependent manner and IL-8 protein degradation in calpain-dependent and proteasome-dependent manners. The MAPK and NF-κB pathways are involved in PKC-induced COX-2 expression, but only the NF-κB pathway is involved in PKC-induced IL-8 expression. Glucosamine attenuates PKC-mediated IκBα phosphorylation, nuclear NF-κB translocation, and NF-κB reporter activation. Both PGE(2) and IL-8 promote cell proliferation and IL-8 induces cell migration; thus, glucosamine appears to suppress PKC-induced cell proliferation and migration. Furthermore, glucosamine significantly inhibits the growth of breast cancer xenografts and this is accompanied by a reduction in COX-2 and IL-8 expression. In conclusion, glucosamine seems to attenuate the inflammatory response in vitro and in vivo and this occurs, at least in part by targeting to the NF-κB signaling pathway, resulting in an inhibition of breast cancer cell growth.

  11. Retinal Cell Death Caused by Sodium Iodate Involves Multiple Caspase-Dependent and Caspase-Independent Cell-Death Pathways

    Directory of Open Access Journals (Sweden)

    Jasmin Balmer

    2015-07-01

    Full Text Available Herein, we have investigated retinal cell-death pathways in response to the retina toxin sodium iodate (NaIO3 both in vivo and in vitro. C57/BL6 mice were treated with a single intravenous injection of NaIO3 (35 mg/kg. Morphological changes in the retina post NaIO3 injection in comparison to untreated controls were assessed using electron microscopy. Cell death was determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL staining. The activation of caspases and calpain was measured using immunohistochemistry. Additionally, cytotoxicity and apoptosis in retinal pigment epithelial (RPE cells, primary retinal cells, and the cone photoreceptor (PRC cell line 661W were assessed in vitro after NaIO3 treatment using the ApoToxGlo™ assay. The 7-AAD/Annexin-V staining was performed and necrostatin (Nec-1 was administered to the NaIO3-treated cells to confirm the results. In vivo, degenerating RPE cells displayed a rounded shape and retracted microvilli, whereas PRCs featured apoptotic nuclei. Caspase and calpain activity was significantly upregulated in retinal sections and protein samples from NaIO3-treated animals. In vitro, NaIO3 induced necrosis in RPE cells and apoptosis in PRCs. Furthermore, Nec-1 significantly decreased NaIO3-induced RPE cell death, but had no rescue effect on treated PRCs. In summary, several different cell-death pathways are activated in retinal cells as a result of NaIO3.

  12. Proteolytic fragmentation of inositol 1,4,5-trisphosphate receptors: a novel mechanism regulating channel activity?

    Science.gov (United States)

    Wang, Liwei; Alzayady, Kamil J; Yule, David I

    2016-06-01

    Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are a family of ubiquitously expressed intracellular Ca(2+) release channels. Regulation of channel activity by Ca(2+) , nucleotides, phosphorylation, protein binding partners and other cellular factors is thought to play a major role in defining the specific spatiotemporal characteristics of intracellular Ca(2+) signals. These properties are, in turn, believed pivotal for the selective and specific physiological activation of Ca(2+) -dependent effectors. IP3 Rs are also substrates for the intracellular cysteine proteases, calpain and caspase. Cleavage of the IP3 R has been proposed to play a role in apoptotic cell death by uncoupling regions important for IP3 binding from the channel domain, leaving an unregulated leaky Ca(2+) pore. Contrary to this hypothesis, we demonstrate following proteolysis that N- and C-termini of IP3 R1 remain associated, presumably through non-covalent interactions. Further, we show that complementary fragments of IP3 R1 assemble into tetrameric structures and retain their ability to be regulated robustly by IP3 . While peptide continuity is clearly not necessary for IP3 -gating of the channel, we propose that cleavage of the IP3 R peptide chain may alter other important regulatory events to modulate channel activity. In this scenario, stimulation of the cleaved IP3 R may support distinct spatiotemporal Ca(2+) signals and activation of specific effectors. Notably, in many adaptive physiological events, the non-apoptotic activities of caspase and calpain are demonstrated to be important, but the substrates of the proteases are poorly defined. We speculate that proteolytic fragmentation may represent a novel form of IP3 R regulation, which plays a role in varied adaptive physiological processes.

  13. Neuroprotective effects of phytocannabinoid-based medicines in experimental models of Huntington's disease.

    Science.gov (United States)

    Sagredo, Onintza; Pazos, M Ruth; Satta, Valentina; Ramos, José A; Pertwee, Roger G; Fernández-Ruiz, Javier

    2011-09-01

    We studied whether combinations of botanical extracts enriched in either Δ(9)-tetrahydrocannabinol (Δ(9)-THC) or cannabidiol (CBD), which are the main constituents of the cannabis-based medicine Sativex, provide neuroprotection in rat models of Huntington's disease (HD). We used rats intoxicated with 3-nitropropionate (3NP) that were given combinations of Δ(9)-THC- and CBD-enriched botanical extracts. The issue was also studied in malonate-lesioned rats. The administration of Δ(9)-THC- and CBD-enriched botanical extracts combined in a ratio of 1:1 as in Sativex attenuated 3NP-induced GABA deficiency, loss of Nissl-stained neurons, down-regulation of CB(1) receptor and IGF-1 expression, and up-regulation of calpain expression, whereas it completely reversed the reduction in superoxide dismutase-1 expression. Similar responses were generally found with other combinations of Δ(9)-THC- and CBD-enriched botanical extracts, suggesting that these effects are probably related to the antioxidant and CB(1) and CB(2) receptor-independent properties of both phytocannabinoids. In fact, selective antagonists for both receptor types, i.e., SR141716 and AM630, respectively, were unable to prevent the positive effects on calpain expression caused in 3NP-intoxicated rats by the 1:1 combination of Δ(9)-THC and CBD. Finally, this combination also reversed the up-regulation of proinflammatory markers such as inducible nitric oxide synthase observed in malonate-lesioned rats. In conclusion, this study provides preclinical evidence in support of a beneficial effect of the cannabis-based medicine Sativex as a neuroprotective agent capable of delaying disease progression in HD, a disorder that is currently poorly managed in the clinic, prompting an urgent need for clinical trials with agents showing positive results in preclinical studies. PMID:21674569

  14. P53-mediated cell cycle arrest and apoptosis through a caspase-3-independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Qiao CUI; Jing-hua YU; Jin-nan WU; Shin-ichi TASHIRO; Satoshi ONODERA; Mutsuhiko MINAMI; Takashi IKEJIMA

    2007-01-01

    Aim: To study the caspase-3-independent mechanisms in oridonin-induced MCF-7 human breast cancer cell apoptosis in vitro. Methods: The viability of oridonin-treated MCF-7 cells was measured by MTT (thiazole blue) assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleoso-mal DNA fragmentation was assayed by agarose gel electrophoresis. The apoptotic ratio was determined by lactate dehydrogenase assay. Cell cycle alternation and mitochondrial membrane potential were measured by flow cytometric analysis. Bax, Bcl-2, caspase-3, caspase-9, heat shock protein (Hsp)90, p53, p-p53, p21, Poly (ADP-ribose) polymerase (PARP), and the inhibitor of caspase-activated Dnase (ICAD) protein expressions were detected by Western blot analysis. Results: Oridonin inhibited cell growth in a time- and dose-dependent manner. Cell cycle was altered through the upregulation of p53 and p21 protein expressions. Pan-caspase inhibitor Z-VAD-fmk and calpain inhibitor Ⅱ both decreased cell death ratio. Nucleosomal DNA fragmentation and the downregulation of △ψmit were detected in oridonin-induced MCF-7 cell apoptosis, which was involved in a postmitochondrial caspase-9-dependent pathway. Decreased Bcl-2 and Hsp90 expression levels and increased Bax and p21 expression levels were positively correlated with elevated levels of phosphorylated p53 phosphorylation. Moreover, PARP was partially cleaved by calpain rather than by capase-3. Conclusion: DNA damage provoked alternations in the mitochondrial and caspase-9 pathways as well as p53-mediated cell cycle arrest, but was not related to caspase-3 activity in oridonin-induced MCF-7 cells.

  15. Redox-control of the alarmin, Interleukin-1α

    Directory of Open Access Journals (Sweden)

    Donald A. McCarthy

    2013-01-01

    Full Text Available The pro-inflammatory cytokine Interleukin-1α (IL-1α has recently emerged as a susceptibility marker for a wide array of inflammatory diseases associated with oxidative stress including Alzheimer's, arthritis, atherosclerosis, diabetes and cancer. In the present study, we establish that expression and nuclear localization of IL-1α are redox-dependent. Shifts in steady-state H2O2 concentrations (SS-[H2O2] resulting from enforced expression of manganese superoxide dismutase (SOD2 drive IL-1α mRNA and protein expression. The redox-dependent expression of IL-1α is accompanied by its increased nuclear localization. Both IL-1α expression and its nuclear residency are abrogated by catalase co-expression. Sub-lethal doses of H2O2 also cause IL-1α nuclear localization. Mutagenesis revealed IL-1α nuclear localization does not involve oxidation of cysteines within its N terminal domain. Inhibition of the processing enzyme calpain prevents IL-1α nuclear localization even in the presence of H2O2. H2O2 treatment caused extracellular Ca2+ influx suggesting oxidants may influence calpain activity indirectly through extracellular Ca2+ mobilization. Functionally, as a result of its nuclear activity, IL-1α overexpression promotes NF-kB activity, but also interacts with the histone acetyl transferase (HAT p300. Together, these findings demonstrate a mechanism by which oxidants impact inflammation through IL-1α and suggest that antioxidant-based therapies may prove useful in limiting inflammatory disease progression.

  16. Lens transcriptome profile during cataract development in Mip-null mice.

    Science.gov (United States)

    Bennett, Thomas M; Zhou, Yuefang; Shiels, Alan

    2016-09-16

    Major intrinsic protein or aquaporin-0 (MIP/AQP0) functions as a water channel and a cell-junction molecule in the vertebrate eye lens. Loss of MIP function in the lens leads to degraded optical quality and cataract formation by pathogenic mechanisms that are unclear. Here we have used microarray-hybridization analysis to detect lens transcriptome changes during cataract formation in mice that are functionally null for MIP (Mip-/-). In newborn Mip-/- lenses (P1) 11 genes were up-regulated and 18 were down-regulated (>2-fold, p=6-fold) in the Mip-/- lens at P1 included those coding for a mitochondrial translocase (Timmdc1), a matrix metallopeptidase (Mmp2), a Rho GTPase-interacting protein (Ubxn11) and a transcription factor (Twist2). Apart from Mip, the most down-regulated genes (>4-fold) in the Mip-/- lens at P1 included those coding for a proteasome sub-unit (Psmd8), a ribonuclease (Pop4), and a heat-shock protein (Hspb1). Lens fiber cell degeneration in the Mip-/- lens was associated with increased numbers of TUNEL-positive cell nuclei and dramatically elevated levels of calpain-mediated proteolysis of αII-spectrin. However red-ox status, measured by glutathione and free-radical levels, was similar to that of wild-type. These data suggest that while relatively few genes (∼1.5% of the transcriptome) were differentially regulated >2-fold in the Mip-/- lens, calpain hyper-activation acts as a terminal pathogenic event during lens fiber cell death and cataract formation. PMID:27524245

  17. Effect of compensatory growth on forms of glycogen, postmortem proteolysis, and meat quality in pigs.

    Science.gov (United States)

    Chaosap, C; Parr, T; Wiseman, J

    2011-07-01

    =0.167). There were no significant effects of the feeding regimen on micro- and milli-calpain large subunit gene expression (for micro-calpain at SL1, P=0.450; at SL2, P=0.171; at SL3, P=0.281; for milli-calpain at SL1, P=0.666; at SL2, P=0.123; at SL3, P=0.617) or the activity of the 2 proteolytic enzymes at any of the slaughter dates (for micro-calpain at SL1, P=0.238; at SL2, P =0.238; at SL3, P=0.222; for milli-calpain at SL1, P=0.296; at SL2, P=0.230; at SL3, P=0.615). In R40 there was a trend (P=0.070) for greater gene expression of caspase 3, whereas in R40A2 the increase was significant (P=0.009) relative to pigs consuming feed ad libitum. However, gene expression of the E3 ligase, MuRF1, at SL3 was less in R40A42 (P=0.019). Although compensatory growth does appear to influence the expression of various proteolytic systems, the changes do not appear to be associated with meat quality as measured by shear force.

  18. Validation of commercial DNA tests for quantitative beef quality traits.

    Science.gov (United States)

    Van Eenennaam, A L; Li, J; Thallman, R M; Quaas, R L; Dikeman, M E; Gill, C A; Franke, D E; Thomas, M G

    2007-04-01

    Associations between 3 commercially available genetic marker panels (GeneSTAR Quality Grade, GeneSTAR Tenderness, and Igenity Tender-GENE) and quantitative beef traits were validated by the US National Beef Cattle Evaluation Consortium. Validation was interpreted to be the independent confirmation of the associations between genetic tests and phenotypes, as claimed by the commercial genotyping companies. Validation of the quality grade test (GeneSTAR Quality Grade) was carried out on 400 Charolais x Angus crossbred cattle, and validation of the tenderness tests (GeneSTAR Tenderness and Igenity Tender-GENE) was carried out on over 1,000 Bos taurus and Bos indicus cattle. The GeneSTAR Quality Grade marker panel is composed of 2 markers (TG5, a SNP upstream from the start of the first exon of thyroglobulin, and QG2, an anonymous SNP) and is being marketed as a test associated with marbling and quality grade. In this validation study, the genotype results from this test were not associated with marbling score; however, the association of substituting favorable alleles of the marker panel with increased quality grade (percentage of cattle grading Choice or Prime) approached significance (P meat tenderness, as assessed by Warner-Bratzler shear force. These marker panels share 2 common mu-calpain SNP, but each has a different calpastatin SNP. In both panels, there were highly significant (P < 0.001) associations of the calpastatin marker and the mu-calpain haplotype with tenderness. The genotypic effects of the 2 tenderness panels were similar to each other, with a 1 kg difference in Warner-Bratzler shear force being observed between the most and least tender genotypes. Unbiased and independent validation studies are important to help build confidence in marker technology and also as a potential source of data required to enable the integration of marker data into genetic evaluations. As DNA tests associated with more beef production traits enter the marketplace, it will

  19. Association of CAPN-10 SNPs with Polycystic Ovary Syndrome%钙蛋白酶10基因多态性与多囊卵巢综合征

    Institute of Scientific and Technical Information of China (English)

    邓妙; 刘元伟; 张红艳; 岑加萍; 张治芬

    2014-01-01

    Polycystic ovary syndrome (PCOS) is a common endocrine disease in women. The phenomenon of familial aggregation suggests the role of genetic factors. Recent studies revealed that insulin resistanc (IR) played an important role in the pathophysiology of PCOS,most of the PCOS patients suffered from IR. Calpain-10 gene(CAPN-10) was the first positional cloned gene that was associated with type-2 diabetes mellitus (T2DM). Its mutation was relevant to IR, which affected the genetic susceptibility of T2DM. Due to the common mechanism of IR, CAPN-10 became an important candidate gene for PCOS. And difference in the genome sequence and modification was the fundamental reason for the difference of genetic susceptibility, in which the variation of single nucleotide polymorphisms (SNP) caused by 90%. Currently, data base of SNP (dbSNP) had listed 521 human CAPN-10 SNP locus. Now we will summarise the current status and progress in between CAPN-10 and PCOS.%多囊卵巢综合征(polycystic ovarian syndrome,PCOS)是女性常见的内分泌疾病,其家族聚集现象提示遗传因素的作用。近年研究认为,胰岛素抵抗(insulin resistance,IR)是PCOS病理生理的中心环节,多数PCOS患者存在IR。钙蛋白酶10(calpain-10,CAPN-10)基因是第1个被定位克隆的与2型糖尿病(type-2 diabetes mellitus,T2DM)有关的基因,该基因的变异与IR的发生有关,并影响T2DM的遗传易感性。因为PCOS与T2DM有共同的IR机制,CAPN-10也成为PCOS的重要候选基因。造成疾病遗传易感性差异的根本原因是基因组序列和修饰上的差异。其中,单核苷酸多态性(single nucleotide polymorphisms,SNP)引起的变异占90%。目前,SNP数据库(data base of SNP,dbSNP)已列出521个人类CAPN-10 SNP位点。综述CAPN-10与PCOS的研究现况,特别是CAPN-10 SNP与PCOS的研究进展。

  20. Eryptosis in lead-exposed workers

    Energy Technology Data Exchange (ETDEWEB)

    Aguilar-Dorado, Itzel-Citlalli [Biochemistry Department, Centro de Investigación y Estudios Avanzados IPN, México, DF (Mexico); Hernández, Gerardo [Section of Methodology of Science, Centro de Investigación y Estudios Avanzados IPN, México, DF (Mexico); Quintanar-Escorza, Martha-Angelica [Faculty of Medicine, UJED, Durango, DGO (Mexico); Maldonado-Vega, María [CIATEC, León, GTO (Mexico); Rosas-Flores, Margarita [Biochemistry Department, Centro de Investigación y Estudios Avanzados IPN, México, DF (Mexico); Calderón-Salinas, José-Víctor, E-mail: jcalder@cinvestav.mx [Biochemistry Department, Centro de Investigación y Estudios Avanzados IPN, México, DF (Mexico)

    2014-12-01

    Eryptosis is a physiological phenomenon in which old and damaged erythrocytes are removed from circulation. Erythrocytes incubated with lead have exhibited major eryptosis. In the present work we found evidence of high levels of eryptosis in lead exposed workers possibly via oxidation. Blood samples were taken from 40 male workers exposed to lead (mean blood lead concentration 64.8 μg/dl) and non-exposed workers (4.2 μg/dl). The exposure to lead produced an intoxication characterized by 88.3% less δ-aminolevulinic acid dehydratase (δALAD) activity in lead exposed workers with respect to non-lead exposed workers. An increment of oxidation in lead exposed workers was characterized by 2.4 times higher thiobarbituric acid-reactive substance (TBARS) concentration and 32.8% lower reduced/oxidized glutathione (GSH/GSSG) ratio. Oxidative stress in erythrocytes of lead exposed workers is expressed in 192% higher free calcium concentration [Ca{sup 2+}]{sub i} and 1.6 times higher μ-calpain activity with respect to non-lead exposed workers. The adenosine triphosphate (ATP) concentration was not significantly different between the two worker groups. No externalization of phosphatidylserine (PS) was found in non-lead exposed workers (< 0.1%), but lead exposed workers showed 2.82% externalization. Lead intoxication induces eryptosis possibly through a molecular pathway that includes oxidation, depletion of reduced glutathione (GSH), increment of [Ca{sup 2+}], μ-calpain activation and externalization of PS in erythrocytes. Identifying molecular signals that induce eryptosis in lead intoxication is necessary to understand its physiopathology and chronic complications. - Graphical abstract: Fig. 1. (A) Blood lead concentration (PbB) and (B) phosphatidylserine externalization on erythrocyte membranes of non-lead exposed (□) and lead exposed workers (■). Values are mean ± SD. *Significantly different (P < 0.001). - Highlights: • Erythrocytes of lead exposed workers

  1. 钙蛋白酶10基因多态性与多囊卵巢综合征%Association of CAPN-10 SNPs with Polycystic Ovary Syndrome

    Institute of Scientific and Technical Information of China (English)

    邓妙; 刘元伟; 张红艳; 岑加萍; 张治芬

    2014-01-01

    Polycystic ovary syndrome (PCOS) is a common endocrine disease in women. The phenomenon of familial aggregation suggests the role of genetic factors. Recent studies revealed that insulin resistanc (IR) played an important role in the pathophysiology of PCOS,most of the PCOS patients suffered from IR. Calpain-10 gene(CAPN-10) was the first positional cloned gene that was associated with type-2 diabetes mellitus (T2DM). Its mutation was relevant to IR, which affected the genetic susceptibility of T2DM. Due to the common mechanism of IR, CAPN-10 became an important candidate gene for PCOS. And difference in the genome sequence and modification was the fundamental reason for the difference of genetic susceptibility, in which the variation of single nucleotide polymorphisms (SNP) caused by 90%. Currently, data base of SNP (dbSNP) had listed 521 human CAPN-10 SNP locus. Now we will summarise the current status and progress in between CAPN-10 and PCOS.%多囊卵巢综合征(polycystic ovarian syndrome,PCOS)是女性常见的内分泌疾病,其家族聚集现象提示遗传因素的作用。近年研究认为,胰岛素抵抗(insulin resistance,IR)是PCOS病理生理的中心环节,多数PCOS患者存在IR。钙蛋白酶10(calpain-10,CAPN-10)基因是第1个被定位克隆的与2型糖尿病(type-2 diabetes mellitus,T2DM)有关的基因,该基因的变异与IR的发生有关,并影响T2DM的遗传易感性。因为PCOS与T2DM有共同的IR机制,CAPN-10也成为PCOS的重要候选基因。造成疾病遗传易感性差异的根本原因是基因组序列和修饰上的差异。其中,单核苷酸多态性(single nucleotide polymorphisms,SNP)引起的变异占90%。目前,SNP数据库(data base of SNP,dbSNP)已列出521个人类CAPN-10 SNP位点。综述CAPN-10与PCOS的研究现况,特别是CAPN-10 SNP与PCOS的研究进展。

  2. Autophagy-associated atrophy and metabolic remodeling of the mouse diaphragm after short-term intermittent hypoxia.

    Directory of Open Access Journals (Sweden)

    Christian Giordano

    Full Text Available Short-term intermittent hypoxia (IH is common in patients with acute respiratory disorders. Although prolonged exposure to hypoxia induces atrophy and increased fatigability of skeletal muscle, the response to short-term IH is less well known. We hypothesized that the diaphragm and limb muscles would adapt differently to short-term IH given that hypoxia stimulates ventilation and triggers a superimposed exercise stimulus in the diaphragm.We determined the structural, metabolic, and contractile properties of the mouse diaphragm after 4 days of IH (8 hours per day, 30 episodes per hour to a FiO2 nadir=6%, and compared responses in the diaphragm to a commonly studied reference limb muscle, the tibialis anterior. Outcome measures included muscle fiber size, assays of muscle proteolysis (calpain, ubiquitin-proteasome, and autophagy pathways, markers of oxidative stress and mitochondrial function, quantification of intramyocellular lipid and lipid metabolism genes, type I myosin heavy chain (MyHC expression, and in vitro contractile properties.After 4 days of IH, the diaphragm alone demonstrated significant atrophy (30% decrease of myofiber size together with increased LC3B-II protein (2.4-fold and mRNA markers of the autophagy pathway (LC3B, Gabarapl1, Bnip3, whereas active calpain and E3 ubiquitin ligases (MuRF1, atrogin-1 were unaffected in both muscles. Succinate dehydrogenase activity was significantly reduced by IH in both muscles. However, only the diaphragm exhibited increased intramyocellular lipid droplets (2.5-fold after IH, along with upregulation of genes linked to activated lipid metabolism. In addition, although the diaphragm showed evidence for acute fatigue immediately following IH, it underwent an adaptive fiber type switch toward slow type I MyHC-expressing fibers, associated with greater intrinsic endurance of the muscle during repetitive stimulation in vitro.Short-term IH induces preferential atrophy in the mouse diaphragm

  3. Oral treatment with the herbal formula B401 protects against aging-dependent neurodegeneration by attenuating oxidative stress and apoptosis in the brain of R6/2 mice

    Directory of Open Access Journals (Sweden)

    Wang SE

    2015-11-01

    Full Text Available Sheue-Er Wang,1,2 Ching-Lung Lin,1 Chih-Hsiang Hsu,1 Shuenn-Jyi Sheu,3 Chung-Hsin Wu1 1Department of Life Science, National Taiwan Normal University, Taipei, 2Department of Pathological Inspection, Saint Paul’s Hospital, Taoyuan, 3Brion Research Institute of Taiwan, Taipei, Taiwan Background: Neurodegeneration is characterized by progressive neurological deficits due to selective neuronal loss in the nervous system. Huntington’s disease (HD is an incurable neurodegenerative disorder. Neurodegeneration in HD patients shows aging-dependent pattern. Our previous study has suggested that a herbal formula B401 may have neuroprotective effects in the brains of R6/2 mice. Objective: To clarify possible mechanisms for neurodegeneration, which improves the understanding the aging process. This study focuses on clarifying neurodegenerative mechanisms and searching potential therapeutic targets in HD patients. Methods: The oxidative stress and apoptosis were compared in the brain tissue between R6/2 HD mice with and without oral B401 treatment. Expressions of proteins for oxidative stress and apoptosis in the brain tissue of R6/2 HD mice were examined by using immunostaining and Western blotting techniques. Results: R6/2 HD mice with oral B401 treatment significantly reduced reactive oxygen species levels in the blood, but markedly increased expressions of superoxide dismutase 2 in the brain tissue. Furthermore, R6/2 HD mice with oral B401 treatment significantly increased expressions of B-cell lymphoma 2 (Bcl-2, but significantly reduced expressions of Bcl-2-associated X protein (Bax, calpain, and caspase-3 in the brain tissue. Conclusion: Our findings provide evidence that the herbal formula B401 can remedy for aging-dependent neurodegeneration of R6/2 mice via suppressing oxidative stress and apoptosis in the brain. We suggest that the herbal formula B401 can be developed as a potential health supplement for ameliorating aging

  4. 牦牛 CAPN3基因的克隆及组织表达特异性%Cloning and Tissue-specific Expression of CAPN3 Gene in Yak

    Institute of Scientific and Technical Information of China (English)

    王英杰; 阎萍; 潘和平; 吴晓云; 李明霞

    2016-01-01

    Using the longissimus of yak(Bos grunniens)back as material, the CDs sequence of yak CAPN3 gene was cloned by RT-PCR, and it was analyzed by bioinformatics. The results indicated that the length of CDs in yak CAPN3 gene was 2 469 bp and encoding 822 amino acid residues. Bioinformatics analysis showed that the protein encoded by CAPN3 was the non-secretory surface protein, containing 35 phosphorylation site, and mainly played a biological role in the cytoplasm and nuclei. The secondary structure was mainly composed of α-helices, random coil, extended chain and β-turn, having the structure domains of CysPc, calpain-Ⅲ and EFh families and no signal peptide. The CAPN3 of yak was the most similar with those of Bos taurus, Ovis aries and Sus scrofa in phylogenetic tree. Real-time PCR analysis revealed the expressions of CAPN3 in varied tissues. There were expressions in all 7 tissues, and they were high in the muscle and pancreas.%以牦牛背最长肌为材料,采用 RT-PCR 法克隆了 CAPN3基因的 CDs 区,并对其进行生物信息学分析。结果表明,牦牛 CAPN3基因的 CDs 区长2469 bp,编码822个氨基酸残基;生物信息学分析显示,其编码的蛋白属于非分泌表面蛋白,含有35个磷酸化位点,主要在细胞质和细胞核中发挥生物学作用。二级结构主要由α-螺旋、无规则卷曲、伸展链和β-转角组成,具有CysPc、calpain-Ⅲ和 EFh 家族蛋白结构域,无信号肽。牦牛 CAPN3基因与黄牛、绵羊和猪在系统发育树上的距离最近。运用实时荧光定量分析 CAPN3在不同组织中的表达量,CAPN3基因在牦牛的7种组织中均有表达,但在背最长肌、胰脏中的表达量较高。

  5. Effect of Different Ages and Postmortem Aging on Tenderness of Cherry Valley Ducks Breast%不同日龄及宰后成熟对樱桃谷鸭嫩度的影响

    Institute of Scientific and Technical Information of China (English)

    邓方; 潘道东; 曹锦轩; 张小涛

    2013-01-01

    This research focused on the changes of tenderness of different ages cherry valley duck meat during postmortem aging.The effects of drip loss,cooking loss,shear force value,pH and calpain activity of duck breast meat were examined,and the degradation of Troponin-T and Desmin,which were heavily correlated with the tenderness of the protein,were also determined in the study.The result showed feed-day had a significant effect on the tenderness of duck meat (P<0.05),and the shear force value was positively correlated with postmortem time.pH and the shear force value of ducks muscle were positively correlation with postmortem time (P<0.05),while the cooking loss had a negative correlation with the shear force.The calpain activity were extremely significant (P<0.01) affected by shear force in the experiment.Meanwhile,the difference of the shear-force change was significant among the three day-age groups(P<0.01).%通过测定不同日龄樱桃谷鸭宰后不同时间的滴水损失、蒸煮损失、pH值、剪切力、钙蛋白酶活性等指标,同时结合SDS-PAGE和蛋白质印迹分析,研究骨骼肌中与肌肉嫩度高度相关的肌钙蛋白(Troponin-T)、肌间线蛋白(Desmin)的降解情况以及日龄对宰后鸭肉嫩度的影响.研究结果表明:随着日龄的增加,宰后肌肉剪切力显著增加(P<0.05);随着宰后成熟时间的延长,宰后肌肉剪切力减小.宰后成熟期间,pH值和肌肉剪切力呈显著正相关(P<0.05),蒸煮损失和肌肉剪切力呈显著负相关(P<0.05),钙蛋白酶酶活力和肌肉剪切力呈极显著正相关(P<0.01).增加日龄使得宰后肌肉剪切力增大,且日龄之间差异性极显著(P<0.01).

  6. Eryptosis in lead-exposed workers

    International Nuclear Information System (INIS)

    Eryptosis is a physiological phenomenon in which old and damaged erythrocytes are removed from circulation. Erythrocytes incubated with lead have exhibited major eryptosis. In the present work we found evidence of high levels of eryptosis in lead exposed workers possibly via oxidation. Blood samples were taken from 40 male workers exposed to lead (mean blood lead concentration 64.8 μg/dl) and non-exposed workers (4.2 μg/dl). The exposure to lead produced an intoxication characterized by 88.3% less δ-aminolevulinic acid dehydratase (δALAD) activity in lead exposed workers with respect to non-lead exposed workers. An increment of oxidation in lead exposed workers was characterized by 2.4 times higher thiobarbituric acid-reactive substance (TBARS) concentration and 32.8% lower reduced/oxidized glutathione (GSH/GSSG) ratio. Oxidative stress in erythrocytes of lead exposed workers is expressed in 192% higher free calcium concentration [Ca2+]i and 1.6 times higher μ-calpain activity with respect to non-lead exposed workers. The adenosine triphosphate (ATP) concentration was not significantly different between the two worker groups. No externalization of phosphatidylserine (PS) was found in non-lead exposed workers (< 0.1%), but lead exposed workers showed 2.82% externalization. Lead intoxication induces eryptosis possibly through a molecular pathway that includes oxidation, depletion of reduced glutathione (GSH), increment of [Ca2+], μ-calpain activation and externalization of PS in erythrocytes. Identifying molecular signals that induce eryptosis in lead intoxication is necessary to understand its physiopathology and chronic complications. - Graphical abstract: Fig. 1. (A) Blood lead concentration (PbB) and (B) phosphatidylserine externalization on erythrocyte membranes of non-lead exposed (□) and lead exposed workers (■). Values are mean ± SD. *Significantly different (P < 0.001). - Highlights: • Erythrocytes of lead exposed workers showed higher PS

  7. Oral treatment with herbal formula B307 alleviates cardiac failure in aging R6/2 mice with Huntington’s disease via suppressing oxidative stress, inflammation, and apoptosis

    Directory of Open Access Journals (Sweden)

    Lin CL

    2015-07-01

    Full Text Available Ching-Lung Lin,1 Sheue-Er Wang,2 Chih-Hsiang Hsu,1 Shuenn-Jyi Sheu,3 Chung-Hsin Wu1 1Department of Life Science, National Taiwan Normal University, Taipei, 2Department of Pathological Inspection, Soeurs de Saint Paul de Chartres Medical Corporate Body, Taoyuan City, 3Brion Research Institute of Taiwan, New Taipei City, Taiwan Abstract: Cardiac failure is often observed in aging patients with Huntington’s disease (HD. However, conventional pharmacological treatments for cardiac failure in HD patients have rarely been studied. Chinese herbal medicines, especially combined herbal formulas, have been widely used to treat cardiac dysfunctions over the centuries. Thus, we assess whether oral treatment with herbal formula B307 can alleviate cardiac failure in transgenic mice with HD. After oral B307 or vehicle treatment for 2 weeks, cardiac function and cardiomyocytes in 12-week-old male R6/2 HD mice and their wild-type littermate controls (WT were examined and then compared via echocardiography, immunohistochemistry, and Western blotting. We found that cardiac performance in aging R6/2 HD mice had significantly deteriorated in comparison with their WT (P<0.01. Cardiac expressions of superoxide dismutase 2 (SOD2 and B-cell lymphoma 2 (Bcl-2 in aging R6/2 HD mice were significantly lower than their WT (P<0.01, but cardiac expressions of tumor necrosis factor alpha (TNF-α, neurotrophin-3 (3-NT, 4-hydroxynonenal (4-HNE, Bcl-2-associated X protein (Bax, calpain, caspase 12, caspase 9, and caspase 3 of aging R6/2 HD mice were significantly higher than their WT (P<0.05. Furthermore, we found that cardiac performance in aging R6/2 HD mice had significantly improved under oral B307 treatment (P<0.05. Cardiac expressions of SOD2 and Bcl-2 of aging R6/2 HD mice were significantly higher under oral B307 treatment (P<0.01, but cardiac expressions of TNF-α, 3-NT, 4-HNE, Bax, calpain, caspase 12, caspase 9, and caspase 3 of aging R6/2 HD mice were significantly

  8. Changes in oxidative stress parameters and neurodegeneration markers in the brain of the senescence-accelerated mice SAMP-8.

    Science.gov (United States)

    Sureda, Francesc X; Gutierrez-Cuesta, Javier; Romeu, Marta; Mulero, Miquel; Canudas, Anna Maria; Camins, Antoni; Mallol, Jordi; Pallàs, Mercè

    2006-04-01

    The senescence-accelerated strains of mice (SAMP) are well-characterized animal models of senescence. Senescence may be related to enhanced production or defective control of reactive oxygen species, which lead to neuronal damage. Therefore, the activity of various oxidative-stress related enzymes was determined in the cortex of 5 months-old senescence-accelerated mice prone-8 (SAMP-8) of both sexes and compared with senescence-accelerated mice-resistant-1 (SAMR-1). Glutathione reductase and peroxidase activities in SAMP-8 male mice were lower than in male SAMR-1, and a decreased catalase activity was found in both male and female SAMP-8 mice, which correlates with the lower catalase expression found by Western blotting. Nissl staining showed marked loss of neuronal cells in the cerebral cortex of five month-old SAMP-8 mice. SAMP-8 mice also had marked astrogliosis and microgliosis. We also found an increase in caspase-3 and calpain activity in the cortex. In addition, we observed morphological changes in the immunostaining of tau protein in SAMP-8, indicative of a loss of their structural function. Altogether, these results show that, at as early as 5 months of age, SAMP-8 mice have cytological and molecular alterations indicative of neurodegeneration in the cerebral cortex and suggestive of altered control of the production of oxidative species and hyper-activation of calcium-dependent enzymes. PMID:16542809

  9. Bicarbonate-dependent secretion and proteolytic processing of recombinant myocilin.

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    José-Daniel Aroca-Aguilar

    Full Text Available Myocilin is an extracellular glycoprotein of poorly understood function. Mutations of this protein are involved in glaucoma, an optic neuropathy characterized by a progressive and irreversible visual loss and frequently associated with elevated intraocular pressure. We previously showed that recombinant myocilin undergoes an intracellular proteolytic processing by calpain II which cleaves the central region of the protein, releasing one N- and one C-terminal fragment. Myocilin cleavage is reduced by glaucoma mutations and it has been proposed to participate in intraocular pressure modulation. To identify possible factors regulating the proteolytic processing of recombinant myocilin, we used a cellular model in which we analyzed how different culture medium parameters (i.e., culture time, cell density, pH, bicarbonate concentration, etc. affect the presence of the extracellular C-terminal fragment. Extracellular bicarbonate depletion associated with culture medium acidification produced a reversible intracellular accumulation of full-length recombinant myocilin and incremented its intracellular proteolytic processing, raising the extracellular C-terminal fragment percentage. It was also determined that myocilin intracellular accumulation depends on its N-terminal region. These data suggest that aqueous humor bicarbonate variations could also modulate the secretion and cleavage of myocilin present in ocular tissues.

  10. Neuronal Nicotinic Receptors as New Targets for Amphetamine-Induced Oxidative Damage and Neurotoxicity

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    Elena Escubedo

    2011-06-01

    Full Text Available Amphetamine derivatives such as methamphetamine (METH and 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy” are widely abused drugs in a recreational context. This has led to concern because of the evidence that they are neurotoxic in animal models and cognitive impairments have been described in heavy abusers. The main targets of these drugs are plasmalemmal and vesicular monoamine transporters, leading to reverse transport and increased monoamine efflux to the synapse. As far as neurotoxicity is concerned, increased reactive oxygen species (ROS production seems to be one of the main causes. Recent research has demonstrated that blockade of a7 nicotinic acetylcholine receptors (nAChR inhibits METH- and MDMA-induced ROS production in striatal synaptosomes which is dependent on calcium and on NO-synthase activation. Moreover, a7 nAChR antagonists (methyllycaconitine and memantine attenuated in vivo the neurotoxicity induced by METH and MDMA, and memantine prevented the cognitive impairment induced by these drugs. Radioligand binding experiments demonstrated that both drugs have affinity to a7 and heteromeric nAChR, with MDMA showing lower Ki values, while fluorescence calcium experiments indicated that MDMA behaves as a partial agonist on a7 and as an antagonist on heteromeric nAChR. Sustained Ca increase led to calpain and caspase-3 activation. In addition, modulatory effects of MDMA on a7 and heteromeric nAChR populations have been found.

  11. Characterisation in vivo of ways of induced deaths by p53, in the male germinal cells

    International Nuclear Information System (INIS)

    The male germinal cells constitute a heterogeneous cell population including pre-meiotic proliferating cells (spermatogonia) and meiotic cells and post meiotic cells in differentiation (spermatocytes and spermatids). We study the involvement in vivo of the p53 protein in the death of these cells with the help of two models, (1) a transgenic model of infertility, MTp53, in which the p53 is over expressed in the differentiated cells and induced their death, (2) the response of these cells to gamma irradiation, where only the spermatogonia die by apoptosis dependent of p53. We showed that the caspases (cysteine-aspartic proteases) are involved in the terminal differentiation of normal germinal cells. But in the MTp53 model, the p53 induces the death of differentiated cells via the activation of calpains and not of caspases. We studied the response of spermatogonia, to gamma irradiation by a transcriptomic approach, by DNA chips and semi-quantitative RT-PCR. we showed that the puma and dr5 genes are induced by the p53 after irradiation. more, the study of mice invalidated for trail ( the dr5 ligand) or for puma, allowed to demonstrate that the two effectors are essential to the activation of intrinsic and extrinsic ways of apoptosis. (N.C.)

  12. Plasma membrane calcium pump regulation by metabolic stress

    Institute of Scientific and Technical Information of China (English)

    Jason; IE; Bruce

    2010-01-01

    The plasma membrane Ca2+-ATPase(PMCA)is an ATPdriven pump that is critical for the maintenance of low resting[Ca2+]i in all eukaryotic cells.Metabolic stress, either due to inhibition of mitochondrial or glycolytic metabolism,has the capacity to cause ATP depletion and thus inhibit PMCA activity.This has potentially fatal consequences,particularly for non-excitable cells in which the PMCA is the major Ca2+efflux pathway.This is because inhibition of the PMCA inevitably leads to cytosolic Ca2+ overload and the consequent cell death.However,the relationship between metabolic stress,ATP depletion and inhibition of the PMCA is not as simple as one would have originally predicted.There is increasing evidence that metabolic stress can lead to the inhibition of PMCA activity independent of ATP or prior to substantial ATP depletion.In particular,there is evidence that the PMCA has its own glycolytic ATP supply that can fuel the PMCA in the face of impaired mitochondrial function.Moreover, membrane phospholipids,mitochondrial membrane potential,caspase/calpain cleavage and oxidative stress have all been implicated in metabolic stress-induced inhibition of the PMCA.The major focus of this review is to challenge the conventional view of ATP-dependent regulation of the PMCA and bring together some of the alternative or additional mechanisms by which metabolic stress impairs PMCA activity resulting in cytosolic Ca2+ overload and cytotoxicity.

  13. Increased expression of stefin B in the nucleus of T98G astrocytoma cells delays caspase activation

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    Tao eSun

    2012-09-01

    Full Text Available Stefin B (cystatin B is an endogenous inhibitor of cysteine proteinases localized in the nucleus and the cytosol. Loss-of-function mutations in the stefin B gene (CSTB gene were reported in patients with Unverricht-Lundborg disease (EPM1. Our previous results showed that thymocytes isolated from stefin B-deficient mice are more sensitive to apoptosis induced by the protein kinase C inhibitor staurosporin (STS than the wild-type control cells. We have also shown that the increased expression of stefin B in the nucleus of T98G astrocytoma cells delayed cell cycle progression through the S phase. In the present study we examined if the nuclear or cytosolic functions of stefin B are responsible for the accelerated induction of apoptosis observed in the cells from stefin B-deficient mice. We have shown that the overexpression of stefin B in the nucleus, but not in the cytosol of astrocytoma T98G cells, delayed caspase-3 and-7 activation. Pretreatment of cells with the pan-caspase inhibitor z-Val-Ala-Asp(OMe-fluoromethylketone completely inhibited caspase activation, while treatment with the inhibitor of calpains- and papain-like cathepsins (2S,3S-trans-epoxysuccinyl-leucylamido-3-methyl-butane ethyl ester did not prevent caspase activation. We concluded that the delay of caspase activation in T98G cells overexpressing stefin B in the nucleus is independent of cathepsin inhibition.

  14. Mechanisms of excitation-contraction uncoupling relevant to activity-induced muscle fatigue.

    Science.gov (United States)

    Lamb, Graham D

    2009-06-01

    If the free [Ca2+] in the cytoplasm of a skeletal muscle fiber is raised substantially for a period of seconds to minutes or to high levels just briefly, it leads to disruption of the normal excitation-contraction (E-C) coupling process and a consequent long-lasting decrease in force production. It appears that the disruption to the coupling occurs at the triad junction, where the voltage-sensor molecules (dihydropyridine receptors) normally interact with and open the Ca2+ release channels (ryanodine receptors) in the adjacent sarcoplasmic reticulum (SR). This disruption results in inadequate release of SR Ca2+ upon stimulation. Such E-C uncoupling may underlie the long-duration low-frequency fatigue that can occur after various types of exercise, as well as possibly being a contributing factor to the muscle weakness in certain muscle diseases. The process or processes causing the disruption of the coupling between the voltage sensors and the release channels is not known with certainty, but might be associated with structural changes at the triad junction, possibly caused by activation of the Ca2+-dependent protease, micro-calpain.

  15. Anthranilate fluorescence marks a calcium-propagated necrotic wave that promotes organismal death in C. elegans.

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    Cassandra Coburn

    2013-07-01

    Full Text Available For cells the passage from life to death can involve a regulated, programmed transition. In contrast to cell death, the mechanisms of systemic collapse underlying organismal death remain poorly understood. Here we present evidence of a cascade of cell death involving the calpain-cathepsin necrosis pathway that can drive organismal death in Caenorhabditis elegans. We report that organismal death is accompanied by a burst of intense blue fluorescence, generated within intestinal cells by the necrotic cell death pathway. Such death fluorescence marks an anterior to posterior wave of intestinal cell death that is accompanied by cytosolic acidosis. This wave is propagated via the innexin INX-16, likely by calcium influx. Notably, inhibition of systemic necrosis can delay stress-induced death. We also identify the source of the blue fluorescence, initially present in intestinal lysosome-related organelles (gut granules, as anthranilic acid glucosyl esters--not, as previously surmised, the damage product lipofuscin. Anthranilic acid is derived from tryptophan by action of the kynurenine pathway. These findings reveal a central mechanism of organismal death in C. elegans that is related to necrotic propagation in mammals--e.g., in excitotoxicity and ischemia-induced neurodegeneration. Endogenous anthranilate fluorescence renders visible the spatio-temporal dynamics of C. elegans organismal death.

  16. Targeting p35/Cdk5 Signalling via CIP-Peptide Promotes Angiogenesis in Hypoxia

    Science.gov (United States)

    Bosutti, Alessandra; Qi, Jie; Pennucci, Roberta; Bolton, David; Matou, Sabine; Ali, Kamela; Tsai, Li-Huei; Krupinski, Jerzy; Petcu, Eugene B.; Montaner, Joan; Al Baradie, Raid; Caccuri, Francesca; Caruso, Arnaldo; Alessandri, Giulio; Kumar, Shant; Rodriguez, Cristina; Martinez-Gonzalez, Jose; Slevin, Mark

    2013-01-01

    Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition. PMID:24098701

  17. Targeting p35/Cdk5 signalling via CIP-peptide promotes angiogenesis in hypoxia.

    Directory of Open Access Journals (Sweden)

    Alessandra Bosutti

    Full Text Available Cyclin-dependent kinase-5 (Cdk5 is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition.

  18. Resveratrol Induces Apoptosis-Like Death and Prevents In Vitro and In Vivo Virulence of Entamoeba histolytica

    Science.gov (United States)

    Pais-Morales, Jonnatan; Betanzos, Abigail; García-Rivera, Guillermina; Chávez-Munguía, Bibiana; Shibayama, Mineko; Orozco, Esther

    2016-01-01

    Entamoeba histolytica causes amoebiasis, an infection that kills 100,000 individuals each year. Metronidazole and its derivatives are currently used against this protozoan, but these drugs present adverse effects on human health. Here, we investigated the effect of resveratrol (a natural compound) on E. histolytica trophozoites viability, as well as its influence on the parasite virulence. Trophozoites growth was arrested by 72 μM resveratrol and the IC50 was determined as 220 μM at 48 h. Cells appeared smaller, rounded and in clusters, with debris-containing vacuoles and with abnormally condensed chromatin. Resveratrol triggered reactive oxygen species production. It caused lipid peroxidation and produced phosphatidylserine externalization and DNA fragmentation this latter evidenced by TUNEL assays. It also provoked an increase of intracellular Ca2+ concentration, activated calpain and decreased superoxide dismutase activity, indicating that an apoptosis-like event occurred; however, autophagy was not detected. Cytopathic activity, phagocytosis, encystment and in vivo virulence were diminished dramatically by pre-incubation of trophozoites with resveratrol, evidencing that resveratrol attenuated the trophozoite virulence in vitro. Interestingly, after the inoculation of virulent trophozoites, animals treated with the drug did not develop or developed very small abscesses. Our findings propose that resveratrol could be an alternative to contend amoebiasis. PMID:26731663

  19. Effects of chronic administration of clenbuterol on contractile properties and calcium homeostasis in rat extensor digitorum longus muscle.

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    Pascal Sirvent

    Full Text Available Clenbuterol, a β2-agonist, induces skeletal muscle hypertrophy and a shift from slow-oxidative to fast-glycolytic muscle fiber type profile. However, the cellular mechanisms of the effects of chronic clenbuterol administration on skeletal muscle are not completely understood. As the intracellular Ca2+ concentration must be finely regulated in many cellular processes, the aim of this study was to investigate the effects of chronic clenbuterol treatment on force, fatigue, intracellular calcium (Ca2+ homeostasis and Ca2+-dependent proteolysis in fast-twitch skeletal muscles (the extensor digitorum longus, EDL, muscle, as they are more sensitive to clenbuterol-induced hypertrophy. Male Wistar rats were chronically treated with 4 mg.kg-1 clenbuterol or saline vehicle (controls for 21 days. Confocal microscopy was used to evaluate sarcoplasmic reticulum Ca2+ load, Ca2+-transient amplitude and Ca2+ spark properties. EDL muscles from clenbuterol-treated animals displayed hypertrophy, a shift from slow to fast fiber type profile and increased absolute force, while the relative force remained unchanged and resistance to fatigue decreased compared to control muscles from rats treated with saline vehicle. Compared to control animals, clenbuterol treatment decreased Ca2+-transient amplitude, Ca2+ spark amplitude and frequency and the sarcoplasmic reticulum Ca2+ load was markedly reduced. Conversely, calpain activity was increased by clenbuterol chronic treatment. These results indicate that chronic treatment with clenbuterol impairs Ca2+ homeostasis and this could contribute to the remodeling and functional impairment of fast-twitch skeletal muscle.

  20. Nisin ZP, a Bacteriocin and Food Preservative, Inhibits Head and Neck Cancer Tumorigenesis and Prolongs Survival.

    Science.gov (United States)

    Kamarajan, Pachiyappan; Hayami, Takayuki; Matte, Bibiana; Liu, Yang; Danciu, Theodora; Ramamoorthy, Ayyalusamy; Worden, Francis; Kapila, Sunil; Kapila, Yvonne

    2015-01-01

    The use of small antimicrobial peptides or bacteriocins, like nisin, to treat cancer is a new approach that holds great promise. Nisin exemplifies this new approach because it has been used safely in humans for many years as a food preservative, and recent laboratory studies support its anti-tumor potential in head and neck cancer. Previously, we showed that nisin (2.5%, low content) has antitumor potential in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. The current studies explored a naturally occurring variant of nisin (nisin ZP; 95%, high content) for its antitumor effects in vitro and in vivo. Nisin ZP induced the greatest level of apoptosis in HNSCC cells compared to low content nisin. HNSCC cells treated with increasing concentrations of nisin ZP exhibited increasing levels of apoptosis and decreasing levels of cell proliferation, clonogenic capacity, and sphere formation. Nisin ZP induced apoptosis through a calpain-dependent pathway in HNSCC cells but not in human oral keratinocytes. Nisin ZP also induced apoptosis dose-dependently in human umbilical vein endothelial cells (HUVEC) with concomitant decreases in vascular sprout formation in vitro and reduced intratumoral microvessel density in vivo. Nisin ZP reduced tumorigenesis in vivo and long-term treatment with nisin ZP extended survival. In addition, nisin treated mice exhibited normal organ histology with no evidence of inflammation, fibrosis or necrosis. In summary, nisin ZP exhibits greater antitumor effects than low content nisin, and thus has the potential to serve as a novel therapeutic for HNSCC.

  1. 氧化应激对心房颤动犬心房肌细胞凋亡及凋亡相关蛋白影响的研究%The effects of oxidative stress on atrial myocardial cells apoptosis and expression of apoptosis protein in atrial fibrillation canines

    Institute of Scientific and Technical Information of China (English)

    盛力; 单鸿波; 刘洁; 李悦; 李为民; 公永太; 杨宝峰; 薛红杰; 刘巍; 初杉; 张莉

    2008-01-01

    目的 观察普罗布考(probucol)对长期心房快速起搏诱发心房颤动(房颤)犬心房肌细胞凋亡及凋亡相关蛋白表达的影响,探讨氧化应激在房颤心房结构重构中的作用.方法 杂种犬20只,随机分为假手术组(n=6)、对照组(n=7)和普罗布考组(n=7).无菌条件下开胸后在犬右心房缝植4对心外膜记录电极,电极尾端经皮下由犬背部穿出;在右心耳缝植螺旋型起搏电极,连接实验用AOO高频起搏器(400次/min),心房快速起搏6周,建立房颤犬模型;假手术组犬仅缝植心外膜记录电极和起搏电极但不起搏;对照组及普罗布考组犬心房快速起搏6周;普罗布考组于起搏前一周开始服用普罗布考(100 mg·kg-1·d-1),直至起搏结束.TUNEL法检测心房肌细胞凋亡情况;免疫组化方法及免疫印记法检测凋亡相关蛋白caspase-3、bcl-2和bax表达情况;免疫组化方法检测calpain Ⅰ表达;比色法检测心房肌总抗氧化能力(T-AOC)、丙二醛(MDA)和抗超氧阴离子(抗O2-)水平;于起搏前、起博6周后,经心外膜电极记录各组犬房颤诱发情况.结果 与假手术组犬相比,对照组犬左、右心房肌凋亡细胞数量显著增加[(44.3±9.7)% vs (1.36±0.70)%,(42.1±11.9)% vs (1.07±0.50)%,P<0.01],心房肌caspase-3、bax和calpain Ⅰ表达明显上调(P<0.01),bcl-2表达显著下调(P<0.01).与对照组犬相比,普罗布考组犬左、右心房肌凋亡细胞数量明显减少[(21.4±5.8)% vs (44.3±9.7)%,(20.1±6.1)% vs (42.1±11.9)%,P<0.01],calpain Ⅰ、caspase-3和bax表达显著下调(P<0.01),bcl-2表达增加(P<0.05).与假手术组犬相比对照组犬心房肌MDA水平明显增加(P<0.01),T-AOC、抗O2-水平明显降低(P<0.01);与对照组犬相比,普罗布考组犬心房肌MDA水平显著降低(P<0.05),T-AOC、抗O2-水平显著增加(P<0.01).对照组和普罗布考组起搏后房颤诱发率和平均持续时间均较起搏前显著增加(P<0.05);起搏后

  2. Triggers, Inhibitors, Mechanisms, and Significance of Eryptosis: The Suicidal Erythrocyte Death

    Directory of Open Access Journals (Sweden)

    Elisabeth Lang

    2015-01-01

    Full Text Available Suicidal erythrocyte death or eryptosis is characterized by erythrocyte shrinkage, cell membrane blebbing, and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca2+ entry, ceramide formation, stimulation of caspases, calpain activation, energy depletion, oxidative stress, and dysregulation of several kinases. Eryptosis is triggered by a wide variety of xenobiotics. It is inhibited by several xenobiotics and endogenous molecules including NO and erythropoietin. The susceptibility of erythrocytes to eryptosis increases with erythrocyte age. Phosphatidylserine exposing erythrocytes adhere to the vascular wall by binding to endothelial CXC-Motiv-Chemokin-16/Scavenger-receptor for phosphatidylserine and oxidized low density lipoprotein (CXCL16. Phosphatidylserine exposing erythrocytes are further engulfed by phagocytosing cells and are thus rapidly cleared from circulating blood. Eryptosis eliminates infected or defective erythrocytes thus counteracting parasitemia in malaria and preventing detrimental hemolysis of defective cells. Excessive eryptosis, however, may lead to anemia and may interfere with microcirculation. Enhanced eryptosis contributes to the pathophysiology of several clinical disorders including metabolic syndrome and diabetes, malignancy, cardiac and renal insufficiency, hemolytic uremic syndrome, sepsis, mycoplasma infection, malaria, iron deficiency, sickle cell anemia, thalassemia, glucose 6-phosphate dehydrogenase deficiency, and Wilson’s disease. Facilitating or inhibiting eryptosis may be a therapeutic option in those disorders.

  3. Dietary protein deficiency reduces lysosomal and nonlysosomal ATP-dependent proteolysis in muscle

    Science.gov (United States)

    Tawa, N. E. Jr; Kettelhut, I. C.; Goldberg, A. L.

    1992-01-01

    When rats are fed a protein deficient (PD) diet for 7 days, rates of proteolysis in skeletal muscle decrease by 40-50% (N. E. Tawa, Jr., and A. L. Goldberg. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E317-325, 1992). To identify the underlying biochemical adaptations, we measured different proteolytic processes in incubated muscles. The capacity for intralysosomal proteolysis, as shown by sensitivity to methylamine or lysosomal protease inhibitors, fell 55-75% in muscles from PD rats. Furthermore, extracts of muscles of PD rats showed 30-70% lower activity of many lysosomal proteases, including cathepsins B, H, and C, and carboxypeptidases A and C, as well as other lysosomal hydrolases. The fall in cathepsin B and proteolysis was evident by 3 days on the PD diet, and both returned to control levels 3 days after refeeding of the normal diet. In muscles maintained under optimal conditions, 80-90% of protein breakdown occurs by nonlysosomal pathways. In muscles of PD rats, this ATP-dependent process was also 40-60% slower. Even though overall proteolysis decreased in muscles of PD rats, their capacity for Ca(2+)-dependent proteolysis increased (by 66%), as did the activity of the calpains (+150-250%). Thus the lysosomal and the ATP-dependent processes decrease coordinately and contribute to the fall in muscle proteolysis in PD animals.

  4. DNA damage, somatic aneuploidy, and malignant sarcoma susceptibility in muscular dystrophies.

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    Wolfgang M Schmidt

    2011-04-01

    Full Text Available Albeit genetically highly heterogeneous, muscular dystrophies (MDs share a convergent pathology leading to muscle wasting accompanied by proliferation of fibrous and fatty tissue, suggesting a common MD-pathomechanism. Here we show that mutations in muscular dystrophy genes (Dmd, Dysf, Capn3, Large lead to the spontaneous formation of skeletal muscle-derived malignant tumors in mice, presenting as mixed rhabdomyo-, fibro-, and liposarcomas. Primary MD-gene defects and strain background strongly influence sarcoma incidence, latency, localization, and gender prevalence. Combined loss of dystrophin and dysferlin, as well as dystrophin and calpain-3, leads to accelerated tumor formation. Irrespective of the primary gene defects, all MD sarcomas share non-random genomic alterations including frequent losses of tumor suppressors (Cdkn2a, Nf1, amplification of oncogenes (Met, Jun, recurrent duplications of whole chromosomes 8 and 15, and DNA damage. Remarkably, these sarcoma-specific genetic lesions are already regularly present in skeletal muscles in aged MD mice even prior to sarcoma development. Accordingly, we show also that skeletal muscle from human muscular dystrophy patients is affected by gross genomic instability, represented by DNA double-strand breaks and age-related accumulation of aneusomies. These novel aspects of molecular pathologies common to muscular dystrophies and tumor biology will potentially influence the strategies to combat these diseases.

  5. Evidence that a panel of neurodegeneration biomarkers predicts vasospasm, infarction, and outcome in aneurysmal subarachnoid hemorrhage.

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    Robert Siman

    Full Text Available Biomarkers for neurodegeneration could be early prognostic measures of brain damage and dysfunction in aneurysmal subarachnoid hemorrhage (aSAH with clinical and medical applications. Recently, we developed a new panel of neurodegeneration biomarkers, and report here on their relationships with pathophysiological complications and outcomes following severe aSAH. Fourteen patients provided serial cerebrospinal fluid samples for up to 10 days and were evaluated by ultrasonography, angiography, magnetic resonance imaging, and clinical examination. Functional outcomes were assessed at hospital discharge and 6-9 months thereafter. Eight biomarkers for acute brain damage were quantified: calpain-derived α-spectrin N- and C-terminal fragments (CCSntf and CCSctf, hypophosphorylated neurofilament H,14-3-3 β and ζ, ubiquitin C-terminal hydrolase L1, neuron-specific enolase, and S100β. All 8 biomarkers rose up to 100-fold in a subset of patients. Better than any single biomarker, a set of 6 correlated significantly with cerebral vasospasm, brain infarction, and poor outcome. Furthermore, CSF levels of 14-3-3β, CCSntf, and NSE were early predictors of subsequent moderate-to-severe vasospasm. These data provide evidence that a panel of neurodegeneration biomarkers may predict lasting brain dysfunction and the pathophysiological processes that lead to it following aSAH. The panel may be valuable as surrogate endpoints for controlled clinical evaluation of treatment interventions and for guiding aSAH patient care.

  6. A novel recombinant 6Aβ15-THc-C chimeric vaccine (rCV02) mitigates Alzheimer’s disease-like pathology, cognitive decline and synaptic loss in aged 3 × Tg-AD mice

    Science.gov (United States)

    Yu, Yun-Zhou; Liu, Si; Wang, Hai-Chao; Shi, Dan-Yang; Xu, Qing; Zhou, Xiao-Wei; Sun, Zhi-Wei; Huang, Pei-Tang

    2016-01-01

    Alzheimer’s disease (AD) is a neurodegenerative disorder that impairs memory and cognition. Targeting amyloid-β (Aβ) may be currently the most promising immunotherapeutic strategy for AD. In this study, a recombinant chimeric 6Aβ15-THc-C immunogen was formulated with alum adjuvant as a novel Aβ B-cell epitope candidate vaccine (rCV02) for AD. We examined its efficacy in preventing the cognitive deficit and synaptic impairment in 3 × Tg-AD mice. Using a toxin-derived carrier protein, the rCV02 vaccine elicited robust Aβ-specific antibodies that markedly reduced AD-like pathology and improved behavioral performance in 3 × Tg-AD mice. Along with the behavioral improvement in aged 3 × Tg-AD mice, rCV02 significantly decreased calpain activation concurrent with reduced soluble Aβ or oligomeric forms of Aβ, probably by preventing dynamin 1 and PSD-95 degradation. Our data support the hypothesis that reducing Aβ levels in rCV02-immunized AD mice increases the levels of presynaptic dynamin 1 and postsynaptic PSD-95 allowing functional recovery of cognition. In conclusion, this novel and highly immunogenic rCV02 shows promise as a new candidate prophylactic vaccine for AD and may be useful for generating rapid and strong Aβ-specific antibodies in AD patients with pre-existing memory Th cells generated after immunization with conventional tetanus toxoid vaccine. PMID:27255752

  7. A novel recombinant 6Aβ15-THc-C chimeric vaccine (rCV02) mitigates Alzheimer's disease-like pathology, cognitive decline and synaptic loss in aged 3 × Tg-AD mice.

    Science.gov (United States)

    Yu, Yun-Zhou; Liu, Si; Wang, Hai-Chao; Shi, Dan-Yang; Xu, Qing; Zhou, Xiao-Wei; Sun, Zhi-Wei; Huang, Pei-Tang

    2016-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder that impairs memory and cognition. Targeting amyloid-β (Aβ) may be currently the most promising immunotherapeutic strategy for AD. In this study, a recombinant chimeric 6Aβ15-THc-C immunogen was formulated with alum adjuvant as a novel Aβ B-cell epitope candidate vaccine (rCV02) for AD. We examined its efficacy in preventing the cognitive deficit and synaptic impairment in 3 × Tg-AD mice. Using a toxin-derived carrier protein, the rCV02 vaccine elicited robust Aβ-specific antibodies that markedly reduced AD-like pathology and improved behavioral performance in 3 × Tg-AD mice. Along with the behavioral improvement in aged 3 × Tg-AD mice, rCV02 significantly decreased calpain activation concurrent with reduced soluble Aβ or oligomeric forms of Aβ, probably by preventing dynamin 1 and PSD-95 degradation. Our data support the hypothesis that reducing Aβ levels in rCV02-immunized AD mice increases the levels of presynaptic dynamin 1 and postsynaptic PSD-95 allowing functional recovery of cognition. In conclusion, this novel and highly immunogenic rCV02 shows promise as a new candidate prophylactic vaccine for AD and may be useful for generating rapid and strong Aβ-specific antibodies in AD patients with pre-existing memory Th cells generated after immunization with conventional tetanus toxoid vaccine. PMID:27255752

  8. Two proteolytic fragments of menin coordinate the nuclear transcription and postsynaptic clustering of neurotransmitter receptors during synaptogenesis between Lymnaea neurons

    Science.gov (United States)

    Getz, Angela M.; Visser, Frank; Bell, Erin M.; Xu, Fenglian; Flynn, Nichole M.; Zaidi, Wali; Syed, Naweed I.

    2016-01-01

    Synapse formation and plasticity depend on nuclear transcription and site-specific protein targeting, but the molecular mechanisms that coordinate these steps have not been well defined. The MEN1 tumor suppressor gene, which encodes the protein menin, is known to induce synapse formation and plasticity in the CNS. This synaptogenic function has been conserved across evolution, however the underlying molecular mechanisms remain unidentified. Here, using central neurons from the invertebrate Lymnaea stagnalis, we demonstrate that menin coordinates subunit-specific transcriptional regulation and synaptic clustering of nicotinic acetylcholine receptors (nAChR) during neurotrophic factor (NTF)-dependent excitatory synaptogenesis, via two proteolytic fragments generated by calpain cleavage. Whereas menin is largely regarded as a nuclear protein, our data demonstrate a novel cytoplasmic function at central synapses. Furthermore, this study identifies a novel synaptogenic mechanism in which a single gene product coordinates the nuclear transcription and postsynaptic targeting of neurotransmitter receptors through distinct molecular functions of differentially localized proteolytic fragments. PMID:27538741

  9. Single nucleotide polymorphisms associated with carcass traits in a population of Brahman and Brahman-influenced steers.

    Science.gov (United States)

    Royer, A M; Shivers, C; Riley, D G; Elzo, M A; Garcia, M D

    2016-01-01

    Brahman cattle are important in tropical regions due to their ability to tolerate excessive heat and parasites. However, Brahman cattle exhibit lower carcass quality characteristics when compared to Bos taurus breeds. The objective of this study was to evaluate potential associations between single nucleotide polymorphisms (SNPs) in six candidate genes for carcass quality and composition traits in a population of Brahman and Brahman-influenced steers. Steers were evaluated through the American Brahman Breeders Association carcass evaluation project in Gonzales, Texas. Carcass traits measured included hot carcass weight, ribeye area, marbling score, yield grade, quality grade, dressing percent, and Warner-Bratzler shear force score. Six previously described candidate genes were chosen for SNP analysis based on their previous association with growth and carcass traits. Candidate genes utilized in the current study included calpastatin (CAST), calpain (CAPN3), thyroglobulin (TG), growth hormone, insulin growth factor 1, and adiponectin. Six unique SNPs from three candidate genes (TG, CAST, and CAPN3) were significantly associated (P carcass quality traits (marbling score and quality grade). A genotypic effect was observed for all significant SNPs, with differing levels of performance observed for animals inheriting different genotypes. Although multiple SNPs in the current study were significantly (P carcass traits, they should be validated in larger populations prior to implementation in selection strategies.

  10. Evolution of EF-hand calcium-modulated proteins. IV. Exon shuffling did not determine the domain compositions of EF-hand proteins

    Science.gov (United States)

    Kretsinger, R. H.; Nakayama, S.

    1993-01-01

    In the previous three reports in this series we demonstrated that the EF-hand family of proteins evolved by a complex pattern of gene duplication, transposition, and splicing. The dendrograms based on exon sequences are nearly identical to those based on protein sequences for troponin C, the essential light chain myosin, the regulatory light chain, and calpain. This validates both the computational methods and the dendrograms for these subfamilies. The proposal of congruence for calmodulin, troponin C, essential light chain, and regulatory light chain was confirmed. There are, however, significant differences in the calmodulin dendrograms computed from DNA and from protein sequences. In this study we find that introns are distributed throughout the EF-hand domain and the interdomain regions. Further, dendrograms based on intron type and distribution bear little resemblance to those based on protein or on DNA sequences. We conclude that introns are inserted, and probably deleted, with relatively high frequency. Further, in the EF-hand family exons do not correspond to structural domains and exon shuffling played little if any role in the evolution of this widely distributed homolog family. Calmodulin has had a turbulent evolution. Its dendrograms based on protein sequence, exon sequence, 3'-tail sequence, intron sequences, and intron positions all show significant differences.

  11. Characterisation in vivo of ways of induced deaths by p53, in the male germinal cells; Caracterisation in vivo des voies de mort induites par la p53, dans les cellules germinales males

    Energy Technology Data Exchange (ETDEWEB)

    Coureuil, M

    2006-10-15

    The male germinal cells constitute a heterogeneous cell population including pre-meiotic proliferating cells (spermatogonia) and meiotic cells and post meiotic cells in differentiation (spermatocytes and spermatids). We study the involvement in vivo of the p53 protein in the death of these cells with the help of two models, (1) a transgenic model of infertility, MTp53, in which the p53 is over expressed in the differentiated cells and induced their death, (2) the response of these cells to gamma irradiation, where only the spermatogonia die by apoptosis dependent of p53. We showed that the caspases (cysteine-aspartic proteases) are involved in the terminal differentiation of normal germinal cells. But in the MTp53 model, the p53 induces the death of differentiated cells via the activation of calpains and not of caspases. We studied the response of spermatogonia, to gamma irradiation by a transcriptomic approach, by DNA chips and semi-quantitative RT-PCR. we showed that the puma and dr5 genes are induced by the p53 after irradiation. more, the study of mice invalidated for trail ( the dr5 ligand) or for puma, allowed to demonstrate that the two effectors are essential to the activation of intrinsic and extrinsic ways of apoptosis. (N.C.)

  12. Differential proteomics reveals multiple components in retrogradely transported axoplasm after nerve injury.

    Science.gov (United States)

    Perlson, Eran; Medzihradszky, Katalin F; Darula, Zsuzsanna; Munno, David W; Syed, Naweed I; Burlingame, Alma L; Fainzilber, Mike

    2004-05-01

    Information on axonal damage is conveyed to neuronal cell bodies by a number of signaling modalities, including the post-translational modification of axoplasmic proteins. Retrograde transport of a subset of such proteins is thought to induce or enhance a regenerative response in the cell body. Here we report the use of a differential 2D-PAGE approach to identify injury-correlated retrogradely transported proteins in nerves of the mollusk Lymnaea. A comprehensive series of gels at different pI ranges allowed resolution of approximately 4000 spots by silver staining, and 172 of these were found to differ between lesioned versus control nerves. Mass spectrometric sequencing of 134 differential spots allowed their assignment to over 40 different proteins, some belonging to a vesicular ensemble blocked by the lesion and others comprising an up-regulated ensemble highly enriched in calpain cleavage products of an intermediate filament termed RGP51 (retrograde protein of 51 kDa). Inhibition of RGP51 expression by RNA interference inhibits regenerative outgrowth of adult Lymnaea neurons in culture. These results implicate regulated proteolysis in the formation of retrograde injury signaling complexes after nerve lesion and suggest that this signaling modality utilizes a wide range of protein components.

  13. Limb girdle muscular dystrophies: The clinicopathological viewpoint

    Directory of Open Access Journals (Sweden)

    Urtizberea J

    2007-01-01

    Full Text Available Limb girdle muscular dystrophies (LGMD are characterized by involvement of the pelvic and shoulder girdles, classically with an onset in the second or third decade and a slow progression as opposed to Duchenne muscular dystrophy. In fact, there are many clinical variants that are related to this broad definition. For the past 13 years and since the discovery of calpain-3 as the underlying defect in LGMD 2A in 1995, a number of different genes have been found to cause LGMD; some of whose encoding proteins are located either in the sarcolemma, nucleus, cytosol or in the extra-cellular matrix. Very little is known regarding a possible common pathogenesis between all these entities. The current nomenclature of LGMDs, although a bit confusing, is still necessary to continue the establishment of homogeneous cohorts of patients and to look for unknown genes. The diagnosis of LGMD is nowadays based on a complementary clinical, immunocytochemical and genetic approach that is best achieved in specialized myology centers. In this context, India can make a significant contribution to improve the routine diagnosis in LGMD patients and to find new LGMD genes in genetic isolates. Therapeutic prospects in LGMD, although quite exciting, remain at a preliminary stage, especially those with gene-therapy orientation.

  14. Upregulation of ICAM-1 Expression on J774.2 Macrophages by Endotoxin Involves Activation of NF-κB but not Protein Tyrosine Kinase: Comparison to Induction of iNOS

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    Hartmut Ruetten

    1999-01-01

    Full Text Available This study compares the signal transduction pathway which leads to the upregulation of intercellular adhesion molecule-1 (ICAM-1 expression with that of the increase in the expression of inducible nitric oxide synthase (iNOS protein and activity caused by endotoxin in cultured J774.2 macrophages. Treatment of J774.2 cells with lipopolysaccharide E. coli (LPS induced a concentration-dependent increase in the expression of ICAM-1 on the cell surface within 4 h and an increase in iNOS protein and activity at 24 h. The upregulation of ICAM-1 expression on J774.2 macrophages caused by LPS was significantly inhibited by pretreatment of the cells with inhibitors of the activation of the nuclear transcription factor NF-κB, such as L-1-tosylamido-2-phenylethylchloromethyl ketone (TPCK, pyrrolidine dithiocarbamate (PDTC, rotenone or calpain inhibitor I, but not by the tyrosine kinase inhibitors, tyrphostin AG126 or genistein. In contrast, genistein or tyrphostin AG126 also prevented the induction of iNOS protein and activity in J774.2 macrophages elicited by LPS. Thus, the increase in the expression of ICAM-1 on J774.2 macrophages by endotoxin involves the activation of NFκB, but not of protein tyrosine kinase.

  15. 5-Lipoxygenase-dependent apoptosis of human lymphocytes in the International Space Station: data from the ROALD experiment.

    Science.gov (United States)

    Battista, Natalia; Meloni, Maria A; Bari, Monica; Mastrangelo, Nicolina; Galleri, Grazia; Rapino, Cinzia; Dainese, Enrico; Agrò, Alessandro Finazzi; Pippia, Proto; Maccarrone, Mauro

    2012-05-01

    The functional adaptation of the immune system to the surrounding environment is also a fundamental issue in space. It has been suggested that a decreased number of lymphocytes might be a cause of immunosuppression, possibly due to the induction of apoptosis. Early activation of 5-lipoxygenase (5-LOX) might play a central role in the initiation of the apoptotic program. The goal of the role of apoptosis in lymphocyte depression (ROALD) experiment, flown on the International Space Station as part of the BIO-4 mission of the European Space Agency, was to ascertain the induction of apoptosis in human lymphocytes under authentic microgravity, and to elucidate the possible involvement of 5-LOX. Our results demonstrate that exposure of human lymphocytes to microgravity for 48 h onboard the ISS remarkably increased apoptotic hallmarks such as DNA fragmentation (∼3-fold compared to ground-based controls) and cleaved-poly (ADP-ribose) polymerase (PARP) protein expression (∼3-fold), as well as mRNA levels of apoptosis-related markers such as p53 (∼3-fold) and calpain (∼4-fold); these changes were paralleled by an early increase of 5-LOX activity (∼2-fold). Our findings provide a molecular background for the immune dysfunction observed in astronauts during space missions, and reveal potential new markers to monitor health status of ISS crew members.

  16. Apoptosis of beta cells in diabetes mellitus.

    Science.gov (United States)

    Anuradha, Rachakatla; Saraswati, Mudigonda; Kumar, Kishore G; Rani, Surekha H

    2014-11-01

    Diabetes mellitus is a multifactorial metabolic disorder characterized by hyperglycemia. Apoptosis in beta cells has been observed in response to diverse stimuli, such as glucose, cytokines, free fatty acids, leptin, and sulfonylureas, leading to the activation of polyol, hexosamine, and diacylglycerol/protein kinase-C (DAG/PKC) pathways that mediate oxidative and nitrosative stress causing the release of different cytokines. Cytokines induce the expression of Fas and tumor necrosis factor-alpha (TNF-α) by activating the transcription factor, nuclear factor-κb, and signal transducer and activator of transcription 1 (STAT-1) in the β cells in the extrinsic pathway of apoptosis. Cytokines produced in beta cells also induce proapoptotic members of the intrinsic pathway of apoptosis. The genetic alterations in apoptosis signaling machinery and the pathogenesis of diabetes include Fas, FasL, Akt, caspases, calpain-10, and phosphatase and tensin homolog (Pten). The other gene products that are involved in diabetes are nitric oxide synthase-2 (NOS2), small ubiquitin-like modifier (SUMO), apolipoprotein CIII (ApoCIII), forkhead box protein O1 (FOXO1), and Kruppel-like zinc finger protein Gli-similar 3 (GLIS3). The gene products having antiapoptotic nature are Bcl-2 and Bcl-XL. Epigenetic mechanisms play an important role in type I and type II diabetes. Further studies on the apoptotic genes and gene products in diabetics may be helpful in pharmacogenomics and individualized treatment along with antioxidants targeting apoptosis in diabetes. PMID:25093391

  17. Neutrophil and Alveolar Macrophage-Mediated Innate Immune Control of Legionella pneumophila Lung Infection via TNF and ROS.

    Science.gov (United States)

    Ziltener, Pascal; Reinheckel, Thomas; Oxenius, Annette

    2016-04-01

    Legionella pneumophila is a facultative intracellular bacterium that lives in aquatic environments where it parasitizes amoeba. However, upon inhalation of contaminated aerosols it can infect and replicate in human alveolar macrophages, which can result in Legionnaires' disease, a severe form of pneumonia. Upon experimental airway infection of mice, L. pneumophila is rapidly controlled by innate immune mechanisms. Here we identified, on a cell-type specific level, the key innate effector functions responsible for rapid control of infection. In addition to the well-characterized NLRC4-NAIP5 flagellin recognition pathway, tumor necrosis factor (TNF) and reactive oxygen species (ROS) are also essential for effective innate immune control of L. pneumophila. While ROS are essential for the bactericidal activity of neutrophils, alveolar macrophages (AM) rely on neutrophil and monocyte-derived TNF signaling via TNFR1 to restrict bacterial replication. This TNF-mediated antibacterial mechanism depends on the acidification of lysosomes and their fusion with L. pneumophila containing vacuoles (LCVs), as well as caspases with a minor contribution from cysteine-type cathepsins or calpains, and is independent of NLRC4, caspase-1, caspase-11 and NOX2. This study highlights the differential utilization of innate effector pathways to curtail intracellular bacterial replication in specific host cells upon L. pneumophila airway infection. PMID:27105352

  18. Eryptosis-inducing activity of bisphenol A and its analogs in human red blood cells (in vitro study).

    Science.gov (United States)

    Maćczak, Aneta; Cyrkler, Monika; Bukowska, Bożena; Michałowicz, Jaromir

    2016-04-15

    Bisphenols are important chemicals that are widely used in the manufacturing of polycarbonates, epoxy resin and thermal paper, and thus the exposure of humans to these substances has been noted. The purpose of this study was to assess eryptotic changes in human erythrocytes exposed (in vitro) to bisphenol A (BPA) and its selected analogs, i.e.,bisphenol F (BPF), bisphenol S (BPS) and bisphenol AF (BPAF). The erythrocytes were incubated with compounds studied at concentrations ranging from 1 to 250μg/mL for 4, 12 or 24h. The results showed that BPA and its analogs increased cytosolic calcium ions level with the strongest effect noted for BPAF. It has also been revealed that all bisphenols analyzed, and BPAF and BPF in particular increased phosphatidylserine translocation in red blood cells, which confirmed that they exhibited eryptotic potential in this cell type. Furthermore, it was shown that BPA and its analogs caused significant increase in calpain and caspase-3 activities, while the strongest effect was noted for BPAF. BPS, which is the main substituent of bisphenol A in polymers and thermal paper production exhibited similar eryptotic potential to BPA. Eryptotic changes in human erythrocytes were provoked by bisphenols at concentrations, which may influence the human body during occupational exposure or subacute poisoning with these compounds.

  19. Violacein induces death of resistant leukaemia cells via kinome reprogramming, endoplasmic reticulum stress and Golgi apparatus collapse.

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    Karla C S Queiroz

    Full Text Available It is now generally recognised that different modes of programmed cell death (PCD are intimately linked to the cancerous process. However, the mechanism of PCD involved in cancer chemoprevention is much less clear and may be different between types of chemopreventive agents and tumour cell types involved. Therefore, from a pharmacological view, it is crucial during the earlier steps of drug development to define the cellular specificity of the candidate as well as its capacity to bypass dysfunctional tumoral signalling pathways providing insensitivity to death stimuli. Studying the cytotoxic effects of violacein, an antibiotic dihydro-indolone synthesised by an Amazon river Chromobacterium, we observed that death induced in CD34(+/c-Kit(+/P-glycoprotein(+/MRP1(+ TF1 leukaemia progenitor cells is not mediated by apoptosis and/or autophagy, since biomarkers of both types of cell death were not significantly affected by this compound. To clarify the working mechanism of violacein, we performed kinome profiling using peptide arrays to yield comprehensive descriptions of cellular kinase activities. Pro-death activity of violacein is actually carried out by inhibition of calpain and DAPK1 and activation of PKA, AKT and PDK, followed by structural changes caused by endoplasmic reticulum stress and Golgi apparatus collapse, leading to cellular demise. Our results demonstrate that violacein induces kinome reprogramming, overcoming death signaling dysfunctions of intrinsically resistant human leukaemia cells.

  20. Lateral mobility of integrin alpha IIb beta 3 (glycoprotein IIb/IIIa) in the plasma membrane of a human megakaryocyte.

    Science.gov (United States)

    Schootemeijer, A; van Willigen, G; van der Vuurst, H; Tertoolen, L G; De Laat, S W; Akkerman, J W

    1997-01-01

    The migration of integrins to sites of cell-cell and cell-matrix contact is thought to be important for adhesion strengthening. We studied the lateral diffusion of integrin alpha IIb beta 3 (glycoprotein IIb/IIIa) in the plasma membrane of a cultured human megakaryocyte by fluorescence recovery after photobleaching of FITC-labelled monovalent Fab fragments directed against the beta 3 subunit. The diffusion of beta 3 on the unstimulated megakaryocyte showed a lateral diffusion coefficient (D) of 0.37 x 10(-9) cm2/s and a mobile fraction of about 50%. Stimulation with ADP (20 microM) or alpha-thrombin (10 U/ml) at 22 degrees C induced transient decreases in both parameters reducing D to 0.21 x 10(-9) cm2/s and the mobile fraction to about 25%. The fall in D was observed within 1 min after stimulation but the fall in mobile fraction showed a lag phase of 5 min. The lag phase was absent in the presence of Calpain I inhibitor, where-as cytochalasin D completely abolished the decreased in mobile fraction. The data are compatible with the concept that cell activation induces anchorage of 50% of the mobile alpha IIb beta 3 (25% of the whole population of receptor) to the cytoplasmic actin filaments, although, as discussed, other rationals are not ruled out. PMID:9031465

  1. Excretory/secretory products from in vitro-cultured Echinococcus granulosus protoscoleces.

    Science.gov (United States)

    Virginio, Veridiana G; Monteiro, Karina M; Drumond, Fernanda; de Carvalho, Marcos O; Vargas, Daiani M; Zaha, Arnaldo; Ferreira, Henrique B

    2012-05-01

    Cystic hydatid disease (CHD) is caused by infection with Echinococcus granulosus metacestodes and affects humans and livestock. Proteins secreted or excreted by protoscoleces, pre-adult worms found in the metacestode, are thought to play fundamental roles in the host-parasite relationship. In this work, we performed an LC-MS/MS proteomic analysis of the excretory-secretory products obtained from the first 48 h of an in vitro culture of the protoscoleces. We identified 32 proteins, including 18 that were never detected previously in metacestode proteomic studies. Among the novel identified excretory-secretory products are antigenic proteins, such as EG19 and P-29 and a calpain protease. We also identified other important protoscolex excretory-secretory products, such as thioredoxin peroxidase and 14-3-3 proteins, which are potentially involved in evasion mechanisms adopted by parasites to establish infection. Several intracellular proteins were found in the excretory-secretory products, revealing a set of identified proteins not previously thought to be exposed at the host-parasite interface. Additionally, immunological analyses established the antigenic profiles of the newly identified excretory-secretory products and revealed, for the first time, the in vitro secretion of the B antigen by protoscoleces. Considering that the excretory-secretory products obtained in vitro might reflect the products released and exposed to the host in vivo, our results provide valuable information on parasite survival strategies in adverse host environments and on the molecular mechanisms underpinning CHD immunopathology.

  2. Crude extract of Rheum palmatum L. Induces cell cycle arrest S phase and apoptosis through mitochondrial-dependent pathways in U-2 OS human osteosarcoma cells.

    Science.gov (United States)

    Lin, Chin-Chung; Lee, Ming-Huei; Lin, Ju-Hwa; Lin, Meng-Liang; Chueh, Fu-Shin; Yu, Chien-Chih; Lin, Jing-Pin; Chou, Yu-Cheng; Hsu, Shu-Chun; Chung, Jing-Gung

    2016-08-01

    Cancer is the second cause of death in children. Osteosarcoma is the most common primary malignancy of solid bone cancer primarily affecting adolescents and young adults. In the Chinese population, the crude extract of Rheum palmatum L. (CERP) has been used for treating different diseases, including SARS, rheumatoid arthritis, coxsackievirus B3, and human colon cancer cell, pancreatic cancer. There are no reports on CERP and human osteosarcoma cells. The present study examined effects of CERP on cytotoxicity including cell cycle distribution and cell death (apoptosis) in U-2 OS human osteosarcoma cells. CERP significantly induced S phase arrest in U-2 OS cells in a dose-dependent. CERP produced DNA damage and DNA condensation. Other effects of CERP were stimulation of ROS and Ca(2+) , mitochondria impairment, and activation of caspase-3, -8, and -9. CERP increased the levels of Bax, Bak, Bad, cyclin B, Fas, PARP, GRP78, GADD153, AIF, Endo G, Calpain-2, p21, and p27, but decreased the levels of Bcl-2, BCL-X, XIAP, Akt, CDC25A, CDK2, Cyclin A, and Cyclin E of U-2 OS cells. It was also observed that CERP promoted the expression of AIF, Endo G, GADD153, and cytochrome c. These results indicate that CERP has anticancer effects in vitro and provide the foundation for in vivo studies of animal models of osteosarcoma. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 957-969, 2016. PMID:25689151

  3. Retinoic Acid Induces Apoptosis of Prostate Cancer DU145 Cells through Cdk5 Overactivation

    Directory of Open Access Journals (Sweden)

    Mei-Chih Chen

    2012-01-01

    Full Text Available Retinoic acid (RA has been believed to be an anticancer drug for a long history. However, the molecular mechanisms of RA actions on cancer cells remain diverse. In this study, the dose-dependent inhibition of RA on DU145 cell proliferation was identified. Interestingly, RA treatment triggered p35 cleavage (p25 formation and Cdk5 overactivation, and all could be blocked by Calpain inhibitor, Calpeptin (CP. Subsequently, RA-triggered DU145 apoptosis detected by sub-G1 phase accumulation and Annexin V staining could also be blocked by CP treatment. Furthermore, RA-triggered caspase 3 activation and following Cdk5 over-activation were destroyed by treatments of both CP and Cdk5 knockdown. In conclusion, we report a new mechanism in which RA could cause apoptosis of androgen-independent prostate cancer cells through p35 cleavage and Cdk5 over-activation. This finding may contribute to constructing a clearer image of RA function and bring RA as a valuable chemoprevention agent for prostate cancer patients.

  4. cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response.

    Directory of Open Access Journals (Sweden)

    María Belén Mestre

    Full Text Available Staphylococcus aureus is a microorganism that causes serious diseases in the human being. This microorganism is able to escape the phagolysosomal pathway, increasing intracellular bacterial survival and killing the eukaryotic host cell to spread the infection. One of the key features of S. aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins. We have shown that the pore-forming toxin α-hemolysin (Hla is the S. aureus-secreted factor responsible for the activation of the autophagic pathway and that this response occurs through a PI3K/Beclin1-independent form. In the present report we demonstrate that cAMP has a key role in the regulation of this autophagic response. Our results indicate that cAMP is able to inhibit the autophagy induced by Hla and that PKA, the classical cAMP effector, does not participate in this regulation. We present evidence that EPAC and Rap2b, through calpain activation, are the proteins involved in the regulation of Hla-induced autophagy. Similar results were obtained in cells infected with different S. aureus strains. Interestingly, in this report we show, for the first time to our knowledge, that both EPAC and Rap2b are recruited to the S. aureus-containing phagosome. We believe that our findings have important implications in understanding innate immune processes involved in intracellular pathogen invasion of the host cell.

  5. Phosphatidylserine externalization and procoagulant activation of erythrocytes induced by Pseudomonas aeruginosa virulence factor pyocyanin.

    Science.gov (United States)

    Qadri, Syed M; Donkor, David A; Bhakta, Varsha; Eltringham-Smith, Louise J; Dwivedi, Dhruva J; Moore, Jane C; Pepler, Laura; Ivetic, Nikola; Nazi, Ishac; Fox-Robichaud, Alison E; Liaw, Patricia C; Sheffield, William P

    2016-04-01

    The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections in multiple hosts by releasing an arsenal of virulence factors such as pyocyanin. Despite numerous reports on the pleiotropic cellular targets of pyocyanin toxicity in vivo, its impact on erythrocytes remains elusive. Erythrocytes undergo an apoptosis-like cell death called eryptosis which is characterized by cell shrinkage and phosphatidylserine (PS) externalization; this process confers a procoagulant phenotype on erythrocytes as well as fosters their phagocytosis and subsequent clearance from the circulation. Herein, we demonstrate that P. aeruginosa pyocyanin-elicited PS exposure and cell shrinkage in erythrocyte while preserving the membrane integrity. Mechanistically, exposure of erythrocytes to pyocyanin showed increased cytosolic Ca(2+) activity as well as Ca(2+) -dependent proteolytic processing of μ-calpain. Pyocyanin further up-regulated erythrocyte ceramide abundance and triggered the production of reactive oxygen species. Pyocyanin-induced increased PS externalization in erythrocytes translated into enhanced prothrombin activation and fibrin generation in plasma. As judged by carboxyfluorescein succinimidyl-ester labelling, pyocyanin-treated erythrocytes were cleared faster from the murine circulation as compared to untreated erythrocytes. Furthermore, erythrocytes incubated in plasma from patients with P. aeruginosa sepsis showed increased PS exposure as compared to erythrocytes incubated in plasma from healthy donors. In conclusion, the present study discloses the eryptosis-inducing effect of the virulence factor pyocyanin, thereby shedding light on a potentially important mechanism in the systemic complications of P. aeruginosa infection. PMID:26781477

  6. Def defines a conserved nucleolar pathway that leads p53 to proteasome-independent degradation

    Institute of Scientific and Technical Information of China (English)

    Ting Tao; Hui Shi; Yihong Guan; Delai Huang; Ye Chen; David P Lane; Jun Chen

    2013-01-01

    p53 protein turnover through the ubiquitination pathway is a vital mechanism in the regulation of its transcriptional activity; however,little is known about p53 turnover through proteasome-independent pathway(s).The digestive organ expansion factor (Def) protein is essential for the development of digestive organs.In zebrafish,loss of function of defselectively upregulates the expression of p53 response genes,which raises a question as to what is the relationship between Def and p53.We report here that Def is a nucleolar protein and that loss of function of defleads to the upregulation of p53 protein,which surprisingly accumulates in the nucleoli.Our extensive studies have demonstrated that Def can mediate the degradation of p53 protein and that this process is independent of the proteasome pathway,but dependent on the activity of Calpain3,a cysteine protease.Our findings define a novel nucleolar pathway that regulates the turnover function of p53,which will advance our understanding of p53's role in organogenesis and tumorigenesis.

  7. The indirect NMDAR inhibitor flupirtine induces sustained post-ischemic recovery, neuroprotection and angioneurogenesis.

    Science.gov (United States)

    Jaeger, Hanna M; Pehlke, Jens R; Kaltwasser, Britta; Kilic, Ertugrul; Bähr, Mathias; Hermann, Dirk M; Doeppner, Thorsten R

    2015-06-10

    N-methyl-D-aspartate receptor (NMDAR) activation induces excitotoxicity, contributing to post-stroke brain injury. Hitherto, NMDAR deactivation failed in clinical trials due to insufficient pre-clinical study designs and drug toxicity. Flupirtine is an indirect NMDAR antagonist being used as analgesic in patients. Taking into account its tolerability profile, we evaluated effects of flupirtine on post-stroke tissue survival, neurological recovery and brain remodeling.Mice were exposed to stroke and intraperitoneally treated with saline (control) or flupirtine at various doses (1-10 mg/kg) and time-points (0-12 hours). Tissue survival and cell signaling were studied on day 2, whereas neurological recovery and tissue remodeling were analyzed until day 84.Flupirtine induced sustained neuroprotection, when delivered up to 9 hours. The latter yielded enhanced neurological recovery that persisted over three months and which was accompanied by enhanced angioneurogenesis. On the molecular level, inhibition of calpain activation was noted, which was associated with increased signal-transducer-and-activator-of-transcription-6 (STAT6) abundance, reduced N-terminal-Jun-kinase and NF-κB activation, as well as reduced proteasomal activity. Consequently, blood-brain-barrier integrity was stabilized, oxidative stress was reduced and brain leukocyte infiltration was diminished.In view of its excellent tolerability, considering its sustained effects on neurological recovery, brain tissue survival and remodeling, flupirtine is an attractive candidate for stroke therapy. PMID:26050199

  8. Single nucleotide polymorphisms associated with carcass traits in a population of Brahman and Brahman-influenced steers.

    Science.gov (United States)

    Royer, A M; Shivers, C; Riley, D G; Elzo, M A; Garcia, M D

    2016-06-21

    Brahman cattle are important in tropical regions due to their ability to tolerate excessive heat and parasites. However, Brahman cattle exhibit lower carcass quality characteristics when compared to Bos taurus breeds. The objective of this study was to evaluate potential associations between single nucleotide polymorphisms (SNPs) in six candidate genes for carcass quality and composition traits in a population of Brahman and Brahman-influenced steers. Steers were evaluated through the American Brahman Breeders Association carcass evaluation project in Gonzales, Texas. Carcass traits measured included hot carcass weight, ribeye area, marbling score, yield grade, quality grade, dressing percent, and Warner-Bratzler shear force score. Six previously described candidate genes were chosen for SNP analysis based on their previous association with growth and carcass traits. Candidate genes utilized in the current study included calpastatin (CAST), calpain (CAPN3), thyroglobulin (TG), growth hormone, insulin growth factor 1, and adiponectin. Six unique SNPs from three candidate genes (TG, CAST, and CAPN3) were significantly associated (P carcass quality traits (marbling score and quality grade). A genotypic effect was observed for all significant SNPs, with differing levels of performance observed for animals inheriting different genotypes. Although multiple SNPs in the current study were significantly (P carcass traits, they should be validated in larger populations prior to implementation in selection strategies.

  9. Two proteolytic fragments of menin coordinate the nuclear transcription and postsynaptic clustering of neurotransmitter receptors during synaptogenesis between Lymnaea neurons.

    Science.gov (United States)

    Getz, Angela M; Visser, Frank; Bell, Erin M; Xu, Fenglian; Flynn, Nichole M; Zaidi, Wali; Syed, Naweed I

    2016-01-01

    Synapse formation and plasticity depend on nuclear transcription and site-specific protein targeting, but the molecular mechanisms that coordinate these steps have not been well defined. The MEN1 tumor suppressor gene, which encodes the protein menin, is known to induce synapse formation and plasticity in the CNS. This synaptogenic function has been conserved across evolution, however the underlying molecular mechanisms remain unidentified. Here, using central neurons from the invertebrate Lymnaea stagnalis, we demonstrate that menin coordinates subunit-specific transcriptional regulation and synaptic clustering of nicotinic acetylcholine receptors (nAChR) during neurotrophic factor (NTF)-dependent excitatory synaptogenesis, via two proteolytic fragments generated by calpain cleavage. Whereas menin is largely regarded as a nuclear protein, our data demonstrate a novel cytoplasmic function at central synapses. Furthermore, this study identifies a novel synaptogenic mechanism in which a single gene product coordinates the nuclear transcription and postsynaptic targeting of neurotransmitter receptors through distinct molecular functions of differentially localized proteolytic fragments. PMID:27538741

  10. Prenatal stress induces long-term effects in cell turnover in the hippocampus-hypothalamus-pituitary axis in adult male rats.

    Directory of Open Access Journals (Sweden)

    Eva Baquedano

    Full Text Available Subchronic gestational stress leads to permanent modifications in the hippocampus-hypothalamus-pituitary-adrenal axis of offspring probably due to the increase in circulating glucocorticoids known to affect prenatal programming. The aim of this study was to investigate whether cell turnover is affected in the hippocampus-hypothalamus-pituitary axis by subchronic prenatal stress and the intracellular mechanisms involved. Restraint stress was performed in pregnant rats during the last week of gestation (45 minutes; 3 times/day. Only male offspring were used for this study and were sacrificed at 6 months of age. In prenatally stressed adults a decrease in markers of cell death and proliferation was observed in the hippocampus, hypothalamus and pituitary. This was associated with an increase in insulin-like growth factor-I mRNA levels, phosphorylation of CREB and calpastatin levels and inhibition of calpain -2 and caspase -8 activation. Levels of the anti-apoptotic protein Bcl-2 were increased and levels of the pro-apoptotic factor p53 were reduced. In conclusion, prenatal restraint stress induces a long-term decrease in cell turnover in the hippocampus-hypothalamus-pituitary axis that might be at least partly mediated by an autocrine-paracrine IGF-I effect. These changes could condition the response of this axis to future physiological and pathophysiological situations.

  11. Advancing a vaccine to prevent human schistosomiasis.

    Science.gov (United States)

    Merrifield, Maureen; Hotez, Peter J; Beaumier, Coreen M; Gillespie, Portia; Strych, Ulrich; Hayward, Tara; Bottazzi, Maria Elena

    2016-06-01

    Several candidate human schistosomiasis vaccines are in different stages of preclinical and clinical development. The major targets are Schistosoma haematobium (urogenitial schistosomiasis) and Schistosoma mansoni (intestinal schistosomiasis) that account for 99% of the world's 252 million cases, with 90% of these cases in Africa. Two recombinant S. mansoni vaccines - Sm-TSP-2 and Sm-14 are in Phase 1 trials, while Smp80 (calpain) is undergoing testing in non-human primates. Sh28GST, also known as Bilhvax is in advanced clinical development for S. haematobium infection. The possibility remains that some of these vaccines may cross-react to target both schistosome species. These vaccines were selected on the basis of their protective immunity in preclinical challenge models, through human immune-epidemiological studies or both. They are being advanced through a combination of academic research institutions, non-profit vaccine product development partnerships, biotechnology companies, and developing country vaccine manufacturers. In addition, new schistosome candidate vaccines are being identified through bioinformatics, OMICs approaches, and moderate throughput screening, although the full potential of reverse vaccinology for schistosomiasis has not yet been realized. The target product profiles of these vaccines vary but many focus on vaccinating children, in some cases following mass treatment with praziquantel, also known as vaccine-linked chemotherapy. Several regulatory pathways have been proposed, some of which rely on World Health Organization prequalification. PMID:27036511

  12. Cloning and characterisation of novel cystatins from elapid snake venom glands.

    Science.gov (United States)

    Richards, Renée; St Pierre, Liam; Trabi, Manuela; Johnson, Lambro A; de Jersey, John; Masci, Paul P; Lavin, Martin F

    2011-04-01

    Snake venoms contain a complex mixture of polypeptides that modulate prey homeostatic mechanisms through highly specific and targeted interactions. In this study we have identified and characterised cystatin-like cysteine-protease inhibitors from elapid snake venoms for the first time. Novel cystatin sequences were cloned from 12 of 13 elapid snake venom glands and the protein was detected, albeit at very low levels, in a total of 22 venoms. One highly conserved isoform, which displayed close sequence identity with family 2 cystatins, was detected in each elapid snake. Crude Austrelaps superbus (Australian lowland copperhead) snake venom inhibited papain, and a recombinant form of A. superbus cystatin inhibited cathepsin L ≅ papain > cathepsin B, with no inhibition observed for calpain or legumain. While snake venom cystatins have truncated N-termini, sequence alignment and structural modelling suggested that the evolutionarily conserved Gly-11 of family 2 cystatins, essential for cysteine protease inhibition, is conserved in snake venom cystatins as Gly-3. This was confirmed by mutagenesis at the Gly-3 site, which increased the dissociation constant for papain by 10(4)-fold. These data demonstrate that elapid snake venom cystatins are novel members of the type 2 family. The widespread, low level expression of type 2 cystatins in snake venom, as well as the presence of only one highly conserved isoform in each species, imply essential housekeeping or regulatory roles for these proteins.

  13. The role of pharmacotherapy in modifying the neurological status of patients with spinal and spinal cord injuries

    Directory of Open Access Journals (Sweden)

    Renato Carlos do Vale Ramos

    2015-12-01

    Full Text Available ABSTRACT The aim here was to conduct a review of the literature on pharmacological therapies for modifying the neurological status of patients with spinal cord injuries. The PubMed database was searched for articles with the terms "spinal cord injury AND methylprednisolone/GM1/apoptosis inhibitor/calpain inhibitor/naloxone/tempol/tirilazad", in Portuguese or in English, published over the last five years. Older studies were included because of their historical importance. The pharmacological groups were divided according to their capacity to interfere with the physiopathological mechanisms of secondary injuries. Use of methylprednisolone needs to be carefully weighed up: other anti-inflammatory agents have shown benefits in humans or in animals. GM1 does not seem to have greater efficacy than methylprednisolone, but longer-term studies are needed. Many inhibitors of apoptosis have shown benefits inin vitro studies or in animals. Naloxone has not shown benefits. Tempol inhibits the main consequences of oxidation at the level of the spinal cord and other antioxidant drugs seem to have an effect superior to that of methylprednisolone. There is an urgent need to find new treatments that improve the neurological status of patients with spinal cord injuries. The benefits from treatment with methylprednisolone have been questioned, with concerns regarding its safety. Other drugs have been studied, and some of these may provide promising alternatives. Additional studies are needed in order to reach conclusions regarding the benefits of these agents in clinical practice.

  14. Inhibition of Setaria cervi protein tyrosine phosphatases by Phenylarsine oxide: A proteomic and biochemical study.

    Science.gov (United States)

    Singh, Neetu; Wadhawan, Mohit; Tiwari, Savitri; Kumar, Ranjeet; Rathaur, Sushma

    2016-07-01

    Phenylarsine oxide (PAO), a specific protein tyrosine phosphatase (PTP) inhibitor significantly decreased the motility and viability of Setaria cervi ultimately leading to its death. The PTP activity present in the cytosolic and detergent soluble fractions as well as on surface of these parasites was significantly inhibited by PAO. A marked alteration in protein spots abundance after proteomic analysis showed 14 down-regulated and 9 upregulated spots in the treated parasites as compared to the control. The PTP inhibition led to increase in the cytosolic and mitochondrial calpain activity in these parasites. PAO also blocked the ATP generation in the parasite depicted by reduced activity of phosphoglycerate kinase and expression of enolase. An increased ROS level, induced lipid peroxidation/protein carbonyl formation and decreased activity of different antioxidant enzymes like thioredoxin reductase, glutathione reductase and glutathione transferases was also observed in the PAO treated parasites. PAO, thus disturbs the overall homeostasis of the filarial parasite by inhibiting PTPs. Thereby suggesting that these molecules could be used as a good chemotherapeutic target for lymphatic filariasis. PMID:26965172

  15. Resveratrol Induces Apoptosis-Like Death and Prevents In Vitro and In Vivo Virulence of Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Jonnatan Pais-Morales

    Full Text Available Entamoeba histolytica causes amoebiasis, an infection that kills 100,000 individuals each year. Metronidazole and its derivatives are currently used against this protozoan, but these drugs present adverse effects on human health. Here, we investigated the effect of resveratrol (a natural compound on E. histolytica trophozoites viability, as well as its influence on the parasite virulence. Trophozoites growth was arrested by 72 μM resveratrol and the IC50 was determined as 220 μM at 48 h. Cells appeared smaller, rounded and in clusters, with debris-containing vacuoles and with abnormally condensed chromatin. Resveratrol triggered reactive oxygen species production. It caused lipid peroxidation and produced phosphatidylserine externalization and DNA fragmentation this latter evidenced by TUNEL assays. It also provoked an increase of intracellular Ca2+ concentration, activated calpain and decreased superoxide dismutase activity, indicating that an apoptosis-like event occurred; however, autophagy was not detected. Cytopathic activity, phagocytosis, encystment and in vivo virulence were diminished dramatically by pre-incubation of trophozoites with resveratrol, evidencing that resveratrol attenuated the trophozoite virulence in vitro. Interestingly, after the inoculation of virulent trophozoites, animals treated with the drug did not develop or developed very small abscesses. Our findings propose that resveratrol could be an alternative to contend amoebiasis.

  16. Resveratrol Induces Apoptosis-Like Death and Prevents In Vitro and In Vivo Virulence of Entamoeba histolytica.

    Science.gov (United States)

    Pais-Morales, Jonnatan; Betanzos, Abigail; García-Rivera, Guillermina; Chávez-Munguía, Bibiana; Shibayama, Mineko; Orozco, Esther

    2016-01-01

    Entamoeba histolytica causes amoebiasis, an infection that kills 100,000 individuals each year. Metronidazole and its derivatives are currently used against this protozoan, but these drugs present adverse effects on human health. Here, we investigated the effect of resveratrol (a natural compound) on E. histolytica trophozoites viability, as well as its influence on the parasite virulence. Trophozoites growth was arrested by 72 μM resveratrol and the IC50 was determined as 220 μM at 48 h. Cells appeared smaller, rounded and in clusters, with debris-containing vacuoles and with abnormally condensed chromatin. Resveratrol triggered reactive oxygen species production. It caused lipid peroxidation and produced phosphatidylserine externalization and DNA fragmentation this latter evidenced by TUNEL assays. It also provoked an increase of intracellular Ca2+ concentration, activated calpain and decreased superoxide dismutase activity, indicating that an apoptosis-like event occurred; however, autophagy was not detected. Cytopathic activity, phagocytosis, encystment and in vivo virulence were diminished dramatically by pre-incubation of trophozoites with resveratrol, evidencing that resveratrol attenuated the trophozoite virulence in vitro. Interestingly, after the inoculation of virulent trophozoites, animals treated with the drug did not develop or developed very small abscesses. Our findings propose that resveratrol could be an alternative to contend amoebiasis.

  17. Expression of genes related to quality of Longissimus dorsi muscle meat in Nellore (Bos indicus) and Canchim (5/8 Bos taurus × 3/8 Bos indicus) cattle.

    Science.gov (United States)

    Giusti, Juliana; Castan, Eduardo; Dal Pai, Maeli; Arrigoni, Mário De Beni; Rodrigues Baldin, Samira; De Oliveira, Henrique Nunes

    2013-06-01

    This study was performed to compare CAPN1, CAPN2, CAST, TG, DGAT1 and LEP gene expressions and correlate them with meat quality traits in two genetic groups (Nellore and Canchim) in order to assess their expression profile and use their expression profile as genetic markers. We analyzed 30 young bulls (1year old), 15 of each genetic group. Samples of the Longissimus dorsi muscle were collected for analysis of: total lipids (TL) and meat tenderness measured as Warner-Bratzler shear force (SF) and myofibrillar fragmentation (MFI) at day of slaughter and 7days of aging. Gene expression profiles were obtained via RT-qPCR. TL and MFI showed differences between breeds, higher MFI in Canchim and higher TL in Nellore. Calpains showed no differential expression between groups, as did DGAT1, TG, and LEP. CAST was expressed more in the Nellore cattle. The only significant within-breed correlation (0.79) between gene expression and meat traits was found for DGAT1 and MFI in Canchim breed. Although the number of animals used in this study was small, the results indicate that the increased expression of CAST in Nellore may reflect tougher meat, but the lack of correlations with the meat traits indicates it is not a promising genetic marker.

  18. Ikaros is degraded by proteasome-dependent mechanism in the early phase of apoptosis induction

    International Nuclear Information System (INIS)

    Research highlights: → Chemotherapeutic drugs or UV treatment reduces Ikaros prior to caspase-3 activation. → Etoposide treatment does not alter the mRNA but shortens the half-life of Ikaros. → MG132 or epoxomicin but not calpeptin inhibits etoposide-induced Ikaros degradation. → Overexpression of Ikaros accelerates etoposide-induced apoptosis in NB4 cells. -- Abstract: Ikaros is an important transcription factor involved in the development and differentiation of hematopoietic cells. In this work, we found that chemotherapeutic drugs or ultraviolet radiation (UV) treatment could reduce the expression of full-length Ikaros (IK1) protein in less than 3 h in leukemic NB4, Kasumi-1 and Jurkat cells, prior to the activation of caspase-3. Etoposide treatment could not alter the mRNA level of IK1 but it could shorten the half-life of IK1. Co-treatment with the proteasome inhibitor MG132 or epoxomicin but not calpain inhibitor calpeptin inhibited etoposide-induced Ikaros downregulation. Overexpression of IK1 could accelerate etoposide-induced apoptosis in NB4 cells, as evidenced by the increase of Annexin V positive cells and the more early activation of caspase 3. To our knowledge, this is the first report to show that upon chemotherapy drugs or UV treatment, IK1 could be degraded via the proteasome system in the early phase of apoptosis induction. These data might shed new insight on the role of IK1 in apoptosis and the post-translational regulation of IK1.

  19. Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria

    KAUST Repository

    Ross, Avena C.

    2013-01-23

    Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multimodule skipping and iteration. Preliminary biochemical analysis of the N-terminal nonribosomal peptide synthetase module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N terminus. © 2012 American Chemical Society.

  20. Defective Kernel 1 (DEK1) is required for three-dimensional growth in Physcomitrella patens.

    Science.gov (United States)

    Perroud, Pierre-François; Demko, Viktor; Johansen, Wenche; Wilson, Robert C; Olsen, Odd-Arne; Quatrano, Ralph S

    2014-08-01

    Orientation of cell division is critical for plant morphogenesis. This is evident in the formation and function of meristems and for morphogenetic transitions. Mosses undergo such transitions: from two-dimensional tip-growing filaments (protonema) to the generation of three-dimensional leaf-like structures (gametophores). The Defective Kernel 1 (DEK1) protein plays a key role in the perception of and/or response to positional cues that specify the formation and function of the epidermal layer in developing seeds of flowering plants. The moss Physcomitrella patens contains the highly conserved DEK1 gene. Using efficient gene targeting, we generated a precise PpDEK1 deletion (∆dek1), which resulted in normal filamentous growth of protonema. Two distinct mutant phenotypes were observed: an excess of buds on the protonema, and abnormal cell divisions in the emerging buds resulting in developmental arrest and the absence of three-dimensional growth. Overexpression of a complete PpDEK1 cDNA, or the calpain domain of PpDEK1 alone, successfully complements both phenotypes. These results in P. patens demonstrate the morphogenetic importance of the DEK1 protein in the control of oriented cell divisions. As it is not for protonema, it will allow dissection of the structure/function relationships of the different domains of DEK1 using gene targeting in null mutant background.

  1. Acquired resistance to innate immune clearance promotes Klebsiella pneumoniae ST258 pulmonary infection

    Science.gov (United States)

    Ahn, Danielle; Peñaloza, Hernán; Wang, Zheng; Wickersham, Matthew; Parker, Dane; Patel, Purvi; Koller, Antonius; Chen, Emily I.; Bueno, Susan M.; Uhlemann, Anne-Catrin; Prince, Alice

    2016-01-01

    Adaptive changes in the genome of a locally predominant clinical isolate of the multidrug-resistant Klebsiella pneumoniae ST258 (KP35) were identified and help to explain the selection of this strain as a successful pulmonary pathogen. The acquisition of 4 new ortholog groups, including an arginine transporter, enabled KP35 to outcompete related ST258 strains lacking these genes. KP35 infection elicited a monocytic response, dominated by Ly6Chi monocytic myeloid-derived suppressor cells that lacked phagocytic capabilities, expressed IL-10, arginase, and antiinflammatory surface markers. In comparison with other K. pneumoniae strains, KP35 induced global changes in the phagocytic response identified with proteomics, including evasion of Ca2+ and calpain activation necessary for phagocytic killing, confirmed in functional studies with neutrophils. This comprehensive analysis of an ST258 K. pneumoniae isolate reveals ongoing genetic adaptation to host microenvironments and innate immune clearance mechanisms that complements its repertoire of antimicrobial resistance genes and facilitates persistence in the lung. PMID:27777978

  2. Granzyme B-dependent proteolysis acts as a switch to enhance the proinflammatory activity of IL-1α.

    LENUS (Irish Health Repository)

    Afonina, Inna S

    2011-10-21

    Granzyme B is a cytotoxic lymphocyte-derived protease that plays a central role in promoting apoptosis of virus-infected target cells, through direct proteolysis and activation of constituents of the cell death machinery. However, previous studies have also implicated granzymes A and B in the production of proinflammatory cytokines, via a mechanism that remains undefined. Here we show that IL-1α is a substrate for granzyme B and that proteolysis potently enhanced the biological activity of this cytokine in vitro as well as in vivo. Consistent with this, compared with full-length IL-1α, granzyme B-processed IL-1α exhibited more potent activity as an immunoadjuvant in vivo. Furthermore, proteolysis of IL-1α within the same region, by proteases such as calpain and elastase, was also found to enhance its biological potency. Thus, IL-1α processing by multiple immune-related proteases, including granzyme B, acts as a switch to enhance the proinflammatory properties of this cytokine.

  3. Calycopterin promotes survival and outgrowth of neuron-like PC12 cells by attenuation of oxidative- and ER-stress-induced apoptosis along with inflammatory response.

    Science.gov (United States)

    Farimani, Mahdi Moridi; Sarvestani, Nazanin Namazi; Ansari, Niloufar; Khodagholi, Fariba

    2011-12-19

    There is mounting evidence implicating the role of oxidative stress induced by reactive oxygen species (ROS) in neurodegenerative disease, including Alzheimer's disease. Herein we investigated the neuroprotective potential of a natural flavonoid, calycopterin, against H(2)O(2)-induced cell death in differentiated PC12 cells. We pretreated PC12 cells with 25, 50, and 100 μM calycopterin followed by the addition of H(2)O(2) as an oxidative stress agent. We measured cell viability by the MTT test and found that 50 μM is the best protective concentration of calycopterin. Moreover, we measured six different parameters of neurite outgrowth. Interestingly, we found that calycopterin not only protects PC12 cells against H(2)O(2)-induced apoptosis but also defends against the destructive effect of oxidative stress on the criteria of neural differentiation. Calycopterin decreased ER stress-associated proteins including calpain and caspase-12, and suppressed ERK, JNK, and p38 MAPK phosphorylation. Moreover, calycopterin inhibited H(2)O(2)-induced nuclear translocation of nuclear factor-κB, a known regulator of a host of genes involved in specific stress and inflammatory responses. This observation was perfectly in agreement with the decrease of COX-2 and TNF-α levels. Calycopterin reduced intracellular ROS levels and increased catalase activity. The protective effect of this compound could represent a promising approach for the treatment of neurodegenerative diseases. PMID:22081883

  4. Neuroprotective effect of Salvia sahendica is mediated by restoration of mitochondrial function and inhibition of endoplasmic reticulum stress.

    Science.gov (United States)

    Shaerzadeh, Fatemeh; Alamdary, Shabnam Zeighamy; Esmaeili, Mohammad Ali; Sarvestani, Nazanin Namazi; Khodagholi, Fariba

    2011-12-01

    Herein, we investigated the protective effect of Salvia sahendica against H(2)O(2)-induced cell death in rat pheochromocytoma (PC12) cells. Our data show that S. sahendica blocks apoptosis pathway by inhibition of cytochrome c release from mitochondria and leakage of calcium from endoplasmic reticulum. It also activates/inactivates two members of Bcl-2 family, Bax and Bcl-2. Bax inhibition and Bcl-2 activation suppress release of cytochrome c from mitochondria that prevents cleavage of caspase-3. Besides S. sahendica suppresses ER stress via attenuation of intracellular levels of calcium. Suppression of ER stress decreased calpain activation and subsequently cleavage of caspase-12. Altogether, these results indicate that S. sahendica protects PC12 cells treated with H(2)O(2) via suppression of upstream factors of apoptosis pathway. While oxidative stress is an early event in Alzheimer disease, it seems that S. sahendica prevents deleterious effects of reactive oxygen species by stabilizing mitochondrial membranes and inhibiting ER stress. PMID:21769643

  5. Bordetella adenylate cyclase toxin mobilizes its beta2 integrin receptor into lipid rafts to accomplish translocation across target cell membrane in two steps.

    Directory of Open Access Journals (Sweden)

    Ladislav Bumba

    2010-05-01

    Full Text Available Bordetella adenylate cyclase toxin (CyaA binds the alpha(Mbeta(2 integrin (CD11b/CD18, Mac-1, or CR3 of myeloid phagocytes and delivers into their cytosol an adenylate cyclase (AC enzyme that converts ATP into the key signaling molecule cAMP. We show that penetration of the AC domain across cell membrane proceeds in two steps. It starts by membrane insertion of a toxin 'translocation intermediate', which can be 'locked' in the membrane by the 3D1 antibody blocking AC domain translocation. Insertion of the 'intermediate' permeabilizes cells for influx of extracellular calcium ions and thus activates calpain-mediated cleavage of the talin tether. Recruitment of the integrin-CyaA complex into lipid rafts follows and the cholesterol-rich lipid environment promotes translocation of the AC domain across cell membrane. AC translocation into cells was inhibited upon raft disruption by cholesterol depletion, or when CyaA mobilization into rafts was blocked by inhibition of talin processing. Furthermore, CyaA mutants unable to mobilize calcium into cells failed to relocate into lipid rafts, and failed to translocate the AC domain across cell membrane, unless rescued by Ca(2+ influx promoted in trans by ionomycin or another CyaA protein. Hence, by mobilizing calcium ions into phagocytes, the 'translocation intermediate' promotes toxin piggybacking on integrin into lipid rafts and enables AC enzyme delivery into host cytosol.

  6. Transcriptional profile of a myotube starvation model of atrophy

    Science.gov (United States)

    Stevenson, Eric J.; Koncarevic, Alan; Giresi, Paul G.; Jackman, Robert W.; Kandarian, Susan C.

    2005-01-01

    Skeletal muscle wasting is a pervasive phenomenon that can result from a wide range of pathological conditions as well as from habitual muscular inactivity. The present work describes a cell-culture condition that induces significant atrophy in skeletal muscle C2C12 myotubes. The failure to replenish differentiation media in mature myotubes leads to rapid atrophy (53% in diameter), which is referred to here as starvation. Affymetrix microarrays were used to develop a transcriptional profile of control (fed) vs. atrophied (nonfed) myotubes. Myotube starvation was characterized by an upregulation of genes involved in translational inhibition, amino acid biosynthesis and transport, and cell cycle arrest/apoptosis, among others. Downregulated genes included several structural and regulatory elements of the extracellular matrix as well as several elements of Wnt/frizzled and TGF-beta signaling pathways. Interestingly, the characteristic transcriptional upregulation of the ubiquitin-proteasome system, calpains, and cathepsins known to occur in multiple in vivo models of atrophy were not seen during myotube starvation. With the exception of the downregulation of extracellular matrix genes, serine protease inhibitor genes, and the upregulation of the translation initiation factor PHAS-I, this model of atrophy in cell culture has a transcriptional profile quite distinct from any study published to date with atrophy in whole muscle. These data show that, although the gross morphology of atrophied muscle fibers may be similar in whole muscle vs. myotube culture, the processes by which this phenotype is achieved differ markedly.

  7. Prenatal Stress Induces Long-Term Effects in Cell Turnover in the Hippocampus-Hypothalamus-Pituitary Axis in Adult Male Rats

    Science.gov (United States)

    Baquedano, Eva; García-Cáceres, Cristina; Diz-Chaves, Yolanda; Lagunas, Natalia; Calmarza-Font, Isabel; Azcoitia, Iñigo; Garcia-Segura, Luis M.; Argente, Jesús; Chowen, Julie A.; Frago, Laura M.

    2011-01-01

    Subchronic gestational stress leads to permanent modifications in the hippocampus-hypothalamus-pituitary-adrenal axis of offspring probably due to the increase in circulating glucocorticoids known to affect prenatal programming. The aim of this study was to investigate whether cell turnover is affected in the hippocampus-hypothalamus-pituitary axis by subchronic prenatal stress and the intracellular mechanisms involved. Restraint stress was performed in pregnant rats during the last week of gestation (45 minutes; 3 times/day). Only male offspring were used for this study and were sacrificed at 6 months of age. In prenatally stressed adults a decrease in markers of cell death and proliferation was observed in the hippocampus, hypothalamus and pituitary. This was associated with an increase in insulin-like growth factor-I mRNA levels, phosphorylation of CREB and calpastatin levels and inhibition of calpain -2 and caspase -8 activation. Levels of the anti-apoptotic protein Bcl-2 were increased and levels of the pro-apoptotic factor p53 were reduced. In conclusion, prenatal restraint stress induces a long-term decrease in cell turnover in the hippocampus-hypothalamus-pituitary axis that might be at least partly mediated by an autocrine-paracrine IGF-I effect. These changes could condition the response of this axis to future physiological and pathophysiological situations. PMID:22096592

  8. Genetic polymorphisms related to meat traits in purebred and crossbred Nelore cattle Polimorfismos genéticos relacionados às características da carne em bovinos Nelore puros e cruzados

    Directory of Open Access Journals (Sweden)

    Rogério Abdallah Curi

    2009-12-01

    Full Text Available The objective of this work was to estimate the allelic and genotypic frequencies of CAST/XmnI, a calpastatin gene polymorphism, and CAPN530, a calpain 1 large subunit gene polymorphism, in different beef genetic groups (Nelore and Nelore x Bos taurus, and to investigate associations between these polymorphisms and carcass and meat traits. Three hundred animals - comprising 114 Nelore, 67 Angus x Nelore, 44 Rubia Gallega x Nelore, 41 Canchim, 19 Brangus three-way cross and 15 Braunvieh three-way cross- were genotyped by PCR-RFLP and phenotyped for rib-eye area (REA, back-fat thickness (BT, intramuscular fat (IF, shear force (SF and myofibrillar fragmentation index (MFI. The occurrence of the two alleles of the CAST/XmnI and CAPN530 single nucleotide polymorphisms (SNPs in a B. indicus breed, which permitted association studies in purebred and crossbred Nelore cattle, was first shown in the present work. No relationship was found between the CAST or CAPN1 SNPs and growth-related traits (REA or fat deposition (BT and IF, since calpastatin and µ-calpain are not physiologically involved with these traits. Moreover, the association results between genotypes and aged meat tenderness (assessed by SF and MFI showed that these markers are useless in assisted selection for purebred Nelore and their crosses with B. taurus.O presente trabalho objetivou estimar, em bovinos de corte de diferentes grupos genéticos (Nelore e Nelore x Bos taurus, as frequências alélicas e genotípicas dos polimorfismos CAST/XmnI, do gene da calpastatina, e CAPN530, do gene da calpaína, bem como avaliar a ocorrência de associações entre esses polimorfismos e características da carcaça e da carne produzida. Trezentos animais - 114 Nelore, 67 Angus x Nelore, 44 Rubia Galega x Nelore, 41 Canchim, 19 tricross Brangus e 15 tricross Braunvieh - foram genotipados por PCR-RFLP e fenotipados para área de olho de lombo (AOL, cobertura de gordura subcutânea (CGS, gordura

  9. Tetanic contraction induces enhancement of fatigability and sarcomeric damage in atrophic skeletal muscle and its underlying molecular mechanisms.

    Science.gov (United States)

    Yu, Zhi-Bin

    2013-11-01

    intracellular resting Ca2+ concentration ([Ca2+]i) in unloaded soleus muscles. High [Ca2+]i activated calpain-1 which induced a higher degradation of desmin. Desmin degradation may loose connections between adjacent myofibrils and further misaligned Z-disc during repeated tetanic contractions. Passive stretch in unloaded muscle could preserve the stability of sarcoplasmic reticulum Ca2+ release channels by means of keeping nNOS activity, and decrease the enhanced protein level and activity of calpain to control levels in unloaded soleus muscles. Therefore, passive stretch restored normal appearance of Z-disc and resisted in part atrophy of unloaded soleus muscles. The above results indicate that enhanced fatigability of high-frequency tetanic contraction is associated to the alteration in K+ channel characteristics, and elevated SERCA activity and slow to fast transition of myosin heavy chain (MHC) isoforms increases fatigability of intermittent tetanic contraction in atrophic soleus muscle. The sarcomeric damage induced by tetanic contraction can be retarded by stretch in atrophic soleus muscles.

  10. The pharmacology of neurotrophic treatment with Cerebrolysin: brain protection and repair to counteract pathologies of acute and chronic neurological disorders.

    Science.gov (United States)

    Masliah, E; Díez-Tejedor, E

    2012-04-01

    Neurotrophic factors are considered as part of the therapeutic strategy for neurological disorders like dementia, stroke and traumatic brain injury. Cerebrolysin is a neuropeptide preparation which mimics the action of endogenous neurotrophic factors on brain protection and repair. In dementia models, Cerebrolysin decreases β-amyloid deposition and microtubule-associated protein tau phosphorylation by regulating glycogen synthase kinase-3β and cyclin-dependent kinase 5 activity, increases synaptic density and restores neuronal cytoarchitecture. These effects protect integrity of the neuronal circuits and thus result in improved cognitive and behavioral performance. Furthermore, Cerebrolysin enhances neurogenesis in the dentate gyrus, the basis for neuronal replacement therapy in neurodegenerative diseases. Experimental studies in stroke animal models have shown that Cerebrolysin stabilizes the structural integrity of cells by inhibition of calpain and reduces the number of apoptotic cells after ischemic lesion. Cerebrolysin induces restorative processes, decreases infarct volume and edema formation and promotes functional recovery. Stroke-induced neurogenesis in the subventricular zone was also promoted by Cerebrolysin, thus supporting the brain's self-repair after stroke. Both, traumatic brain and spinal cord injury conditions stimulate the expression of natural neurotrophic factors to promote repair and regeneration processes -axonal regeneration, neuronal plasticity and neurogenesis- that is considered to be crucial for the future recovery. Neuroprotective effects of Cerebrolysin on experimentally induced traumatic spinal cord injury have shown that Cerebrolysin prevents apoptosis of lesioned motoneurons and promotes functional recovery. This section summarizes the most relevant data on the pharmacology of Cerebrolysin obtained from in vitro assays (biochemical and cell cultures) and in vivo animal models of acute and chronic neurological disorders. PMID

  11. SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat

    Science.gov (United States)

    Goswami, Suneha; Kumar, Ranjeet R.; Dubey, Kavita; Singh, Jyoti P.; Tiwari, Sachidanand; Kumar, Ashok; Smita, Shuchi; Mishra, Dwijesh C.; Kumar, Sanjeev; Grover, Monendra; Padaria, Jasdeep C.; Kala, Yugal K.; Singh, Gyanendra P.; Pathak, Himanshu; Chinnusamy, Viswanathan; Rai, Anil; Praveen, Shelly; Rai, Raj D.

    2016-01-01

    Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger's sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD, and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs). We observed eight different types of post-translational modifications (PTMs) in the DEPs corresponds to the cloned ESTs-147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH) in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant), as compared to HD2329 (thermosusceptible) during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat—a novel step toward the development of

  12. Pyrrolidine dithiocarbamate-zinc(II) and -copper(II) complexes induce apoptosis in tumor cells by inhibiting the proteasomal activity.

    Science.gov (United States)

    Milacic, Vesna; Chen, Di; Giovagnini, Lorena; Diez, Alejandro; Fregona, Dolores; Dou, Q Ping

    2008-08-15

    Zinc and copper are trace elements essential for proper folding, stabilization and catalytic activity of many metalloenzymes in living organisms. However, disturbed zinc and copper homeostasis is reported in many types of cancer. We have previously demonstrated that copper complexes induced proteasome inhibition and apoptosis in cultured human cancer cells. In the current study we hypothesized that zinc complexes could also inhibit the proteasomal chymotrypsin-like activity responsible for subsequent apoptosis induction. We first showed that zinc(II) chloride was able to inhibit the chymotrypsin-like activity of a purified 20S proteasome with an IC(50) value of 13.8 microM, which was less potent than copper(II) chloride (IC(50) 5.3 microM). We then compared the potencies of a pyrrolidine dithiocarbamate (PyDT)-zinc(II) complex and a PyDT-copper(II) complex to inhibit cellular proteasomal activity, suppress proliferation and induce apoptosis in various human breast and prostate cancer cell lines. Consistently, zinc complex was less potent than copper complex in inhibiting the proteasome and inducing apoptosis. Additionally, zinc and copper complexes appear to use somewhat different mechanisms to kill tumor cells. Zinc complexes were able to activate calpain-, but not caspase-3-dependent pathway, while copper complexes were able to induce activation of both proteases. Furthermore, the potencies of these PyDT-metal complexes depend on the nature of metals and also on the ratio of PyDT to the metal ion within the complex, which probably affects their stability and availability for interacting with and inhibiting the proteasome in tumor cells. PMID:18501397

  13. Altered Calcium Handling in Reperfusion Injury.

    Science.gov (United States)

    Bompotis, Georgios C; Deftereos, Spyridon; Angelidis, Christos; Choidis, Efthymios; Panagopoulou, Vasiliki; Kaoukis, Andreas; Vassilikos, Vassilios P; Cleman, Michael W; Giannopoulos, Georgios

    2016-01-01

    Coronary Heart Disease (CHD) is the major mortality cause in the Western Hemisphere. Reinstituting blood flow in the acutely occluded coronary vessel became the standard intervention to prevent Myocardial Infarct (MI) progression. Ever since their conception, thrombolysis, Percutaneous Coronary Intervention (PCI) and Coronary Artery Bypass Grafting (CABG) have been at the forefront of CHD treatment, limiting MI size. However, it quickly became apparent that after a period of ischemia, reperfusion itself sets off a cascade of events leading to cell injury. It seems that cellular changes in the ischemic period, prime the cell for a loss of homeostasis once blood flow returns. Loss of calcium (Ca(2+)) regulation has been found to be a main culprit in both ischemia and reperfusion. Indeed, sarcoplasmic Ca(2+) overload during reperfusion is related to hypercontracture, proteolysis and mitochondrial failure--the so-called Reperfusion Injury (RI). Ca(2+) channels of the sarcolemma (SL) (L-Type Ca((2+)) Channels, Sodium / Calcium Exchanger) initiate Ca(2+) flux and those of the Sarcoplasmic Reticulum (SR) (Ca(2+) ATPase, Ca(2+) release channel) sustain the rise in intracellular Ca(2+) concentration. Ensuing interplay between Ca(2+), SR, mitochondria, myofilaments and proteolytic cascades i.e. calpain activation, results in cell injury. Novel insight about this interplay and details about the extent by which each of these players contributes to the RI, may allow scientists to devise and design proper interventions that ultimately reduce RI in clinical practice. The present article reviews the literature about key subcellular players participating in the sustained rise of cardiac myocyte cytosolic Ca(2+) during ischemia and reperfusion.

  14. Role of plasma membrane calcium ATPase 2 in spinal cord pathology

    Institute of Scientific and Technical Information of China (English)

    Amanda; Kathleen; Fakira; Stella; Elkabes

    2010-01-01

    A number of studies have indicated that plasma membrane calcium ATPases(PMCAs) are expressed in the brain and spinal cord and could play important roles not only in the maintenance of cellular calcium homeostasis but also in the survival and function of central nervous system cells under pathological conditions.The different regional and cellular distributions of the various PMCA isoforms and splice variants in the nervous system and the diverse phenotypes of PMCA knockout mice support the notion that each isoform might play a distinct role. Especially in the spinal cord,the survival of neurons and,in particular,motor neurons could be dependent on PMCA2.This is indicated by the knockdown of PMCA2 in pure spinal cord neuronal cultures that leads to cell death via a decrease in collapsing response mediator protein 1 levels.Moreover,the progressive decline in the number of motor neurons in PMCA2-null mice andheterozygous mice further supports this notion.Therefore,the reported reduction in PMCA2 mRNA and protein levels in the inflamed spinal cord of mice affected by experimental autoimmune encephalomyelitis(EAE) ,an animal model of multiple sclerosis,and after spinal cord contusion injury,suggests that changes in PMCA2 expression could be a cause of neuronal pathology and death during inflammation and injury.Glutamate excitotoxicity mediated via kainate receptors has been implicated in the neuropathology of both EAE and spinal cord injury,and has been identified as a trigger that reduces PMCA2 levels in pure spinal cord neuronal cultures through degradation of the pump by calpain without affecting PMCA2 transcript levels.It remains to be determined which other stimuli modulate PMCA2 mRNA expression in the aforementioned pathological conditions of the spinal cord.

  15. Predicting the Proteins of Angomonas deanei, Strigomonas culicis and Their Respective Endosymbionts Reveals New Aspects of the Trypanosomatidae Family

    Science.gov (United States)

    Motta, Maria Cristina Machado; Martins, Allan Cezar de Azevedo; de Souza, Silvana Sant’Anna; Catta-Preta, Carolina Moura Costa; Silva, Rosane; Klein, Cecilia Coimbra; de Almeida, Luiz Gonzaga Paula; de Lima Cunha, Oberdan; Ciapina, Luciane Prioli; Brocchi, Marcelo; Colabardini, Ana Cristina; de Araujo Lima, Bruna; Machado, Carlos Renato; de Almeida Soares, Célia Maria; Probst, Christian Macagnan; de Menezes, Claudia Beatriz Afonso; Thompson, Claudia Elizabeth; Bartholomeu, Daniella Castanheira; Gradia, Daniela Fiori; Pavoni, Daniela Parada; Grisard, Edmundo C.; Fantinatti-Garboggini, Fabiana; Marchini, Fabricio Klerynton; Rodrigues-Luiz, Gabriela Flávia; Wagner, Glauber; Goldman, Gustavo Henrique; Fietto, Juliana Lopes Rangel; Elias, Maria Carolina; Goldman, Maria Helena S.; Sagot, Marie-France; Pereira, Maristela; Stoco, Patrícia H.; de Mendonça-Neto, Rondon Pessoa; Teixeira, Santuza Maria Ribeiro; Maciel, Talles Eduardo Ferreira; de Oliveira Mendes, Tiago Antônio; Ürményi, Turán P.; de Souza, Wanderley; Schenkman, Sergio; de Vasconcelos, Ana Tereza Ribeiro

    2013-01-01

    Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism

  16. Dexamethasone protected human glioblastoma U87MG cells from temozolomide induced apoptosis by maintaining Bax:Bcl-2 ratio and preventing proteolytic activities

    Directory of Open Access Journals (Sweden)

    Patel Sunil J

    2004-12-01

    Full Text Available Abstract Background Glioblastoma is the deadliest and most prevalent brain tumor. Dexamethasone (DXM is a commonly used steroid for treating glioblastoma patients for alleviation of vasogenic edema and pain prior to treatment with chemotherapeutic drugs. Temozolomide (TMZ, an alkylating agent, has recently been introduced in clinical trials for treating glioblastoma. Here, we evaluated the modulatory effect of DXM on TMZ induced apoptosis in human glioblastoma U87MG cells. Results Freshly grown cells were treated with different doses of DXM or TMZ for 6 h followed by incubation in a drug-free medium for 48 h. Wright staining and ApopTag assay showed no apoptosis in cells treated with 40 μM DXM but considerable amounts of apoptosis in cells treated with 100 μM TMZ. Apoptosis in TMZ treated cells was associated with an increase in intracellular free [Ca2+], as determined by fura-2 assay. Western blot analyses showed alternations in the levels of Bax (pro-apoptotic and Bcl-2 (anti-apoptotic proteins resulting in increased Bax:Bcl-2 ratio in TMZ treated cells. Western blot analyses also detected overexpression of calpain and caspase-3, which cleaved 270 kD α-spectrin at specific sites for generation of 145 and 120 kD spectrin break down products (SBDPs, respectively. However, 1-h pretreatment of cells with 40 μM DXM dramatically decreased TMZ induced apoptosis, decreasing Bax:Bcl-2 ratio and SBDPs. Conclusion Our results revealed an antagonistic effect of DXM on TMZ induced apoptosis in human glioblastoma U87MG cells, implying that treatment of glioblastoma patients with DXM prior to chemotherapy with TMZ might result in an undesirable clinical outcome.

  17. Nisin ZP, a Bacteriocin and Food Preservative, Inhibits Head and Neck Cancer Tumorigenesis and Prolongs Survival.

    Directory of Open Access Journals (Sweden)

    Pachiyappan Kamarajan

    Full Text Available The use of small antimicrobial peptides or bacteriocins, like nisin, to treat cancer is a new approach that holds great promise. Nisin exemplifies this new approach because it has been used safely in humans for many years as a food preservative, and recent laboratory studies support its anti-tumor potential in head and neck cancer. Previously, we showed that nisin (2.5%, low content has antitumor potential in head and neck squamous cell carcinoma (HNSCC in vitro and in vivo. The current studies explored a naturally occurring variant of nisin (nisin ZP; 95%, high content for its antitumor effects in vitro and in vivo. Nisin ZP induced the greatest level of apoptosis in HNSCC cells compared to low content nisin. HNSCC cells treated with increasing concentrations of nisin ZP exhibited increasing levels of apoptosis and decreasing levels of cell proliferation, clonogenic capacity, and sphere formation. Nisin ZP induced apoptosis through a calpain-dependent pathway in HNSCC cells but not in human oral keratinocytes. Nisin ZP also induced apoptosis dose-dependently in human umbilical vein endothelial cells (HUVEC with concomitant decreases in vascular sprout formation in vitro and reduced intratumoral microvessel density in vivo. Nisin ZP reduced tumorigenesis in vivo and long-term treatment with nisin ZP extended survival. In addition, nisin treated mice exhibited normal organ histology with no evidence of inflammation, fibrosis or necrosis. In summary, nisin ZP exhibits greater antitumor effects than low content nisin, and thus has the potential to serve as a novel therapeutic for HNSCC.

  18. Emulsion-core and polyelectrolyte-shell nanocapsules: biocompatibility and neuroprotection against SH-SY5Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Piotrowski, Marek, E-mail: ncpiotro@cyf-kr.edu.pl; Szczepanowicz, Krzysztof [Polish Academy of Sciences, Jerzy Haber Institute of Catalysis and Surface Chemistry (Poland); Jantas, Danuta; Leśkiewicz, Monika; Lasoń, Władysław [Polish Academy of Sciences, Institute of Pharmacology (Poland); Warszyński, Piotr [Polish Academy of Sciences, Jerzy Haber Institute of Catalysis and Surface Chemistry (Poland)

    2013-11-15

    The emulsion-core and polyelectrolyte-coated nanocapsules, designed as water-insoluble neuroprotective drug delivery system, were synthesized using layer-by-layer saturation method. The isopropyl myristate was used as oil phase and docusate sodium salt as emulsifier. For the polyelectrolyte shell preparation, synthetic polyelectrolytes, cationic (PDADMAC, PAH, and PLL) and anionic (PGA) were used. The particle size and zeta potential of nanocapsules were characterized by the dynamic light scattering. The average size of synthesized nanocapsules ranged from ∼80 to ∼100 nm. Zeta potential values ranged from less than approximately −30 mV for the polyanion layers to greater than approximately +30 mV for the polycation layers. Biocompatibilities of the synthesized nanocarriers were evaluated against SH-SY5Y human neuroblastoma cells using various biochemical assays. The results obtained show that synthesized nanocapsules coated with PLL and PGA were nontoxic to SH-SY5Y cells, and they were used as nanocarriers for model neuroprotective drug (a calpain inhibitor MDL 28170). The neuroprotective action of the encapsulated MDL 28170 against hydrogen peroxide-induced oxidative stress cytotoxicity was evaluated in the same cell line. The results showed that nanoencapsulated form of MDL 28170 were biocompatible and protected SH-SY5Y cells against the H{sub 2}O{sub 2} (0.5 mM/24 h)-induced damage in 20–40 times lower concentrations than those of the same drug added directly to the culture medium. These data suggest that the nanoscale carriers of neuroprotective drugs might serve as novel promising therapeutic agents for oxidative stress-related neurodegenerative processes.

  19. Involvement of Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

    International Nuclear Information System (INIS)

    Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide distribution in more than 6000 high plants including medicinal herbs or teas. The aim of this study is to investigate the signal pathway involved in PAs-induced hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9 activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL in a time (8-48 h)- and concentration (1-100 μM)-dependent manner. Furthermore, inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited clivorine-induced apoptosis and rescued clivorine-decreased cell viability. Polyubiquitination of Bcl-xL was detected after incubation with 100 μM clivorine for 40 h in the presence of proteasome specific inhibitor MG132, indicating possible degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell viability. All the other tested PAs such as senecionine, isoline and monocrotaline decreased mouse hepatocytes viability in a concentration-dependent manner. Clivorine (10 μM) induced caspase-3 activation and decreased Bcl-xL was also confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus leading to the activation of mitochondrial-mediated apoptotic pathway

  20. Genetic Risk Factors for Type 2 Diabetes with Pharmacologic Intervention in African-American Patients with Schizophrenia or Schizoaffective Disorder

    Science.gov (United States)

    MR, Irvin; HW, Wiener; RP, Perry; RM, Savage; CP, Go R

    2009-01-01

    An increased prevalence of type 2 diabetes (T2D) in schizophrenia (SCZ) patients has been observed. Exposure to antipsychotics (APs) has been shown to induce metabolic dysregulation in some patients but not all treated patients. We hypothesized important candidate genes for T2D may increase risk for T2D in African-American patients with SCZ or schizoaffective disorder. The PAARTNERS study comprises African-American families with at least one proband with SCZ or schizoaffective disorder. The current study of PAARTNERS SCZ and schizoaffective disorder cases (N=820) examined single nucleotide polymorphisms (SNPs) within select T2D candidate genes including transcription factor like 7 (TCF7L2), calpain 10 (CAPN10), and ectoenzyme nucleotide pyrophosphate phosphodiesterase 1 (ENNP1) for association with prevalent T2D. We report association of TCF7L2 (rs7903146) with T2D under both an additive and recessive model for the risk allele T. Specifically, the odds ratio (OR) for having T2D was 1.4 (p=0.03) under an additive model and 2.4 (p=0.004) under a recessive model. We also report a marginally significant TCF7L2 by AP treatment interaction that should be investigated in future studies. CAPN10 (rs3792267) was marginally associated with T2D with OR=1.5 (p=0.08) when considering the model GG vs. AG/AA with risk allele G. ENPP1 (rs1044498) was not associated with T2D. We conclude TCF7L2, a risk factor for T2D in the general population, is also a risk factor for T2D in African-American patients with SCZ or schizoaffective disorder. Research is needed to determine if T2D associated polymorphisms are of interest in the pharmacogenetics and future treatment choices of antipsychotics in African-American patients. PMID:19643578

  1. Small interfering RNA targeting mcl-1 enhances proteasome inhibitor-induced apoptosis in various solid malignant tumors

    Directory of Open Access Journals (Sweden)

    Zhou Wei

    2011-11-01

    Full Text Available Abstract Background Targeting the ubiquitin-proteasome pathway is a promising approach for anticancer strategies. Recently, we found Bik accumulation in cancer cell lines after they were treated with bortezomib. However, recent evidence indicates that proteasome inhibitors may also induce the accumulation of anti-apoptotic Bcl-2 family members. The current study was designed to analyze the levels of several anti-apoptotic members of Bcl-2 family in different human cancer cell lines after they were treated with proteasome inhibitors. Methods Different human cancer cell lines were treated with proteasome inhibitors. Western blot were used to investigate the expression of Mcl-1 and activation of mitochondrial apoptotic signaling. Cell viability was investigated using SRB assay, and induction of apoptosis was measured using flow cytometry. Results We found elevated Mcl-1 level in human colon cancer cell lines DLD1, LOVO, SW620, and HCT116; human ovarian cancer cell line SKOV3; and human lung cancer cell line H1299, but not in human breast cancer cell line MCF7 after they were treated with bortezomib. This dramatic Mcl-1 accumulation was also observed when cells were treated with other two proteasome inhibitors, MG132 and calpain inhibitor I (ALLN. Moreover, our results showed Mcl-1 accumulation was caused by stabilization of the protein against degradation. Reducing Mcl-1 accumulation by Mcl-1 siRNA reduced Mcl-1 accumulation and enhanced proteasome inhibitor-induced cell death and apoptosis, as evidenced by the increased cleavage of caspase-9, caspase-3, and poly (ADP-ribose polymerase. Conclusions Our results showed that it was not only Bik but also Mcl-1 accumulation during the treatment of proteasome inhibitors, and combining proteasome inhibitors with Mcl-1 siRNA would enhance the ultimate anticancer effect suggesting this combination might be a more effective strategy for cancer therapy.

  2. Outer mitochondrial membrane localization of apoptosis-inducing factor: mechanistic implications for release

    Directory of Open Access Journals (Sweden)

    Seong‑Woon Yu

    2009-11-01

    Full Text Available Poly(ADP-ribose polymerase-1-dependent cell death (known as parthanatos plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor, but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders.

  3. SSH analysis of endosperm transcripts and characterization of heat stress regulated expressed sequence tags in bread wheat

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    Suneha Goswami

    2016-08-01

    Full Text Available Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h wheat cv. HD2985 by suppression subtractive hybridization (SSH. We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger’s sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs. Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs. We observed eight different types of post-translational modifications (PTMs in the DEPs corresponds to the cloned ESTs—147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant, as compared to HD2329 (thermosusceptible during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat – a novel step towards the development of

  4. SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat

    Science.gov (United States)

    Goswami, Suneha; Kumar, Ranjeet R.; Dubey, Kavita; Singh, Jyoti P.; Tiwari, Sachidanand; Kumar, Ashok; Smita, Shuchi; Mishra, Dwijesh C.; Kumar, Sanjeev; Grover, Monendra; Padaria, Jasdeep C.; Kala, Yugal K.; Singh, Gyanendra P.; Pathak, Himanshu; Chinnusamy, Viswanathan; Rai, Anil; Praveen, Shelly; Rai, Raj D.

    2016-01-01

    Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger's sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD, and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs). We observed eight different types of post-translational modifications (PTMs) in the DEPs corresponds to the cloned ESTs-147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH) in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant), as compared to HD2329 (thermosusceptible) during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat—a novel step toward the development of

  5. Evidence that the blood biomarker SNTF predicts brain imaging changes and persistent cognitive dysfunction in mild TBI patients

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    Robert eSiman

    2013-11-01

    Full Text Available Although mild traumatic brain injury (mTBI, or concussion, is not typically associated with abnormalities on computed tomography (CT, it nevertheless causes persistent cognitive dysfunction for many patients. Consequently, new prognostic methods for mTBI are needed to identify at-risk cases, especially at an early and potentially treatable stage. Here, we quantified plasma levels of the neurodegeneration biomarker calpain-cleaved alphaII-spectrin N-terminal fragment (SNTF from 38 participants with CT-negative mTBI, orthopedic injury (OI and normal uninjured controls (age range 12-30 years, and compared them with findings from diffusion tensor magnetic resonance imaging (DTI and long-term cognitive assessment. SNTF levels were at least twice the lower limit of detection in 7 of 17 mTBI cases and in 3 of 13 OI cases, but in none of the uninjured controls. An elevation in plasma SNTF corresponded with significant differences in fractional anisotropy and the apparent diffusion coefficient in the corpus callosum and uncinate fasciculus measured by DTI. Furthermore, increased plasma SNTF on the day of injury correlated significantly with cognitive impairment that persisted for at least 3 months, both across all study participants and also among the mTBI cases by themselves. The elevation in plasma SNTF in the subset of OI cases, accompanied by corresponding white matter and cognitive abnormalities, raises the possibility of identifying undiagnosed cases of mTBI. These data suggest that the blood level of SNTF on the day of a CT-negative mTBI may identify a subset of patients at risk of white matter damage and persistent disability. SNTF could have prognostic and diagnostic utilities in the assessment and treatment of mTBI.

  6. Diagnostic protein biomarkers for severe, moderate and mild traumatic brain injury

    Science.gov (United States)

    Streeter, Jackson; Hayes, Ronald L.; Wang, Kevin K. W.

    2011-06-01

    Traumatic Brain Injury (TBI) is a major problem in military and civilian medicine. Yet, there are no simple non-invasive diagnostics for TBI. Our goal is to develop and clinically validate blood-based biomarker assays for the diagnosis, prognosis and management of mild, moderate and severe TBI patients. These assays will ultimately be suitable for deployment to far-forward combat environments. Using a proteomic and systems biology approach, we identified over 20 candidate biomarkers for TBI and developed robust ELISAs for at least 6 candidate biomarkers, including Ubiquitin C-terminal hydrolase- L1 (UCH-L1), Glial Fibrillary Acidic Protein (GFAP) and a 145 kDa breakdown products of αII-spectrin (SBDP 145) generated by calpain proteolysis. In a multi-center feasibility study (Biomarker Assessment For Neurotrauma Diagnosis And Improved Triage System (BANDITS), we analyzed CSF and blood samples from 101 adult patients with severe TBI [Glasgow Coma Scale (GCS) <= 8] at 6 sites and analyzed 27 mild TBI patients and 5 moderate TBI patients [GCS 9-15] from 2 sites in a pilot study. We identified that serum levels of UCH-L1, GFAP and SBDP145 have strong diagnostic and prognostic properties for severe TBI over controls. Similarly initial post-TBI serum levels (< 6 h) of UCH-L1 and GFAP have diagnostic characteristics for moderate and mild TBI. We are now furthering assay production, refining assay platforms (both benchtop and point-ofcare/ handheld) and planning a pivotal clinical study to seek FDA approval of these TBI diagnostic assays.

  7. Nuclear α Spectrin Differentially Affects Monoubiquitinated Versus Non-Ubiquitinated FANCD2 Function After DNA Interstrand Cross-Link Damage.

    Science.gov (United States)

    Zhang, Pan; Sridharan, Deepa; Lambert, Muriel W

    2016-03-01

    Nonerythroid α spectrin (αIISp) and the Fanconi anemia (FA) protein, FANCD2, play critical roles in DNA interstrand cross-link (ICL) repair during S phase. Both are needed for recruitment of repair proteins, such as XPF, to sites of damage and repair of ICLs. However, the relationship between them in ICL repair and whether αIISp is involved in FANCD2's function in repair is unclear. The present studies show that, after ICL formation, FANCD2 disassociates from αIISp and localizes, before αIISp, at sites of damage in nuclear foci. αIISp and FANCD2 foci do not co-localize, in contrast to our previous finding that αIISp and the ICL repair protein, XPF, co-localize and follow a similar time course for formation. Knock-down of αIISp has no effect on monoubiquitination of FANCD2 (FANCD2-Ub) or its localization to chromatin or foci, though it leads to decreased ICL repair. Studies using cells from FA patients, defective in ICL repair and αIISp, have elucidated an important role for αIISp in the function of non-Ub FANCD2. In FA complementation group A (FA-A) cells, in which FANCD2 is not monoubiquitinated and does not form damage-induced foci, we demonstrate that restoration of αIISp levels to normal, by knocking down the protease μ-calpain, leads to formation of non-Ub FANCD2 foci after ICL damage. Since restoration of αIISp levels in FA-A cells restores DNA repair and cell survival, we propose that αIISp is critical for recruitment of non-Ub FANCD2 to sites of damage, which has an important role in the repair response and ICL repair. PMID:26297932

  8. The influence of chronic nicotine treatment on proteins expressed in the mouse hippocampus and cortex.

    Science.gov (United States)

    Matsuura, Kenji; Otani, Mieko; Takano, Masaoki; Kadoyama, Keiichi; Matsuyama, Shogo

    2016-06-01

    Chronic treatment with nicotine, the primary psychoactive substance in tobacco smoke, affects central nervous system functions, such as synaptic plasticity. Here, to clarify the effects of chronic nicotine treatment on the higher brain functions, proteomic analysis of the hippocampus and cortex of mice treated for 6 months with nicotine was performed using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry. There was significant change in the expression of 16 proteins and one phosphoprotein in the hippocampus (increased tubulin β-5, atp5b, MDH1, cytochrome b-c1 complex subunit 1, Hsc70, dynamin, profilin-2, 4-aminobutyrate aminotransferase, mitochondrial isoform 1 precursor, calpain small subunit 1, and vacuolar adenosine triphosphatase subunit B and decreased γ-actin, α-tubulin isotype M-α-2, putative β-actin, tubulin β-2A, NDUFA10, and G6PD) and 24 proteins and two phosphoproteins in the cortex (increased spectrin α chain, non-erythrocytic 1 isoform 1, tubulin β-5, γ-actin, creatine kinase B-type, LDH-B, secernin-1, UCH-L1, 14-3-3 γ, type II peroxiredoxin 1, PEBP-1, and unnamed protein product and decreased tubulin α-1C, α-internexin, γ-enolase, PDHE1-B, DPYL2, vacuolar adenosine triphosphatase subunit A, vacuolar adenosine triphosphatase subunit B, TCTP, NADH dehydrogenase Fe-S protein 1, protein disulfide-isomerase A3, hnRNP H2, γ-actin, atp5b, and unnamed protein product). Additionally, Western blotting validated the changes in dynamin, Hsc70, MDH1, NDUFA10, α-internexin, tubulin β-5 chain, and secernin-1. Thus, these findings indicate that chronic nicotine treatment changes the expression of proteins and phosphoproteins in the hippocampus and cortex. We propose that effect of smoking on higher brain functions could be mediated by alterations in expression levels of these proteins. PMID:26988295

  9. Protective effects of resveratrol in experimental retinal detachment.

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    Wei Huang

    Full Text Available BACKGROUND: Oxidative stress is one of the major factors that trigger photoreceptor apoptosis. To investigate whether resveratrol, a potent antioxidant and small molecule activator of the FoxO pathway, would be neuroprotective against photoreceptor cell death in a rodent model of retinal detachment. METHODS: Retinal detachment was created in adult Brown Norway rats by subretinal injection of sodium hyaluronate. The animals were treated daily with vehicle or resveratrol (20 mg/kg intraperitoneal injection. Photoreceptor death was assessed by counting the number of apoptotic cells with TdT-dUTP terminal nick-end labeling (TUNEL and measurement of the outer nuclear layer (ONL thickness 3 days after RD. Changes in expression of FoxO1a, FoxO3a, and FoxO4 were analyzed by western blot. The activity of caspase 3, caspase 8, caspase 9, spectrin and their cleavage forms were studied. RESULTS: Three days after retinal detachment, caspase 3, caspase 8 and caspase 9 were significantly activated in the detached retina. Spectrin cleavage products at 120 and 145 kDa were also detected. Both caspase and calpain activation are involved in apoptotic photoreceptor cell death in detached retinas. Treatment with resveratrol increases FoxO1a, FoxO3a, and FoxO4 protein expression in detached retinas only. Resveratrol treatment decreases activation of intrinsic and extrinsic caspase apoptotic pathways triggered by RD. The number of TUNEL-positive cells decreases from 1301±51 cells/mm(2 in control groups to 430±35 cells/mm(2 in treatment groups (p<0.05. Resveratrol treatment also demonstrates 59% less ONL thickness loss compared to controls. CONCLUSIONS: Resveratrol treatment up-regulates the FoxO family and blocks Caspase3, 8, and 9 activation. Resveratrol has the potential to be used as a novel therapeutic agent for preventing vision loss in diseases characterized by photoreceptor detachment.

  10. Novel quinazolinone MJ-29 triggers endoplasmic reticulum stress and intrinsic apoptosis in murine leukemia WEHI-3 cells and inhibits leukemic mice.

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    Chi-Cheng Lu

    Full Text Available The present study was to explore the biological responses of the newly compound, MJ-29 in murine myelomonocytic leukemia WEHI-3 cells in vitro and in vivo fates. We focused on the in vitro effects of MJ-29 on ER stress and mitochondria-dependent apoptotic death in WEHI-3 cells, and to hypothesize that MJ-29 might fully impair the orthotopic leukemic mice. Our results indicated that a concentration-dependent decrease of cell viability was shown in MJ-29-treated cells. DNA content was examined utilizing flow cytometry, whereas apoptotic populations were determined using annexin V/PI, DAPI staining and TUNEL assay. Increasing vital factors of mitochondrial dysfunction by MJ-29 were further investigated. Thus, MJ-29-provaked apoptosis of WEHI-3 cells is mediated through the intrinsic pathway. Importantly, intracellular Ca(2+ release and ER stress-associated signaling also contributed to MJ-29-triggered cell apoptosis. We found that MJ-29 stimulated the protein levels of calpain 1, CHOP and p-eIF2α pathways in WEHI-3 cells. In in vivo experiments, intraperitoneal administration of MJ-29 significantly improved the total survival rate, enhanced body weight and attenuated enlarged spleen and liver tissues in leukemic mice. The infiltration of immature myeloblastic cells into splenic red pulp was reduced in MJ-29-treated leukemic mice. Moreover, MJ-29 increased the differentiations of T and B cells but decreased that of macrophages and monocytes. Additionally, MJ-29-stimulated immune responses might be involved in anti-leukemic activity in vivo. Based on these observations, MJ-29 suppresses WEHI-3 cells in vitro and in vivo, and it is proposed that this potent and selective agent could be a new chemotherapeutic candidate for anti-leukemia in the future.

  11. Antifeedant effects of proteinase inhibitors on feeding behaviors of adult western corn rootworm (Diabrotica virgifera virgifera).

    Science.gov (United States)

    Kim, Jae Hak; Mullin, Christopher A

    2003-04-01

    Low-molecular-weight peptidyl proteinase inhibitors (PIs) including leupeptin, calpain inhibitor I, and calpeptin were found to be potent antifeedants for adult western corn rootworm (WCR) against the phagostimulation of cucurbitacin B (Cuc B) or a corn pollen extract (CPE). Leupeptin was the strongest (ED50 = 0.36 and 0.55 nmol/disk for Cuc B and CPE, respectively) among PIs tested with an antifeedant potency much stronger than the steroid progesterone (ED50 = 2.29 and 5.05 nmol/disk for Cuc B and CPE, respectively), but slightly less than the reference alkaloid, strychnine (ED50 = 0.17 and 0.37 nmol/disk for Cuc B and CPE, respectively). All active PIs contain a di- or tripeptidyl aldehyde moiety, indicating that PIs exert their antifeedant effects by covalent interaction with putative sulfhydryl (SH) groups on taste receptors as do these PIs with cysteine proteinases. However, opposite inhibition potency against Cuc B versus CPE by two thiol-group reducing agents, DTT and L-cysteine, and the results with other cysteine-modifying reagents obscure the net functional role of SH groups at WCR taste chemoreceptors. Surprisingly, the model phagostimulant for diabroticites, Cuc B, was more easily counteracted by these feeding deterrents than the stimulants present in CPE. Three-dimensional structure-antifeedant relationships for the PIs suggest that a novel taste chemoreception mechanism exists for these peptidyl aldehydes or that they fit partially into a strychnine binding pocket on protein chemoreceptors. Favorable economic benefit may be achieved if PIs are discovered to be useful in adult WCR control, since both pre- and postingestive sites would be targeted. PMID:12775144

  12. Repair of traumatic plasmalemmal damage to neurons and other eukar yotic cells

    Institute of Scientific and Technical Information of China (English)

    George D. Bittner; Christopher S. Spaeth§; Andrew D. Poon; Zachary S. Burgess; Christopher H. McGill

    2016-01-01

    The repair (sealing) of plasmalemmal damage, consisting of small holes to complete transections, is criti-cal for cell survival, especially for neurons that rarely regenerate cell bodies. We ifrst describe and evaluate different measures of cell sealing. Some measures, including morphological/ultra-structural observations, membrane potential, and input resistance, provide very ambiguous assessments of plasmalemmal sealing. In contrast, measures of ionic current lfow and dye barriers can, if appropriately used, provide more ac-curate assessments. We describe the effects of various substances (calcium, calpains, cytoskeletal proteins, ESCRT proteins, mUNC-13, NSF, PEG) and biochemical pathways (PKA, PKC, PLC, Epac, cytosolic ox-idation) on plasmalemmal sealing probability, and suggest that substances, pathways, and cellular events associated with plasmalemmal sealing have undergone a very conservative evolution. During sealing, calcium ion inlfux mobilizes vesicles and other membranous structures (lysosomes, mitochondria, etc.) in a continuous fashion to form a vesicular plug that gradually restricts diffusion of increasingly smaller molecules and ions over a period of seconds to minutes. Furthermore, we find no direct evidence that sealing occurs through the collapse and fusion of severed plasmalemmal lealfets, or in a single step involv-ing the fusion of one large wound vesicle with the nearby, undamaged plasmalemma. We describe how increases in perikaryal calcium levels following axonal transection account for observations that cell body survival decreases the closer an axon is transected to the perikaryon. Finally, we speculate on relationships between plasmalemmal sealing, Wallerian degeneration, and the ability of polyethylene glycol (PEG) to seal cell membranes and rejoin severed axonal ends–an important consideration for the future treatment of trauma to peripheral nerves. A better knowledge of biochemical pathways and cytoplasmic structures in-volved in

  13. Molecular mechanisms of 6-hydroxydopamine-induced cytotoxicity in PC12 cells: involvement of hydrogen peroxide-dependent and -independent action.

    Science.gov (United States)

    Saito, Yoshiro; Nishio, Keiko; Ogawa, Yoko; Kinumi, Tomoya; Yoshida, Yasukazu; Masuo, Yoshinori; Niki, Etsuo

    2007-03-01

    The neurotoxin 6-hydroxydopamine (6-OHDA) has been widely used to generate an experimental model of Parkinson's disease. It has been reported that reactive oxygen species (ROS), such as the superoxide anion and hydrogen peroxide (H2O2), generated from 6-OHDA are involved in its cytotoxicity; however, the contribution and role of ROS in 6-OHDA-induced cell death have not been fully elucidated. In the present study using PC12 cells, we observed the generation of 50 microM H2O2 from a lethal concentration of 100 microM 6-OHDA within a few minutes, and compared the sole effect of H2O2 with 6-OHDA. Catalase, an H2O2-removing enzyme, completely abolished the cytotoxic effect of H2O2, while a significant but partial protective effect was observed against 6-OHDA. 6-OHDA induced peroxiredoxin oxidation, cytochrome c release, and caspase-3 activation. Catalase exhibited a strong inhibitory effect against the peroxiredoxin oxidation, and cytochrome c release induced by 6-OHDA; however, caspase-3 activation was not effectively inhibited by catalase. On the other hand, 6-OHDA-induced caspase-3 activation was inhibited in the presence of caspase-8, caspase-9, and calpain inhibitors. These results suggest that the H2O2 generated from 6-OHDA plays a pivotal role in 6-OHDA-induced peroxiredoxin oxidation, and cytochrome c release, while H2O2- and cytochrome c-independent caspase activation pathways are involved in 6-OHDA-induced neurotoxicity. These findings may contribute to explain the importance of generated H2O2 and secondary products as a second messenger of 6-OHDA-induced cell death signal linked to Parkinson's disease.

  14. Effect of single nucleotide polymorphisms of CAPN1 and CAST genes on meat traits in Nellore beef cattle (Bos indicus) and in their crosses with Bos taurus.

    Science.gov (United States)

    Curi, R A; Chardulo, L A L; Mason, M C; Arrigoni, M D B; Silveira, A C; de Oliveira, H N

    2009-08-01

    The objectives of this work were to study the segregation of single nucleotide polymorphisms of the calpain 1, large subunit (CAPN1) and calpastatin (CAST) genes in Nellore (Bos indicus) and Nellore xBos taurus beef cattle, as well as to evaluate their effects on meat traits. For this, 300 animals, including 114 Nellore, 67 Angus x Nellore, 44 Rubia Gallega x Nellore, 41 Canchim, 19 Brangus three-way crosses and 15 Braunvieh three-way crosses, were genotyped for the CAPN4751 [AF_248054.2:g.6545C>T (GenBank accession AF248054.2)] and CAST/DdeI [AF_159246.1:g.2959A>G (GenBank accession AF159246.1)] polymorphisms and phenotyped for Ribeye Area, Backfat Thickness, Intramuscular Fat, Shear Force (SF) and Myofibrillar Fragmentation Index (MFI). In relation to the CAPN4751 polymorphism, a frequency of 10.5% was observed for the C allele in the Nellore breed. In the total sample of studied animals, a significant association was found between genotypes and meat tenderness, assessed by SF (P = 0.005) and MFI (P = 0.008), with genotype CT being more favourable than TT. For the CAST/DdeI polymorphism, a frequency of 55.7% was found for the A allele in the Nellore breed. In the total sample, a significant association was observed between genotypes and meat tenderness - SF (P = 0.004) and MFI (P = 0.006), with genotype AA being more favourable than AG. The relationship between genotypes and aged meat tenderness in confluence with the distribution of favourable alleles shows great potential for application of the CAPN4751 and CAST/DdeI polymorphisms in the genetic improvement of the Nellore breed, whilst contributing to the validation, in this breed and in its crosses with B. taurus, of the association results previously described in the literature.

  15. Maleic Acid--but Not Structurally Related Methylmalonic Acid--Interrupts Energy Metabolism by Impaired Calcium Homeostasis.

    Science.gov (United States)

    Tuncel, Ali Tunç; Ruppert, Thorsten; Wang, Bei-Tzu; Okun, Jürgen Günther; Kölker, Stefan; Morath, Marina Alexandra; Sauer, Sven Wolfgang

    2015-01-01

    Maleic acid (MA) has been shown to induce Fanconi syndrome via disturbance of renal energy homeostasis, though the underlying pathomechanism is still under debate. Our study aimed to examine the pathomechanism underlying maleic acid-induced nephrotoxicity. Methylmalonic acid (MMA) is structurally similar to MA and accumulates in patients affected with methymalonic aciduria, a defect in the degradation of branched-chain amino acids, odd-chain fatty acids and cholesterol, which is associated with the development of tubulointerstitial nephritis resulting in chronic renal failure. We therefore used MMA application as a control experiment in our study and stressed hPTECs with MA and MMA to further validate the specificity of our findings. MMA did not show any toxic effects on proximal tubule cells, whereas maleic acid induced concentration-dependent and time-dependent cell death shown by increased lactate dehydrogenase release as well as ethidium homodimer and calcein acetoxymethyl ester staining. The toxic effect of MA was blocked by administration of single amino acids, in particular L-alanine and L-glutamate. MA application further resulted in severe impairment of cellular energy homeostasis on the level of glycolysis, respiratory chain, and citric acid cycle resulting in ATP depletion. As underlying mechanism we could identify disturbance of calcium homeostasis. MA toxicity was critically dependent on calcium levels in culture medium and blocked by the extra- and intracellular calcium chelators EGTA and BAPTA-AM respectively. Moreover, MA-induced cell death was associated with activation of calcium-dependent calpain proteases. In summary, our study shows a comprehensive pathomechanistic concept for MA-induced dysfunction and damage of human proximal tubule cells. PMID:26086473

  16. Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice

    Science.gov (United States)

    Grill, Mischala A.; Bales, Mark A.; Fought, Amber N.; Rosburg, Kristopher C.; Munger, Stephanie J.; Antin, Parker B.

    2003-01-01

    Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.

  17. Predicting the proteins of Angomonas deanei, Strigomonas culicis and their respective endosymbionts reveals new aspects of the trypanosomatidae family.

    Directory of Open Access Journals (Sweden)

    Maria Cristina Machado Motta

    Full Text Available Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei and Strigomonas culicis (first known as Blastocrithidia culicis, respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine

  18. Reducing C-terminal-truncated alpha-synuclein by immunotherapy attenuates neurodegeneration and propagation in Parkinson's disease-like models.

    Science.gov (United States)

    Games, Dora; Valera, Elvira; Spencer, Brian; Rockenstein, Edward; Mante, Michael; Adame, Anthony; Patrick, Christina; Ubhi, Kiren; Nuber, Silke; Sacayon, Patricia; Zago, Wagner; Seubert, Peter; Barbour, Robin; Schenk, Dale; Masliah, Eliezer

    2014-07-01

    Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are common neurodegenerative disorders of the aging population, characterized by progressive and abnormal accumulation of α-synuclein (α-syn). Recent studies have shown that C-terminus (CT) truncation and propagation of α-syn play a role in the pathogenesis of PD/DLB. Therefore, we explored the effect of passive immunization against the CT of α-syn in the mThy1-α-syn transgenic (tg) mouse model, which resembles the striato-nigral and motor deficits of PD. Mice were immunized with the new monoclonal antibodies 1H7, 5C1, or 5D12, all directed against the CT of α-syn. CT α-syn antibodies attenuated synaptic and axonal pathology, reduced the accumulation of CT-truncated α-syn (CT-α-syn) in axons, rescued the loss of tyrosine hydroxylase fibers in striatum, and improved motor and memory deficits. Among them, 1H7 and 5C1 were most effective at decreasing levels of CT-α-syn and higher-molecular-weight aggregates. Furthermore, in vitro studies showed that preincubation of recombinant α-syn with 1H7 and 5C1 prevented CT cleavage of α-syn. In a cell-based system, CT antibodies reduced cell-to-cell propagation of full-length α-syn, but not of the CT-α-syn that lacked the 118-126 aa recognition site needed for antibody binding. Furthermore, the results obtained after lentiviral expression of α-syn suggest that antibodies might be blocking the extracellular truncation of α-syn by calpain-1. Together, these results demonstrate that antibodies against the CT of α-syn reduce levels of CT-truncated fragments of the protein and its propagation, thus ameliorating PD-like pathology and improving behavioral and motor functions in a mouse model of this disease. PMID:25009275

  19. Dopaminergic lesioning impairs adult hippocampal neurogenesis by distinct modification of α-synuclein.

    Science.gov (United States)

    Schlachetzki, Johannes C M; Grimm, Thomas; Schlachetzki, Zinayida; Ben Abdallah, Nada M B; Ettle, Benjamin; Vöhringer, Patrizia; Ferger, Boris; Winner, Beate; Nuber, Silke; Winkler, Jürgen

    2016-01-01

    Nonmotor symptoms of cognitive and affective nature are present in premotor and motor stages of Parkinson's disease (PD). Neurogenesis, the generation of new neurons, persists throughout the mammalian life span in the hippocampal dentate gyrus. Adult hippocampal neurogenesis may be severely affected in the course of PD, accounting for some of the neuropsychiatric symptoms such as depression and cognitive impairment. Two important PD-related pathogenic factors have separately been attributed to contribute to both PD and adult hippocampal neurogenesis: dopamine depletion and accumulation of α-synuclein (α-syn). In the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model, altered neurogenesis has been linked merely to a reduced dopamine level. Here, we seek to determine whether a distinct endogenous α-syn expression pattern is associated, possibly contributing to the hippocampal neurogenic deficit. We observed a persistent reduction of striatal dopamine and a loss of tyrosine hydroxylase-expressing neurons in the substantia nigra pars compacta in contrast to a complete recovery of tyrosine hydroxylase-immunoreactive dopaminergic fibers within the striatum. However, dopamine levels in the hippocampus were significantly decreased. Survival of newly generated neurons was significantly reduced and paralleled by an accumulation of truncated, membrane-associated, insoluble α-syn within the hippocampus. Specifically, the presence of truncated α-syn species was accompanied by increased activity of calpain-1, a calcium-dependent protease. Our results further substantiate the broad effects of dopamine loss in PD-susceptible brain nuclei, gradually involved in the PD course. Our findings also indicate a detrimental synergistic interplay between dopamine depletion and posttranslational modification of α-syn, contributing to impaired hippocampal plasticity in PD. PMID:26451750

  20. Somatostatin receptor 1 and 5 double knockout mice mimic neurochemical changes of Huntington's disease transgenic mice.

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    Padmesh S Rajput

    Full Text Available BACKGROUND: Selective degeneration of medium spiny neurons and preservation of medium sized aspiny interneurons in striatum has been implicated in excitotoxicity and pathophysiology of Huntington's disease (HD. However, the molecular mechanism for the selective sparing of medium sized aspiny neurons and vulnerability of projection neurons is still elusive. The pathological characteristic of HD is an extensive reduction of the striatal mass, affecting caudate putamen. Somatostatin (SST positive neurons are selectively spared in HD and Quinolinic acid/N-methyl-D-aspartic acid induced excitotoxicity, mimic the model of HD. SST plays neuroprotective role in excitotoxicity and the biological effects of SST are mediated by five somatostatin receptor subtypes (SSTR1-5. METHODS AND FINDINGS: To delineate subtype selective biological responses we have here investigated changes in SSTR1 and 5 double knockout mice brain and compared with HD transgenic mouse model (R6/2. Our study revealed significant loss of dopamine and cAMP regulated phosphoprotein of 32 kDa (DARPP-32 and comparable changes in SST, N-methyl-D-aspartic acid receptors subtypes, calbindin and brain nitric oxide synthase expression as well as in key signaling proteins including calpain, phospho-extracellular-signal-regulated kinases1/2, synapsin-IIa, protein kinase C-α and calcineurin in SSTR1/5(-/- and R6/2 mice. Conversely, the expression of somatostatin receptor subtypes, enkephalin and phosphatidylinositol 3-kinases were strain specific. SSTR1/5 appears to be important in regulating NMDARs, DARPP-32 and signaling molecules in similar fashion as seen in HD transgenic mice. CONCLUSIONS: This is the first comprehensive description of disease related changes upon ablation of G- protein coupled receptor gene. Our results indicate that SST and SSTRs might play an important role in regulation of neurodegeneration and targeting this pathway can provide a novel insight in understanding the

  1. Cytotoxic effects of curcumin in human retinal pigment epithelial cells.

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    Margrit Hollborn

    Full Text Available BACKGROUND: Curcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE cells in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Cellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 µM and strongly reduced by curcumin above 50 µM. Curcumin at 50 µM increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 µM and delayed apoptosis (above 1 µM. The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein. CONCLUSION: It is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (∼10 µM has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as

  2. Neuronal calcium signaling, mitochondrial dysfunction and Alzheimer’s disease

    Science.gov (United States)

    Supnet, Charlene; Bezprozvanny, Ilya

    2016-01-01

    Alzheimer disease (AD) is the most common neurodegenerative disorder that affects millions of ageing people worldwide. AD is characterized by extensive synaptic and neuronal loss which lead to impaired memory and cognitive decline. The cause of pathology in AD is not completely understood and no effective therapy so far has been developed. The accumulation of toxic amyloid-beta 42 (Aβ42) peptide oligomers and aggregates in AD brain has been proposed to be primarily responsible for the pathology of the disease, an idea dubbed ‘amyloid hypothesis’ of AD etiology. In addition to increase in Aβ42 levels, disturbances in neuronal calcium (Ca2+) signaling and alterations in expression levels of Ca2+ signaling proteins have been observed in animal models of familial AD and in studies of postmortem brain samples from sporadic AD patients. Based on these evidence ‘Ca2+ hypothesis of AD’ has been proposed. In particular, familal AD has been linked with enhanced Ca2+ release from the endoplasmic reticulum (ER) and elevated cytosolic Ca2+ levels. The augmented cytosolic Ca2+ levels can trigger signaling cascades that affect synaptic stability and function and can be detrimental to neuronal health, such as Ca2+-dependent phosphatase calcineurin and Ca2+-dependent proteases calpains. Here we review the latest results supporting ‘Ca2+ hypothesis’ of AD pathogenesis. We further argue that over long period of time supranormal cytosolic Ca2+ signaling can impaire mitochondrial function in AD neurons. We conclude that inhibitors and stablizers of neuronal Ca2+ signaling and mitochondrial function may have a therapeutic potential for treatment of AD. We discuss latest and planned AD therapeutic trials of agents targeting Ca2+ channels and mitochodria. PMID:20413848

  3. Implementing meta-analysis from genome-wide association studies for pork quality traits.

    Science.gov (United States)

    Bernal Rubio, Y L; Gualdrón Duarte, J L; Bates, R O; Ernst, C W; Nonneman, D; Rohrer, G A; King, D A; Shackelford, S D; Wheeler, T L; Cantet, R J C; Steibel, J P

    2015-12-01

    Pork quality plays an important role in the meat processing industry. Thus, different methodologies have been implemented to elucidate the genetic architecture of traits affecting meat quality. One of the most common and widely used approaches is to perform genome-wide association (GWA) studies. However, a limitation of many GWA in animal breeding is the limited power due to small sample sizes in animal populations. One alternative is to implement a meta-analysis of GWA (MA-GWA) combining results from independent association studies. The objective of this study was to identify significant genomic regions associated with meat quality traits by performing MA-GWA for 8 different traits in 3 independent pig populations. Results from MA-GWA were used to search for genes possibly associated with the set of evaluated traits. Data from 3 pig data sets (U.S. Meat Animal Research Center, commercial, and Michigan State University Pig Resource Population) were used. A MA was implemented by combining -scores derived for each SNP in every population and then weighting them using the inverse of estimated variance of SNP effects. A search for annotated genes retrieved genes previously reported as candidates for shear force (calpain-1 catalytic subunit [] and calpastatin []), as well as for ultimate pH, purge loss, and cook loss (protein kinase, AMP-activated, γ 3 noncatalytic subunit []). In addition, novel candidate genes were identified for intramuscular fat and cook loss (acyl-CoA synthetase family member 3 mitochondrial []) and for the objective measure of muscle redness, CIE a* (glycogen synthase 1, muscle [] and ferritin, light polypeptide []). Thus, implementation of MA-GWA allowed integration of results for economically relevant traits and identified novel genes to be tested as candidates for meat quality traits in pig populations.

  4. Apoptosis and necrosis in vaccinia virus-infected HeLa G and BSC-40 cells.

    Science.gov (United States)

    Liskova, Jana; Knitlova, Jarmila; Honner, Richard; Melkova, Zora

    2011-09-01

    In most cells, vaccinia virus (VACV) infection is considered to cause a lytic cell death, an equivalent of necrosis. However, upon infection of the epithelial cell lines HeLa G and BSC-40 with VACV strain Western Reserve (WR), we have previously observed an increased activation of and activity attributable to caspases, a typical sign of apoptosis. In this paper, we have further analyzed the type of cell death in VACV-infected cells HeLa G and BSC-40. In a cell-based flow cytometric assay, we showed a specific activation of caspase-2 and 4 in HeLa G and BSC-40 cells infected with VACV, strain WR, while we did not find any effects of inhibitors of calpain and cathepsin D and E. The actual activity of the two caspases, but also of caspase-3, was then confirmed in lysates of infected HeLa G, but not in BSC-40 cells. Accordingly, poly(ADP)-ribose polymerase (PARP) cleavage was found increased only in infected HeLa G cells. Consequently, we have determined morphological features of apoptosis and/or activity of the executioner caspase-3 in infected HeLa G cells in situ, while only a background apoptosis was observed in infected BSC-40 cells. Finally, vaccination strains Dryvax and Praha were found to induce apoptosis in both HeLa G and BSC-40 cells, as characterized morphologically and by PARP cleavage. These findings may be important for understanding the differences in VACV-host interactions and post-vaccination complications in different individuals.

  5. 青光眼视神经保护离我们有多远%Glaucoma neuroprotection-how far is it from a dream to reality

    Institute of Scientific and Technical Information of China (English)

    葛坚

    2008-01-01

    Although the durgs, such as Memantine, Calpain, Erythropoietin, have demonstrated exciting results for neuroprotection in laboratories, the phase Ⅲ clinical trial of Memantine failed to prove such activity. So far, none of neuroprotection drugs has been approved by FDA for clinical use with the failure of Memantine clinical trail indicating that the gap between basic science research and clinical application in glaucomatous optic neuroprotection remains to be filled. This paper offers a new insight into the field of neuroprotecion in glaucoma. To make the dream of optic neuroprotection to reality, we have to implement new perspective strategies to integrate technologies and findings from the researches of human genomonics, proteomics, stem cells, and gene-transferred animal models.%尽管在基础研究中,Memantine、Calpain、促红细胞生成素等药物均显现出喜人的青光眼视神经保护潜能,但随着Memantine等Ⅲ期临床试验的失败,目前仍无一种视神经保护药物能够通过美国食品药品管理局的审核批准并投入临床应用,使临床青光眼视神经保护的研究面临近乎尴尬的困境.青光眼视神经保护的迫切性要求我们必须从新的视角审视问题,充分利用人类基因组计划、蛋白质组计划、干细胞和转基因动物研究成果,对其实施分期靶点干预,以使青光眼视神经保护的梦想变成现实.

  6. Association of CAPN10 SNPs and haplotypes with polycystic ovary syndrome among South Indian Women.

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    Shilpi Dasgupta

    Full Text Available Polycystic Ovary Syndrome (PCOS is known to be characterized by metabolic disorder in which hyperinsulinemia and peripheral insulin resistance are central features. Given the physiological overlap between PCOS and type-2 diabetes (T2DM, and calpain 10 gene (CAPN10 being a strong candidate for T2DM, a number of studies have analyzed CAPN10 SNPs among PCOS women yielding contradictory results. Our study is first of its kind to investigate the association pattern of CAPN10 polymorphisms (UCSNP-44, 43, 56, 19 and 63 with PCOS among Indian women. 250 PCOS cases and 299 controls from Southern India were recruited for this study. Allele and genotype frequencies of the SNPs were determined and compared between the cases and controls. Results show significant association of UCSNP-44 genotype CC with PCOS (p = 0.007 with highly significant odds ratio when compared to TC (OR = 2.51, p = 0.003, 95% CI = 1.37-4.61 as well as TT (OR = 1.94, p = 0.016, 95% CI = 1.13-3.34. While the haplotype carrying the SNP-44 and SNP-19 variants (21121 exhibited a 2 fold increase in the risk for PCOS (OR = 2.37, p = 0.03, the haplotype containing SNP-56 and SNP-19 variants (11221 seems to have a protective role against PCOS (OR = 0.20, p = 0.004. Our results support the earlier evidence for a possible role of UCSNP-44 of the CAPN10 gene in the manifestation of PCOS.

  7. Critical role of astrocytic interleukin-17 A in post-stroke survival and neuronal differentiation of neural precursor cells in adult mice.

    Science.gov (United States)

    Lin, Y; Zhang, J-C; Yao, C-Y; Wu, Y; Abdelgawad, A F; Yao, S-L; Yuan, S-Y

    2016-01-01

    The brain and the immune system interact in complex ways after ischemic stroke, and the long-term effects of immune response associated with stroke remain controversial. As a linkage between innate and adaptive immunity, interleukin-17 A (IL-17 A) secreted from gamma delta (γδ) T cells has detrimental roles in the pathogenesis of acute ischemic stroke. However, to date, the long-term actions of IL-17 A after stroke have not been investigated. Here, we found that IL-17 A showed two distinct peaks of expression in the ischemic hemisphere: the first occurring within 3 days and the second on day 28 after stroke. Our data also showed that astrocyte was the major cellular source of IL-17 A that maintained and augmented subventricular zone (SVZ) neural precursor cells (NPCs) survival, neuronal differentiation, and subsequent synaptogenesis and functional recovery after stroke. IL-17 A also promoted neuronal differentiation in cultured NPCs from the ischemic SVZ. Furthermore, our in vitro data revealed that in primary astrocyte cultures activated astrocytes released IL-17 A via p38 mitogen-activated protein kinase (MAPK). Culture media from reactive astrocytes increased neuronal differentiation of NSCs in vitro. Blockade of IL-17 A with neutralizing antibody prevented this effect. In addition, after screening for multiple signaling pathways, we revealed that the p38 MAPK/calpain 1 signaling pathway was involved in IL-17 A-mediated neurogenesis in vivo and in vitro. Thus, our results reveal a previously uncharacterized property of astrocytic IL-17 A in the maintenance and augment of survival and neuronal differentiation of NPCs, and subsequent synaptogenesis and spontaneous recovery after ischemic stroke. PMID:27336717

  8. SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat.

    Science.gov (United States)

    Goswami, Suneha; Kumar, Ranjeet R; Dubey, Kavita; Singh, Jyoti P; Tiwari, Sachidanand; Kumar, Ashok; Smita, Shuchi; Mishra, Dwijesh C; Kumar, Sanjeev; Grover, Monendra; Padaria, Jasdeep C; Kala, Yugal K; Singh, Gyanendra P; Pathak, Himanshu; Chinnusamy, Viswanathan; Rai, Anil; Praveen, Shelly; Rai, Raj D

    2016-01-01

    Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger's sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD, and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs). We observed eight different types of post-translational modifications (PTMs) in the DEPs corresponds to the cloned ESTs-147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH) in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant), as compared to HD2329 (thermosusceptible) during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat-a novel step toward the development of "climate-smart" wheat

  9. Potentiation of NMDA receptor-dependent cell responses by extracellular high mobility group box 1 protein.

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    Marco Pedrazzi

    Full Text Available BACKGROUND: Extracellular high mobility group box 1 (HMGB1 protein can operate in a synergistic fashion with different signal molecules promoting an increase of cell Ca(2+ influx. However, the mechanisms responsible for this effect of HMGB1 are still unknown. PRINCIPAL FINDINGS: Here we demonstrate that, at concentrations of agonist per se ineffective, HMGB1 potentiates the activation of the ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR in isolated hippocampal nerve terminals and in a neuroblastoma cell line. This effect was abolished by the NMDA channel blocker MK-801. The HMGB1-facilitated NMDAR opening was followed by activation of the Ca(2+-dependent enzymes calpain and nitric oxide synthase in neuroblastoma cells, resulting in an increased production of NO, a consequent enhanced cell motility, and onset of morphological differentiation. We have also identified NMDAR as the mediator of HMGB1-stimulated murine erythroleukemia cell differentiation, induced by hexamethylenebisacetamide. The potentiation of NMDAR activation involved a peptide of HMGB1 located in the B box at the amino acids 130-139. This HMGB1 fragment did not overlap with binding sites for other cell surface receptors of HMGB1, such as the advanced glycation end products or the Toll-like receptor 4. Moreover, in a competition assay, the HMGB1((130-139 peptide displaced the NMDAR/HMGB1 interaction, suggesting that it comprised the molecular and functional site of HMGB1 regulating the NMDA receptor complex. CONCLUSION: We propose that the multifunctional cytokine-like molecule HMGB1 released by activated, stressed, and damaged or necrotic cells can facilitate NMDAR-mediated cell responses, both in the central nervous system and in peripheral tissues, independently of other known cell surface receptors for HMGB1.

  10. Maleic Acid--but Not Structurally Related Methylmalonic Acid--Interrupts Energy Metabolism by Impaired Calcium Homeostasis.

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    Ali Tunç Tuncel

    Full Text Available Maleic acid (MA has been shown to induce Fanconi syndrome via disturbance of renal energy homeostasis, though the underlying pathomechanism is still under debate. Our study aimed to examine the pathomechanism underlying maleic acid-induced nephrotoxicity. Methylmalonic acid (MMA is structurally similar to MA and accumulates in patients affected with methymalonic aciduria, a defect in the degradation of branched-chain amino acids, odd-chain fatty acids and cholesterol, which is associated with the development of tubulointerstitial nephritis resulting in chronic renal failure. We therefore used MMA application as a control experiment in our study and stressed hPTECs with MA and MMA to further validate the specificity of our findings. MMA did not show any toxic effects on proximal tubule cells, whereas maleic acid induced concentration-dependent and time-dependent cell death shown by increased lactate dehydrogenase release as well as ethidium homodimer and calcein acetoxymethyl ester staining. The toxic effect of MA was blocked by administration of single amino acids, in particular L-alanine and L-glutamate. MA application further resulted in severe impairment of cellular energy homeostasis on the level of glycolysis, respiratory chain, and citric acid cycle resulting in ATP depletion. As underlying mechanism we could identify disturbance of calcium homeostasis. MA toxicity was critically dependent on calcium levels in culture medium and blocked by the extra- and intracellular calcium chelators EGTA and BAPTA-AM respectively. Moreover, MA-induced cell death was associated with activation of calcium-dependent calpain proteases. In summary, our study shows a comprehensive pathomechanistic concept for MA-induced dysfunction and damage of human proximal tubule cells.

  11. Gliedergürtelmuskeldystrophien

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    Finsterer J

    2005-01-01

    Full Text Available Gliedergürtelmuskeldystrophien (LGMDs sind charakterisiert durch progrediente Paresen und Atrophien im Becken- und Schultergürtelbereich, gelegentlich mit Ausbreitung nach distal, auf die bulbäre Muskulatur oder die Atemhilfsmuskulatur. LGMDs sind genetisch heterogen und werden derzeit in 6 autosomal dominante (LGMD1A–F und 10 autosomal rezessive Formen (LGMD2A–J unterteilt. Die LGMDs werden durch Mutationen in Genen, die für Myotilin (5q31, LGMD1A, Lamin A/C (1q11-q21.2, LGMD1B, Caveolin-3 (3p25, LGMD1C, unbekannte Proteine (7q, LGMD1D, 6q23, LGMD1E, 7q32.1-32.2, LGMD1F, Calpain-3 (15q15.1-21.1, LGMD2A, Dysferlin (2p13.3-13.1, LGMD2B, gamma-Sarkoglykan (13q12, LGMD2C, alpha-Sarkoglykan (17q12- q21.3, LGMD2D, beta-Sarkoglykan (4q12, LGMD2E, delta-Sarkoglykan (5q33-q34, LGMD2F, Telethonin (17q11-q12, LGMD2G, die E3-Ubiquitin-Ligase (9q31-q34.1, LGMD2H, das Fukutin-related Protein (19q13.3, LGMD2I oder Titin (2q31, LGMD2J kodieren, verursacht. Der Krankheitsbeginn liegt zwischen der frühen Kindheit und dem mittleren Erwachsenenalter. Das Geschlechterverhältnis ist ausgeglichen. Langsam und rasch progrediente Verläufe kommen vor. Die Lebenserwartung ist normal oder stark eingeschränkt. Eine Herzbeteiligung wird häufig beobachtet. Die Diagnose basiert auf Anamnese, Neurostatus, Blutchemie, Elektromyographie, Muskelbiopsie sowie DNA-Analyse. Derzeit steht keine kausale, sondern lediglich eine symptomatische Therapie zur Verfügung.

  12. Activity-dependent regulation of the K/Cl transporter KCC2 membrane diffusion, clustering, and function in hippocampal neurons.

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    Chamma, Ingrid; Heubl, Martin; Chevy, Quentin; Renner, Marianne; Moutkine, Imane; Eugène, Emmanuel; Poncer, Jean Christophe; Lévi, Sabine

    2013-09-25

    The neuronal K/Cl transporter KCC2 exports chloride ions and thereby influences the efficacy and polarity of GABA signaling in the brain. KCC2 is also critical for dendritic spine morphogenesis and the maintenance of glutamatergic transmission in cortical neurons. Because KCC2 plays a pivotal role in the function of central synapses, it is of particular importance to understand the cellular and molecular mechanisms underlying its regulation. Here, we studied the impact of membrane diffusion and clustering on KCC2 function. KCC2 forms clusters in the vicinity of both excitatory and inhibitory synapses. Using quantum-dot-based single-particle tracking on rat primary hippocampal neurons, we show that KCC2 is slowed down and confined at excitatory and inhibitory synapses compared with extrasynaptic regions. However, KCC2 escapes inhibitory synapses faster than excitatory synapses, reflecting stronger molecular constraints at the latter. Interfering with KCC2-actin interactions or inhibiting F-actin polymerization releases diffusion constraints on KCC2 at excitatory but not inhibitory synapses. Thus, F-actin constrains KCC2 diffusion at excitatory synapses, whereas KCC2 is confined at inhibitory synapses by a distinct mechanism. Finally, increased neuronal activity rapidly increases the diffusion coefficient and decreases the dwell time of KCC2 at excitatory synapses. This effect involves NMDAR activation, Ca(2+) influx, KCC2 S940 dephosphorylation and calpain protease cleavage of KCC2 and is accompanied by reduced KCC2 clustering and ion transport function. Thus, activity-dependent regulation of KCC2 lateral diffusion and clustering allows for a rapid regulation of chloride homeostasis in neurons.

  13. ER stress in retinal degeneration in S334ter Rho rats.

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    Vishal M Shinde

    Full Text Available The S334ter rhodopsin (Rho rat (line 4 bears the rhodopsin gene with an early termination codon at residue 334 that is a model for several such mutations found in human patients with autosomal dominant retinitis pigmentosa (ADRP. The Unfolded Protein Response (UPR is implicated in the pathophysiology of several retinal disorders including ADRP in P23H Rho rats. The aim of this study was to examine the onset of UPR gene expression in S334ter Rho retinas to determine if UPR is activated in ADRP animal models and to investigate how the activation of UPR molecules leads to the final demise of S334ter Rho photoreceptors. RT-PCR was performed to evaluate the gene expression profiles for the P10, P12, P15, and P21 stages of the development and progression of ADRP in S334ter Rho photoreceptors. We determined that during the P12-P15 period, ER stress-related genes are strongly upregulated in transgenic retinas, resulting in the activation of the UPR that was confirmed using western blot analysis and RT-PCR. The activation of UPR was associated with the increased expression of JNK, Bik, Bim, Bid, Noxa, and Puma genes and cleavage of caspase-12 that together with activated calpains presumably compromise the integrity of the mitochondrial MPTP, leading to the release of pro-apoptotic AIF1 into the cytosol of S334ter Rho photoreceptor cells. Therefore, two major cross-talking pathways, the UPR and mitochondrial MPTP occur in S334ter-4 Rho retina concomitantly and eventually promote the death of the photoreceptor cells.

  14. CGP37157, an inhibitor of the mitochondrial Na+/Ca2+ exchanger, protects neurons from excitotoxicity by blocking voltage-gated Ca2+ channels.

    Science.gov (United States)

    Ruiz, A; Alberdi, E; Matute, C

    2014-04-10

    Inhibition of the mitochondrial Na(+)/Ca(2+) exchanger (NCLX) by CGP37157 is protective in models of neuronal injury that involve disruption of intracellular Ca(2+) homeostasis. However, the Ca(2+) signaling pathways and stores underlying neuroprotection by that inhibitor are not well defined. In the present study, we analyzed how intracellular Ca(2+) levels are modulated by CGP37157 (10 μM) during NMDA insults in primary cultures of rat cortical neurons. We initially assessed the presence of NCLX in mitochondria of cultured neurons by immunolabeling, and subsequently, we analyzed the effects of CGP37157 on neuronal Ca(2+) homeostasis using cameleon-based mitochondrial Ca(2+) and cytosolic Ca(2+) ([Ca(2+)]i) live imaging. We observed that NCLX-driven mitochondrial Ca(2+) exchange occurs in cortical neurons under basal conditions as CGP37157 induced a decrease in [Ca(2)]i concomitant with a Ca(2+) accumulation inside the mitochondria. In turn, CGP37157 also inhibited mitochondrial Ca(2+) efflux after the stimulation of acetylcholine receptors. In contrast, CGP37157 strongly prevented depolarization-induced [Ca(2+)]i increase by blocking voltage-gated Ca(2+) channels (VGCCs), whereas it did not induce depletion of ER Ca(2+) stores. Moreover, mitochondrial Ca(2+) overload was reduced as a consequence of diminished Ca(2+) entry through VGCCs. The decrease in cytosolic and mitochondrial Ca(2+) overload by CGP37157 resulted in a reduction of excitotoxic mitochondrial damage, characterized here by a reduction in mitochondrial membrane depolarization, oxidative stress and calpain activation. In summary, our results provide evidence that during excitotoxicity CGP37157 modulates cytosolic and mitochondrial Ca(2+) dynamics that leads to attenuation of NMDA-induced mitochondrial dysfunction and neuronal cell death by blocking VGCCs.

  15. Plasma membrane Ca2+-ATPases:Targets of oxidative stress in brain aging and neurodegeneration

    Institute of Scientific and Technical Information of China (English)

    Asma; Zaidi

    2010-01-01

    The plasma membrane Ca2+-ATPase(PMCA)pumps play an important role in the maintenance of precise levels of intracellular Ca2+[Ca2+]i,essential to the functioning of neurons.In this article,we review evidence showing age-related changes of the PMCAs in synaptic plasma membranes(SPMs).PMCA activity and protein levels in SPMs diminish progressively with increasing age. The PMCAs are very sensitive to oxidative stress and undergo functional and structural changes when exposed to oxidants of physiological relevance.The major signatures of oxidative modification in the PMCAs are rapid inactivation,conformational changes,aggregation, internalization from the plasma membrane and proteolytic degradation.PMCA proteolysis appears to be mediated by both calpains and caspases.The predominance of one proteolytic pathway vs the other,the ensuing pattern of PMCA degradation and its consequence on pump activity depends largely on the type of insult,its intensity and duration.Experimental reduction of PMCA expression not only alters the dynamics of cellular Ca2+ handling but also has a myriad of downstream conse-quences on various aspects of cell function,indicating a broad role of these pumps.Age-and oxidation-related down-regulation of the PMCAs may play an important role in compromised neuronal function in the aging brain and its several-fold increased susceptibility to neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease,and stroke.Therapeutic approaches that protect the PMCAs and stabilize[Ca2+]i homeostasis may be capable of slowing and/or preventing neuronal degeneration.The PMCAs are therefore emerging as a new class of drug targets for therapeutic interventions in various chronic degenerative disorders.

  16. The pharmacology of neurotrophic treatment with Cerebrolysin: brain protection and repair to counteract pathologies of acute and chronic neurological disorders.

    Science.gov (United States)

    Masliah, E; Díez-Tejedor, E

    2012-04-01

    Neurotrophic factors are considered as part of the therapeutic strategy for neurological disorders like dementia, stroke and traumatic brain injury. Cerebrolysin is a neuropeptide preparation which mimics the action of endogenous neurotrophic factors on brain protection and repair. In dementia models, Cerebrolysin decreases β-amyloid deposition and microtubule-associated protein tau phosphorylation by regulating glycogen synthase kinase-3β and cyclin-dependent kinase 5 activity, increases synaptic density and restores neuronal cytoarchitecture. These effects protect integrity of the neuronal circuits and thus result in improved cognitive and behavioral performance. Furthermore, Cerebrolysin enhances neurogenesis in the dentate gyrus, the basis for neuronal replacement therapy in neurodegenerative diseases. Experimental studies in stroke animal models have shown that Cerebrolysin stabilizes the structural integrity of cells by inhibition of calpain and reduces the number of apoptotic cells after ischemic lesion. Cerebrolysin induces restorative processes, decreases infarct volume and edema formation and promotes functional recovery. Stroke-induced neurogenesis in the subventricular zone was also promoted by Cerebrolysin, thus supporting the brain's self-repair after stroke. Both, traumatic brain and spinal cord injury conditions stimulate the expression of natural neurotrophic factors to promote repair and regeneration processes -axonal regeneration, neuronal plasticity and neurogenesis- that is considered to be crucial for the future recovery. Neuroprotective effects of Cerebrolysin on experimentally induced traumatic spinal cord injury have shown that Cerebrolysin prevents apoptosis of lesioned motoneurons and promotes functional recovery. This section summarizes the most relevant data on the pharmacology of Cerebrolysin obtained from in vitro assays (biochemical and cell cultures) and in vivo animal models of acute and chronic neurological disorders.

  17. Exercise Ameliorates Renal Cell Apoptosis in Chronic Kidney Disease by Intervening in the Intrinsic and the Extrinsic Apoptotic Pathways in a Rat Model

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    Kuan-Chou Chen

    2013-01-01

    Full Text Available We hypothesized that doxorubicin (DR induced chronic kidney disease (CKD could trigger the intrinsic and the extrinsic renal cell apoptotic pathways, while treadmill exercise could help prevent adverse effects. Male Sprague-Dawley rats were subjected to treadmill running exercise at a speed of 30 m/min, 30 or 60 min/day, 3 times per week, for a total period of 11 weeks. The physiological and biochemical parameters were seen substantially improved (DR-CKD control, 30 min, 60 min exercise: the ratio of kidney weight/body weight (0.89, 0.74, and 0.72; the WBC (1.35, 1.08, and 1.42 × 104 cells/μL; RBC (5.30, 6.38, and 6.26 × 106 cells/μL; the platelet count (15.1, 12.8, and 11.3 × 105/μL; serum cholesterol (659, 360, and 75 mg/dL; serum triglyceride (542, 263, and 211 mg/dL; BUN (37, 25, and 22 mg/dL. Bcl-2 and intramitochondrial cytochrome c were upregulated, while the levels of Bax, SOD, MDA, cleaved caspases 9, 3, 8, 12, and calpain were all downregulated in DRCKD groups with exercise. CHOP (GADD153 and GRP78 were totally unaffected. FAS (CD95 was only slightly suppressed in the 60 min exercise DRCKD group. Conclusively, exercise can ameliorate CKD through the regulation of the intrinsic and extrinsic apoptosis pathways. The 60 min exercise yields more beneficial effect than the 30 min counterpart.

  18. Chapter Three - Ubiquitination and Protein Turnover of G-Protein-Coupled Receptor Kinases in GPCR Signaling and Cellular Regulation.

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    Penela, P

    2016-01-01

    G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and β-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse "effector" functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. β-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes. PMID:27378756

  19. Protein defects in neuromuscular diseases

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    Vainzof M.

    2003-01-01

    Full Text Available Muscular dystrophies are a heterogeneous group of genetically determined progressive disorders of the muscle with a primary or predominant involvement of the pelvic or shoulder girdle musculature. The clinical course is highly variable, ranging from severe congenital forms with rapid progression to milder forms with later onset and a slower course. In recent years, several proteins from the sarcolemmal muscle membrane (dystrophin, sarcoglycans, dysferlin, caveolin-3, from the extracellular matrix (alpha2-laminin, collagen VI, from the sarcomere (telethonin, myotilin, titin, nebulin, from the muscle cytosol (calpain 3, TRIM32, from the nucleus (emerin, lamin A/C, survival motor neuron protein, and from the glycosylation pathway (fukutin, fukutin-related protein have been identified. Mutations in their respective genes are responsible for different forms of neuromuscular diseases. Protein analysis using Western blotting or immunohistochemistry with specific antibodies is of the utmost importance for the differential diagnosis and elucidation of the physiopathology of each genetic disorder involved. Recent molecular studies have shown clinical inter- and intra-familial variability in several genetic disorders highlighting the importance of other factors in determining phenotypic expression and the role of possible modifying genes and protein interactions. Developmental studies can help elucidate the mechanism of normal muscle formation and thus muscle regeneration. In the last fifteen years, our research has focused on muscle protein expression, localization and possible interactions in patients affected by different forms of muscular dystrophies. The main objective of this review is to summarize the most recent findings in the field and our own contribution.

  20. Synergistic efficacy of sorafenib and genistein in growth inhibition by down regulating angiogenic and survival factors and increasing apoptosis through upregulation of p53 and p21 in malignant neuroblastoma cells having N-Myc amplification or non-amplification.

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    Roy Choudhury, Subhasree; Karmakar, Surajit; Banik, Naren L; Ray, Swapan K

    2010-12-01

    Neuroblastoma is an extracranial, solid, and heterogeneous malignancy in children. The conventional therapeutic modalities are mostly ineffective and thus new therapeutic strategies for malignant neuroblastoma are urgently warranted. We examined the synergistic efficacy of combination of sorafenib (SF) and genistein (GST) in human malignant neuroblastoma SK-N-DZ (N-Myc amplified) and SH-SY5Y (N-Myc non-amplified) cell lines. MTT assay showed dose-dependent decrease in cell viability and the combination therapy more prominently inhibited the cell proliferation in both cell lines than either treatment alone. Apoptosis was confirmed morphologically by Wright staining. Flow cytometric analysis of cell cycle phase distribution and Annexin V-FITC/PI staining showed increase in subG1 DNA content and early apoptosis, respectively, after treatment with the combination of drugs. Apoptosis was further confirmed by scanning electron microscopy. Combination therapy showed activation of caspase-8, cleavage of Bid to tBid, increase in p53 and p21 expression, down regulation of anti-apoptotic Mcl-1, and increase in Bax:Bcl-2 ratio to trigger apoptosis. Down regulation of MDR, hTERT, N-Myc, VEGF, FGF-2, NF-κB, p-Akt, and c-IAP2 indicated suppression of angiogenic and survival pathways. Mitochondrial release of cytochrome c and Smac into cytosol indicated involvement of mitochondia in apoptosis. Increases in proteolytic activities of calpain and caspase-3 were also confirmed. Our results suggested that combination of SF and GST inhibited angiogenic and survival factors and increased apoptosis via receptor and mitochondria mediated pathways in both neuroblastoma SK-N-DZ and SH-SY5Y cell lines. Thus, this combination of drugs could be a potential therapeutic strategy against human malignant neuroblastoma cells having N-Myc amplification or non-amplification. PMID:19777160

  1. Association of CAPN1 and CAST gene polymorphisms with meat tenderness in Bos taurus beef cattle from Argentina

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    Pablo Corva

    2007-01-01

    Full Text Available The activity of the calpains/calpastatin proteolytic system is closely related to the postmortem tenderization of meat. We investigated the association between beef tenderness and single nucleotide polymorphism (SNP markers on the CAPN1 gene (SNP316, alleles C/G; SNP530 alleles A/G and the CAST gene 3' untranslated region (SNP2870, alleles A/G. We sampled nine slaughter groups comprising 313 steers which had been reared in beef production systems in Argentina between 2002 and 2004 from crosses between Angus, Hereford and Limousin cattle. Minor allele frequencies for the markers were 0.27 to 0.46 (C, 0.02 to 0.18 (A, and 0.24 to 0.53 (A, respectively. The presence of CAPN1 markers had significant effects on meat shear force but no detectable effects were demonstrated for the CAST marker. The shear force of meat from steers with the SNP316 CC genotype was 11% lower than for the SNP316 CG genotype and 17% lower than for the SNP316 GG genotype. There were very few steers with the SNP530 AA genotype and, contrary to previous studies, meat from steers with the SNP530 GG genotype showed an 11.5% higher shear force than that from steers with the SNP530 GA genotype. Final body weight, carcass weight and rib eye area were not affected by any of the markers. These results support the concept that CAPN1 variants are associated with tenderness across a wide range of beef production systems.

  2. Association of selected SNP with carcass and taste panel assessed meat quality traits in a commercial population of Aberdeen Angus-sired beef cattle

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    Williams John L

    2009-06-01

    Full Text Available Abstract Background The purpose of this study was to evaluate the effects of eight single nucleotide polymorphisms (SNP, previously associated with meat and milk quality traits in cattle, in a population of 443 commercial Aberdeen Angus-cross beef cattle. The eight SNP, which were located within five genes: μ-calpain (CAPN1, calpastatin (CAST, leptin (LEP, growth hormone receptor (GHR and acylCoA:diacylglycerol acyltransferase 1 (DGAT1, are included in various commercial tests for tenderness, fatness, carcass composition and milk yield/quality. Methods A total of 27 traits were examined, 19 relating to carcass quality, such as carcass weight and fatness, one mechanical measure of tenderness, and the remaining seven were sensory traits, such as flavour and tenderness, assessed by a taste panel. Results An SNP in the CAPN1 gene, CAPN316, was significantly associated with tenderness measured by both the tenderometer and the taste panel as well as the weight of the hindquarter, where animals inheriting the CC genotype had more tender meat and heavier hindquarters. An SNP in the leptin gene, UASMS2, significantly affected overall liking, where animals with the TT genotype were assigned higher scores by the panellists. The SNP in the GHR gene was significantly associated with odour, where animals inheriting the AA genotype produced steaks with an intense odour when compared with the other genotypes. Finally, the SNP in the DGAT1 gene was associated with sirloin weight after maturation and fat depth surrounding the sirloin, with animals inheriting the AA genotype having heavier sirloins and more fat. Conclusion The results of this study confirm some previously documented associations. Furthermore, novel associations have been identified which, following validation in other populations, could be incorporated into breeding programmes to improve meat quality.

  3. The association of CAPN1 316 marker genotypes with growth and meat quality traits of steers finished on pasture

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    María C. Miquel

    2009-01-01

    Full Text Available The objective of this paper was to determine the association of a SNP in the µ-calpain gene at position 316 with growth and quality of meat traits of steers grown on pasture. Fifty-nine Brangus and 20 Angus steers were genotyped for CAPN1 316. Warner Bratzler shear force was measured in l. lumborum samples after a 7-day aging period. A multivariate analysis of variance was performed, including shear force (WBSF, final weight (FW, average daily gain (ADG, backfat thickness (BFT, average monthly fat thickness gain (AMFTG, rib-eye area (REA, and beef rib-eye depth (RED as dependent variables. The CAPN1 316 genotype was statistically significant. Univariate analyses were done with these variables. The marker genotype was statistically significant (p < 0.05 for WBSF (kg: CC: 4.41 ± 0.57; CG: 5.58 ± 0.20; GG: 6.29 ± 0.18, FW (kg: CC: 360.23 ± 14.71; CG: 381.34 ± 5.26; GG: 399.23 ± 4.68, and ADG (kg/d: CC: 0.675 ± 0.046; CG: 0.705 ± 0.016; GG: 0.765 ± 0.014 Shear force, final weight and average daily gain were significantly different according to the CAPN1 316 marker genotypes. The marker genotype was statistically significant in the multivariate analysis (p = 0.001. The first characteristic root explained 89% of the differences among genotypes. WBSF, FW and ADG were the most important traits in the first vector, indicating that animals with the marker genotype for lowest WBSF also have the lowest FW and ADG.

  4. Alterations of proteins in MDCK cells during acute potassium deficiency.

    Science.gov (United States)

    Peerapen, Paleerath; Ausakunpipat, Nardtaya; Chanchaem, Prangwalai; Thongboonkerd, Visith

    2016-06-01

    Chronic K(+) deficiency can cause hypokalemic nephropathy associated with metabolic alkalosis, polyuria, tubular dilatation, and tubulointerstitial injury. However, effects of acute K(+) deficiency on the kidney remained unclear. This study aimed to explore such effects by evaluating changes in levels of proteins in renal tubular cells during acute K(+) deficiency. MDCK cells were cultivated in normal K(+) (NK) (K(+)=5.3 mM), low K(+) (LK) (K(+)=2.5 mM), or K(+) depleted (KD) (K(+)=0 mM) medium for 24 h and then harvested. Cellular proteins were resolved by two-dimensional gel electrophoresis (2-DE) and visualized by SYPRO Ruby staining (5 gels per group). Spot matching and quantitative intensity analysis revealed a total 48 protein spots that had significantly differential levels among the three groups. Among these, 46 and 30 protein spots had differential levels in KD group compared to NK and LK groups, respectively. Comparison between LK and NK groups revealed only 10 protein spots that were differentially expressed. All of these differentially expressed proteins were successfully identified by Q-TOF MS and/or MS/MS analyses. The altered levels of heat shock protein 90 (HSP90), ezrin, lamin A/C, tubulin, chaperonin-containing TCP1 (CCT1), and calpain 1 were confirmed by Western blot analysis. Global protein network analysis showed three main functional networks, including 1) cell growth and proliferation, 2) cell morphology, cellular assembly and organization, and 3) protein folding in which the altered proteins were involved. Further investigations on these networks may lead to better understanding of pathogenic mechanisms of low K(+)-induced renal injury.

  5. Effect of single nucleotide polymorphisms of CAPN1 and CAST genes on meat traits in Nellore beef cattle (Bos indicus) and in their crosses with Bos taurus.

    Science.gov (United States)

    Curi, R A; Chardulo, L A L; Mason, M C; Arrigoni, M D B; Silveira, A C; de Oliveira, H N

    2009-08-01

    The objectives of this work were to study the segregation of single nucleotide polymorphisms of the calpain 1, large subunit (CAPN1) and calpastatin (CAST) genes in Nellore (Bos indicus) and Nellore xBos taurus beef cattle, as well as to evaluate their effects on meat traits. For this, 300 animals, including 114 Nellore, 67 Angus x Nellore, 44 Rubia Gallega x Nellore, 41 Canchim, 19 Brangus three-way crosses and 15 Braunvieh three-way crosses, were genotyped for the CAPN4751 [AF_248054.2:g.6545C>T (GenBank accession AF248054.2)] and CAST/DdeI [AF_159246.1:g.2959A>G (GenBank accession AF159246.1)] polymorphisms and phenotyped for Ribeye Area, Backfat Thickness, Intramuscular Fat, Shear Force (SF) and Myofibrillar Fragmentation Index (MFI). In relation to the CAPN4751 polymorphism, a frequency of 10.5% was observed for the C allele in the Nellore breed. In the total sample of studied animals, a significant association was found between genotypes and meat tenderness, assessed by SF (P = 0.005) and MFI (P = 0.008), with genotype CT being more favourable than TT. For the CAST/DdeI polymorphism, a frequency of 55.7% was found for the A allele in the Nellore breed. In the total sample, a significant association was observed between genotypes and meat tenderness - SF (P = 0.004) and MFI (P = 0.006), with genotype AA being more favourable than AG. The relationship between genotypes and aged meat tenderness in confluence with the distribution of favourable alleles shows great potential for application of the CAPN4751 and CAST/DdeI polymorphisms in the genetic improvement of the Nellore breed, whilst contributing to the validation, in this breed and in its crosses with B. taurus, of the association results previously described in the literature. PMID:19392828

  6. The association of CAPN1 316 marker genotypes with growth and meat quality traits of steers finished on pasture.

    Science.gov (United States)

    Miquel, María C; Villarreal, Edgardo; Mezzadra, Carlos; Melucci, Lilia; Soria, Liliana; Corva, Pablo; Schor, Alejandro

    2009-07-01

    The objective of this paper was to determine the association of a SNP in the μ-calpain gene at position 316 with growth and quality of meat traits of steers grown on pasture. Fifty-nine Brangus and 20 Angus steers were genotyped for CAPN1 316. Warner Bratzler shear force was measured in l. lumborum samples after a 7-day aging period. A multivariate analysis of variance was performed, including shear force (WBSF), final weight (FW), average daily gain (ADG), backfat thickness (BFT), average monthly fat thickness gain (AMFTG), rib-eye area (REA), and beef rib-eye depth (RED) as dependent variables. The CAPN1 316 genotype was statistically significant. Univariate analyses were done with these variables. The marker genotype was statistically significant (p < 0.05) for WBSF (kg: CC: 4.41 ± 0.57; CG: 5.58 ± 0.20; GG: 6.29 ± 0.18), FW (kg: CC: 360.23 ± 14.71; CG: 381.34 ± 5.26; GG: 399.23 ± 4.68), and ADG (kg/d: CC: 0.675 ± 0.046; CG: 0.705 ± 0.016; GG: 0.765 ± 0.014) Shear force, final weight and average daily gain were significantly different according to the CAPN1 316 marker genotypes. The marker genotype was statistically significant in the multivariate analysis (p = 0.001). The first characteristic root explained 89% of the differences among genotypes. WBSF, FW and ADG were the most important traits in the first vector, indicating that animals with the marker genotype for lowest WBSF also have the lowest FW and ADG. PMID:21637511

  7. Novel Therapeutic and Prevention Approaches for Schistosomiasis: Review

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    Rashika A.F. El Ridi

    2013-09-01

    Full Text Available Schistosomiasis is a debilitating disease affecting approximately 600 million people in 74 developing countries, with 800 million, mostly children at risk. To circumvent the threat of having praziquantel (PZQ as the only drug used for treatment, several PZQ derivatives were synthesized, and drugs destined for other parasites were used with success. A plethora of plant-derived oils and extracts were found to effectively kill juvenile and adult schistosomes, yet none was progressed to pre- and clinical studies except an oleo-gum resin extracted from the stem of Commiphora molmol, myrrh, which action was challenged in several trials. We have proposed an essential fatty acid, a component of our diet and cells, the polyunsaturated fatty acid arachidonic acid (ARA as a remedy for schistosomiasis, due to its ability to activate the parasite tegument-bound neutral sphingomyelinase, with subsequent hydrolysis of the apical lipid bilayer sphingomyelin molecules, allowing access of specific antibody molecules, and eventual worm attrition. This concept was convincingly supported using larval and adult Schistosoma mansoni and Schistosoma haematobium worms in in vitro experiments, and in vivo studies in inbred mice and outbred hamsters. Even if ARA proves to be an entirely effective and safe therapy for schistosomiasis, it will not prevent reinfection, and accordingly, the need for developing an effective vaccine remains an urgent priority. Our studies have supported the status of S. mansoni calpain, glutathione-S-transferase, aldolase, triose phosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, enolase, and 2-cys peroxiredoxin as vaccine candidates, as they are larval excreted-secreted products and, contrary to the surface membrane molecules, are entirely accessible to the host immune system effector elements. We have proposed that the use of these molecules, in conjunction with Th2 cytokines-inducing adjuvants for recruiting and activating

  8. Neuron-microglia crosstalk up-regulates neuronal FGF-2 expression which mediates neuroprotection against excitotoxicity via JNK1/2.

    Science.gov (United States)

    Figueiredo, Catarina; Pais, Teresa F; Gomes, João R; Chatterjee, Sukalyan

    2008-10-01

    Glial cells and neurons are in constant reciprocal signalling both under physiological and neuropathological conditions. Microglial activation is often associated with neuronal death during inflammation of the CNS, although microglial cells are also known to exert a neuroprotective role. In this work, we investigated the interplay between cerebellar granule neurons (CGN) and microglia in the perspective of CGN survival to an excitotoxic stimulus, quinolinic acid (QA), a catabolite of the tryptophan degradation pathway. We observed that CGN succumb to QA challenge via extracellular signal regulated kinase 1 and 2 (ERK) activation. Our data with transgenic mice expressing the natural inhibitor of calpains, calpastatin, indicate that together with cathepsins they mediate QA-induced toxicity acting downstream of the mitogen-activated protein kinase kinase-ERK pathway. Microglial cells are not only resistant to QA but can rescue neurons from QA-mediated toxicity when they are mixed in culture with neurons or by using mixed culture-conditioned medium (MCCM). This effect is mediated via fibroblast growth factor-2 (FGF-2) present in MCCM. FGF-2 is transcriptionally up-regulated in neurons and secreted in the MCCM as a result of neuron-microglia crosstalk. The neuroprotection is associated with the retention of cathepsins in the lysosomes and with transactivation of inducible heat-shock protein 70 downstream of FGF-2. Furthermore, FGF-2 upon release by neurons activates c-jun N-terminal kinase 1 and 2 pathway which also contributes to neuronal survival. We suggest that FGF-2 plays a pivotal role in neuroprotection against QA as an outcome of neuron-microglia interaction.

  9. An IP-10 (CXCL10-derived peptide inhibits angiogenesis.

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    Cecelia C Yates-Binder

    Full Text Available Angiogenesis plays a critical role in processes such as organ development, wound healing, and tumor growth. It requires well-orchestrated integration of soluble and matrix factors and timely recognition of such signals to regulate this process. Previous work has shown that newly forming vessels express the chemokine receptor CXC receptor 3 (CXCR3 and, activation by its ligand IP-10 (CXCL10, both inhibits development of new vasculature and causes regression of newly formed vessels. To identify and develop new therapeutic agents to limit or reverse pathological angiogenesis, we identified a 21 amino acid fragment of IP-10, spanning the α-helical domain residues 77-98, that mimic the actions of the whole IP-10 molecule on endothelial cells. Treatment of the endothelial cells with the 22 amino acid fragment referred to as IP-10p significantly inhibited VEGF-induced endothelial motility and tube formation in vitro, properties critical for angiogenesis. Using a Matrigel plug assay in vivo, we demonstrate that IP-10p both prevented vessel formation and induced involution of nascent vessels. CXCR3 neutralizing antibody was able to block the inhibitory effects of the IP-10p, demonstrating specificity of the peptide. Inhibition of endothelial function by IP-10p was similar to that described for IP-10, secondary to CXCR3-mediated increase in cAMP production, activation of PKA inhibiting cell migration, and inhibition of VEGF-mediated m-calpain activation. IP-10p provides a novel therapeutic agent that inhibits endothelial cell function thus, allowing for the modulation of angiogenesis.

  10. Blockade of KCa3.1 potassium channels protects against cisplatin-induced acute kidney injury.

    Science.gov (United States)

    Chen, Cheng-Lung; Liao, Jiunn-Wang; Hu, Oliver Yoa-Pu; Pao, Li-Heng

    2016-09-01

    Tubular cell apoptosis significantly contributes to cisplatin-induced acute kidney injury (AKI) pathogenesis. Although KCa3.1, a calcium-activated potassium channel, participates in apoptosis, its involvement in cisplatin-induced AKI is unknown. Here, we found that cisplatin treatment triggered an early induction of KCa3.1 expression associated with HK-2 cell apoptosis, the development of renal tubular damage, and apoptosis in mice. Treatment with the highly selective KCa3.1 blocker TRAM-34 suppressed cisplatin-induced HK-2 cell apoptosis. We further assessed whether KCa3.1 mediated cisplatin-induced AKI in genetic knockout and pharmacological blockade mouse models. KCa3.1 deficiency reduced renal function loss, renal tubular damage, and the induction of the apoptotic marker caspase-3 in the kidneys of cisplatin-treated KCa3.1 (-/-) mice. Pharmacological blockade of KCa3.1 by TRAM-34 similarly attenuated cisplatin-induced AKI in mice. Furthermore, we dissected the mechanisms underlying cisplatin-induced apoptosis reduction via KCa3.1 blockade. We found that KCa3.1 blockade attenuated cytochrome c release and the increase in the intrinsic apoptotic mediators Bax, Bak, and caspase-9 after cisplatin treatment. KCa3.1 blocking inhibited the cisplatin-induced activation of the endoplasmic reticulum (ER) stress mediator caspase-12, which is independent of calcium-dependent protease m-calpain activation. Taken together, KCa3.1 blockade protects against cisplatin-induced AKI through the attenuation of apoptosis by interference with intrinsic apoptotic and ER stress-related mediators, providing a potential target for the prevention of cisplatin-induced AKI. PMID:26438401

  11. MARCKS is a natively unfolded protein with an inaccessible actin-binding site: evidence for long-range intramolecular interactions.

    Science.gov (United States)

    Tapp, Hazel; Al-Naggar, Iman M; Yarmola, Elena G; Harrison, Alexis; Shaw, Gerry; Edison, Arthur S; Bubb, Michael R

    2005-03-18

    Myristoylated alanine-rich C kinase substrate (MARCKS) is an unfolded protein that contains well characterized actin-binding sites within the phosphorylation site domain (PSD), yet paradoxically, we now find that intact MARCKS does not bind to actin. Intact MARCKS also does not bind as well to calmodulin as does the PSD alone. Myristoylation at the N terminus alters how calmodulin binds to MARCKS, implying that, despite its unfolded state, the distant N terminus influences binding events at the PSD. We show that the free PSD binds with site specificity to MARCKS, suggesting that long-range intramolecular interactions within MARCKS are also possible. Because of the unusual primary sequence of MARCKS with an overall isoelectric point of 4.2 yet a very basic PSD (overall charge of +13), we speculated that ionic interactions between oppositely charged domains of MARCKS were responsible for long-range interactions within MARCKS that sterically influence binding events at the PSD and that explain the observed differences between properties of the PSD and MARCKS. Consistent with this hypothesis, chemical modifications of MARCKS that neutralize negatively charged residues outside of the PSD allow the PSD to bind to actin and increase the affinity of MARCKS for calmodulin. Similarly, both myristoylation of MARCKS and cleavage of MARCKS by calpain are shown to increase the availability of the PSD so as to activate its actin-binding activity. Because abundant evidence supports the conclusion that MARCKS is an important protein in regulating actin dynamics, our data imply that post-translational modifications of MARCKS are necessary and sufficient to regulate actin-binding activity. PMID:15640140

  12. Subcellular localization and functional domain studies of DEFECTIVE KERNEL1 in maize and Arabidopsis suggest a model for aleurone cell fate specification involving CRINKLY4 and SUPERNUMERARY ALEURONE LAYER1.

    Science.gov (United States)

    Tian, Qing; Olsen, Lene; Sun, Beimeng; Lid, Stein Erik; Brown, Roy C; Lemmon, Betty E; Fosnes, Kjetil; Gruis, Darren Fred; Opsahl-Sorteberg, Hilde-Gunn; Otegui, Marisa S; Olsen, Odd-Arne

    2007-10-01

    DEFECTIVE KERNEL1 (DEK1), which consists of a membrane-spanning region (DEK1-MEM) and a calpain-like Cys proteinase region (DEK1-CALP), is essential for aleurone cell formation at the surface of maize (Zea mays) endosperm. Immunolocalization and FM4-64 dye incubation experiments showed that DEK1 and CRINKLY4 (CR4), a receptor kinase implicated in aleurone cell fate specification, colocalized to plasma membrane and endosomes. SUPERNUMERARY ALEURONE LAYER1 (SAL1), a negative regulator of aleurone cell fate encoding a class E vacuolar sorting protein, colocalized with DEK1 and CR4 in endosomes. Immunogold localization, dual-axis electron tomography, and diffusion of fluorescent dye tracers showed that young aleurone cells established symplastic subdomains through plasmodesmata of larger dimensions than those connecting starchy endosperm cells and that CR4 preferentially associated with plasmodesmata between aleurone cells. Genetic complementation experiments showed that DEK1-CALP failed to restore wild-type phenotypes in maize and Arabidopsis thaliana dek1 mutants, and DEK1-MEM also failed to restore wild-type phenotypes in Arabidopsis dek1-1 mutants. Instead, ectopic expression of DEK1-MEM under the control of the cauliflower mosaic virus 35S promoter gave a dominant negative phenotype. These data suggest a model for aleurone cell fate specification in which DEK1 perceives and/or transmits a positional signal, CR4 promotes the lateral movement of aleurone signaling molecules between aleurone cells, and SAL1 maintains the proper plasma membrane concentration of DEK1 and CR4 proteins via endosome-mediated recycling/degradation.

  13. Limb-girdle muscular dystrophy subtypes First-reported cohort from northeastern China*

    Institute of Scientific and Technical Information of China (English)

    Omar Abdulmonem Mahmood; Xinmei Jiang; Qi Zhang

    2013-01-01

    The relative frequencies of different subtypes of limb-girdle muscular dystrophies vary widely among different populations. We estimated the percentage of limb-girdle muscular dystrophy sub-types in Chinese people based on 68 patients with limb-girdle muscular dystrophy from the Myology Clinic, Neurology Department, First Hospital of Jilin University, China. A diagnosis of calpainopathy was made in 12 cases (17%), and dysferlin deficiency in 10 cases (15%). Two biopsies revealedα-sarcoglycan deficiency (3%), and two others revealed a lack of caveolin-3 (3%). A diagnosis of unclassified limb-girdle muscular dystrophy was made in the remaining patients (62%). The ap-pearances of calpain 3-and dysferlin-deficient biopsies were similar, though rimmed vacuoles were unique to dysferlinopathy, while inflammatory infiltrates were present in both these limb-girdle muscular dystrophy type 2D biopsies. Macrophages were detected in seven dysferlinopathy biop-sies. The results of this study suggest that the distribution of limb-girdle muscular dystrophy sub-types in the Han Chinese population is similar to that reported in the West. The less necrotic, re-generating and inflammatory appearance of limb-girdle muscular dystrophy type 2A, but with more lobulated fibers, supports the idea that calpainopathy is a less active, but more chronic disease than dysferlinopathy. Unusual features indicated an extended limb-girdle muscular dystrophy disease spectrum. The use of acid phosphatase stain should be considered in suspected dysferlinopathies. To the best of our knowledge, this is the first report to define the relative proportions of the various forms of limb-girdle muscular dystrophy in China, based on protein testing.

  14. NF-κB in acute pancreatitis: Mechanisms and therapeutic potential.

    Science.gov (United States)

    Jakkampudi, Aparna; Jangala, Ramaiah; Reddy, B Ratnakar; Mitnala, Sasikala; Nageshwar Reddy, D; Talukdar, Rupjyoti

    2016-01-01

    The incidence of acute pancreatitis (AP) is increasing globally and mortality could be high among patients with organ failure and infected necrosis. The predominant factors responsible for the morbidity and mortality of AP are systemic inflammatory response syndrome and multiorgan dysfunction. Even though preclinical studies have shown antisecretory agents (somatostatin), antioxidants (S-adenosyl methionine [SAM], selenium), protease inhibitors, platelet activating factor inhibitor (Lexipafant), and anti-inflammatory immunomodulators (eg. prostaglandin E, indomethacin) to benefit AP in terms of reducing the severity and/or mortality, most of these agents have shown heterogeneous results in clinical studies. Several years of experimental studies have implicated nuclear factor-kappa B (NF-κB) activation as an early and central event in the progression of inflammation in AP. In this manuscript, we review the literature on the role of NF-κB in the pathogenesis of AP, its early intraacinar activation, and how it results in progression of the disease. We also discuss why anti-protease, antisecretory, and anti-inflammatory agents are unlikely to be effective in clinical acute pancreatitis. NF-κB, being a central molecule that links the initial acinar injury to systemic inflammation and perpetuate the inflammation, we propose that more studies be focussed towards targeted inhibition of NF-κB activity. Direct NF-κB inhibition strategies have already been attempted in patients with various cancers. So far, peroxisome proliferator activator receptor gamma (PPAR-γ) ligand, pyrrolidine dithiocarbamate (PDTC), proteasome inhibitor and calpain I inhibitor have been shown to have direct inhibitory effects on NF-κB activation in experimental AP. PMID:27282980

  15. Detecting loci under recent positive selection in dairy and beef cattle by combining different genome-wide scan methods.

    Directory of Open Access Journals (Sweden)

    Yuri Tani Utsunomiya

    Full Text Available As the methodologies available for the detection of positive selection from genomic data vary in terms of assumptions and execution, weak correlations are expected among them. However, if there is any given signal that is consistently supported across different methodologies, it is strong evidence that the locus has been under past selection. In this paper, a straightforward frequentist approach based on the Stouffer Method to combine P-values across different tests for evidence of recent positive selection in common variations, as well as strategies for extracting biological information from the detected signals, were described and applied to high density single nucleotide polymorphism (SNP data generated from dairy and beef cattle (taurine and indicine. The ancestral Bovinae allele state of over 440,000 SNP is also reported. Using this combination of methods, highly significant (P<3.17×10(-7 population-specific sweeps pointing out to candidate genes and pathways that may be involved in beef and dairy production were identified. The most significant signal was found in the Cornichon homolog 3 gene (CNIH3 in Brown Swiss (P = 3.82×10(-12, and may be involved in the regulation of pre-ovulatory luteinizing hormone surge. Other putative pathways under selection are the glucolysis/gluconeogenesis, transcription machinery and chemokine/cytokine activity in Angus; calpain-calpastatin system and ribosome biogenesis in Brown Swiss; and gangliosides deposition in milk fat globules in Gyr. The composite method, combined with the strategies applied to retrieve functional information, may be a useful tool for surveying genome-wide selective sweeps and providing insights in to the source of selection.

  16. Anti-Inflammatory Effects of Spirulina platensis Extract via the Modulation of Histone Deacetylases

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    Tho X. Pham

    2016-06-01

    Full Text Available We previously demonstrated that the organic extract of Spirulina platensis (SPE, an edible blue-green alga, possesses potent anti-inflammatory effects. In this study, we investigated if the regulation of histone deacetylases (HDACs play a role in the anti-inflammatory effect of SPE in macrophages. Treatment of macrophages with SPE rapidly and dose-dependently reduced HDAC2, 3, and 4 proteins which preceded decreases in their mRNA levels. Degradation of HDAC4 protein was attenuated in the presence of inhibitors of calpain proteases, lysosomal acidification, and Ca2+/calmodulin-dependent protein kinase II, respectively, but not a proteasome inhibitor. Acetylated histone H3 was increased in SPE-treated macrophages to a similar level as macrophages treated with a pan-HDAC inhibitor, with concomitant inhibition of inflammatory gene expression upon LPS stimulation. Knockdown of HDAC3 increased basal and LPS-induced pro-inflammatory gene expression, while HDAC4 knockdown increased basal expression of interleukin-1β (IL-1β, but attenuated LPS-induced inflammatory gene expression. Chromatin immunoprecipitation showed that SPE decreased p65 binding and H3K9/K14 acetylation at the Il-1β and tumor necrosis factor α (Tnfα promoters. Our results suggest that SPE increased global histone H3 acetylation by facilitating HDAC protein degradation, but decreases histone H3K9/K14 acetylation and p65 binding at the promoters of Il-1β and Tnfα to exert its anti-inflammatory effect.

  17. Gallic acid induces necroptosis via TNF-α signaling pathway in activated hepatic stellate cells.

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    Ya Ju Chang

    Full Text Available Gallic acid (3, 4, 5-trihydroxybenzoic acid, GA, a natural phenolic acid widely found in gallnuts, tea leaves and various fruits, possesses several bioactivities against inflammation, oxidation, and carcinogenicity. The beneficial effect of GA on the reduction of animal hepatofibrosis has been indicated due to its antioxidative property. However, the cytotoxicity of GA autoxidation causing cell death has also been reported. Herein, we postulated that GA might target activated hepatic stellate cells (aHSCs, the cell type responsible for hepatofibrosis, to mitigate the process of fibrosis. The molecular cytotoxic mechanisms that GA exerted on aHSCs were then analyzed. The results indicated that GA elicited aHSC programmed cell death through TNF-α-mediated necroptosis. GA induced significant oxidative stress through the suppression of catalase activity and the depletion of glutathione (GSH. Elevated oxidative stress triggered the production of TNF-α facilitating the undergoing of necroptosis through the up-regulation of key necroptotic regulatory proteins TRADD and receptor-interacting protein 3 (RIP3, and the inactivation of caspase-8. Calmodulin and calpain-1 activation were engaged, which promoted subsequent lysosomal membrane permeabilization (LMP. The TNF-α antagonist (SPD-304 and the RIP1 inhibitor (necrostatin-1, Nec-1 confirmed GA-induced TNFR1-mediated necroptosis. The inhibition of RIP1 by Nec-1 diverted the cell death from necroptosis to apoptosis, as the activation of caspase 3 and the increase of cytochrome c. Collectively, this is the first report indicating that GA induces TNF signaling-triggered necroptosis in aHSCs, which may offer an alternative strategy for the amelioration of liver fibrosis.

  18. Truncated N-terminal huntingtin fragment with expanded-polyglutamine (htt552-100Q)suppresses brain-derived neurotrophic factor transcription in astrocytes

    Institute of Scientific and Technical Information of China (English)

    Linhui Wang; Fang Lin; Jin Wang; Junchao Wu; Rong Han; Lujia Zhu; Guoxing Zhang; Marian DiFiglia; Zhenghong Qin

    2012-01-01

    Although huntingtin (htt) can be cleaved at many sites by caspases,calpains,and aspartyl proteases,amino acid (aa) 552 was defined as a preferred site for cleavage in human Huntington disease (HD) brains in vivo.To date,the normal function of wild-type N-terminal htt fragment 1-552 aa (htt552) and its pathological roles of mutant htt552 are still unknown.Although mutant htt (mhtt) is also expressed in astrocytes,whether and how mhtt contributes to the neurodegeneration through astrocytes in HD remains largely unknown.In this study,a glia HD model,using an adenoviral vector to express wild-type htt552 (htt552-18Q) and its mutation (htt552-100Q) in rat primary cortical astrocytes,was generated to investigate the influence of htt552 on the transcription of brainderived neurotrophic factor (BDNF). Results from enzyme linked immunosorbent assay showed that the level of BDNF in astrocyte-conditioned medium was decreased in the astrocytes expressing htt552-100Q.Quantitative real-time polymerase chain reaction demonstrated that htt552-100Q reduced the transcripts of the BDNF Ⅲ and Ⅳ, hence, repressed the transcription of BDNF.Furthermore,immunofluorescence showed that aggregates formed by htt552-100Q entrapped transcription factors cAMP-response element-binding protein and stimulatory protein 1,which might account for the reduction of BDNF transcription.These findings suggest that mhtt552 reduces BDNF transcription in astrocytes,which might contribute to the neuronal dysfunction in HD.

  19. Inflammatory and protein metabolism signaling responses in human skeletal muscle after burn injury.

    Science.gov (United States)

    Merritt, Edward K; Cross, James M; Bamman, Marcas M

    2012-01-01

    Severe burn injuries lead to a prolonged hypercatabolic state resulting in dramatic loss of skeletal muscle mass. Postburn muscle loss is well documented but the molecular signaling cascade preceding atrophy is not. The purpose of this study is to determine the response to burn injury of signaling pathways driving muscle inflammation and protein metabolism. Muscle biopsies were collected in the early flow phase after burn injury from the vastus lateralis of a noninjured leg in patients with 20 to 60% TBSA burns and compared with uninjured, matched controls. Circulating levels of proinflammatory cytokines were also compared. Immunoblotting was performed to determine the protein levels of key signaling components for translation initiation, proteolysis, and tumor necrosis factor/nuclear factor kappa B (NFκB)and interleukin (IL)-6/STAT3 signaling. Burn subjects had significantly higher levels of circulating proinflammatory cytokines, with no difference in muscle STAT3 activity and lower NFκB activity. No differences were found in any translational signaling components. Regarding proteolytic signaling in burn, calpain-2 was 47% higher, calpastatin tended to be lower, and total ubiquitination was substantially higher. Surprisingly, a systemic proinflammatory response 3 to 10 days postburn did not lead to elevated muscle STAT3 or NFκB signaling. Signaling molecules governing translation initiation were unaffected, whereas indices of calcium-mediated proteolysis and ubiquitin-proteasome activity were upregulated. These novel findings are the first in humans to suggest that the net catabolic effect of burn injury in skeletal muscle (ie, atrophy) may be mediated, at least during the early flow phase, almost entirely by an increased proteolytic activity in the absence of suppressed protein synthesis signaling.

  20. Repair of traumatic plasmalemmal damage to neurons and other eukaryotic cells.

    Science.gov (United States)

    Bittner, George D; Spaeth, Christopher S; Poon, Andrew D; Burgess, Zachary S; McGill, Christopher H

    2016-07-01

    The repair (sealing) of plasmalemmal damage, consisting of small holes to complete transections, is critical for cell survival, especially for neurons that rarely regenerate cell bodies. We first describe and evaluate different measures of cell sealing. Some measures, including morphological/ultra-structural observations, membrane potential, and input resistance, provide very ambiguous assessments of plasmalemmal sealing. In contrast, measures of ionic current flow and dye barriers can, if appropriately used, provide more accurate assessments. We describe the effects of various substances (calcium, calpains, cytoskeletal proteins, ESCRT proteins, mUNC-13, NSF, PEG) and biochemical pathways (PKA, PKC, PLC, Epac, cytosolic oxidation) on plasmalemmal sealing probability, and suggest that substances, pathways, and cellular events associated with plasmalemmal sealing have undergone a very conservative evolution. During sealing, calcium ion influx mobilizes vesicles and other membranous structures (lysosomes, mitochondria, etc.) in a continuous fashion to form a vesicular plug that gradually restricts diffusion of increasingly smaller molecules and ions over a period of seconds to minutes. Furthermore, we find no direct evidence that sealing occurs through the collapse and fusion of severed plasmalemmal leaflets, or in a single step involving the fusion of one large wound vesicle with the nearby, undamaged plasmalemma. We describe how increases in perikaryal calcium levels following axonal transection account for observations that cell body survival decreases the closer an axon is transected to the perikaryon. Finally, we speculate on relationships between plasmalemmal sealing, Wallerian degeneration, and the ability of polyethylene glycol (PEG) to seal cell membranes and rejoin severed axonal ends - an important consideration for the future treatment of trauma to peripheral nerves. A better knowledge of biochemical pathways and cytoplasmic structures involved in