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Sample records for calpain 3-deficient mice

  1. Alteration of Sarcoplasmic Reticulum Ca2+ Release in Skeletal Muscle from Calpain 3-Deficient Mice

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    Govindan Dayanithi

    2009-01-01

    Full Text Available Mutations of Ca2+-activated proteases (calpains cause muscular dystrophies. Nevertheless, the specific role of calpains in Ca2+ signalling during the onset of dystrophies remains unclear. We investigated Ca2+ handling in skeletal cells from calpain 3-deficient mice. [Ca2+]i responses to caffeine, a ryanodine receptor (RyR agonist, were decreased in −/− myotubes and absent in −/− myoblasts. The −/− myotubes displayed smaller amplitudes of the Ca2+ transients induced by cyclopiazonic acid in comparison to wild type cells. Inhibition of L-type Ca2+ channels (LCC suppressed the caffeine-induced [Ca2+]i responses in −/− myotubes. Hence, the absence of calpain 3 modifies the sarcoplasmic reticulum (SR Ca2+ release, by a decrease of the SR content, an impairment of RyR signalling, and an increase of LCC activity. We propose that calpain 3-dependent proteolysis plays a role in activating support proteins of intracellular Ca2+ signalling at a stage of cellular differentiation which is crucial for skeletal muscle regeneration.

  2. Calpain mediated cisplatin-induced ototoxicity in mice*

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    Liang Chang; Aimei Wang

    2013-01-01

    Ototoxic drug-induced apoptosis of inner ear cel s has been shown to be associated with calpain expression. Cisplatin has severe ototoxicity, and can induce cochlear cel apoptosis. This study assumed that cisplatin activated calpain expression in apoptotic cochlear cel s. A mouse model of cisplatin-induced ototoxicity was established by intraperitoneal injection with cisplatin (2.5, 3.5, 4.5, 5.5 mg/kg). Immunofluorescence staining, image analysis and western blotting were used to detect the expression of calpain 1 and calpain 2 in the mouse cochlea. At the same time, the auditory brainstem response was measured to observe the change in hearing. Results revealed that after intraperitoneal injection with cisplatin for 5 days, the auditory brainstem response threshold shifts increased in mice. Calpain 1 and calpain 2 expression significantly increased in outer hair cel s, the spiral ganglion and stria vascularis. Calpain 2 protein expression markedly increased with an in-creased dose of cisplatin. Results suggested that calpain 1 and calpain 2 mediated cispla-tin-induced ototoxicity in BALB/c mice. During this process, calpain 2 plays a leading role.

  3. Inner ear dysfunction in caspase-3 deficient mice

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    Woo Minna

    2011-10-01

    Full Text Available Abstract Background Caspase-3 is one of the most downstream enzymes activated in the apoptotic pathway. In caspase-3 deficient mice, loss of cochlear hair cells and spiral ganglion cells coincide closely with hearing loss. In contrast with the auditory system, details of the vestibular phenotype have not been characterized. Here we report the vestibular phenotype and inner ear anatomy in the caspase-3 deficient (Casp3-/- mouse strain. Results Average ABR thresholds of Casp3-/- mice were significantly elevated (P Casp3+/- mice and Casp3+/+ mice at 3 months of age. In DPOAE testing, distortion product 2F1-F2 was significantly decreased (P Casp3-/- mice, whereas Casp3+/- and Casp3+/+ mice showed normal and comparable values to each other. Casp3-/- mice were hyperactive and exhibited circling behavior when excited. In lateral canal VOR testing, Casp3-/- mice had minimal response to any of the stimuli tested, whereas Casp3+/- mice had an intermediate response compared to Casp3+/+ mice. Inner ear anatomical and histological analysis revealed gross hypomorphism of the vestibular organs, in which the main site was the anterior semicircular canal. Hair cell numbers in the anterior- and lateral crista, and utricle were significantly smaller in Casp3-/- mice whereas the Casp3+/- and Casp3+/+ mice had normal hair cell numbers. Conclusions These results indicate that caspase-3 is essential for correct functioning of the cochlea as well as normal development and function of the vestibule.

  4. Spontaneous metastasis in matrix metalloproteinase 3-deficient mice

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    Juncker-Jensen, Anna; Rømer, John; Pennington, Caroline J;

    2009-01-01

    in tumorigenesis and metastatic growth. In this model the stromal expression of MMP-3 mRNA resembles the predominant MMP-3 expression pattern observed in human ductal breast carcinomas. We studied a cohort of 63 PyMT transgenic mice, either deficient for MMP-3 or wild-type controls. The degree of metastasis did...... not differ significantly between the two groups of mice, although the median lung metastasis volume was more than threefold increased in MMTV-PyMT mice deficient in MMP-3. Likewise, primary tumor growth rate and lymph node metastasis were not significantly affected by MMP-3-deficiency. By comparing m......RNA levels in MMP-3-deficient PyMT tumors with PyMT wild-type tumors we excluded compensatory transcriptional changes of other MMPs or their specific inhibitors. Thus, we conclude that genetic ablation of MMP-3 does not significantly affect tumor growth and metastasis in the MMTV-PyMT model....

  5. Calpain-2 compensation promotes angiotensin II-induced ascending and abdominal aortic aneurysms in calpain-1 deficient mice.

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    Venkateswaran Subramanian

    Full Text Available Recently, we demonstrated that angiotensin II (AngII-infusion profoundly increased both aortic protein and activity of calpains, calcium-activated cysteine proteases, in mice. In addition, pharmacological inhibition of calpain attenuated AngII-induced abdominal aortic aneurysm (AA in mice. Recent studies have shown that AngII infusion into mice leads to aneurysmal formation localized to the ascending aorta. However, the precise functional contribution of calpain isoforms (-1 or -2 in AngII-induced abdominal AA formation is not known. Similarly, a functional role of calpain in AngII-induced ascending AA remains to be defined. Using BDA-410, an inhibitor of calpains, and calpain-1 genetic deficient mice, we examined the relative contribution of calpain isoforms in AngII-induced ascending and abdominal AA development.To investigate the relative contribution of calpain-1 and -2 in development of AngII-induced AAs, male LDLr -/- mice that were either calpain-1 +/+ or -/- were fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min for 4 weeks. Calpain-1 deficiency had no significant effect on body weight or blood pressure during AngII infusion. Moreover, calpain-1 deficiency showed no discernible effects on AngII-induced ascending and abdominal AAs. Interestingly, AngII infusion induced increased expression of calpain-2 protein, thus compensating for total calpain activity in aortas of calpain-1 deficient mice. Oral administration of BDA-410, a calpain inhibitor, along with AngII-infusion significantly attenuated AngII-induced ascending and abdominal AA formation in both calpain-1 +/+ and -/- mice as compared to vehicle administered mice. Furthermore, BDA-410 administration attenuated AngII-induced aortic medial hypertrophy and macrophage accumulation. Western blot and immunostaining analyses revealed BDA-410 administration attenuated AngII-induced C-terminal fragmentation of filamin A, an actin binding cytoskeletal protein in aorta.Calpain

  6. Impaired bone formation in Pdia3 deficient mice.

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    Yun Wang

    Full Text Available 1α,25-Dihydroxyvitamin D3 [1α,25(OH2D3] is crucial for normal skeletal development and bone homeostasis. Protein disulfide isomerase family A, member 3 (PDIA3 mediates 1α,25(OH2D3 initiated-rapid membrane signaling in several cell types. To understand its role in regulating skeletal development, we generated Pdia3-deficient mice and examined the physiologic consequence of Pdia3-disruption in embryos and Pdia3+/- heterozygotes at different ages. No mice homozygous for the Pdia3-deletion were found at birth nor were there embryos after E12.5, indicating that targeted disruption of the Pdia3 gene resulted in early embryonic lethality. Pdia3-deficiency also resulted in skeletal manifestations as revealed by µCT analysis of the tibias. In comparison to wild type mice, Pdia3 heterozygous mice displayed expanded growth plates associated with decreased tether formation. Histomorphometry also showed that the hypertrophic zone in Pdia3+/- mice was more cellular than seen in wild type growth plates. Metaphyseal trabecular bone in Pdia3+/- mice exhibited an age-dependent phenotype with lower BV/TV and trabecular numbers, which was most pronounced at 15 weeks of age. Bone marrow cells from Pdia3+/- mice exhibited impaired osteoblastic differentiation, based on reduced expression of osteoblast markers and mineral deposition compared to cells from wild type animals. Collectively, our findings provide in vivo evidence that PDIA3 is essential for normal skeletal development. The fact that the Pdia3+/- heterozygous mice share a similar growth plate and bone phenotype to nVdr knockout mice, suggests that PDIA3-mediated rapid membrane signaling might be an alternative mechanism responsible for 1α,25(OH2D3's actions in regulating skeletal development.

  7. m-Calpain is required for preimplantation embryonic development in mice

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    Williams Karen

    2006-01-01

    Full Text Available Abstract Background μ-calpain and m-calpain are ubiquitously expressed proteases implicated in cellular migration, cell cycle progression, degenerative processes and cell death. These heterodimeric enzymes are composed of distinct catalytic subunits, encoded by Capn1 (μ-calpain or Capn2 (m-calpain, and a common regulatory subunit encoded by Capn4. Disruption of the mouse Capn4 gene abolished both μ-calpain and m-calpain activity, and resulted in embryonic lethality, thereby suggesting essential roles for one or both of these enzymes during mammalian embryogenesis. Disruption of the Capn1 gene produced viable, fertile mice implying that either m-calpain could compensate for the loss of μ-calpain, or that the loss of m-calpain was responsible for death of Capn4-/- mice. Results To distinguish between the alternatives described above, we deleted an essential coding region in the mouse Capn2 gene in embryonic stems cells and transmitted this mutant allele through the mouse germline. Breeding of heterozygous animals failed to produce homozygous mutant live offspring or implanted embryos. A nested PCR genotyping protocol was established, and homozygous preimplantation mutant embryos were detected at the morula but not at the blastocyts stage. Conclusion We conclude that homozygous disruption of the Capn2 gene results in pre-implantation embryonic lethality between the morula and blastocyst stage. This establishes that μ-calpain and m-calpain have distinct functions, and that m-calpain is vital for development of the preimplantation murine embryo.

  8. Altered neuronal architecture and plasticity in the visual cortex of adult MMP-3-deficient mice.

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    Aerts, Jeroen; Nys, Julie; Moons, Lieve; Hu, Tjing-Tjing; Arckens, Lutgarde

    2015-09-01

    Matrix metalloproteinases (MMPs) are Zn(2+)-dependent endopeptidases considered to be essential for normal brain development and neuroplasticity by modulating extracellular matrix proteins, receptors, adhesion molecules, growth factors and cytoskeletal proteins. Specifically, MMP-3 has recently been implicated in synaptic plasticity, hippocampus-dependent learning and neuronal development and migration in the cerebellum. However, the function(s) of this enzyme in the neocortex is understudied. Therefore, we explored the phenotypical characteristics of the neuronal architecture and the capacity for experience-dependent cortical plasticity in the visual cortex of adult MMP-3-deficient (MMP-3(-/-)) mice. Golgi-Cox stainings revealed a significant reduction in apical dendritic length and an increased number of apical obliques for layer V pyramidal neurons in the visual cortex of adult MMP-3(-/-) mice compared to wild-type (WT) animals. In addition, a significant upregulation of both phosphorylated and non-phosphorylated neurofilament protein (NF)-high, phosphorylated NF-medium, NF-low and α-internexin was detected in the visual cortex of MMP-3(-/-) mice. To assess the effect of MMP-3 deficiency on cortical plasticity, we monocularly enucleated adult MMP-3(-/-) mice and analyzed the reactivation of the contralateral visual cortex 7 weeks post-enucleation. In contrast to previous results in C57Bl/6J adult mice, activity remained confined to the binocular zone and did not expand into the monocular regions indicative for an aberrant open-eye potentiation. Permanent hypoactivity in the monocular cortex lateral and medial to V1 also indicated a lack of cross-modal plasticity. These observations demonstrate that genetic inactivation of MMP-3 has profound effects on the structural integrity and plasticity response of the visual cortex of adult mice.

  9. AMPD3-deficient mice exhibit increased erythrocyte ATP levels but anemia not improved due to PK deficiency.

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    Cheng, Jidong; Morisaki, Hiroko; Toyama, Keiko; Ikawa, Masahito; Okabe, Masaru; Morisaki, Takayuki

    2012-11-01

    AMP deaminase (AMPD) catalyzes AMP to IMP and plays an important role in energy charge and nucleotide metabolism. Human AMPD3 deficiency is a type of erythrocyte-specific enzyme deficiency found in individuals without clinical symptoms, although an increased level of ATP in erythrocytes has been reported. To better understand the physiological and pathological roles of AMPD3 deficiency, we established a line of AMPD3-deficient [A3(-/-)] mice. No AMPD activity and a high level of ATP were observed in erythrocytes of these mice, similar to human RBC-AMPD3 deficiency, while other characteristics were unremarkable. Next, we created AMPD3 and pyruvate kinase (PK) double-deficient [PKA(-/-,-/-)] mice by mating A3(-/-) mice with CBA-Pk-1slc/Pk-1slc mice [PK(-/-)], a spontaneous PK-deficient strain showing hemolytic anemia. In PKA(-/-,-/-) mice, the level of ATP in red blood cells was increased 1.5 times as compared to PK(-/-) mice, although hemolytic anemia in those animals was not improved. In addition, we observed osmotic fragility of erythrocytes in A3(-/-) mice under fasting conditions. In contrast, the ATP level in erythrocytes was elevated in A3(-/-) mice as compared to the control. In conclusion, AMPD3 deficiency increases the level of ATP in erythrocytes, but does not improve anemia due to PK deficiency and leads to erythrocyte dysfunction.

  10. Complement C3-Deficient Mice Fail to Display Age-Related Hippocampal Decline.

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    Shi, Qiaoqiao; Colodner, Kenneth J; Matousek, Sarah B; Merry, Katherine; Hong, Soyon; Kenison, Jessica E; Frost, Jeffrey L; Le, Kevin X; Li, Shaomin; Dodart, Jean-Cosme; Caldarone, Barbara J; Stevens, Beth; Lemere, Cynthia A

    2015-09-23

    The complement system is part of the innate immune response responsible for removing pathogens and cellular debris, in addition to helping to refine CNS neuronal connections via microglia-mediated pruning of inappropriate synapses during brain development. However, less is known about the role of complement during normal aging. Here, we studied the role of the central complement component, C3, in synaptic health and aging. We examined behavior as well as electrophysiological, synaptic, and neuronal changes in the brains of C3-deficient male mice (C3 KO) compared with age-, strain-, and gender-matched C57BL/6J (wild-type, WT) control mice at postnatal day 30, 4 months, and 16 months of age. We found the following: (1) region-specific and age-dependent synapse loss in aged WT mice that was not observed in C3 KO mice; (2) age-dependent neuron loss in hippocampal CA3 (but not in CA1) that followed synapse loss in aged WT mice, neither of which were observed in aged C3 KO mice; and (3) significantly enhanced LTP and cognition and less anxiety in aged C3 KO mice compared with aged WT mice. Importantly, CA3 synaptic puncta were similar between WT and C3 KO mice at P30. Together, our results suggest a novel and prominent role for complement protein C3 in mediating aged-related and region-specific changes in synaptic function and plasticity in the aging brain. Significance statement: The complement cascade, part of the innate immune response to remove pathogens, also plays a role in synaptic refinement during brain development by the removal of weak synapses. We investigated whether complement C3, a central component, affects synapse loss during aging. Wild-type (WT) and C3 knock-out (C3 KO) mice were examined at different ages. The mice were similar at 1 month of age. However, with aging, WT mice lost synapses in specific brain regions, especially in hippocampus, an area important for memory, whereas C3 KO mice were protected. Aged C3 KO mice also performed better on

  11. Helicobacter Infection Is Required for Inflammation and Colon Cancer in Smad3-Deficient Mice

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    Maggio-Price, Lillian; Treuting, Piper; Zeng, Weiping; Tsang, Mark; Bielefeldt-Ohmann, Helle; Iritani, Brian M.

    2017-01-01

    Accumulating evidence suggests that intestinal microbial organisms may play an important role in triggering and sustaining inflammation in individuals afflicted with inflammatory bowel disease (IBD). Moreover, individuals with IBD are at increased risk for developing colorectal cancer, suggesting that chronic inflammation may initiate genetic or epigenetic changes associated with cancer development. We tested the hypothesis that bacteria may contribute to the development of colon cancer by synergizing with defective transforming growth factor-β (TGF-β) signaling, a pathway commonly mutated in human colon cancer. Although others have reported that mice deficient in the TGF-β signaling molecule SMAD3 develop colon cancer, we found that SMAD3-deficient mice maintained free of the Gram-negative enterohepatic bacteria Helicobacter spp. for up to 9 months do not develop colon cancer. Furthermore, infection of SMAD3−/− mice with Helicobacter triggers colon cancer in 50% to 66% of the animals. Using real-time PCR, we found that Helicobacter organisms concentrate in the cecum, the preferred site of tumor development. Mucinous adenocarcinomas develop 5 to 30 weeks after infection and are preceded by an early inflammatory phase, consisting of increased proliferation of epithelial cells; increased numbers of cyclooxygenase-2–positive cells, CD4+ T cells, macrophages; and increased MHC class II expression. Colonic tissue revealed increased transcripts for the oncogene c-myc and the proinflammatory cytokines interleukin-1α (IL-1α), IL-1β, IL-6, IFN-γ, and tumor necrosis factor-α, some of which have been implicated in colon cancer. These results suggest that bacteria may be important in triggering colorectal cancer, notably in the context of gene mutations in the TGF-β signaling pathway, one of the most commonly affected cellular pathways in colorectal cancer in humans. PMID:16424015

  12. Leiomodin-3-deficient mice display nemaline myopathy with fast-myofiber atrophy

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    Lei Tian

    2015-06-01

    Full Text Available Nemaline myopathy (NM is one of the most common forms of congenital myopathy, and affects either fast myofibers, slow myofibers, or both. However, an animal model for congenital myopathy with fast-myofiber-specific atrophy is not available. Furthermore, mutations in the leiomodin-3 (LMOD3 gene have recently been identified in a group of individuals with NM. However, it is not clear how loss of LMOD3 leads to NM. Here, we report a mouse mutant in which the piggyBac (PB transposon is inserted into the Lmod3 gene and disrupts its expression. Lmod3PB/PB mice show severe muscle weakness and postnatal growth retardation. Electron microscopy and immunofluorescence studies of the mutant skeletal muscles revealed the presence of nemaline bodies, a hallmark of NM, and disorganized sarcomeric structures. Interestingly, Lmod3 deficiency caused muscle atrophy specific to the fast fibers. Together, our results show that Lmod3 is required in the fast fibers for sarcomere integrity, and this study offers the first NM mouse model with muscle atrophy that is specific to fast fibers. This model could be a valuable resource for interrogating myopathy pathogenesis and developing therapeutics for NM as well as other pathophysiological conditions with preferential atrophy of fast fibers, including cancer cachexia and sarcopenia.

  13. Characterization of connexin30.3-deficient mice suggests a possible role of connexin30.3 in olfaction.

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    Zheng-Fischhöfer, Qingyi; Schnichels, Marc; Dere, Ekrem; Strotmann, Jörg; Loscher, Nadine; McCulloch, Fiona; Kretz, Markus; Degen, Joachim; Reucher, Harald; Nagy, James I; Peti-Peterdi, Janos; Huston, Joseph P; Breer, Heinz; Willecke, Klaus

    2007-12-01

    We have generated connexin30.3-deficient mice in which the coding region of the connexin30.3 gene was replaced by the lacZ reporter gene. The expression pattern of this connexin was characterized using beta-galactosidase staining and immunoblot analyses. In skin, beta-galactosidase/connexin30.3 protein was expressed in the spinous and granulous layers of the epidermis. Specific beta-galactosidase/connexin30.3 expression was also detected in the thin ascending limb of Henle's loop in the kidney. In addition, we found beta-galactosidase/connexin30.3 in progenitor cells of the olfactory epithelium and in a subpopulation of cells in the apical layer of the vomeronasal organ. Connexin30.3-deficient mice were fertile and displayed no abnormalities in the skin or in the chemosensory systems. Furthermore, they showed normal auditory thresholds as measured by brain stem evoked potentials. These mice did, however, exhibit reduced behavioural responses to a vanilla scent.

  14. Complement C3 deficiency attenuates chronic hypoxia-induced pulmonary hypertension in mice.

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    Eileen M Bauer

    Full Text Available Evidence suggests a role of both innate and adaptive immunity in the development of pulmonary arterial hypertension. The complement system is a key sentry of the innate immune system and bridges innate and adaptive immunity. To date there are no studies addressing a role for the complement system in pulmonary arterial hypertension.Immunofluorescent staining revealed significant C3d deposition in lung sections from IPAH patients and C57Bl6/J wild-type mice exposed to three weeks of chronic hypoxia to induce pulmonary hypertension. Right ventricular systolic pressure and right ventricular hypertrophy were increased in hypoxic vs. normoxic wild-type mice, which were attenuated in C3-/- hypoxic mice. Likewise, pulmonary vascular remodeling was attenuated in the C3-/- mice compared to wild-type mice as determined by the number of muscularized peripheral arterioles and morphometric analysis of vessel wall thickness. The loss of C3 attenuated the increase in interleukin-6 and intracellular adhesion molecule-1 expression in response to chronic hypoxia, but not endothelin-1 levels. In wild-type mice, but not C3-/- mice, chronic hypoxia led to platelet activation as assessed by bleeding time, and flow cytometry of platelets to determine cell surface P-selectin expression. In addition, tissue factor expression and fibrin deposition were increased in the lungs of WT mice in response to chronic hypoxia. These pro-thrombotic effects of hypoxia were abrogated in C3-/- mice.Herein, we provide compelling genetic evidence that the complement system plays a pathophysiologic role in the development of PAH in mice, promoting pulmonary vascular remodeling and a pro-thrombotic phenotype. In addition we demonstrate C3d deposition in IPAH patients suggesting that complement activation plays a role in the development of PAH in humans.

  15. Pitx3 deficient mice as a genetic animal model of co-morbid depressive disorder and parkinsonism.

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    Kim, Kyoung-Shim; Kang, Young-Mi; Kang, Young; Park, Tae-Shin; Park, Hye-Yeon; Kim, Yoon-Jung; Han, Baek-Soo; Kim, Chun-Hyung; Lee, Chul-Ho; Ardayfio, Paul A; Han, Pyung-Lim; Jung, Bong-Hyun; Kim, Kwang-Soo

    2014-03-13

    Approximately 40-50% of all patients with Parkinson׳s disease (PD) show symptoms and signs of depressive disorders, for which neither pathogenic understanding nor rational treatment are available. Using Pit3x-deficient mice, a model for selective nigrostriatal dopaminergic neurodegeneration, we tested depression-related behaviors and acute stress responses to better understand how a nigrostriatal dopaminergic deficit increases the prevalence of depressive disorders in PD patients. Pitx3-deficient mice showed decreased sucrose consumption and preference in the two-bottle free-choice test of anhedonia. Acute restraint stress increased c-Fos (known as a neuronal activity marker) expression levels in various brain regions, including the prefrontal cortex, striatum, nucleus accumbens, and paraventricular nucleus of the hypothalamus (PVN), in both Pitx3+/+ and -/- mice. However, the stress-induced increases in c-Fos levels in the cortex, dorsal striatum, and PVN were significantly greater in Pitx3-/- than +/+ mice, suggesting that signs of depressive disorders in parkinsonism are related to altered stress vulnerability. Based on these results, we propose that Pitx3-/- mice may serve as a useful genetic animal model for co-morbid depressive disorder and parkinsonism.

  16. Ghrelin Inhibition Restores Glucose Homeostasis in Hepatocyte Nuclear Factor-1α (MODY3)-Deficient Mice.

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    Brial, François; Lussier, Carine R; Belleville, Karine; Sarret, Philippe; Boudreau, François

    2015-09-01

    Hepatocyte nuclear factor-1α (HNF1α) is a transcription factor expressed in tissues of endoderm origin. Mutations in HNF1A are associated with maturity-onset diabetes of the young 3 (MODY3). Mice deficient for Hnf1α are hyperglycemic, with their pancreatic β-cells being defective in glucose-sensing insulin secretion. The specific mechanisms involved in this defect are unclear. Gut hormones control glucose homeostasis. Our objective was to explore whether changes in these hormones play a role in glucose homeostasis in the absence of Hnf1α. An increase in ghrelin gene transcript and a decrease in glucose-dependent insulinotropic polypeptide (GIP) gene transcripts were observed in the gut of Hnf1α-null mice. These changes correlated with an increase of ghrelin and a decrease of GIP-labeled cells. Ghrelin serological levels were significantly induced in Hnf1α-null mice. Paradoxically, GIP levels were also induced in these mice. Treatment of Hnf1α-null mice with a ghrelin antagonist led to a recovery of the diabetic symptoms. We conclude that upregulation of ghrelin in the absence of Hnf1α impairs insulin secretion and can be reversed by pharmacological inhibition of ghrelin/GHS-R interaction. These observations open up on future strategies to counteract ghrelin action in a program that could become beneficial in controlling non-insulin-dependent diabetes.

  17. Effect of Complement Component C3 Deficiency on Experimental Lyme Borreliosis in Mice

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    Lawrenz, Matthew B.; Wooten, R. Mark; Zachary, James F.; Drouin, Scott M.; Weis, Janis J.; Wetsel, Rick A.; Norris, Steven J.

    2003-01-01

    Mice deficient in complement component C3 (C3−/−) and syngeneic C57BL/6 control mice were challenged with Borrelia burgdorferi to determine the role of complement in immune clearance and joint histopathology during experimental Lyme borreliosis. Tibiotarsal joint, ear, and heart tissues were monitored for spirochete numbers at 2, 4, 8, and 12 weeks postinoculation with 105 B. burgdorferi B31 clone 5A4 by using quantitative real-time PCR. The spirochete load in joint and ear tissue remained hi...

  18. Exacerbation of facial motoneuron loss after facial nerve axotomy in CCR3-deficient mice

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    Derek A Wainwright

    2009-12-01

    Full Text Available We have previously demonstrated a neuroprotective mechanism of FMN (facial motoneuron survival after facial nerve axotomy that is dependent on CD4+ Th2 cell interaction with peripheral antigen-presenting cells, as well as CNS (central nervous system-resident microglia. PACAP (pituitary adenylate cyclase-activating polypeptide is expressed by injured FMN and increases Th2-associated chemokine expression in cultured murine microglia. Collectively, these results suggest a model involving CD4+ Th2 cell migration to the facial motor nucleus after injury via microglial expression of Th2-associated chemokines. However, to respond to Th2-associated chemokines, Th2 cells must express the appropriate Th2-associated chemokine receptors. In the present study, we tested the hypothesis that Th2-associated chemokine receptors increase in the facial motor nucleus after facial nerve axotomy at timepoints consistent with significant T-cell infiltration. Microarray analysis of Th2-associated chemokine receptors was followed up with real-time PCR for CCR3, which indicated that facial nerve injury increases CCR3 mRNA levels in mouse facial motor nucleus. Unexpectedly, quantitative- and co-immunofluorescence revealed increased CCR3 expression localizing to FMN in the facial motor nucleus after facial nerve axotomy. Compared with WT (wild-type, a significant decrease in FMN survival 4 weeks after axotomy was observed in CCR3−/− mice. Additionally, compared with WT, a significant decrease in FMN survival 4 weeks after axotomy was observed in Rag2−/− (recombination activating gene-2-deficient mice adoptively transferred CD4+ T-cells isolated from CCR3−/− mice, but not in CCR3−/− mice adoptively transferred CD4+ T-cells derived from WT mice. These results provide a basis for further investigation into the co-operation between CD4+ T-cell- and CCR3-mediated neuroprotection after FMN injury.

  19. Exacerbation of Facial Motoneuron Loss after Facial Nerve Axotomy in CCR3-Deficient Mice

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    Derek A Wainwright

    2009-11-01

    Full Text Available We have previously demonstrated a neuroprotective mechanism of FMN (facial motoneuron survival after facial nerve axotomy that is dependent on CD4+ Th2 cell interaction with peripheral antigen-presenting cells, as well as CNS (central nervous system-resident microglia. PACAP (pituitary adenylate cyclase-activating polypeptide is expressed by injured FMN and increases Th2-associated chemokine expression in cultured murine microglia. Collectively, these results suggest a model involving CD4+ Th2 cell migration to the facial motor nucleus after injury via microglial expression of Th2-associated chemokines. However, to respond to Th2-associated chemokines, Th2 cells must express the appropriate Th2-associated chemokine receptors. In the present study, we tested the hypothesis that Th2-associated chemokine receptors increase in the facial motor nucleus after facial nerve axotomy at timepoints consistent with significant T-cell infiltration. Microarray analysis of Th2-associated chemokine receptors was followed up with real-time PCR for CCR3, which indicated that facial nerve injury increases CCR3 mRNA levels in mouse facial motor nucleus. Unexpectedly, quantitative- and co-immunofluorescence revealed increased CCR3 expression localizing to FMN in the facial motor nucleus after facial nerve axotomy. Compared with WT (wild-type, a significant decrease in FMN survival 4 weeks after axotomy was observed in CCR3–/– mice. Additionally, compared with WT, a significant decrease in FMN survival 4 weeks after axotomy was observed in Rag2 –/– (recombination activating gene-2-deficient mice adoptively transferred CD4+ T-cells isolated from CCR3–/– mice, but not in CCR3–/– mice adoptively transferred CD4+ T-cells derived from WT mice. These results provide a basis for further investigation into the co-operation between CD4+ T-cell- and CCR3-mediated neuroprotection after FMN injury.

  20. Absence of strong strain effects in behavioral analyses of Shank3-deficient mice

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    Elodie Drapeau

    2014-06-01

    Full Text Available Haploinsufficiency of SHANK3, caused by chromosomal abnormalities or mutations that disrupt one copy of the gene, leads to a neurodevelopmental syndrome called Phelan-McDermid syndrome, symptoms of which can include absent or delayed speech, intellectual disability, neurological changes and autism spectrum disorders. The SHANK3 protein forms a key structural part of the post-synaptic density. We previously generated and characterized mice with a targeted disruption of Shank3 in which exons coding for the ankyrin-repeat domain were deleted and expression of full-length Shank3 was disrupted. We documented specific deficits in synaptic function and plasticity, along with reduced reciprocal social interactions, in Shank3 heterozygous mice. Changes in phenotype owing to a mutation at a single locus are quite frequently modulated by other loci, most dramatically when the entire genetic background is changed. In mice, each strain of laboratory mouse represents a distinct genetic background and alterations in phenotype owing to gene knockout or transgenesis are frequently different across strains, which can lead to the identification of important modifier loci. We have investigated the effect of genetic background on phenotypes of Shank3 heterozygous, knockout and wild-type mice, using C57BL/6, 129SVE and FVB/Ntac strain backgrounds. We focused on observable behaviors with the goal of carrying out subsequent analyses to identify modifier loci. Surprisingly, there were very modest strain effects over a large battery of analyses. These results indicate that behavioral phenotypes associated with Shank3 haploinsufficiency are largely strain-independent.

  1. Enhancement of leptin receptor signaling by SOCS3 deficiency induces development of gastric tumors in mice.

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    Inagaki-Ohara, K; Mayuzumi, H; Kato, S; Minokoshi, Y; Otsubo, T; Kawamura, Y I; Dohi, T; Matsuzaki, G; Yoshimura, A

    2014-01-01

    Leptin acts on its receptor (ObR) in the hypothalamus to inhibit food intake and energy expenditure. Leptin and ObR are also expressed in the gastrointestinal tract; however, the physiological significance of leptin signaling in the gut remains uncertain. Suppressor of cytokine signaling 3 (SOCS3) is a key negative feedback regulator of ObR-mediated signaling in the hypothalamus. We now show that gastrointestinal epithelial cell-specific SOCS3 conditional knockout (T3b-SOCS3 cKO) mice developed gastric tumors by enhancing leptin production and the ObRb/signal transducer and activator of transcription 3 (STAT3) signaling pathway. All T3b-SOCS3 cKO mice developed tumors in the stomach but not in the bowels by 2 months of age, even though the SOCS3 deletion occurred in both the epithelium of stomach and bowels. The tumors developed in the absence of the inflammatory response and all cKO mice died within 6 months. These tumors displayed pathology and molecular alterations, such as an increase in MUC2 (Mucin 2, oligomeric mucus/gel-forming) and TFF3 (trefoil factor 3), resembling human intestinal-type gastric tumors. Administration of antileptin antibody to T3b-SOCS3 cKO mice reduced hyperplasia of gastric mucosa, which is the step of the initiation of gastric tumor. These data suggest that SOCS3 is an antigastric tumor gene that suppresses leptin overexpression and ObRb/STAT3 hyperactivation, supporting the hypothesis that the leptin/ObRb/STAT3 axis accelerates tumorigenesis and that it may represent a new therapeutic target for the treatment of gastric cancer.

  2. Severe Developmental B Lymphopoietic Defects in Foxp3-Deficient Mice are Refractory to Adoptive Regulatory T Cell Therapy.

    Science.gov (United States)

    Riewaldt, Julia; Düber, Sandra; Boernert, Marie; Krey, Martina; Dembinski, Marcin; Weiss, Siegfried; Garbe, Annette I; Kretschmer, Karsten

    2012-01-01

    The role of Foxp3-expressing regulatory T (T(reg)) cells in tolerance and autoimmunity is well-established. However, although of considerable clinical interest, the role of T(reg) cells in the regulation of hematopoietic homeostasis remains poorly understood. Thus, we analysed B and T lymphopoiesis in the scurfy (Sf) mouse model of T(reg) cell deficiency. In these experiments, the near-complete block of B lymphopoiesis in the BM of adolescent Sf mice was attributed to autoimmune T cells. We could exclude a constitutive lympho-hematopoietic defect or a B cell-intrinsic function of Foxp3. Efficient B cell development in the BM early in ontogeny and pronounced extramedullary B lymphopoietic activity resulted in a peripheral pool of mature B cells in adolescent Sf mice. However, marginal zone B and B-1a cells were absent throughout ontogeny. Developmental B lymphopoietic defects largely correlated with defective thymopoiesis. Importantly, neonatal adoptive T(reg) cell therapy suppressed exacerbated production of inflammatory cytokines and restored thymopoiesis but was ineffective in recovering defective B lymphopoiesis, probably due to a failure to compensate production of stroma cell-derived IL-7 and CXCL12. Our observations on autoimmune-mediated incapacitation of the BM environment in Foxp3-deficient mice will have direct implications for the rational design of BM transplantation protocols for patients with severe genetic deficiencies in functional Foxp3(+) T(reg) cells.

  3. 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...

  4. Immune complex glomerulonephritis following bone marrow transplantation in C3 deficient mice.

    Directory of Open Access Journals (Sweden)

    Thomas R Welch

    Full Text Available BACKGROUND: The role of circulating complement in host defense and immune disease is well established. Although a number of cells and tissues are capable of synthesizing complement components locally, the importance of such local synthesis in immune disease has been difficult to establish. METHODOLOGY/PRINCIPAL FINDINGS: We used bone marrow transplantation (BMT between C3 knockout (C3KO and wild type (WT mice to construct animals that were discordant for systemic (hepatic and local (monocytic C3 synthetic capacity. An immune complex glomerulonephritis (GN was then induced using intraperitoneal injections of horse spleen apoferritin (HSA with a lipopolysaccharide (LPS adjuvant. All HSA/LPS animals developed a proliferative GN with glomerular infiltration by monocytes. By sensitive ELISA, monocyte C3 synthesis could be detected in C3KO animals transplanted with WT bone marrow cells. Despite this, there were no significant differences among groups of mice in measures of clinical (proteinuria, renal function or histologic (glomerular cellularity, crescents disease severity. CONCLUSIONS/SIGNIFICANCE: In this model of GN, local synthesis of C3 by infiltrating cells does not appear to be of pathologic importance.

  5. Proximal giant neurofilamentous axonopathy in mice genetically engineered to resist calpain and caspase cleavage of α-2 spectrin

    Science.gov (United States)

    Kassa, R.; Monterroso, V.; Wentzell, J.; Ramos, A.L.; Couchi, E.; Lecomte, MC; Iordanov, M; Kretzschmar, D.; Nicolas, G.; Tshala-Katumbay, D.

    2012-01-01

    We use 1,2-diacetylbenzene (1,2-DAB) to probe molecular mechanisms of proximal giant neurofilamentous axonopathy (PGNA), a pathological hallmark of amyotrophic lateral sclerosis. The spinal cord proteome of rodents displaying 1,2-DAB-PGNA suggests a reduction in the abundance of αII-spectrin (Spna2), a key protein in the maintenance of axonal integrity. Protein immunoblotting indicates that this reduction is due Spna2 degradation. We investigated the importance of such degradation in 1,2-DAB-PGNA. Spna2 mutant mice lacking a calpain- and/or caspase-sensitive domain (CSD), thus hypothetically resistant to 1,2-DAB, and wild-type littermates, were treated with 1,2-DAB, 35 mg/kg/day, or saline-control, for 3 weeks. 1,2-DAB induced motor weakness and PGNA irrespective of the genotype. Spna2-calpain breakdown products were not detected in mutant mice, which displayed a normal structure of the nervous system under saline-treatment. Intriguingly, treatment with 1,2-DAB reduced the abundance of the caspase-specific 120 kDa Spna2 breakdown products. Our findings indicate that degradation of Spna2 by calpain- and/or caspase is not central to the pathogenesis of 1,2-DAB axonopathy. In addition, the Spna2-CSD seems to be not required for the maintenance of the cytoskeleton integrity. Our conceptual framework offers opportunities to study the role of calpain-caspase cross-talk, including that of the protease degradomics, in models of axonal degeneration. PMID:22212489

  6. Proximal giant neurofilamentous axonopathy in mice genetically engineered to resist calpain and caspase cleavage of α-II spectrin.

    Science.gov (United States)

    Kassa, R; Monterroso, V; Wentzell, J; Ramos, A L; Couchi, E; Lecomte, M C; Iordanov, M; Kretzschmar, D; Nicolas, G; Tshala-Katumbay, D

    2012-07-01

    We use 1,2-diacetylbenzene (1,2-DAB) to probe molecular mechanisms of proximal giant neurofilamentous axonopathy (PGNA), a pathological hallmark of amyotrophic lateral sclerosis. The spinal cord proteome of rodents displaying 1,2-DAB PGNA suggests a reduction in the abundance of α-II spectrin (Spna2), a key protein in the maintenance of axonal integrity. Protein immunoblotting indicates that this reduction is due to Spna2 degradation. We investigated the importance of such degradation in 1,2-DAB PGNA. Spna2 mutant mice lacking a calpain- and/or caspase-sensitive domain (CSD), thus hypothetically resistant to 1,2-DAB, and wild-type littermates, were treated with 1,2-DAB, 35 mg/kg/day, or saline control, for 3 weeks. 1,2-DAB induced motor weakness and PGNA, irrespective of the genotype. Spna2-calpain breakdown products were not detected in mutant mice, which displayed a normal structure of the nervous system under saline treatment. Intriguingly, treatment with 1,2-DAB reduced the abundance of the caspase-specific 120-kDa Spna2 breakdown products. Our findings indicate that degradation of Spna2 by calpain- and/or caspase is not central to the pathogenesis of 1,2-DAB axonopathy. In addition, the Spna2-CSD seems to be not required for the maintenance of the cytoskeleton integrity. Our conceptual framework offers opportunities to study the role of calpain-caspase cross talk, including that of the protease degradomics, in models of axonal degeneration.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. ClC-3 deficiency protects preadipocytes against apoptosis induced by palmitate in vitro and in type 2 diabetes mice.

    Science.gov (United States)

    Huang, Yun-Ying; Huang, Xiong-Qin; Zhao, Li-Yan; Sun, Fang-Yun; Chen, Wen-Liang; Du, Jie-Yi; Yuan, Feng; Li, Jie; Huang, Xue-Lian; Liu, Jie; Lv, Xiao-Fei; Guan, Yong-Yuan; Chen, Jian-Wen; Wang, Guan-Lei

    2014-11-01

    Palmitate, a common saturated free fatty acid (FFA), has been demonstrated to induce preadipocyte apoptosis in the absence of adipogenic stimuli, suggesting that preadipocytes may be prone to apoptosis under adipogenic insufficient conditions, like type 2 diabetes mellitus (T2DM). ClC-3, encoding Cl(-) channel or Cl(-)/H(+) antiporter, is critical for cell fate choices of proliferation versus apoptosis under diseased conditions. However, it is unknown whether ClC-3 is related with preadipocyte apoptosis induced by palmitate or T2DM. Palmitate, but not oleate, induced apoptosis and increase in ClC-3 protein expression and endoplasmic reticulum (ER) stress in 3T3-L1 preadipocyte. ClC-3 specific siRNA attenuated palmitate-induced apoptosis and increased protein levels of Grp78, ATF4, CHOP and phosphorylation of JNK1/2, whereas had no effects on increased phospho-PERK and phospho-eIF2α protein expression. Moreover, the enhanced apoptosis was shown in preadipocytes from high-sucrose/fat, low-dose STZ induced T2DM mouse model with hyperglycemia, hyperlipidemia (elevated serum TG and FFA levels) and insulin resistance. ClC-3 knockout significantly attenuated preadipocyte apoptosis and the above metabolic disorders in T2DM mice. These data demonstrated that ClC-3 deficiency prevent preadipocytes against palmitate-induced apoptosis via suppressing ER stress, and also suggested that ClC-3 may play a role in regulating cellular apoptosis and disorders of glucose and lipid metabolism during T2DM.

  9. Alkaline ceramidase 3 deficiency aggravates colitis and colitis-associated tumorigenesis in mice by hyperactivating the innate immune system.

    Science.gov (United States)

    Wang, K; Xu, R; Snider, A J; Schrandt, J; Li, Y; Bialkowska, A B; Li, M; Zhou, J; Hannun, Y A; Obeid, L M; Yang, V W; Mao, C

    2016-03-03

    Increasing studies suggest that ceramides differing in acyl chain length and/or degree of unsaturation have distinct roles in mediating biological responses. However, still much remains unclear about regulation and role of distinct ceramide species in the immune response. Here, we demonstrate that alkaline ceramidase 3 (Acer3) mediates the immune response by regulating the levels of C18:1-ceramide in cells of the innate immune system and that Acer3 deficiency aggravates colitis in a murine model by augmenting the expression of pro-inflammatory cytokines in myeloid and colonic epithelial cells (CECs). According to the NCBI Gene Expression Omnibus (GEO) database, ACER3 is downregulated in immune cells in response to lipopolysaccharides (LPS), a potent inducer of the innate immune response. Consistent with these data, we demonstrated that LPS downregulated both Acer3 mRNA levels and its enzymatic activity while elevating C(18:1)-ceramide, a substrate of Acer3, in murine immune cells or CECs. Knocking out Acer3 enhanced the elevation of C(18:1)-ceramide and the expression of pro-inflammatory cytokines in immune cells and CECs in response to LPS challenge. Similar to Acer3 knockout, treatment with C(18:1)-ceramide, but not C18:0-ceramide, potentiated LPS-induced expression of pro-inflammatory cytokines in immune cells. In the mouse model of dextran sulfate sodium-induced colitis, Acer3 deficiency augmented colitis-associated elevation of colonic C(18:1)-ceramide and pro-inflammatory cytokines. Acer3 deficiency aggravated diarrhea, rectal bleeding, weight loss and mortality. Pathological analyses revealed that Acer3 deficiency augmented colonic shortening, immune cell infiltration, colonic epithelial damage and systemic inflammation. Acer3 deficiency also aggravated colonic dysplasia in a mouse model of colitis-associated colorectal cancer. Taken together, these results suggest that Acer3 has an important anti-inflammatory role by suppressing cellular or tissue C(18

  10. Characteristics of mitochondrial calpains.

    Science.gov (United States)

    Ozaki, Taku; Tomita, Hiroshi; Tamai, Makoto; Ishiguro, Sei-Ichi

    2007-09-01

    Calpains are considered to be cytoplasmic enzymes, although several studies have shown that calpain-like protease activities also exist in mitochondria. We partially purified mitochondrial calpain from swine liver mitochondria and characterized. Only one type of mitochondrial calpain was detected by the column chromatographies. The mitochondrial calpain was stained with anti-mu-calpain and calpain small subunit antibodies. The susceptibility of mitochondrial calpain to calpain inhibitors and the optimum pH differ from those of cytosolic mu- and m-calpains. The Ca(2+)-dependency of mitochondrial calpain was similar to that of cytosolic mu-calpain. Therefore, we named the protease mitochondrial mu-like calpain. In zymogram analysis, two types of caseinolytic enzymes existed in mitochondria and showed different mobilities from cytosolic mu- and m-calpains. The upper major band was stained with anti-mu-calpain and calpain small subunit antibodies (mitochondrial calpain I, mitochondrial mu-like calpain). The lower band was stained only with anti-calpain small subunit antibody (mitochondrial calpain II, unknown mitochondrial calpain). Calpastatin was not detected in mitochondrial compartments. The mitochondrial calpain processed apoptosis-inducing factor (AIF) to truncated AIF (tAIF), releasing tAIF into the intermembrane space. These results indicate that mitochondrial calpain, which differs from mu- and m-calpains, seems to be a ubiquitous calpain and may play a role in mitochondrial apoptotic signalling.

  11. A novel role for APOBEC3: Susceptibility to sexual transmission of murine acquired immunodeficiency virus (mAIDS is aggravated in APOBEC3 deficient mice

    Directory of Open Access Journals (Sweden)

    Jones Philip H

    2012-06-01

    Full Text Available Abstract Background APOBEC3 proteins are host factors that restrict infection by retroviruses like HIV, MMTV, and MLV and are variably expressed in hematopoietic and non-hematopoietic cells, such as macrophages, lymphocytes, dendritic, and epithelia cells. Previously, we showed that APOBEC3 expressed in mammary epithelia cells function to limit milk-borne transmission of the beta-retrovirus, mouse mammary tumor virus. In this present study, we used APOBEC3 knockout mice and their wild type counterpart to query the role of APOBEC3 in sexual transmission of LP-BM5 MLV – the etiological agent of murine AIDs (mAIDs. Results We show that mouse APOBEC3 is expressed in murine genital tract tissues and gametes and that genital tract tissue of APOBEC3-deficient mice are more susceptible to infection by LP-BM5 virus. APOBEC3 expressed in genital tract tissues most likely plays a role in decreasing virus transmission via the sexual route, since mice deficient in APOBEC3 gene have higher genitalia and seminal plasma virus load and sexually transmit the virus more efficiently to their partners compared to APOBEC3+ mice. Moreover, we show that female mice sexually infected with LP-BM5 virus transmit the virus to their off-spring in APOBEC3-dependent manner. Conclusion Our data indicate that genital tissue intrinsic APOBEC3 restricts genital tract infection and limits sexual transmission of LP-BM5 virus.

  12. Gastric tumor development in Smad3-deficient mice initiates from forestomach/glandular transition zone along the lesser curvature.

    Science.gov (United States)

    Nam, Ki Taek; O'Neal, Ryan; Lee, Yeo Song; Lee, Yong Chan; Coffey, Robert J; Goldenring, James R

    2012-06-01

    SMAD proteins are downstream effectors of the TGF-β signaling pathway. Smad3-null mice develop colorectal cancer by 6 months of age. In this study, we have examined whether the loss of Smad3 promotes gastric neoplasia in mice. The stomachs of Smad3⁻/⁻ mice were compared with age-matched Smad3 heterozygous and wild-type mice. E-cadherin, Ki-67, phosphoSTAT3, and TFF2/SP expression was analyzed by immunohistochemisty. The short hairpin RNA (ShRNA)-mediated knockdown of Smad3 in AGS and MKN28 cells was also performed. In addition, we examined alterations in DCLK1-expressing cells. Smad3⁻/⁻ mouse stomachs at 6 months of age revealed the presence of exophytic growths along the lesser curvature in the proximal fundus. Six-month-old Smad3⁻/⁻ mouse stomachs showed metaplastic columnar glands initiating from the transition zone junction between the forestomach and the glandular epithelium along the lesser curvature. Ten-month-old Smad3⁻/⁻ mice all exhibited invasive gastric neoplastic changes with increased Ki-67, phosphoSTAT3 expression, and aberrant cytosolic E-cadherin staining in papillary glands within the invading submucosal gland. The shRNA-mediated knockdown of Smad3 in AGS and MKN28 cells promoted the expression of phosphoSTAT3. DCLK1-expressing cells, which also stained for the tuft cell marker acetylated-α-tubulin, were observed in 10-month-old Smad3⁻/⁻ mice within tumors and in fundic invasive lesions. In conclusion, Smad3-null mice develop gastric tumors in the fundus, which arise from the junction between the forestomach and the glandular epithelium and progress to prominent invasive tumors over time. Smad3-null mice represent a novel model of fundic gastric tumor initiated from forestomach/glandular transition zone along the lesser curvature.

  13. TLR3 deficiency renders astrocytes permissive to herpes simplex virus infection and facilitates establishment of CNS infection in mice

    DEFF Research Database (Denmark)

    Reinert, Line; Harder, Louis Andreas; Holm, Christian;

    2012-01-01

    , it is not known what cell type mediates the role of TLR3 in the immunological control of HSV, and it is not known whether TLR3 sensing occurs prior to or after CNS entry. Here, we show that in mice TLR3 provides early control of HSV-2 infection immediately after entry into the CNS by mediating type I IFN...

  14. Role of the Na+/H+ exchanger 3 in angiotensin II-induced hypertension in NHE3-deficient mice with transgenic rescue of NHE3 in small intestines.

    Science.gov (United States)

    Li, Xiao C; Shull, Gary E; Miguel-Qin, Elisa; Chen, Fang; Zhuo, Jia L

    2015-11-01

    The role of Na(+/)H(+) exchanger 3 (NHE3) in the kidney in angiotensin II (ANG II)-induced hypertension remains unknown. The present study used global NHE3-deficient mice with transgenic rescue of the Nhe3 gene in small intestines (tgNhe3(-/-)) to test the hypothesis that genetic deletion of NHE3 selectively in the kidney attenuates ANG II-induced hypertension. Six groups of wild-type (tgNhe3(+/+)) and tgNhe3(-/-) mice were infused with either vehicle or ANG II (1.5 mg/kg/day, i.p., 2 weeks, or 10 nmol/min, i.v., 30 min), treated with or without losartan (20 mg/kg/day, p.o.) for 2 weeks. Basal systolic blood pressure (SBP) and mean intra-arterial blood pressure (MAP) were significantly lower in tgNhe3(-/-) mice (P excretion, urinary Na(+) excretion, urinary K(+) excretion, and urinary Cl(-) excretion were significantly lower in tgNhe3(-/-) mice (P < 0.01). These responses were associated with significantly elevated plasma ANG II and aldosterone levels, and marked upregulation in aquaporin 1, the Na(+)/HCO3 cotransporter, the α1 subunit isoform of Na(+)/K(+)-ATPase, protein kinase Cα, MAP kinases ERK1/2, and glycogen synthase kinase 3 α/β in the renal cortex of tgNhe3(-/-) mice (P < 0.01). ANG II infusion markedly increased SBP and MAP and renal cortical transporter and signaling proteins in tgNhe3(+/+), as expected, but all of these responses to ANG II were attenuated in tgNhe3(-/-) mice (P < 0.01). These results suggest that NHE3 in the kidney is necessary for maintaining normal blood pressure and fully developing ANG II-dependent hypertension.

  15. Calpain 8/nCL-2 and calpain 9/nCL-4 constitute an active protease complex, G-calpain, involved in gastric mucosal defense.

    Directory of Open Access Journals (Sweden)

    Shoji Hata

    2010-07-01

    Full Text Available Calpains constitute a superfamily of Ca2+-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8(-/- and Capn9(-/- mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8(-/- stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9(-/-. Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed "G-calpain," in which both were essential for activity. This is the first example of a "hybrid" calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S "knock-in" (Capn8(CS/CS mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8(-/- stomach, that of the Capn8(CS/CS mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric

  16. Regulation and physiological roles of the calpain system in muscular disorders

    OpenAIRE

    2012-01-01

    Calpains, a family of Ca2+-dependent cytosolic cysteine proteases, can modulate their substrates' structure and function through limited proteolytic activity. In the human genome, there are 15 calpain genes. The most-studied calpains, referred to as conventional calpains, are ubiquitous. While genetic studies in mice have improved our understanding about the conventional calpains' physiological functions, especially those essential for mammalian life as in embryogenesis, many reports have poi...

  17. Complement C3 deficiency leads to accelerated amyloid beta plaque deposition and neurodegeneration and modulation of the microglia/macrophage phenotype in amyloid precursor protein transgenic mice.

    Science.gov (United States)

    Maier, Marcel; Peng, Ying; Jiang, Liying; Seabrook, Timothy J; Carroll, Michael C; Lemere, Cynthia A

    2008-06-18

    Complement factor C3 is the central component of the complement system and a key inflammatory protein activated in Alzheimer's disease (AD). Previous studies demonstrated that inhibition of C3 by overexpression of soluble complement receptor-related protein y in an AD mouse model led to reduced microgliosis, increased amyloid beta (Abeta) plaque burden, and neurodegeneration. To further address the role of C3 in AD pathology, we generated a complement C3-deficient amyloid precursor protein (APP) transgenic AD mouse model (APP;C3(-/-)). Brains were analyzed at 8, 12, and 17 months of age by immunohistochemical and biochemical methods and compared with age-matched APP transgenic mice. At younger ages (8-12 months), no significant neuropathological differences were observed between the two transgenic lines. In contrast, at 17 months of age, APP;C3(-/-) mice showed significant changes of up to twofold increased total Abeta and fibrillar amyloid plaque burden in midfrontal cortex and hippocampus, which correlated with (1) significantly increased Tris-buffered saline (TBS)-insoluble Abeta(42) levels and reduced TBS-soluble Abeta(42) and Abeta(40) levels in brain homogenates, (2) a trend for increased Abeta levels in the plasma, (3) a significant loss of neuronal-specific nuclear protein-positive neurons in the hippocampus, and (4) differential activation of microglia toward a more alternative phenotype (e.g., significantly increased CD45-positive microglia, increased brain levels of interleukins 4 and 10, and reduced levels of CD68, F4/80, inducible nitric oxide synthase, and tumor necrosis factor). Our results suggest a beneficial role for complement C3 in plaque clearance and neuronal health as well as in modulation of the microglia phenotype.

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

    Science.gov (United States)

    Machado, Vanessa M; Morte, Maria I; Carreira, Bruno P; Azevedo, Maria M; Takano, Jiro; Iwata, Nobuhisa; Saido, Takaomi C; Asmussen, Hannelore; Horwitz, Alan R; Carvalho, Caetana M; Araújo, Inês M

    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 models. On the other hand, after stroke, neural stem cells (NSC) increase their proliferation and newly formed neuroblasts migrate towards the site of injury. However, the process of forming new neurons after injury is not efficient and finding ways to improve it may help with recovery after lesion. Understanding the role of calpains in the process of neurogenesis may therefore open a new window for the treatment of stroke. We investigated the involvement of calpains in NSC proliferation and neuroblast migration in two highly neurogenic regions in the mouse brain, the dentate gyrus (DG) and the subventricular zone (SVZ). We used mice that lack calpastatin, the endogenous calpain inhibitor, and calpains were also modulated directly, using calpeptin, a pharmacological calpain inhibitor. Calpastatin deletion impaired both NSC proliferation and neuroblast migration. Calpain inhibition increased NSC proliferation, migration speed and migration distance in cells from the SVZ. Overall, our work suggests that calpains are important for neurogenesis and encourages further research on their neurogenic role. Prospective therapies targeting calpain activity may improve the formation of new neurons following stroke, in addition to affording neuroprotection.

  19. Calpains, mitochondria, and apoptosis.

    Science.gov (United States)

    Smith, Matthew A; Schnellmann, Rick G

    2012-10-01

    Mitochondrial activity is critical for efficient function of the cardiovascular system. In response to cardiovascular injury, mitochondrial dysfunction occurs and can lead to apoptosis and necrosis. Calpains are a 15-member family of Ca(2+)-activated cysteine proteases localized to the cytosol and mitochondria, and several have been shown to regulate apoptosis and necrosis. For example, in endothelial cells, Ca(2+) overload causes mitochondrial calpain 1 cleavage of the Na(+)/Ca(2+) exchanger leading to mitochondrial Ca(2+) accumulation. Also, activated calpain 1 cleaves Bid, inducing cytochrome c release and apoptosis. In renal cells, calpains 1 and 2 promote apoptosis and necrosis by cleaving cytoskeletal proteins, which increases plasma membrane permeability and cleavage of caspases. Calpain 10 cleaves electron transport chain proteins, causing decreased mitochondrial respiration and excessive activation, or inhibition of calpain 10 activity induces mitochondrial dysfunction and apoptosis. In cardiomyocytes, calpain 1 activates caspase 3 and poly-ADP ribose polymerase during tumour necrosis factor-α-induced apoptosis, and calpain 1 cleaves apoptosis-inducing factor after Ca(2+) overload. Many of these observations have been elucidated with calpain inhibitors, but most calpain inhibitors are not specific for calpains or a specific calpain family member, creating more questions. The following review will discuss how calpains affect mitochondrial function and apoptosis within the cardiovascular system.

  20. [Physiological importance of calpains in gastric mucosal defense].

    Science.gov (United States)

    Hata, Shoji; Sorimachi, Hiroyuki

    2011-06-01

    The continuous and/or improper ingestion of irritants, including alcohol, NSAIDs, and Helicobacter pylori, often leads to serious gastropathies, affecting a wide range of people. A complex gastric defense system works to protect against these threats, for example by secreting mucus. Recently, by analysis of gene targeting mice for two gastrointestinal-tract-specific calpains, calpain-8 and calpain-9, we have demonstrated that they are cooperatively involved in the mucosal defense against stress-induced gastropathies. Calpains-8 and -9 are members of Ca2+ -dependent intracellular proteases comprising a superfamily in almost all eukaryotes, and form a functional complex, "G-calpain", expressed specifically in the mucus-producing cells. In this review, we show our recent results on calpains -8 and -9, and discuss gastric mucosal defense mechanisms involving them.

  1. Calpains in muscle wasting.

    Science.gov (United States)

    Bartoli, Marc; Richard, Isabelle

    2005-10-01

    Calpains are intracellular nonlysosomal Ca(2+)-regulated cysteine proteases. They mediate regulatory cleavages of specific substrates in a large number of processes during the differentiation, life and death of the cell. The purpose of this review is to synthesize our current understanding of the participation of calpains in muscle atrophy. Muscle tissue expresses mainly three different calpains: the ubiquitous calpains and calpain 3. The participation of the ubiquitous calpains in the initial degradation of myofibrillar proteins occurring in muscle atrophy as well as in the necrosis process accompanying muscular dystrophies has been well characterized. Inactivating mutations in the calpain 3 gene are responsible for limb-girdle muscular dystrophy type 2A and calpain 3 has been found to be downregulated in different atrophic situations, suggesting that it has to be absent for the atrophy to occur. The fact that similar regulations of calpain activities occur during exercise as well as in atrophy led us to propose that the calpains control cytoskeletal modifications needed for muscle plasticity.

  2. Complement C3-Deficiency Leads to Accelerated Aβ Plaque Deposition and Neurodegeneration, and Modulation of the Microglia/Macrophage Phenotype in APP Transgenic Mice

    OpenAIRE

    Maier, Marcel; Peng, Ying; Jiang, Liying; Seabrook, Timothy J.; Carroll, Michael C.; Lemere, Cynthia A.

    2008-01-01

    Complement factor C3 is the central component of the complement system and a key inflammatory protein activated in Alzheimer's disease (AD). Previous studies demonstrated that inhibition of C3 by overexpression of sCrry in an AD mouse model led to reduced microgliosis, increased Aβ plaque burden and neurodegeneration. To further address the role of C3 in AD pathology, we generated a complement C3-deficient APP transgenic AD mouse model (APP;C3−/−). Brains were analyzed at 8, 12 and 17 months ...

  3. Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease.

    Science.gov (United States)

    Trinchese, Fabrizio; Fa', Mauro; Liu, Shumin; Zhang, Hong; Hidalgo, Ariel; Schmidt, Stephen D; Yamaguchi, Hisako; Yoshii, Narihiko; Mathews, Paul M; Nixon, Ralph A; Arancio, Ottavio

    2008-08-01

    Calpains are calcium-dependent enzymes that determine the fate of proteins through regulated proteolytic activity. Calpains have been linked to the modulation of memory and are key to the pathogenesis of Alzheimer disease (AD). When abnormally activated, calpains can also initiate degradation of proteins essential for neuronal survival. Here we show that calpain inhibition through E64, a cysteine protease inhibitor, and the highly specific calpain inhibitor BDA-410 restored normal synaptic function both in hippocampal cultures and in hippocampal slices from the APP/PS1 mouse, an animal model of AD. Calpain inhibition also improved spatial-working memory and associative fear memory in APP/PS1 mice. These beneficial effects of the calpain inhibitors were associated with restoration of normal phosphorylation levels of the transcription factor CREB and involved redistribution of the synaptic protein synapsin I. Thus, calpain inhibition may prove useful in the alleviation of memory loss in AD.

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

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    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.

  5. [Calpains and cardiac diseases].

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    Perrin, C; Vergely, C; Rochette, L

    2004-09-01

    Calpains are a large family of cytosolic cysteine proteases composed of at least fourteen distinct isoforms. The family can be divided into two groups on the basis of distribution: ubiquitous and tissue-specific. Our current knowledge about calpains properties apply mainly to the ubiquitous isozymes, micro- and milli-calpain (classic calpains). These forms are activated after autolysis. Translocation and subsequent interactions with phospholipids of these enzymes increase their activity. Calpains are able to cleave a subset of substrates, as enzymes, structural and signalling proteins. Cardiac pathologies, such as heart failure, atrial fibrillation or clinical states particularly ischemia reperfusion, are associated with an increase of cytosolic calcium and in this regards, calpain activation has been evoked as one of the mediators leading to myocardial damage. Calpain activities have been shown to be increased in hearts experimentally subjected to ischemia reperfusion or during hypertrophy, but also in atrial tissue harvested from patients suffering from atrial fibrillations. These activities have been related to an increase of the proteolysis of different myocardial components, particularly, troponins, which are major regulators of the contraction of cardiomyocytes. Moreover, recent works have demonstrated that calpains are involved in the development of myocardial cell death by necrosis or apoptosis.

  6. Calpains promote neutrophil recruitment and bacterial clearance in an acute bacterial peritonitis model.

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    Kumar, Vijay; Everingham, Stephanie; Hall, Christine; Greer, Peter A; Craig, Andrew W B

    2014-03-01

    Activation of the innate immune system is critical for clearance of bacterial pathogens to limit systemic infections and host tissue damage. Here, we report a key role for calpain proteases in bacterial clearance in mice with acute peritonitis. Using transgenic mice expressing Cre recombinase primarily in innate immune cells (fes-Cre), we generated conditional capns1 knockout mice. Consistent with capns1 being essential for stability and function of the ubiquitous calpains (calpain-1, calpain-2), peritoneal cells from these mice had reduced levels of calpain-2/capns1, and reduced proteolysis of their substrate selenoprotein K. Using an acute bacterial peritonitis model, we observed impaired bacterial killing within the peritoneum and development of bacteremia in calpain knockout mice. These defects correlated with significant reductions in IL-1α release, neutrophil recruitment, and generation of reactive oxygen species in calpain knockout mice with acute bacterial peritonitis. Peritoneal macrophages from calpain knockout mice infected with enterobacteria ex vivo, were competent in phagocytosis of bacteria, but showed impaired clearance of intracellular bacteria compared with control macrophages. Together, these results implicate calpains as key mediators of effective innate immune responses to acute bacterial infections, to prevent systemic dissemination of bacteria that can lead to sepsis.

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

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    Vanessa Mendes Machado

    2015-02-01

    Full Text Available 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 models. On the other hand, after stroke, neural stem cells increase their proliferation and newly formed neuroblasts migrate towards the site of injury. However, the process of forming new neurons after injury is not efficient and finding ways to improve it may help with recovery after lesion. Understanding the role of calpains in the process of neurogenesis may therefore open a new window for the treatment of stroke. We investigated the involvement of calpains in neural stem cell proliferation and neuroblast migration in two highly neurogenic regions in the mouse brain, the dentate gyrus and the subventricular zone. We used mice that lack calpastatin, the endogenous calpain inhibitor, and calpains were also modulated directly, using calpeptin, a pharmacological calpain inhibitor. Calpastatin deletion impaired both neural stem cell proliferation and neuroblast migration. Calpain inhibition increased neural stem cell proliferation, migration speed and migration distance in cells from the subventricular zone. Overall, our work suggests that calpains are important for neurogenesis and warrant further research on how they influence the formation of new neurons. Prospective therapies targeting calpain activity not only may afford neuroprotection following stroke, but also benefit the formation and survival of new neurons.

  8. Calpain/SHP-1 interaction by honokiol dampening peritoneal dissemination of gastric cancer in nu/nu mice.

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    Shing Hwa Liu

    Full Text Available BACKGROUND: Honokiol, a small-molecular weight natural product, has previously been reported to activate apoptosis and inhibit gastric tumorigenesis. Whether honokiol inhibits the angiogenesis and metastasis of gastric cancer cells remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: We tested the effects of honokiol on angiogenic activity and peritoneal dissemination using in vivo, ex vivo and in vitro assay systems. The signaling responses in human gastric cancer cells, human umbilical vascular endothelial cells (HUVECs, and isolated tumors were detected and analyzed. In a xenograft gastric tumor mouse model, honokiol significantly inhibited the peritoneal dissemination detected by PET/CT technique. Honokiol also effectively attenuated the angiogenesis detected by chick chorioallantoic membrane assay, mouse matrigel plug assay, rat aortic ring endothelial cell sprouting assay, and endothelial cell tube formation assay. Furthermore, honokiol effectively enhanced signal transducer and activator of transcription (STAT-3 dephosphorylation and inhibited STAT-3 DNA binding activity in human gastric cancer cells and HUVECs, which was correlated with the up-regulation of the activity and protein expression of Src homology 2 (SH2-containing tyrosine phosphatase-1 (SHP-1. Calpain-II inhibitor and siRNA transfection significantly reversed the honokiol-induced SHP-1 activity. The decreased STAT-3 phosphorylation and increased SHP-1 expression were also shown in isolated peritoneal metastatic tumors. Honokiol was also capable of inhibiting VEGF generation, which could be reversed by SHP-1 siRNA transfection. CONCLUSIONS/SIGNIFICANCE: Honokiol increases expression and activity of SPH-1 that further deactivates STAT3 pathway. These findings also suggest that honokiol is a novel and potent inhibitor of angiogenesis and peritoneal dissemination of gastric cancer cells, providing support for the application potential of honokiol in gastric cancer therapy.

  9. Grainyhead-like 3 (Grhl3) deficiency in brain leads to altered locomotor activity and decreased anxiety-like behaviours in aged mice.

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    Dworkin, Sebastian; Auden, Alana; Partridge, Darren D; Daglas, Maria; Medcalf, Robert L; Mantamadiotis, Theo; Georgy, Smitha R; Darido, Charbel; Jane, Stephen M; Ting, Stephen B

    2016-12-01

    The highly conserved Grainyhead-like (Grhl) family of transcription factors, comprising three members in vertebrates (Grhl1-3), play critical regulatory roles during embryonic development, cellular proliferation and apoptosis. Although loss of Grhl function leads to multiple neural abnormalities in numerous animal models, a comprehensive analysis of Grhl expression and function in the mammalian brain has not been reported. Here we show that only Grhl3 expression is detectable in the embryonic mouse brain; particularly within the habenula, an organ known to modulate repressive behaviours. Using both Grhl3-knockout mice (Grhl3(-/-) ), and brain-specific conditional deletion of Grhl3 in adult mice (Nestin-Cre/Grhl3(flox/flox) ), we performed histological expression analyses and behavioural tests to assess long-term effects of Grhl3 loss on motor co-ordination, spatial memory, anxiety and stress. We found that complete deletion of Grhl3 did not lead to noticeable structural or cell-intrinsic defects in the embryonic brain, however aged Grhl3 conditional knockout (cKO) mice showed enlarged lateral ventricles and displayed marked changes in motor function and behaviours suggestive of decreased fear and anxiety. We conclude that loss of Grhl3 in the brain leads to significant alterations in locomotor activity and decreased self-inhibition, and as such, these mice may serve as a novel model of human conditions of impulsive behaviour or hyperactivity. This article is protected by copyright. All rights reserved.

  10. Activation of calpains, calpastatin and spectrin cleavage in the brain during the pathology of fatal murine cerebral malaria.

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    Shukla, Meena; Rajgopal, Yadavalli; Babu, Phanithi Prakash

    2006-01-01

    Neuronal calpains appear to be activated uncontrollably by sustained elevation of cytosolic calcium levels under pathological conditions as well as neurodegenerative diseases. In the present study, we have characterized calpain activation in cytosolic extract of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). Pathology of FMCM resulted in the increase in activity of calpains in both cerebral cortex and cerebellum. Western blot analysis revealed an increase in the levels of mu-calpain (calpain-1) in the cytosolic fraction of infected cerebral cortex and cerebellum although a decrease in the level of m-calpain was observed in the cytosolic fraction of infected cerebellum and cerebral cortex. Calpain activation was further confirmed by monitoring the formation of calpain-specific spectrin breakdown products (SBDP). Protease-specific SBDP revealed the formation of calpain-generated 150kDa product in the infected cerebral cortex and cerebellum. The specific signature fragment of calpain activation and spectrin breakdown after Plasmodium berghei ANKA infection provide a strong evidence of the role of calpains during the cell death in cerebral cortex and cerebellum. Given the role of calpains in neurodegeneration and cell death, our results strongly suggest that calpains are important mediators of cell injury and neurological sequelae associated with FMCM.

  11. Calpains: markers of tumor aggressiveness?

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    Roumes, Hélène; Leloup, Ludovic; Dargelos, Elise; Brustis, Jean-Jacques; Daury, Laetitia; Cottin, Patrick

    2010-05-15

    Rhabdomyosarcoma (RMS) are soft-tissue sarcoma commonly encountered in childhood. RMS cells can acquire invasive behavior and form metastases. The metastatic dissemination implicates many proteases among which are mu-calpain and m-calpain. Study of calpain expression and activity underline the deregulation of calpain activity in RMS. Analysis of kinetic characteristics of RMS cells, compared to human myoblasts LHCN-M2 cells, shows an important migration velocity in RMS cells. One of the major results of this study is the positive linear correlation between calpain activity and migration velocity presenting calpains as a marker of tumor aggressiveness. The RMS cytoskeleton is disorganized. Specifying the role of mu- and m-calpain using antisense oligonucleotides led to show that both calpains up-regulate alpha- and beta-actin in ARMS cells. Moreover, the invasive behavior of these cells is higher than that of LHCN-M2 cells. However, it is similar to that of non-treated LHCN-M2 cells, when calpains are inhibited. In summary, calpains may be involved in the anarchic adhesion, migration and invasion of RMS. The direct relationship between calpain activity and migration velocities or invasive behavior indicates that calpains could be considered as markers of tumor aggressiveness and as potential targets for limiting development of RMS tumor as well as their metastatic behavior.

  12. Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice.

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    Kook, Seunghyi; Wang, Ping; Young, Lisa R; Schwake, Michael; Saftig, Paul; Weng, Xialian; Meng, Ying; Neculai, Dante; Marks, Michael S; Gonzales, Linda; Beers, Michael F; Guttentag, Susan

    2016-04-15

    The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients.

  13. Calpain activity is essential in skin wound healing and contributes to scar formation.

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    Dany Nassar

    Full Text Available Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST, a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT. CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFβ-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on

  14. Calpains: an elaborate proteolytic system.

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    Ono, Yasuko; Sorimachi, Hiroyuki

    2012-01-01

    Calpain is an intracellular Ca(2+)-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02). Recent expansion of sequence data across the species definitively shows that calpain has been present throughout evolution; calpains are found in almost all eukaryotes and some bacteria, but not in archaebacteria. Fifteen genes within the human genome encode a calpain-like protease domain. Interestingly, some human calpains, particularly those with non-classical domain structures, are very similar to calpain homologs identified in evolutionarily distant organisms. Three-dimensional structural analyses have helped to identify calpain's unique mechanism of activation; the calpain protease domain comprises two core domains that fuse to form a functional protease only when bound to Ca(2+)via well-conserved amino acids. This finding highlights the mechanistic characteristics shared by the numerous calpain homologs, despite the fact that they have divergent domain structures. In other words, calpains function through the same mechanism but are regulated independently. This article reviews the recent progress in calpain research, focusing on those studies that have helped to elucidate its mechanism of action. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

  15. Role of Calpain in Apoptosis

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    Hamid Reza Momeni

    2011-01-01

    Full Text Available Apoptosis, a form of programmed cell death that occurs under physiologicalas well as pathological conditions, is characterized by morphological and biochemicalfeatures. While the importance of caspases in apoptosis is established,several noncaspase proteases (Ca2+-dependent proteases such as calpain mayplay a role in the execution of apoptosis. The calpain family consists of twomajor isoforms, calpain I and calpain II which require μM and mM Ca2+ concentrationsto initiate their activity. An increase in intracellular Ca2+ level isthought to trigger a cascade of biochemical processes including calpain activation.Once activated, calpains degrade membrane, cytoplasmic and nuclear substrates,leading to the breakdown of cellular architecture and finally apoptosis.The activation of calpain has been implicated in neuronal apoptosis followingspinal cord injuries and neurodegenerative diseases. This review focuses oncalpain with an emphasis on its key role in the proteolysis of cellular proteinsubstrates following apoptosis.

  16. The inhibition of calpains ameliorates vascular restenosis through MMP2/TGF-β1 pathway.

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    Tang, Lianghu; Pei, Haifeng; Yang, Yi; Wang, Xiong; Wang, Ting; Gao, Erhe; Li, De; Yang, Yongjian; Yang, Dachun

    2016-07-25

    Restenosis limits the efficacy of vascular percutaneous intervention, in which vascular smooth muscle cell (VSMC) proliferation and activation of inflammation are two primary causal factors. Calpains influence VSMC proliferation and collagen synthesis. However, the roles of calpastatin and calpains in vascular restenosis remain unclear. Here, restenosis was induced by ligating the left carotid artery, and VSMCs were pretreated with platelet-derived growth factor (PDGF)-BB. Adenovirus vector carrying MMP2 sequence and specific small interfering RNA against calpain-1/2 were introduced. Finally, restenosis enhanced the expression of calpain-1/2, but reduced calpastatin content. In calpastatin transgenic mice, lumen narrowing was attenuated gradually and peaked on days 14-21. Cell proliferation and migration as well as collagen synthesis were inhibited in transgenic mice, and expression of calpain-1/2 and MMP2/transforming growth factor-β1 (TGF-β1). Consistently, in VSMCs pretreated with PDGF-BB, calpastatin induction and calpains inhibition suppressed the proliferation and migration of VSMCs and collagen synthesis, and reduced expression of calpain-1/2 and MMP2/TGF-β1. Moreover, simvastatin improved restenosis indicators by suppressing the HIF-1α/calpains/MMP2/TGF-β1 pathway. However, MMP2 supplementation eliminated the vascular protection of calpastatin induction and simvastatin. Collectively, calpains inhibition plays crucial roles in vascular restenosis by preventing neointimal hyperplasia at the early stage via suppression of the MMP2/TGF-β1 pathway.

  17. The expression of calpain Ⅱ in the retina of rds mice%细胞因子Ⅱ在遗传性视网膜色素变性的转基因小鼠视网膜的表达研究

    Institute of Scientific and Technical Information of China (English)

    陈颖; 张清炯; 谭智群

    2001-01-01

    ObjectiveTo study some possible apoptosis mechanisms of the process of retinal degeneration in rds mice.MethodsThe retina of rds and C3B mice were collected at different time periods to form the experimental and control groups respectively.By using immunohistochemical technique,the expressions of calpain in the retina of rds and C3B mice at different time periods were analyzed.ResultsThe expression of calpain could be seen in the ganglionic layer of rds mice in the early stage(1 week),on the inner nuclear at about 2 weeks, outer nuclear layer at 4~5 weeks.The intense and scope increase with the growth of retina.After 4 weeks,the expression of calpain Ⅱ showed all of the rest of retina.ConclusionHigh calcium concentration activated the expression of Calpain Ⅱ.Therefore,high calcium concentration may be a factor in increasing the apoptosis of the retinal cells in the rds mice.The apoptosis may start from the ganglion cell layer in the rds mice.%目的 探讨细胞因子Ⅱ(calpain Ⅱ)在视网膜色素变性过程中表达的变化,以此窥视遗传性视网膜色素变性的转基因小鼠(rds小鼠)发病的某些可能因素?。方法 以C3B小鼠为对照,选择不同生长时期的rds小鼠的视网膜组织,用免疫组化。方法 检测Calpain Ⅱ在不同生长阶段的小鼠模型视网膜中的是否表达及表达情况?。结果 1周即可见Calpain Ⅱ在rds小鼠视网膜神经节细胞层的表达,2周左右内核层也见表达,4周后外核层也开始出现由强至弱的calpain Ⅱ强阳性表达,最终至全部视网膜剩余组织中均可见calpain Ⅱ的表达?。结论  只有高钙才能激活calpain Ⅱ的表达,研究中calpain Ⅱ参与了rds小鼠视网膜细胞的变性死亡?因此,高钙是rds小鼠视网膜变性的一个可能因素

  18. 1,25-Vitamin D3 Deficiency Induces Albuminuria.

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    Sonneveld, Ramon; Hoenderop, Joost G J; Stavenuiter, Andrea W D; Ferrantelli, Evelina; Baltissen, Marijke P A; Dijkman, Henry B; Florquin, Sandrine; Rops, Angelique L; Wetzels, Jack F M; Berden, Jo H M; van der Vlag, Johan; Nijenhuis, Tom

    2016-04-01

    Vitamin D plays an important role in renal (patho)physiology. Patients with glomerular diseases have an injured renal filtration barrier, leading to proteinuria and reduced renal function. An impaired renal function also leads to 1,25-vitamin D3 deficiency as a result of reduced renal 1α-hydroxylase activity. Vitamin D treatment to reduce proteinuria remains controversial, although there is an inverse correlation between vitamin D levels and proteinuria. Herein, we showed that 1,25-vitamin D3-deficient 25-hydroxy-vitamin-D3-1α-hydroxylase knockout mice and 1,25-vitamin D3-deficient rats develop podocyte injury and renal dysfunction. Glomerular injury was characterized by proteinuria and partial podocyte foot process effacement. Expression of nephrin, podocin, desmin, and transient receptor potential channel C6 in the podocyte was significantly altered in 1,25-vitamin D3-deficient animals. Supplementation with 1,25-vitamin D3 or 1,25-vitamin D2 prevented podocyte effacement or reversed glomerular and tubulointerstitial damage in 1,25-vitamin D3-deficient animals, thereby preserving and restoring renal function, respectively. The effect of 1,25-vitamin D3 deficiency and 1,25-vitamin D3 and 1,25-vitamin D2 repletion on proteinuria could not be explained by hypocalcemia, changes in parathyroid hormone, or fibroblast growth factor 23. This study demonstrates that 1,25-vitamin D3 deficiency directly leads to renal injury in rodents. Translated to human subjects, this would underline the need for early vitamin D supplementation in patients with glomerular disease and chronic renal insufficiency, which might inhibit or potentially reverse renal injury.

  19. Calpains, skeletal muscle function and exercise.

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    Murphy, Robyn M

    2010-03-01

    1. Skeletal muscle fibres contain ubiquitous (mu-calpain and m-calpain) and muscle-specific (calpain-3) Ca(2+)-dependent proteases. The physiological roles of the calpains are not well understood, although ubiquitous calpains have been associated with apoptosis and myogenesis and calpain-3 is likely involved in sarcomeric remodelling. A defect in the expression of calpain-3 results in limb-girdle muscular dystrophy Type 2A. 2. At resting [Ca(2+)](i), calpains are present predominantly in their full-length, unautolysed/unactivated forms. Once activated, mu-calpain and calpain-3 appear in their autolysed forms and this measurement can be used to determine when in vivo activation occurs. Endogenously expressed mu-calpain and calpain-3 are activated within a physiological [Ca(2+)] range in a Ca(2+)- and time-dependent manner. 3. In skeletal muscle, mu-calpain is a freely diffusible protein that binds rapidly when [Ca(2+)](i) is increased. Calpain-3 is tightly bound in skeletal muscle fibres at the N2A line of the large elastic protein titin. 4. Overall, neither mu-calpain nor calpain-3 are activated immediately following sprint, endurance or eccentric exercise, despite the frequent episodes of high cytoplasmic [Ca(2+)] that would occur during these types of muscle contractions. Importantly, however, a substantial proportion of calpain-3, but not mu-calpain, is activated 24 h after a single bout of eccentric exercise. 5. In vitro studies have shown that calpain-3 becomes activated if exposed for a prolonged period of time (> 1 h) to resting cytoplasmic [Ca(2+)] that are approximately two- to fourfold higher than normal. This suggests that the small but sustained increase in [Ca(2+)](i) that likely occurs after eccentric contractions is both high and long enough to result in calpain-3 activation and supports the role for calpain-3 in sarcomeric remodelling.

  20. Calpains mediate axonal cytoskeleton disintegration during Wallerian degeneration.

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    Ma, Marek; Ferguson, Toby A; Schoch, Kathleen M; Li, Jian; Qian, Yaping; Shofer, Frances S; Saatman, Kathryn E; Neumar, Robert W

    2013-08-01

    In both the central nervous system (CNS) and peripheral nervous system (PNS), transected axons undergo Wallerian degeneration. Even though Augustus Waller first described this process after transection of axons in 1850, the molecular mechanisms may be shared, at least in part, by many human diseases. Early pathology includes failure of synaptic transmission, target denervation, and granular disintegration of the axonal cytoskeleton (GDC). The Ca(2+)-dependent protease calpains have been implicated in GDC but causality has not been established. To test the hypothesis that calpains play a causal role in axonal and synaptic degeneration in vivo, we studied transgenic mice that express human calpastatin (hCAST), the endogenous calpain inhibitor, in optic and sciatic nerve axons. Five days after optic nerve transection and 48 h after sciatic nerve transection, robust neurofilament proteolysis observed in wild-type controls was reduced in hCAST transgenic mice. Protection of the axonal cytoskeleton in sciatic nerves of hCAST mice was nearly complete 48 h post-transection. In addition, hCAST expression preserved the morphological integrity of neuromuscular junctions. However, compound muscle action potential amplitudes after nerve transection were similar in wild-type and hCAST mice. These results, in total, provide direct evidence that calpains are responsible for the morphological degeneration of the axon and synapse during Wallerian degeneration.

  1. Calpain-1 deletion impairs mGluR-dependent LTD and fear memory extinction

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    Zhu, Guoqi; Briz, Victor; Seinfeld, Jeff; Liu, Yan; Bi, Xiaoning; Baudry, Michel

    2017-01-01

    Recent studies indicate that calpain-1 is required for the induction of long-term potentiation (LTP) elicited by theta-burst stimulation in field CA1 of hippocampus. Here we determined the contribution of calpain-1 in another type of synaptic plasticity, the long-term depression (LTD) elicited by activation of type-I metabotropic glutamate receptors (mGluR-LTD). mGluR-LTD was associated with calpain-1 activation following T-type calcium channel opening, and resulted in the truncation of a regulatory subunit of PP2A, B56α. This signaling pathway was required for both the early and late phase of Arc translation during mGluR-LTD, through a mechanism involving mTOR and ribosomal protein S6 activation. In contrast, in hippocampal slices from calpain-1 knock-out (KO) mice, application of the mGluR agonist, DHPG, did not result in B56α truncation, increased Arc synthesis and reduced levels of membrane GluA1-containing AMPA receptors. Consistently, mGluR-LTD was impaired in calpain-1 KO mice, and the impairment could be rescued by phosphatase inhibitors, which also restored Arc translation in response to DHPG. Furthermore, calpain-1 KO mice exhibited impairment in fear memory extinction to tone presentation. These results indicate that calpain-1 plays a critical role in mGluR-LTD and is involved in many forms of synaptic plasticity and learning and memory. PMID:28202907

  2. Calpains Released by T Lymphocytes Cleave TLR2 To Control IL-17 Expression.

    Science.gov (United States)

    Perez, Joëlle; Dansou, Boris; Hervé, Roxane; Levi, Charlène; Tamouza, Houda; Vandermeersch, Sophie; Demey-Thomas, Emmanuelle; Haymann, Jean-Philippe; Zafrani, Lara; Klatzmann, David; Boissier, Marie-Christophe; Letavernier, Emmanuel; Baud, Laurent

    2016-01-01

    Calpains are intracellular proteases that play a key role in inflammation/immunity. Rare studies show that they are partially externalized. However, the mechanism of this secretion and the functions of exteriorized calpains remain poorly understood. In this study, we found that mouse and human lymphocytes secreted calpains through an ABCA1-driven process. In turn, extracellular calpains inhibited IL-17A expression. We were able to attribute this function to a cleavage of the TLR2 extracellular domain, which prevented TLR2-induced transcription of molecules essential for IL-17A induction. Calpain exteriorization and TLR2 cleavage were critical for the control of IL-17A expression by low doses of IL-2. By using newly developed transgenic mice in which extracellular calpains are specifically inactivated, we provide evidence for the relevance of calpain externalization in vivo in regulating IL-17A expression and function in experimental sterile peritonitis and autoimmune arthritis, respectively. Thus, this study identifies calpain exteriorization as a potential target for immune modulation.

  3. Calpains and Coronary Vascular Disease.

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    Potz, Brittany A; Sabe, Ashraf A; Abid, M Ruhul; Sellke, Frank W

    2016-01-01

    Despite many advances in percutaneous and surgical interventions in the treatment of coronary artery disease (CAD), up to one-third of patients are still either not candidates or receive suboptimal revascularization. Calpains are a class of calcium-activated non-lysosomal cysteine proteases that serve as a proteolytic unit for cellular homeostasis. Uncontrolled activation of calpain has been found to be involved in the pathogenesis of myocardial reperfusion injury, cardiac hypertrophy, myocardial stunning and cardiac ischemia. Inhibition of calpains has been shown to significantly attenuate myocardial stunning and reduced infarct size after ischemia-reperfusion. Calpain inhibition therefore serves as a potential medical therapy for patients suffering from a number of diseases, including CAD.

  4. 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.

  5. Disruption of calpain reduces lipotoxicity-induced cardiac injury by preventing endoplasmic reticulum stress

    Science.gov (United States)

    Li, Shengcun; Zhang, Lulu; Ni, Rui; Cao, Ting; Zheng, Dong; Xiong, Sidong; Greer, Peter A.; Fan, Guo-Chang; Peng, Tianqing

    2016-01-01

    Diabetes and obesity are prevalent in westernized countries. In both conditions, excessive fatty acid uptake by cardiomyocytes induces cardiac lipotoxicity, an important mechanism contributing to diabetic cardiomyopathy. This study investigated the effect of calpain disruption on cardiac lipotoxicity. Cardiac-specific capns1 knockout mice and their wild-type littermates (male, age of 4 weeks) were fed a high fat diet (HFD) or normal diet for 20 weeks. HFD increased body weight, altered blood lipid profiles and impaired glucose tolerance comparably in both capns1 knockout mice and their wild-type littermates. Calpain activity, cardiomyocyte cross-sectional areas, collagen deposition and triglyceride were significantly increased in HFD-fed mouse hearts, and these were accompanied by myocardial dysfunction and up-regulation of hypertrophic and fibrotic collagen genes as well as pro-inflammatory cytokines. These effects of HFD were attenuated by disruption of calpain in capns1 knockout mice. Mechanistically, deletion of capns1 in HFD-fed mouse hearts and disruption of calpain with calpain inhibitor-III, silencing of capn1, or deletion of capns1 in palmitate-stimulated cardiomyocytes prevented endoplasmic reticulum stress, apoptosis, cleavage of caspase-12 and junctophilin-2, and pro-inflammatory cytokine expression. Pharmacological inhibition of endoplasmic reticulum stress diminished palmitate-induced apoptosis and pro-inflammatory cytokine expression in cardiomyocytes. In summary, disruption of calpain prevents lipotoxicity-induced apoptosis in cardiomyocytes and cardiac injury in mice fed a HFD. The role of calpain is mediated, at least partially, through endoplasmic reticulum stress. Thus, calpain/endoplasmic reticulum stress may represent a new mechanism and potential therapeutic targets for cardiac lipotoxicity. PMID:27523632

  6. The calpains: modular designs and functional diversity.

    Science.gov (United States)

    Croall, Dorothy E; Ersfeld, Klaus

    2007-01-01

    The calpain family is named for the calcium dependence of the papain-like, thiol protease activity of the well-studied ubiquitous vertebrate enzymes calpain-1 (mu-calpain) and calpain-2 (m-calpain). Proteins showing sequence relatedness to the catalytic core domains of these enzymes are included in this ancient and diverse eukaryotic protein family. Calpains are examples of highly modular organization, with several varieties of amino-terminal or carboxy-terminal modules flanking a conserved core. Acquisition of the penta-EF-hand module involved in calcium binding (and the formation of heterodimers for some calpains) seems to be a relatively late event in calpain evolution. Several alternative mechanisms for binding calcium and associating with membranes/phospholipids are found throughout the family. The gene family is expanded in mammals, trypanosomes and ciliates, with up to 26 members in Tetrahymena, for example; in striking contrast to this, only a single calpain gene is present in many other protozoa and in plants. The many isoforms of calpain and their multiple splice variants complicate the discussion and analysis of the family, and challenge researchers to ascertain the relationships between calpain gene sequences, protein isoforms and their distinct or overlapping functions. In mammals and plants it is clear that a calpain plays an essential role in development. There is increasing evidence that ubiquitous calpains participate in a variety of signal transduction pathways and function in important cellular processes of life and death. In contrast to relatively promiscuous degradative proteases, calpains cleave only a restricted set of protein substrates and use complex substrate-recognition mechanisms, involving primary and secondary structural features of target proteins. The detailed physiological significance of both proteolytically active calpains and those lacking key catalytic residues requires further study.

  7. Inhibition of calpain attenuates encephalitogenicity of MBP-specific T cells

    Science.gov (United States)

    Guyton, Mary K.; Das, Arabinda; Inoue, Jun; Azuma, Mitsuyoshi; Ray, Swapan K.; Brahmachari, Saurav; Banik, Naren L.

    2009-01-01

    Multiple sclerosis (MS) is a T cell-mediated autoimmune disease of the CNS, possessing both immune and neurodegenerative events that lead to disability. Adoptive transfer (AT) of myelin basic protein (MBP)-specific T cells into naïve female SJL/J mice results in a relapsing-remitting (RR) form of experimental autoimmune encephalomyelitis (EAE). Blocking the mechanisms by which MBP-specific T cells are activated before AT may help characterize the immune arm of MS and offer novel targets for therapy. One such target is calpain, which is involved in activation of T cells, migration of immune cells into the CNS, degradation of axonal and myelin proteins, and neuronal apoptosis. Thus, the hypothesis that inhibiting calpain in MBP-specific T cells would diminish their encephalitogenicity in RR-EAE mice was tested. Incubating MBP-specific T cells with the calpain inhibitor SJA6017 before AT markedly suppressed the ability of these T cells to induce clinical symptoms of RR-EAE. These reductions correlated with decreases in demyelination, inflammation, axonal damage, and loss of oligodendrocytes and neurons. Also, calpain:calpastatin ratio, production of tBid, and Bax:Bcl-2 ratio, and activities of calpain and caspases, and internucleosomal DNA fragmentation were attenuated. Thus, these data suggest calpain as a promising target for treating EAE and MS. PMID:19627443

  8. Understanding the substrate specificity of conventional calpains.

    Science.gov (United States)

    Sorimachi, Hiroyuki; Mamitsuka, Hiroshi; Ono, Yasuko

    2012-09-01

    Calpains are intracellular Ca(2+)-dependent Cys proteases that play important roles in a wide range of biological phenomena via the limited proteolysis of their substrates. Genetic defects in calpain genes cause lethality and/or functional deficits in many organisms, including humans. Despite their biological importance, the mechanisms underlying the action of calpains, particularly of their substrate specificities, remain largely unknown. Studies show that certain sequence preferences influence calpain substrate recognition, and some properties of amino acids have been related successfully to substrate specificity and to the calpains' 3D structure. The full spectrum of this substrate specificity, however, has not been clarified using standard sequence analysis algorithms, e.g., the position-specific scoring-matrix method. More advanced bioinformatics techniques were used recently to identify the substrate specificities of calpains and to develop a predictor for calpain cleavage sites, demonstrating the potential of combining empirical data acquisition and machine learning. This review discusses the calpains' substrate specificities, introducing the benefits of bioinformatics applications. In conclusion, machine learning has led to the development of useful predictors for calpain cleavage sites, although the accuracy of the predictions still needs improvement. Machine learning has also elucidated information about the properties of calpains' substrate specificities, including a preference for sequences over secondary structures and the existence of a substrate specificity difference between two similar conventional calpains, which has never been indicated biochemically.

  9. 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

    2015-11-06

    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.

  10. 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

    . 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...... caspase and calpain activity was reduced markedly in EPO-treated mice....

  11. 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.

  12. Over-expression of calpastatin inhibits calpain activation and attenuates post-infarction myocardial remodeling.

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    Tingqiao Ye

    Full Text Available Calpain is activated following myocardial infarction and ablation of calpastatin (CAST, an endogenous inhibitor of calpains, promotes left ventricular remodeling after myocardial infarction (MI. The present study aimed to investigate the effect of transgenic over-expression of CAST on the post-infarction myocardial remodeling process.We established transgenic mice (TG ubiquitously over-expressing human CAST protein and produced MI in TG mice and C57BL/6J wild-type (WT littermates.The CAST protein expression was profoundly upregulated in the myocardial tissue of TG mice compared with WT littermates (P < 0.01. Overexpression of CAST significantly reduced the infarct size (P < 0.01 and blunted MI-induced interventricular hypertrophy, global myocardial fibrosis and collagen I and collagen III deposition, hypotension and hemodynamic disturbances at 21 days after MI. Moreover, the MI-induced up-regulation and activation of calpains were obviously attenuated in CAST TG mice. MI-induced down-regulation of CAST was partially reversed in TG mice. Additionally, the MI-caused imbalance of matrix metalloproteinases and their inhibitors was improved in TG mice.Transgenic over-expression of CAST inhibits calpain activation and attenuates post-infarction myocardial remodeling.

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

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    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. Neuroprotective strategies against calpain-mediated neurodegeneration

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    Yildiz-Unal A

    2015-02-01

    Full Text Available Aysegul Yildiz-Unal,1 Sirin Korulu,2 Arzu Karabay3 1Department of Molecular Biology and Genetics, Faculty of Science, Mugla Sitki Koçman University, Kötekli, Mugla, Turkey; 2Department of Molecular Biology and Genetics, Istanbul Arel University, Istanbul Turkey; 3Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Maslak, Istanbul, Turkey Abstract: Calpains are calcium-dependent proteolytic enzymes that have deleterious effects on neurons upon their pathological over-activation. According to the results of numerous studies to date, there is no doubt that abnormal calpain activation triggers activation and progression of apoptotic processes in neurodegeneration, leading to neuronal death. Thus, it is very crucial to unravel all the aspects of calpain-mediated neurodegeneration in order to protect neurons through eliminating or at least minimizing its lethal effects. Protecting neurons against calpain-activated apoptosis basically requires developing effective, reliable, and most importantly, therapeutically applicable approaches to succeed. From this aspect, the most significant studies focusing on preventing calpain-mediated neurodegeneration include blocking the N-methyl-d-aspartate (NMDA-type glutamate receptor activities, which are closely related to calpain activation; directly inhibiting calpain itself via intrinsic or synthetic calpain inhibitors, or inhibiting its downstream processes; and utilizing the neuroprotectant steroid hormone estrogen and its receptors. In this review, the most remarkable neuroprotective strategies for calpain-mediated neurodegeneration are categorized and summarized with respect to their advantages and disadvantages over one another, in terms of their efficiency and applicability as a therapeutic regimen in the treatment of neurodegenerative diseases. Keywords: calpain, neurodegeneration, neuroprotection, calpain inhibitors, NMDAR, Speedy/RINGO

  15. Calpain-6 deficiency promotes skeletal muscle development and regeneration.

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    Kazuo Tonami

    Full Text Available Calpains are Ca(2+-dependent modulator Cys proteases that have a variety of functions in almost all eukaryotes. There are more than 10 well-conserved mammalian calpains, among which eutherian calpain-6 (CAPN6 is unique in that it has amino acid substitutions at the active-site Cys residue (to Lys in humans, strongly suggesting a loss of proteolytic activity. CAPN6 is expressed predominantly in embryonic muscles, placenta, and several cultured cell lines. We previously reported that CAPN6 is involved in regulating microtubule dynamics and actin reorganization in cultured cells. The physiological functions of CAPN6, however, are still unclear. Here, to elucidate CAPN6's in vivo roles, we generated Capn6-deficient mice, in which a lacZ expression cassette was integrated into the Capn6 gene. These Capn6-deficient mouse embryos expressed lacZ predominantly in skeletal muscles, as well as in cartilage and the heart. Histological and biochemical analyses showed that the CAPN6 deficiency promoted the development of embryonic skeletal muscle. In primary cultured skeletal muscle cells that were induced to differentiate into myotubes, Capn6 expression was detected in skeletal myocytes, and Capn6-deficient cultures showed increased differentiation. Furthermore, we found that CAPN6 was expressed in the regenerating skeletal muscles of adult mice after cardiotoxin-induced degeneration. In this experimental system, Capn6-deficient mice exhibited more advanced skeletal-muscle regeneration than heterozygotes or wild-type mice at the same time point. These results collectively showed that a loss of CAPN6 promotes skeletal muscle differentiation during both development and regeneration, suggesting a novel physiological function of CAPN6 as a suppressor of skeletal muscle differentiation.

  16. 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...

  17. 半乳糖凝集素3基因敲除对小鼠皮肤肿瘤生长的影响%The inhibition of skin tumor growth in Galecit n-3-deficient mice

    Institute of Scientific and Technical Information of China (English)

    张蒙; 张杰武; 孔令宇

    2014-01-01

    Objective To observe the effect of Galectin -3 gene knock out to the mouse skin tumor growth and discuss the mechanism of inhibition of the mouse cutaneous tumor induced by 12 -o -tetrade-canoylphorbol-13-acetate which caused by Galectin -3 knock out.Methods The DMBA +TPA multi-step induced skin tumor in mice model were used to establish the skin cancer model .The control group was the same age wild type mice .We observed the inhibition of the mouse tumor growth by Galectin -3 knock out .In situ tumor cells were collected and cultured on soft agar for colony formation assay .The side population of the situ cancer cells was analyzed quantitatively by flow cytometry .Results 1.Compared with wild type mice group(group A), Galectin-3 knock out mice group ( group B) displayed a significant delay of the appearance of tumor .The tumor incidence and the average number of tumor per mice between group A and group B had obvious difference ( P<0.01).2.In vitro,data of soft agar colony formation assay showed that colony formation rate in group A are signif -icantly higher than group B(P<0.01).3.The collection and separation of A and B group in situ tumor cells ,u-sing flow cytometry instrument for in situ tumor cell side population quantitative analysis ,group A compared with group B had obvious difference(P<0.01).Conclusion The knock out of the Galectin -3 gene reduces the skin cancer stem cells ,inhibits tumor cell proliferation ,depresses chemical carcinogenesis of mice skin .Galectin-3 gene may become the new target for skin cancer chemotherapy .%目的:观察半乳糖凝集素3(Gal3)基因敲除对小鼠皮肤肿瘤生长产生的影响,探讨Gal3基因敲除抑制DMBA+TPA诱导小鼠皮肤肿瘤生长的机制。方法采用DMBA+TPA多步骤诱导小鼠皮肤癌模型方法建立皮肤癌发生模型。以同龄野生型小鼠为对照组观察Gal3基因敲除对小鼠肿瘤生长产生的影响,通过采集分离原位癌细胞进行体外软琼脂克隆

  18. Critical role of calpain in inflammation

    Science.gov (United States)

    Ji, Jingjing; Su, Lei; Liu, Zhifeng

    2016-01-01

    Calpains are a family of cysteine proteases, implicated in a wide range of cellular calcium-regulated functions. Evidence from previous studies using an inhibitor of calpain indicates that calpain activation is involved in the process of numerous inflammation-associated diseases. As a result of in-depth studies, calpains have been proposed to influence the process of inflammation via a variety of mechanisms. The aim of the present study is to provide an overview of recent reports regarding the role of calpain in the process of inflammation, including regulation of immune cell migration, modulation of the activation of inflammatory mediators, degradation of certain associated proteins and induction of cell apoptosis. Understanding these mechanisms may contribute to the investigation of novel therapeutic targets for inflammation-associated diseases. PMID:28101338

  19. Calpains expression during Xenopus laevis development.

    Science.gov (United States)

    Moudilou, E N; Mouterfi, N; Exbrayat, J-M; Brun, C

    2010-10-01

    Calpains are cytoplasmic proteases activated by calcium, implicated in cell differentiation and apoptosis. The best characterized enzymes are calpains 1-3. The aim of this work was to localize calpains 1-3 during the development of Xenopus laevis in order to clarify the function of these three proteases. For the first time, we detected the localization of the three proteases at the protein level between one-cell stage and adult age. Their expression was weak at early stages, then increased at tadpole stage and decreased through metamorphosis and adult life. The calpain's expression was maximal during the period characterized by the appearance of organs and modelling process. These observations suggest that calpains play a crucial role during development.

  20. Retinoic acid counteracts developmental defects in the substantia nigra caused by Pitx3 deficiency

    NARCIS (Netherlands)

    Jacobs, Frank M. J.; Smits, Simone M.; Noorlander, Cornelle W.; von Oerthel, Lars; van der Linden, Annemarie J. A.; Burbach, J. Peter H.; Smidt, Marten P.

    2007-01-01

    Selective neuronal loss in the substantia nigra (SNc), as described for Parkinson's disease (PD) in humans and for Pitx3 deficiency in mice, highlights the existence of neuronal subpopulations. As yet unknown subset-specific gene cascades might underlie the observed differences in neuronal vulnerabi

  1. 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.

  2. Conventional calpains and programmed cell death.

    Science.gov (United States)

    Łopatniuk, Paulina; Witkowski, Jacek M

    2011-01-01

    The evidence on the crucial role of a family of calcium-dependent cysteine proteases called calpains in programmed cell death is rich and still growing. However, understanding of the mechanisms of their functions in apoptosis is not full yet. Calpains have been implicated in both physiological and pathological cell death control, especially in various malignancies, but also in the immune system development and function. There is also growing evidence on calpain involvement in apoptosis execution in certain pathological conditions of the central nervous system, in cardiovascular diseases, etc. Understanding of the clinical significance of calpain activation pathways, after intense studies of the influence of calpain activity on drug-induced apoptosis, seems especially important lately, as calpains have become noticed as potential therapeutic targets. To allow pharmacological targeting of these enzymes, thorough knowledge of their patterns of activation and further interactions with already known apoptotic pathways is necessary. A comprehensive summary of both well established and recently obtained information in the field is an important step that may lead to future advances in the use of calpain-targeted agents in the clinic.

  3. Implications of calpains in health and diseases.

    Science.gov (United States)

    Chakraborti, Sajal; Alam, Md Nur; Paik, Dibyendu; Shaikh, Soni; Chakraborti, Tapati

    2012-10-01

    The number of mammalian calpain protease family members has grown as many as 15 till recent count. Although initially described as a cytosolic protease, calpains have now been found in almost all subcellular locations i.e., from mitochondria to endoplasmic reticulum and from caveolae to Golgi bodies. Importantly, some calpains do not possess the 28 kDa regulatory subunit and have only the 80 kDa catalytic subunit. In some instances, the 80 kDa subunit by itself confers the calpain proteolytic activity. Calpains have been shown to be involved in a number of physiological processes such as cell cycle progression, remodeling of cytoskeletal-cell membrane attachments, signal transduction, gene expression and apoptosis. Recent studies have linked calpain deficiencies or it's over production with a variety of diseases, such as muscular dystrophies, gastropathy, diabetes, Alzheimer's and Parkinson's diseases, atherosclerosis and pulmonary hypertension. Herein, we present a brief overview on some implications of calpains on human health and some diseases.

  4. Calpains and cancer: friends or enemies?

    Science.gov (United States)

    Moretti, Daniele; Del Bello, Barbara; Allavena, Giulia; Maellaro, Emilia

    2014-12-15

    Calpains are a complex family of ubiquitous or tissue-specific cysteine proteases that proteolyze a variety of substrates (leading to their degradation or functional modulation) and are implicated in several pathophysiological phenomena. In tumor cell biology, calpains are implicated in a triple way: they are involved in different processes crucial for tumor progression, including cell proliferation, apoptotic cell death, survival mechanisms, migration and invasiveness; they have aberrant expression in several human cancers; a variety of anticancer drugs induce cytotoxicity through activation of calpains or the latter can influence response to therapy. This review covers established and recent literature showing these diverse aspects in tumor cells.

  5. NLRP3 deficiency ameliorates neurovascular damage in experimental ischemic stroke.

    Science.gov (United States)

    Yang, Fan; Wang, Ziying; Wei, Xinbing; Han, Huirong; Meng, Xianfang; Zhang, Yan; Shi, Weichen; Li, Fengli; Xin, Tao; Pang, Qi; Yi, Fan

    2014-04-01

    Although the innate immune response to induce postischemic inflammation is considered as an essential step in the progression of cerebral ischemia injury, the role of innate immunity mediator NLRP3 in the pathogenesis of ischemic stroke is unknown. In this study, focal ischemia was induced by middle cerebral artery occlusion in NLRP3(-/-), NOX2(-/-), or wild-type (WT) mice. By magnetic resonance imaging (MRI), Evans blue permeability, and electron microscopic analyses, we found that NLRP3 deficiency ameliorated cerebral injury in mice after ischemic stroke by reducing infarcts and blood-brain barrier (BBB) damage. We further showed that the contribution of NLRP3 to neurovascular damage was associated with an autocrine/paracrine pattern of NLRP3-mediated interleukin-1β (IL-1β) release as evidenced by increased brain microvessel endothelial cell permeability and microglia-mediated neurotoxicity. Finally, we found that NOX2 deficiency improved outcomes after ischemic stroke by mediating NLRP3 signaling. This study for the first time shows the contribution of NLRP3 to neurovascular damage and provides direct evidence that NLRP3 as an important target molecule links NOX2-mediated oxidative stress to neurovascular damage in ischemic stroke. Pharmacological targeting of NLRP3-mediated inflammatory response at multiple levels may help design a new approach to develop therapeutic strategies for prevention of deterioration of cerebral function and for the treatment of stroke.

  6. Differences in mRNA expression of calpains, calpastatin isoforms and calpain/calpastatin ratios among bovine skeletal muscles.

    Science.gov (United States)

    Muroya, Susumu; Neath, Kate E; Nakajima, Ikuyo; Oe, Mika; Shibata, Masahiro; Ojima, Koichi; Chikuni, Koichi

    2012-03-01

    Messenger RNA (mRNA) expression of calpain-1 (µ-calpain), -2 (m-calpain), -3 (p94), small subunit (calpain-4; 28 kDa), and three types of calpastatin (CSTN) isoform were investigated for 10 skeletal muscles of Holstein cattle by real-time and/or semi-quantitative reverse transcription polymerase chain reaction. Noticeably, effect of muscle type was observed on 28 kDa expression (P Calpain-1/CSTN I, calpain-2/CSTN I in LT and PM were higher than that in TN (P Calpain-3/CSTN-I and -III in LT and/or PM showed higher values than that in TN (P calpain and CSTN expressions are regulated by muscle type, suggesting especially by muscle fiber type. Calpains/CSTN-I ratios, especially 28 kDa/CSTN-I, may account for higher extent of post mortem proteolysis previously observed in LT and PM muscles.

  7. Regulation of TET Protein Stability by Calpains

    Directory of Open Access Journals (Sweden)

    Yu Wang

    2014-01-01

    Full Text Available DNA methylation at the fifth position of cytosine (5mC is an important epigenetic modification that affects chromatin structure and gene expression. Recent studies have established a critical function of the Ten-eleven translocation (Tet family of proteins in regulating DNA methylation dynamics. Three Tet genes have been identified in mammals, and they all encode for proteins capable of oxidizing 5mC as part of the DNA demethylation process. Although regulation of Tet expression at the transcriptional level is well documented, how TET proteins are regulated at posttranslational level is poorly understood. In this study, we report that all three TET proteins are direct substrates of calpains, a family of calcium-dependent proteases. Specifically, calpain1 mediates TET1 and TET2 turnover in mouse ESCs, and calpain2 regulates TET3 level during differentiation. This study provides evidence that TET proteins are subject to calpain-mediated degradation.

  8. Regulation of TET protein stability by calpains.

    Science.gov (United States)

    Wang, Yu; Zhang, Yi

    2014-01-30

    DNA methylation at the fifth position of cytosine (5mC) is an important epigenetic modification that affects chromatin structure and gene expression. Recent studies have established a critical function of the Ten-eleven translocation (Tet) family of proteins in regulating DNA methylation dynamics. Three Tet genes have been identified in mammals, and they all encode for proteins capable of oxidizing 5mC as part of the DNA demethylation process. Although regulation of Tet expression at the transcriptional level is well documented, how TET proteins are regulated at posttranslational level is poorly understood. In this study, we report that all three TET proteins are direct substrates of calpains, a family of calcium-dependent proteases. Specifically, calpain1 mediates TET1 and TET2 turnover in mouse ESCs, and calpain2 regulates TET3 level during differentiation. This study provides evidence that TET proteins are subject to calpain-mediated degradation.

  9. MSH3-deficiency initiates EMAST without oncogenic transformation of human colon epithelial cells.

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    Christoph Campregher

    Full Text Available BACKGROUND/AIM: Elevated microsatellite instability at selected tetranucleotide repeats (EMAST is a genetic signature in certain cases of sporadic colorectal cancer and has been linked to MSH3-deficiency. It is currently controversial whether EMAST is associated with oncogenic properties in humans, specifically as cancer development in Msh3-deficient mice is not enhanced. However, a mutator phenotype is different between species as the genetic positions of repetitive sequences are not conserved. Here we studied the molecular effects of human MSH3-deficiency. METHODS: HCT116 and HCT116+chr3 (both MSH3-deficient and primary human colon epithelial cells (HCEC, MSH3-wildtype were stably transfected with an EGFP-based reporter plasmid for the detection of frameshift mutations within an [AAAG]17 repeat. MSH3 was silenced by shRNA and changes in protein expression were analyzed by shotgun proteomics. Colony forming assay was used to determine oncogenic transformation and double strand breaks (DSBs were assessed by Comet assay. RESULTS: Despite differential MLH1 expression, both HCT116 and HCT116+chr3 cells displayed comparable high mutation rates (about 4×10(-4 at [AAAG]17 repeats. Silencing of MSH3 in HCECs leads to a remarkable increased frameshift mutations in [AAAG]17 repeats whereas [CA]13 repeats were less affected. Upon MSH3-silencing, significant changes in the expression of 202 proteins were detected. Pathway analysis revealed overexpression of proteins involved in double strand break repair (MRE11 and RAD50, apoptosis, L1 recycling, and repression of proteins involved in metabolism, tRNA aminoacylation, and gene expression. MSH3-silencing did not induce oncogenic transformation and DSBs increased 2-fold. CONCLUSIONS: MSH3-deficiency in human colon epithelial cells results in EMAST, formation of DSBs and significant changes of the proteome but lacks oncogenic transformation. Thus, MSH3-deficiency alone is unlikely to drive human colon

  10. Impact of RTN3 deficiency on expression of BACE1 and amyloid deposition.

    Science.gov (United States)

    Shi, Qi; Ge, Yingying; Sharoar, Md Golam; He, Wanxia; Xiang, Rong; Zhang, Zhuohua; Hu, Xiangyou; Yan, Riqiang

    2014-10-15

    Reticulon 3 (RTN3) has previously been shown to interact with BACE1 and negatively regulate BACE1 activity. To what extent RTN3 deficiency affects BACE1 activity is an intriguing question. In this study, we aimed to address this by generating RTN3-null mice. Mice with complete deficiency of RTN3 grow normally and have no obviously discernible phenotypes. Morphological analyses of RTN3-null mice showed no significant alterations in cellular structure, although RTN3 is recognized as a protein contributing to the shaping of tubular endoplasmic reticulum. Biochemical analysis revealed that RTN3 deficiency increased protein levels of BACE1. This elevation of BACE1 levels correlated with enhanced processing of amyloid precursor protein at the β-secretase site. We also demonstrated that RTN3 deficiency in Alzheimer's mouse models facilitates amyloid deposition, further supporting an in vivo role of RTN3 in the regulation of BACE1 activity. Since it has been shown that RTN3 monomer is reduced in brains of Alzheimer's patients, our results suggest that long-lasting reduction of RTN3 levels has adverse effects on BACE1 activity and may contribute to Alzheimer's pathogenesis.

  11. 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.

  12. [The role of calpains in the regulation of synaptic function].

    Science.gov (United States)

    Karpenko, M N; Tikhomirova, M S

    2014-04-01

    Calpains are calcium-activated neutral cysteine proteases, involved in the regulation of a number of physiological functions. Substrates of calpains include receptors, kinases, phosphatases, cytoskeleton and synaptosomal proteins. Some of them undergo complete degradation, though most of the substrates are subjected to limited proteolysis, which results in proteins having new properties. In the following review, we discuss involvement of calpains in the regulation of synapse structure and function. Namely, calpains participate in the regulation of synthesis, release and reuptake of neurotransmitters, modulation of receptors, stabilization or destabilization of the neuronal cytoskeleton. However, uncontrolled hyperactivation of calpains leads to dysregulation of these processes causing neuronal death.

  13. 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

  14. 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.

  15. Dietary vitamin D3 deficiency alters intestinal mucosal defense and increases susceptibility to Citrobacter rodentium-induced colitis.

    Science.gov (United States)

    Ryz, Natasha R; Lochner, Arion; Bhullar, Kirandeep; Ma, Caixia; Huang, Tina; Bhinder, Ganive; Bosman, Else; Wu, Xiujuan; Innis, Sheila M; Jacobson, Kevan; Vallance, Bruce A

    2015-11-01

    Vitamin D deficiency affects more that 1 billion people worldwide. Although thought to increase risk of bacterial infections, the importance of vitamin D on host defense against intestinal bacterial pathogens is currently unclear since injection of the active form of vitamin D, 1,25(OH)2D3, increased susceptibility to the enteric bacterial pathogen Citrobacter rodentium by suppressing key immune/inflammatory factors. To further characterize the role of vitamin D during bacteria-induced colitis, we fed weanling mice either vitamin D3-deficient or vitamin D3-sufficient diets for 5 wk and then challenged them with C. rodentium. Vitamin D3-deficient mice lost significantly more body weight, carried higher C. rodentium burdens, and developed worsened histological damage. Vitamin D3-deficient mice also suffered greater bacterial translocation to extra-intestinal tissues, including mesenteric lymph nodes, spleen, and liver. Intestinal tissues of infected vitamin D3-deficient mice displayed increased inflammatory cell infiltrates as well as significantly higher gene transcript levels of inflammatory mediators TNF-α, IL-1β, IL-6, TGF-β, IL-17A, and IL-17F as well as the antimicrobial peptide REG3γ. Notably, these exaggerated inflammatory responses accelerated the loss of commensal microbes and were associated with an impaired ability to detoxify bacterial lipopolysaccharide. Overall, these studies show that dietary-induced vitamin D deficiency exacerbates intestinal inflammatory responses to infection, also impairing host defense.

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

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    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.

  17. Structure-function relationships in calpains.

    Science.gov (United States)

    Campbell, Robert L; Davies, Peter L

    2012-11-01

    Calpains are a family of complex multi-domain intracellular enzymes that share a calcium-dependent cysteine protease core. These are not degradative enzymes, but instead carry out limited cleavage of target proteins in response to calcium signalling. Selective cutting of cytoskeletal proteins to facilitate cell migration is one such function. The two most abundant and extensively studied members of this family in mammals, calpains 1 and 2, are heterodimers of an isoform-specific 80 kDa large subunit and a common 28 kDa small subunit. Structures of calpain-2, both Ca2+-free and bound to calpastatin in the activated Ca2+-bound state, have provided a wealth of information about the enzyme's structure-function relationships and activation. The main association between the subunits is the pairing of their C-terminal penta-EF-hand domains through extensive intimate hydrophobic contacts. A lesser contact is made between the N-terminal anchor helix of the large subunit and the penta-EF-hand domain of the small subunit. Up to ten Ca2+ ions are co-operatively bound during activation. The anchor helix is released and individual domains change their positions relative to each other to properly align the active site. Because calpains 1 and 2 require ~30 and ~350 μM Ca2+ ions for half-maximal activation respectively, it has long been argued that autoproteolysis, subunit dissociation, post-translational modifications or auxiliary proteins are needed to activate the enzymes in the cell, where Ca2+ levels are in the nanomolar range. In the absence of robust support for these mechanisms, it is possible that under normal conditions calpains are transiently activated by high Ca2+ concentrations in the microenvironment of a Ca2+ influx, and then return to an inactive state ready for reactivation.

  18. Calpain免疫BALB/c鼠后IFN-γ和IF-4水平的动态变化%The kinetics of cytokine IFN-γand IF-4 expression in BAIB/c mice immunized with calpain recombinant protein

    Institute of Scientific and Technical Information of China (English)

    林琳; 周建辉; 黄伦芳; 陈雪艳; 耿艺介; 黄达娜; 张仁利

    2009-01-01

    目的 探讨弓形虫疫苗候选分子-钙离子激活的中性蛋白激酶(calpain)的保护性免疫力和免疫保护性机制.方法 用重组纯化的Calpaln抗原免疫BALB/c小鼠,RT-PCR分析Calpain抗原免疫BALB/c小鼠一氧化氮激酶(iNos.eNos.nNos)mRNA的表达;ELISA分析免疫小鼠体外培养脾细胞产生的细胞因子IFN-γ和IL-4的动态变化.结果 重组Calpain抗原免疫小鼠后.RT-PCR测定重组Calpain抗原免疫1周小鼠iNos mRNA的表达显著性的增强,培养24h免疫鼠脾细胞上清中IFN-γ浓度显著性的高于对照组,而IL-4的产生在免役组与对照组差异无统计学意义.结论 Calpain抗原能够诱导IFN-γ的产生,提示Calpain诱导Th1的免疫应答反应来抗弓形虫的感染.

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

    OpenAIRE

    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...

  20. 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

  1. Low levels of inorganic lead noncompetitively inhibit mu-calpain.

    Science.gov (United States)

    Audesirk, T; Pedersen, C; Audesirk, G; Kern, M

    1998-11-16

    Calpain is a ubiquitous calcium-dependent cysteine protease, whose cytoskeletal protein substrates suggest that it may be important in neuronal differentiation. Lead (Pb2+) is known to substitute for Ca2+ in a variety of intracellular processes, and interferes with the development of hippocampal neurons in vitro. We found that free Pb2+ at 1 nM does not activate calpain in the absence of Ca2+. Pb2+ inhibited the activity of calpain; the degree of calpain inhibition was dependent on an interaction between concentrations of both Ca2+ and Pb2+. In the presence of 1 microM free Ca2+, 10 pM free Pb2+ reduced calpain activity, but in the presence of 100 microM free Ca2+, 1 nM free Pb2+ failed to inhibit calpain. This provides evidence that Pb2+ competes for the Ca2+ binding sites on calpain. In the presence of 40 microM free Ca2+, 1 nM free Pb2+ significantly reduces Vmax without altering Km, suggesting that Pb2+ acts as a noncompetitive inhibitor of calpain. Inhibition of calpain is one mechanism by which Pb2+ may interfere with neuronal development.

  2. 1,25-Vitamin D3 Deficiency Induces Albuminuria

    NARCIS (Netherlands)

    Sonneveld, R.; Hoenderop, J.G.; Stavenuiter, A.W.; Ferrantelli, E.; Baltissen, M.P.A.; Dijkman, H.B.; Florquin, S.; Rops, A.; Wetzels, J.F.M.; Berden, J.H.M.; Vlag, J. van der; Nijenhuis, T.

    2016-01-01

    Vitamin D plays an important role in renal (patho)physiology. Patients with glomerular diseases have an injured renal filtration barrier, leading to proteinuria and reduced renal function. An impaired renal function also leads to 1,25-vitamin D3 deficiency as a result of reduced renal 1alpha-hydroxy

  3. Massive expansion of the calpain gene family in unicellular eukaryotes

    Directory of Open Access Journals (Sweden)

    Zhao Sen

    2012-09-01

    Full Text Available Abstract Background Calpains are Ca2+-dependent cysteine proteases that participate in a range of crucial cellular processes. Dysfunction of these enzymes may cause, for instance, life-threatening diseases in humans, the loss of sex determination in nematodes and embryo lethality in plants. Although the calpain family is well characterized in animal and plant model organisms, there is a great lack of knowledge about these genes in unicellular eukaryote species (i.e. protists. Here, we study the distribution and evolution of calpain genes in a wide range of eukaryote genomes from major branches in the tree of life. Results Our investigations reveal 24 types of protein domains that are combined with the calpain-specific catalytic domain CysPc. In total we identify 41 different calpain domain architectures, 28 of these domain combinations have not been previously described. Based on our phylogenetic inferences, we propose that at least four calpain variants were established in the early evolution of eukaryotes, most likely before the radiation of all the major supergroups of eukaryotes. Many domains associated with eukaryotic calpain genes can be found among eubacteria or archaebacteria but never in combination with the CysPc domain. Conclusions The analyses presented here show that ancient modules present in prokaryotes, and a few de novo eukaryote domains, have been assembled into many novel domain combinations along the evolutionary history of eukaryotes. Some of the new calpain genes show a narrow distribution in a few branches in the tree of life, likely representing lineage-specific innovations. Hence, the functionally important classical calpain genes found among humans and vertebrates make up only a tiny fraction of the calpain family. In fact, a massive expansion of the calpain family occurred by domain shuffling among unicellular eukaryotes and contributed to a wealth of functionally different genes.

  4. Calpains are downstream effectors of bax-dependent excitotoxic apoptosis.

    Science.gov (United States)

    D'Orsi, Beatrice; Bonner, Helena; Tuffy, Liam P; Düssmann, Heiko; Woods, Ina; Courtney, Michael J; Ward, Manus W; Prehn, Jochen H M

    2012-02-01

    Excitotoxicity resulting from excessive Ca(2+) influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca(2+) levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16-E18) murine cortical neurons that (1) underwent excitotoxic necrosis, characterized by immediate deregulation of Ca(2+) homeostasis, a persistent depolarization of mitochondrial membrane potential (Δψ(m)), and insensitivity to bax-gene deletion, (2) underwent excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca(2+) increases, sensitivity to bax gene deletion, and delayed Δψ(m) depolarization and Ca(2+) deregulation, or (3) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis but did not influence excitotoxic necrosis. Calpeptin failed to exert a protective effect in bax-deficient neurons but protected bid-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy using a calpain-sensitive Förster resonance energy transfer probe. We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injury. Oxygen/glucose deprivation-induced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during bax-dependent apoptosis and in this setting function as downstream cell-death executioners.

  5. Bacterial calpains and the evolution of the calpain (C2) family of peptidases.

    Science.gov (United States)

    Rawlings, Neil D

    2015-11-02

    Homologues of calpain, often thought to be an essential, cytoplasmic, calcium-dependent cysteine endopeptidase found exclusively in eukaryotes, have been found in bacterial proteomes. The homologues lack calcium-binding sites, have differing domain architectures, and can be secreted or membrane-associated. Homologues are rare and occur in a minority of bacterial phyla and often in a minority of species in a genus. However, the differences in domain architecture argue against a recent, horizontal gene transfer from a eukaryote. From analysis of a phylogenetic tree and absence of homologues in archaea, calpains in eukaryotes may be derived from genes horizontally transferred from a bacterium.

  6. 1,25(OH)2D3 Deficiency Induces Colon Inflammation via Secretion of Senescence-Associated Inflammatory Cytokines.

    Science.gov (United States)

    Liu, Yun; Chen, Lulu; Zhi, Chunchun; Shen, Ming; Sun, Weiwei; Miao, Dengshun; Yuan, Xiaoqin

    2016-01-01

    Epidemiological studies showed that 1,25-Dihydroxyvitamin D[1,25(OH)2D3] insufficiency appears to be associated with aging and colon cancer while underlying biological mechanisms remain largely unknown. Inflammatory bowel disease is one of the risk factors for colon cancer. In this study, we investigated whether 1,25(OH)2D3 deficiency has an impact on the colon of 25-hydroxyvitamin D 1α-hydroxylase knockout [Cyp27b1(-/-)] mice fed on a rescue diet (high calcium, phosphate, and lactose) from weaning to 10 months of age. We found that 1,25(OH)2D3 deficient mice displayed significant colon inflammation phenotypes including shortened colon length, thinned and disordered mucosal structure, and inflammatory cell infiltration. DNA damage, cellular senescence and the production of senescence-associated inflammatory cytokines were also increased significantly in the colon of Cyp27b1(-/-)mice. Furthermore, the levels of ROS in the colon were increased significantly, whereas the expression levels of antioxidative genes were down-regulated dramatically in the colon of Cyp27b1(-/-)mice. Taken together, our results demonstrated that 1,25(OH)2D3 deficiency could induce colon inflammation, which may result from increased oxidative stress and DNA damage, subsequently, induced cell senescence and overproduction of senescence-associated secretory factors. Therefore, our findings suggest that 1,25(OH)2D3 may play an important role in preventing the development and progression of colon inflammation and colon cancer.

  7. A conserved role for calpains during myoblast fusion.

    Science.gov (United States)

    Buffolo, Marcio; Batista Possidonio, Ana Claudia; Mermelstein, Claudia; Araujo, Helena

    2015-07-01

    Myoblast fusion is a key step during skeletal muscle differentiation as it enables the formation of contractile fibers. Calpains have been implicated in some aspects of myogenesis in mammals, but whether they exert a conserved function during myoblast fusion has not been investigated. Here, we studied Calpain function in two models of myogenesis: in vitro analysis of chick myogenic cultures and in vivo analysis of Drosophila melanogaster muscle development. First we showed that Calpain A is important for fly muscle function. In addition, Calpain A knockdown reduced lateral body wall muscle length and width, as well as the number of nuclei in dorsal oblique muscles, consistent with fewer cells fusing to form fibers. Treatment of chick cultures with a selective Calpain inhibitor led to the formation of thinner myotubes containing a reduced number of nuclei, consistent with decreased myoblast fusion. Dynamic changes in IκBα labeling and transfection with a dominant-negative IκBα suggest a role for the NFκB pathway during chick myogenesis and a possible role of Calpains in attenuating NFκB signals that restrict myoblast fusion. Our data suggest that different model organisms may be used to study the role of Calpains in regular myogenesis and Calpain-related muscular degenerative disorders.

  8. The role of calpains in myocardial remodelling and heart failure.

    Science.gov (United States)

    Letavernier, Emmanuel; Zafrani, Lara; Perez, Joëlle; Letavernier, Béatrice; Haymann, Jean-Philippe; Baud, Laurent

    2012-10-01

    Calpains are cytosolic calcium-activated cysteine proteases. Recently, they have been proposed to influence signal transduction processes leading to myocardial remodelling and heart failure. In this review, we will first describe some of these molecular mechanisms. Calpains may contribute to myocardial hypertrophy and inflammation, mainly through the activation of transcription factors such as NF-κB. They play an important role in the fibrosis process partly by activating transforming growth factor β. They are also implicated in cell death as they cause the breakdown of sarcolemma and sarcomeres. Nevertheless, a key to understanding the molecular basis of calpain-mediated myocardial remodelling likely lies in the identification of mechanisms involved in calpain secretion, since cytosolic and extracellular proteases would have different functions. Finally, we will provide an overview of the available evidence that calpains are indeed actively involved in the common causes of heart failure, including hypertension, diabetes, atherosclerosis, ischaemia-reperfusion, atrial fibrillation, congestive failure, and mechanical unloading.

  9. Smad3 Deficiency Ameliorates Hepatic Fibrogenesis through the Expression of Senescence Marker Protein-30, an Antioxidant-Related Protein

    Directory of Open Access Journals (Sweden)

    Da-Hee Jeong

    2013-12-01

    Full Text Available Smad3 is a key mediator of the transforming growth factor (TGF-β1 signaling pathway that plays central role in inflammation and fibrosis. In present study, we evaluated the effect of Smad3 deficiency in Smad3−/− mice with carbon tetrachloride (CCl4-induced liver fibrosis. The animals were received CCl4 or olive oil three times a week for 4 weeks. Histopathological analyses were performed to evaluate the fibrosis development in the mice. Alteration of protein expression controlled by Smad3 was examined using a proteomic analysis. CCl4-induced liver fibrosis was rarely detected in Smad3−/− mice compared to Smad3+/+. Proteomic analysis revealed that proteins related to antioxidant activities such as senescence marker protein-30 (SMP30, selenium-binding proteins (SP56 and glutathione S-transferases (GSTs were up-regulated in Smad3−/− mice. Western blot analysis confirmed that SMP30 protein expression was increased in Smad3−/− mice. And SMP30 levels were decreased in CCl4-treated Smad3+/+ and Smad3−/− mice. These results indicate that Smad3 deficiency influences the proteins level related to antioxidant activities during early liver fibrosis. Thus, we suggest that Smad3 deteriorate hepatic injury by inhibitor of antioxidant proteins as well as mediator of TGF-β1 signaling.

  10. Contribution of calpains to myocardial ischaemia/reperfusion injury.

    Science.gov (United States)

    Inserte, Javier; Hernando, Victor; Garcia-Dorado, David

    2012-10-01

    Loss of calcium (Ca(2+)) homeostasis contributes through different mechanisms to cell death occurring during the first minutes of reperfusion. One of them is an unregulated activation of a variety of Ca(2+)-dependent enzymes, including the non-lysosomal cysteine proteases known as calpains. This review analyses the involvement of the calpain family in reperfusion-induced cardiomyocyte death. Calpains remain inactive before reperfusion due to the acidic pHi and increased ionic strength in the ischaemic myocardium. However, inappropriate calpain activation occurs during myocardial reperfusion, and subsequent proteolysis of a wide variety of proteins contributes to the development of contractile dysfunction and necrotic cell death by different mechanisms, including increased membrane fragility, further impairment of Na(+) and Ca(2+) handling, and mitochondrial dysfunction. Recent studies demonstrating that calpain inhibition contributes to the cardioprotective effects of preconditioning and postconditioning, and the beneficial effects obtained with new and more selective calpain inhibitors added at the onset of reperfusion, point to the potential cardioprotective value of therapeutic strategies designed to prevent calpain activation.

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

    DEFF Research Database (Denmark)

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

    2004-01-01

    The intermediate filament protein, desmin, was purified from pork longissimus dorsi and incubated with either P-calpain, m-calpain or cathepsin B. Proteolysis of desmin was followed using SDS-PAGE and Western blotting. After incubation of desmin with the proteases, cleavage sites on the desmin...... a 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...

  12. FGFR3 Deficiency Causes Multiple Chondroma-like Lesions by Upregulating Hedgehog Signaling.

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    Siru Zhou

    2015-06-01

    Full Text Available Most cartilaginous tumors are formed during skeletal development in locations adjacent to growth plates, suggesting that they arise from disordered endochondral bone growth. Fibroblast growth factor receptor (FGFR3 signaling plays essential roles in this process; however, the role of FGFR3 in cartilaginous tumorigenesis is not known. In this study, we found that postnatal chondrocyte-specific Fgfr3 deletion induced multiple chondroma-like lesions, including enchondromas and osteochondromas, adjacent to disordered growth plates. The lesions showed decreased extracellular signal-regulated kinase (ERK activity and increased Indian hedgehog (IHH expression. The same was observed in Fgfr3-deficient primary chondrocytes, in which treatment with a mitogen-activated protein kinase (MEK inhibitor increased Ihh expression. Importantly, treatment with an inhibitor of IHH signaling reduced the occurrence of chondroma-like lesions in Fgfr3-deficient mice. This is the first study reporting that the loss of Fgfr3 function leads to the formation of chondroma-like lesions via downregulation of MEK/ERK signaling and upregulation of IHH, suggesting that FGFR3 has a tumor suppressor-like function in chondrogenesis.

  13. NLRP3 Deficiency Improves Angiotensin II-Induced Hypertension But Not Fetal Growth Restriction During Pregnancy.

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    Shirasuna, Koumei; Karasawa, Tadayoshi; Usui, Fumitake; Kobayashi, Motoi; Komada, Tadanori; Kimura, Hiroaki; Kawashima, Akira; Ohkuchi, Akihide; Taniguchi, Shun'ichiro; Takahashi, Masafumi

    2015-11-01

    Preeclampsia is a pregnancy-specific syndrome characterized by elevated blood pressure, proteinuria, and intrauterine growth restriction (IUGR). Although sterile inflammation appears to be involved, its pathogenesis remains unclear. Recent evidence indicates that sterile inflammation is mediated through the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes, composed of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1. Here we investigated the role of the NLRP3 inflammasomes in the pathogenesis of preeclampsia using Nlrp3(-/-) and Asc(-/-) (Nlrp3 and Asc deficient) pregnant mice. During pregnancy in mice, continuous infusion of high-dose angiotensin II (AngII) induced hypertension, proteinuria, and IUGR, whereas infusion of low-dose AngII caused hypertension alone. AngII-induced hypertension was prevented in Nlrp3(-/-) mice but not in Asc(-/-), indicating that NLRP3 contributes to gestational hypertension independently of ASC-mediated inflammasomes. Although NLRP3 deficiency had no effect on IUGR, it restored the IL-6 up-regulation in the placenta and kidney of AngII-infused mice. Furthermore, treatment with hydralazine prevented the development of gestational hypertension but not IUGR or IL-6 expression in the placenta and kidney. These findings demonstrate that NLRP3 contributes to the development of gestational hypertension independently of the inflammasomes and that IUGR and kidney injury can occur independent of blood pressure elevation during pregnancy.

  14. Small Intestine but Not Liver Lysophosphatidylcholine Acyltransferase 3 (Lpcat3) Deficiency Has a Dominant Effect on Plasma Lipid Metabolism.

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    Kabir, Inamul; Li, Zhiqiang; Bui, Hai H; Kuo, Ming-Shang; Gao, Guangping; Jiang, Xian-Cheng

    2016-04-01

    Lysophosphatidylcholine acyltransferase 3 (Lpcat3) is involved in phosphatidylcholine remodeling in the small intestine and liver. We investigated lipid metabolism in inducible intestine-specific and liver-specificLpcat3gene knock-out mice. We producedLpcat3-Flox/villin-Cre-ER(T2)mice, which were treated with tamoxifen (at days 1, 3, 5, and 7), to deleteLpcat3specifically in the small intestine. At day 9 after the treatment, we found that Lpcat3 deficiency in enterocytes significantly reduced polyunsaturated phosphatidylcholines in the enterocyte plasma membrane and reduced Niemann-Pick C1-like 1 (NPC1L1), CD36, ATP-binding cassette transporter 1 (ABCA1), and ABCG8 levels on the membrane, thus significantly reducing lipid absorption, cholesterol secretion through apoB-dependent and apoB-independent pathways, and plasma triglyceride, cholesterol, and phospholipid levels, as well as body weight. Moreover, Lpcat3 deficiency does not cause significant lipid accumulation in the small intestine. We also utilized adenovirus-associated virus-Cre to depleteLpcat3in the liver. We found that liver deficiency only reduces plasma triglyceride levels but not other lipid levels. Furthermore, there is no significant lipid accumulation in the liver. Importantly, small intestine Lpcat3 deficiency has a much bigger effect on plasma lipid levels than that of liver deficiency. Thus, inhibition of small intestine Lpcat3 might constitute a novel approach for treating hyperlipidemia.

  15. Calpain-dependent disruption of nucleo-cytoplasmic transport in ALS motor neurons

    Science.gov (United States)

    Yamashita, Takenari; Aizawa, Hitoshi; Teramoto, Sayaka; Akamatsu, Megumi; Kwak, Shin

    2017-01-01

    Nuclear dysfunction in motor neurons has been hypothesized to be a principal cause of amyotrophic lateral sclerosis (ALS) pathogenesis. Here, we investigated the mechanism by which the nuclear pore complex (NPC) is disrupted in dying motor neurons in a mechanistic ALS mouse model (adenosine deaminase acting on RNA 2 (ADAR2) conditional knockout (AR2) mice) and in ALS patients. We showed that nucleoporins (Nups) that constituted the NPC were cleaved by activated calpain via a Ca2+-permeable AMPA receptor-mediated mechanism in dying motor neurons lacking ADAR2 expression in AR2 mice. In these neurons, nucleo-cytoplasmic transport was disrupted, and the level of the transcript elongation enzyme RNA polymerase II phosphorylated at Ser2 was significantly decreased. Analogous changes were observed in motor neurons lacking ADAR2 immunoreactivity in sporadic ALS patients. Therefore, calpain-dependent NPC disruption may participate in ALS pathogenesis, and inhibiting Ca2+-mediated cell death signals may be a therapeutic strategy for ALS. PMID:28045133

  16. Calpain-catalyzed proteolysis of human dUTPase specifically removes the nuclear localization signal peptide.

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    Zoltán Bozóky

    Full Text Available BACKGROUND: Calpain proteases drive intracellular signal transduction via specific proteolysis of multiple substrates upon Ca(2+-induced activation. Recently, dUTPase, an enzyme essential to maintain genomic integrity, was identified as a physiological calpain substrate in Drosophila cells. Here we investigate the potential structural/functional significance of calpain-activated proteolysis of human dUTPase. METHODOLOGY/PRINCIPAL FINDINGS: Limited proteolysis of human dUTPase by mammalian m-calpain was investigated in the presence and absence of cognate ligands of either calpain or dUTPase. Significant proteolysis was observed only in the presence of Ca(II ions, inducing calpain action. The presence or absence of the dUTP-analogue α,β-imido-dUTP did not show any effect on Ca(2+-calpain-induced cleavage of human dUTPase. The catalytic rate constant of dUTPase was unaffected by calpain cleavage. Gel electrophoretic analysis showed that Ca(2+-calpain-induced cleavage of human dUTPase resulted in several distinctly observable dUTPase fragments. Mass spectrometric identification of the calpain-cleaved fragments identified three calpain cleavage sites (between residues (4SE(5; (7TP(8; and (31LS(32. The cleavage between the (31LS(32 peptide bond specifically removes the flexible N-terminal nuclear localization signal, indispensable for cognate localization. CONCLUSIONS/SIGNIFICANCE: Results argue for a mechanism where Ca(2+-calpain may regulate nuclear availability and degradation of dUTPase.

  17. 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

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    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

  18. Rbfox1 downregulation and altered calpain 3 splicing by FRG1 in a mouse model of Facioscapulohumeral muscular dystrophy (FSHD.

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    Mariaelena Pistoni

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (FRG1 in mice, frogs, and worms perturbs muscle development and causes FSHD-like phenotypes. FRG1 has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in FRG1 mice. We find that splicing perturbations parallel the responses of different muscles to FRG1 over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of FRG1-affected splicing events. Rbfox1 knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the Rbfox1 RNA and decreases its stability. Consistent with this, Rbfox1 expression is down-regulated in mice and cells over-expressing FRG1 as well as in FSHD patients. Among the genes affected is Calpain 3, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In FRG1 mice and FSHD patients, the Calpain 3 isoform lacking exon 6 (Capn3 E6- is increased. Finally, Rbfox1 knockdown and over-expression of Capn3 E6- inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of FRG1, reducing Rbfox1 levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.

  19. Calpain Inhibition Reduces Axolemmal Leakage in Traumatic Axonal Injury

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    János Sándor

    2009-12-01

    Full Text Available Calcium-induced, calpain-mediated proteolysis (CMSP has recently been implicated to the pathogenesis of diffuse (traumatic axonal injury (TAI. Some studies suggested that subaxolemmal CMSP may contribute to axolemmal permeability (AP alterations observed in TAI. Seeking direct evidence for this premise we investigated whether subaxolemmal CMSP may contribute to axolemmal permeability alterations (APA and pre-injury calpain-inhibition could reduce AP in a rat model of TAI. Horseradish peroxidase (HRP, a tracer that accumulates in axons with APA was administered one hour prior to injury into the lateral ventricle; 30 min preinjury a single tail vein bolus injection of 30 mg/kg MDL-28170 (a calpain inhibitor or its vehicle was applied in Wistar rats exposed to impact acceleration brain injury. Histological detection of traumatically injured axonal segments accumulating HRP and statistical analysis revealed that pre-injury administration of the calpain inhibitor MDL-28170 significantly reduced the average length of HRP-labeled axonal segments. The axono-protective effect of pre-injury calpain inhibition recently demonstrated with classical immunohistochemical markers of TAI was further corroborated in this experiment; significant reduction of the length of labeled axons in the drug-treated rats implicate CMSP in the progression of altered AP in TAI.

  20. Calpain-6 confers atherogenicity to macrophages by dysregulating pre-mRNA splicing

    Science.gov (United States)

    Tonami, Kazuo; Hata, Shoji; Aiuchi, Toshihiro; Lei, Xiao-Feng; Kim-Kaneyama, Joo-ri; Takeya, Motohiro; Itabe, Hiroyuki; Kurihara, Hiroki; Miyazaki, Akira

    2016-01-01

    Macrophages contribute to the development of atherosclerosis through pinocytotic deposition of native LDL–derived cholesterol in macrophages in the vascular wall. Inhibiting macrophage-mediated lipid deposition may have protective effects in atheroprone vasculature, and identifying mechanisms that potentiate this process may inform potential therapeutic interventions for atherosclerosis. Here, we report that dysregulation of exon junction complex–driven (EJC-driven) mRNA splicing confers hyperpinocytosis to macrophages during atherogenesis. Mechanistically, we determined that inflammatory cytokines induce an unconventional nonproteolytic calpain, calpain-6 (CAPN6), which associates with the essential EJC-loading factor CWC22 in the cytoplasm. This association disturbs the nuclear localization of CWC22, thereby suppressing the splicing of target genes, including those related to Rac1 signaling. CAPN6 deficiency in LDL receptor–deficient mice restored CWC22/EJC/Rac1 signaling, reduced pinocytotic deposition of native LDL in macrophages, and attenuated macrophage recruitment into the lesions, generating an atheroprotective phenotype in the aorta. In macrophages, the induction of CAPN6 in the atheroma interior limited macrophage movements, resulting in a decline in cell clearance from the lesions. Consistent with this finding, we observed that myeloid CAPN6 contributed to atherogenesis in a murine model of bone marrow transplantation. Furthermore, macrophages from advanced human atheromas exhibited increased CAPN6 induction and impaired CWC22 nuclear localization. Together, these results indicate that CAPN6 promotes atherogenicity in inflamed macrophages by disturbing CWC22/EJC systems. PMID:27525442

  1. Role of the calpain on the development of diabetes mellitus and its chronic complications.

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    Wan, Ting-Ting; Li, Xiu-Fen; Sun, Yan-Ming; Li, Yan-Bo; Su, Ying

    2015-08-01

    Diabetes mellitus (DM) is associated with acute and chronic complications that cause major morbidity and significant mortality. Calpains, a family of Ca(2+)-dependent cytosolic cysteine proteases, can modulate their substrates' structure and function through limited proteolytic activity. Calpain is a ubiquitous calcium-sensitive protease that is essential for normal physiologic function. However, alterations in calcium homeostasis lead to pathologic activation of calpain in diabetes mellitus. Since not much is known on the relationship between calpain and diabetes mellitus, this review outlines the contribution of calpain to chronic complications of diabetes mellitus, such as diabetic cardiomyopathy, diabetic nephropathy and diabetic retinopathy.

  2. Elevated Expression of Calpain-4 Predicts Poor Prognosis in Patients with Gastric Cancer after Gastrectomy

    Science.gov (United States)

    Peng, Peike; Min, Lingqiang; Song, Shushu; Zhao, Junjie; Li, Lili; Yang, Caiting; Shao, Miaomiao; Zhang, Mingming; Wu, Hao; Zhang, Jie; Li, Can; Wang, Xuefei; Wang, Hongshan; Qin, Jing; Ruan, Yuanyuan; Gu, Jianxin

    2016-01-01

    Calpain-4 belongs to the calpain family of calcium-dependent cysteine proteases, and functions as a small regulatory subunit of the calpains. Recent evidence indicates that calpain-4 plays critical roles in tumor migration and invasion. However, the roles of calpain-4 in gastric tumorigenesis remain poorly understood. Herein, we examined calpain-4 expression by immunohistochemical staining on tissue microarrays containing tumor samples of 174 gastric cancer patients between 2004 and 2008 at a single center. The Kaplan-Meier method was used to compare survival curves, and expression levels were correlated to clinicopathological factors and overall survival. Our data demonstrated that calpain-4 was generally increased in gastric cancer cell lines and primary tumor tissues. High expression of calpain-4 was positively associated with vessel invasion, lymph node metastasis, and advanced TNM (Tumor Node Metastasis) stage. Multivariate analysis identified calpain-4 as an independent prognostic factor for poor prognosis. A predictive nomogram integrating calpain-4 expression with other independent prognosticators was constructed, which generated a better prognostic value for overall survival of gastric cancer patients than a TNM staging system. In conclusion, calpain-4 could be regarded as a potential prognosis indicator for clinical outcomes in gastric cancer. PMID:27689993

  3. Elevated Expression of Calpain-4 Predicts Poor Prognosis in Patients with Gastric Cancer after Gastrectomy

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    Peike Peng

    2016-09-01

    Full Text Available Calpain-4 belongs to the calpain family of calcium-dependent cysteine proteases, and functions as a small regulatory subunit of the calpains. Recent evidence indicates that calpain-4 plays critical roles in tumor migration and invasion. However, the roles of calpain-4 in gastric tumorigenesis remain poorly understood. Herein, we examined calpain-4 expression by immunohistochemical staining on tissue microarrays containing tumor samples of 174 gastric cancer patients between 2004 and 2008 at a single center. The Kaplan-Meier method was used to compare survival curves, and expression levels were correlated to clinicopathological factors and overall survival. Our data demonstrated that calpain-4 was generally increased in gastric cancer cell lines and primary tumor tissues. High expression of calpain-4 was positively associated with vessel invasion, lymph node metastasis, and advanced TNM (Tumor Node Metastasis stage. Multivariate analysis identified calpain-4 as an independent prognostic factor for poor prognosis. A predictive nomogram integrating calpain-4 expression with other independent prognosticators was constructed, which generated a better prognostic value for overall survival of gastric cancer patients than a TNM staging system. In conclusion, calpain-4 could be regarded as a potential prognosis indicator for clinical outcomes in gastric cancer.

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

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    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.

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

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

    2014-11-07

    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.

  6. Impairment of survival signaling and efferocytosis in TRPC3-deficient macrophages

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    Tano, Jean-Yves; Smedlund, Kathryn; Lee, Robert [Department of Physiology and Pharmacology and the Center for Diabetes and Endocrine Research, University of Toledo College of Medicine, Health Science Campus, 3000 Arlington Av, Toledo, OH 43614 (United States); Abramowitz, Joel; Birnbaumer, Lutz [Laboratory of Membrane Signaling, Department of Signal Transduction, National Institute of Environmental Health Science, Research Triangle Park, NC 23709 (United States); Vazquez, Guillermo, E-mail: Guillermo.Vazquez@utoledo.edu [Department of Physiology and Pharmacology and the Center for Diabetes and Endocrine Research, University of Toledo College of Medicine, Health Science Campus, 3000 Arlington Av, Toledo, OH 43614 (United States)

    2011-07-08

    Highlights: {yields} We examined the role of TRPC3 channel in macrophage survival, apoptosis and efferocytic properties. {yields} TRPC3-deficient macrophages exhibit impaired survival signaling, increased apoptosis and impaired efferocytosis. {yields} These findings suggest that macrophage TRPC3 is an essential component for macrophage survival and clearance of apoptotic cells. -- Abstract: We have recently shown that in macrophages proper operation of the survival pathways phosphatidylinositol-3-kinase (PI3K)/AKT and nuclear factor kappa B (NFkB) has an obligatory requirement for constitutive, non-regulated Ca{sup 2+} influx. In the present work we examined if Transient Receptor Potential Canonical 3 (TRPC3), a member of the TRPC family of Ca{sup 2+}-permeable cation channels, contributes to the constitutive Ca{sup 2+} influx that supports macrophage survival. We used bone marrow-derived macrophages obtained from TRPC3{sup -/-} mice to determine the activation status of survival signaling pathways, apoptosis and their efferocytic properties. Treatment of TRPC3{sup +/+} macrophages with the pro-apoptotic cytokine TNF{alpha} induced time-dependent phosphorylation of I{kappa}B{alpha}, AKT and BAD, and this was drastically reduced in TRPC3{sup -/-} macrophages. Compared to TRPC3{sup +/+} cells TRPC3{sup -/-} macrophages exhibited reduced constitutive cation influx, increased apoptosis and impaired efferocytosis. The present findings suggest that macrophage TRPC3, presumably through its constitutive function, contributes to survival signaling and efferocytic properties.

  7. Evolutionary and physical linkage between calpains and penta-EF-hand Ca2+-binding proteins.

    Science.gov (United States)

    Maki, Masatoshi; Maemoto, Yuki; Osako, Yohei; Shibata, Hideki

    2012-04-01

    The name calpain was historically given to a protease that is activated by Ca(2+) and whose primary structure contains a Ca(2+)-binding penta-EF-hand (PEF) as well as a calpain cysteine protease (CysPc) domain and a C2-domain-like (C2L) domain. In the human genome, CysPc domains are found in 15 genes, but only nine of them encode PEF domains. Fungi and budding yeasts have calpain-like sequences that lack the PEF domain, and each protein (designated PalB and Rim13, respectively) is orthologous to human calpain-7, indicating that the calpain-7 orthologs are evolutionarily more conserved than classical calpains possessing PEF domains. An N-terminal region of calpain-7 has a tandem repeat of microtubule-interacting and transport domains that interact with a subset of endosomal sorting complex required for transport (ESCRT) III proteins. In addition to calpains, PEF domains are found in other Ca(2+)-binding proteins including ALG-2 that associates with ALIX (an ESCRT-III accessory protein) and TSG101 (an ESCRT-I subunit). Phylogenetic comparison of dissected domain structures of calpains and experimentally confirmed protein-protein interaction networks imply that there is an evolutionary and physical linkage between mammalian calpains and PEF proteins involving the ESCRT system.

  8. Partial autolysis of μ/m-calpain during post mortem aging of chicken muscle.

    Science.gov (United States)

    Zhao, Liang; Jiang, Nanqi; Li, Miaozhen; Huang, Ming; Zhou, Guanghong

    2016-12-01

    The objective of this study was to investigate changes occurring in μ/m-calpain in post mortem chicken muscles and to determine the origin of the unknown bands found in calpain casein zymography. The unknown bands were reported with slightly greater mobility compared to conventional μ/m-calpain bands in casein zymography. Identification of these bands was accomplished using native polyacrylamide gel electrophoresis, liquid chromatography tandem mass spectrometry and with protein phosphatase treatment. Results showed that the unknown bands were corresponding to μ/m-calpain, and dephosphorylation by protein phosphatase did not change their appearance. The calpain samples were then incubated with various concentrations of Ca(2+) to determine the relationship between changes in μ/m-calpain and the appearance of the unknown bands. The products of μ/m-calpain partial autolysis were found to be consistent with the appearance of the unknown bands. Therefore, the appearance of these bands did not result from phosphorylation of μ/m-calpain as previously hypothesized, but from partial autolysis of μ/m-calpain. Also their presence suggests that μ/m-calpain undergoes partial autolysis during aging which may play certain roles in meat quality improvement.

  9. 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.

  10. Calpain-2-mediated PTEN degradation contributes to BDNF-induced stimulation of dendritic protein synthesis.

    Science.gov (United States)

    Briz, Victor; Hsu, Yu-Tien; Li, Yi; Lee, Erin; Bi, Xiaoning; Baudry, Michel

    2013-03-06

    Memory consolidation has been suggested to be protein synthesis dependent. Previous data indicate that BDNF-induced dendritic protein synthesis is a key event in memory formation through activation of the mammalian target of rapamycin (mTOR) pathway. BDNF also activates calpain, a calcium-dependent cysteine protease, which has been shown to play a critical role in learning and memory. This study was therefore directed at testing the hypothesis that calpain activity is required for BDNF-stimulated local protein synthesis, and at identifying the underlying molecular mechanism. In rat hippocampal slices, cortical synaptoneurosomes, and cultured neurons, BDNF-induced mTOR pathway activation and protein translation were blocked by calpain inhibition. BDNF treatment rapidly reduced levels of hamartin and tuberin, negative regulators of mTOR, in a calpain-dependent manner. Treatment of brain homogenates with purified calpain-1 and calpain-2 truncated both proteins. BDNF treatment increased phosphorylation of both Akt and ERK, but only the effect on Akt was blocked by calpain inhibition. Levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a phosphatase that inactivates Akt, were decreased following BDNF treatment, and calpain inhibition reversed this effect. Calpain-2, but not calpain-1, treatment of brain homogenates resulted in PTEN degradation. In cultured cortical neurons, knockdown of calpain-2, but not calpain-1, by small interfering RNA completely suppressed the effect of BDNF on mTOR activation. Our results reveal a critical role for calpain-2 in BDNF-induced mTOR signaling and dendritic protein synthesis via PTEN, hamartin, and tuberin degradation. This mechanism therefore provides a link between proteolysis and protein synthesis that might contribute to synaptic plasticity.

  11. Distinct regulatory functions of calpain 1 and 2 during neural stem cell self-renewal and differentiation.

    Directory of Open Access Journals (Sweden)

    Daniela M Santos

    Full Text Available Calpains are calcium regulated cysteine proteases that have been described in a wide range of cellular processes, including apoptosis, migration and cell cycle regulation. In addition, calpains have been implicated in differentiation, but their impact on neural differentiation requires further investigation. Here, we addressed the role of calpain 1 and calpain 2 in neural stem cell (NSC self-renewal and differentiation. We found that calpain inhibition using either the chemical inhibitor calpeptin or the endogenous calpain inhibitor calpastatin favored differentiation of NSCs. This effect was associated with significant changes in cell cycle-related proteins and may be regulated by calcium. Interestingly, calpain 1 and calpain 2 were found to play distinct roles in NSC fate decision. Calpain 1 expression levels were higher in self-renewing NSC and decreased with differentiation, while calpain 2 increased throughout differentiation. In addition, calpain 1 silencing resulted in increased levels of both neuronal and glial markers, β-III Tubulin and glial fibrillary acidic protein (GFAP. Calpain 2 silencing elicited decreased levels of GFAP. These results support a role for calpain 1 in repressing differentiation, thus maintaining a proliferative NSC pool, and suggest that calpain 2 is involved in glial differentiation.

  12. 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.

  13. [Advances in the research of the relationship between calpains and post-burn skeletal muscle wasting].

    Science.gov (United States)

    Ma, Li; Chai, Jia-ke

    2013-06-01

    Calpains are intracellular nonlysosomal Ca(2+-) regulated cysteine proteases, widely located in the tissues of most mammals. Skeletal muscle tissue mainly expresses m-calpain, µ-caplain, n-calpain, and their endogenous inhibitor calpastatin. They are closely related to the cell apoptosis, cytoskeleton formation, cell cycles, etc. Calpains are also considered to be participating in the protein degradation process. Severe burns are typically followed by hypermetabolic responses that are characterized by hyperdynamic circulatory responses with increased proteolysis and cell apoptosis. Recently, overloading of Ca(2+) in skeletal muscle cells, which activates the calpains is observed after a serious burn. This paper aims to review the current research of the relationship between calpains and post-burn skeletal muscle wasting from the perspectives of structure, function, and physiological activities.

  14. Increased Calpain Correlates with Th1 Cytokine Profile in PBMCs from MS Patients

    Science.gov (United States)

    Imam, Sarah A.; Guyton, Mary K.; Haque, Azizul; Vandenbark, Arthur; Tyor, William R.; Ray, Swapan K.; Banik, Naren L.

    2007-01-01

    Multiple sclerosis (MS) is a devastating autoimmune demyelinating disease of the CNS. This study investigated whether expression and activity of the calcium-activated protease calpain correlated with Th1/Th2 dysregulation in MS patients during states of relapse and remission. Calpain expression and activity were significantly increased in peripheral blood mononuclear cells (PBMCs) from MS patients, compared to controls, with the highest expression and activity noted during relapse. Th1 cytokines were highest and Th2 cytokines were lowest in MS patients during relapse. Treatment with calpain inhibitor, calpeptin, decreased Th1 cytokines in PBMCs from MS patients. Calpain inhibitor also reduced degradation of myelin basic protein (MBP) by inhibiting the calpain secreted from MBP-specific T cells. Taken together, these results suggested calpain involvement in Th1/Th2 dysregulation in MS patients. PMID:17765980

  15. 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

    2001-11-09

    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.

  16. Role of calpain-10 in the development of diabetes mellitus and its complications.

    Science.gov (United States)

    Pánico, Pablo; Salazar, Ana María; Burns, Anna L; Ostrosky-Wegman, Patricia

    2014-02-01

    Calpain activity has been implicated in several cellular processes such as cell signaling, apoptosis, exocytosis, mitochondrial metabolism and cytoskeletal remodeling. Evidence has indicated that the impairment of calpain expression and the activity of different calpain family members are involved in diverse pathologies. Calpain-10 has been implicated in the development of type 2 diabetes, and polymorphisms in the CAPN10 gene have been associated with an increased risk of developing this disease. The present work focused on the molecular biology of calpain-10, supporting its key participation in glucose metabolism. Current knowledge regarding the role of calpain-10 in the development of type 2 diabetes mellitus and diabetes-related diseases is additionally reviewed.

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

    Directory of Open Access Journals (Sweden)

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

    2008-01-01

    Full Text Available 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 expression of calpain 10, μ-calpain, and m-calpain in skeletal muscles were determined by RT-PCR, using β-actin as internal standard. We found exercise is useful for BW lossing, especially in the obese rats. The BW difference between OS and OE rats (69 g vs. 18.2 g was more significantly than that between LS and LE rats (41.8 g vs. 28.7g. The calpain 10 expression of LS rats (0.965 was lower than that of LE rats (1.006, whereas those of OS and OE were comparable. The μ- or m-calpain expressions of sedentary groups (OS, LS was significantly higher than those of exercise groups (OE, LE. The μ-calpain expression (1.13/0.92 and m-calpain expression (1.01/0.99 of OS/LS rats was significantly higher than those of OE/LE rats [1.07/0.9 (μ-calpain; 0.97/0.95 (m-calpain]. We concluded that the μ- or m-calpains in skeletal muscle are regulated by exercise in both lean and obese Zucker rats. Exercise and BW controlling might improve the physiopathology of obesity and diabetes. Both μ- or m-calpains might become useful markers for prognoses of diabetes.

  18. 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

  19. Epistasis Between Calpain 1 and Its Inhibitor Calpastatin Within Breeds of Cattle

    OpenAIRE

    Barendse, W.; Harrison, B. E.; Hawken, R. J.; Ferguson, D. M.; Thompson, J.M.; Thomas, M. B.; Bunch, R. J.

    2007-01-01

    The calpain gene family and its inhibitors have diverse effects, many related to protein turnover, which appear to affect a range of phenotypes such as diabetes, exercise-induced muscle injury, and pathological events associated with degenerative neural diseases in humans, fertility, longevity, and postmortem effects on meat tenderness in livestock species. The calpains are inhibited by calpastatin, which binds directly to calpain. Here we report the direct measurement of epistatic interactio...

  20. 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

    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...

  1. Suppression of cancer cell migration and invasion by protein phosphatase 2A through dephosphorylation of mu- and m-calpains.

    Science.gov (United States)

    Xu, Lijun; Deng, Xingming

    2006-11-17

    The mu- and m-calpains are major members of the calpain family that play an essential role in regulating cell motility. We have recently discovered that nicotine-activated protein kinase C iota enhances calpain phosphorylation in association with enhanced calpain activity and accelerated migration and invasion of human lung cancer cells. Here we found that specific disruption of protein phosphatase 2A (PP2A) activity by expression of SV40 small tumor antigen up-regulates phosphorylation of mu- and m-calpains whereas C2-ceramide, a potent PP2A activator, reduces nicotine-induced calpain phosphorylation, suggesting that PP2A may function as a physiological calpain phosphatase. PP2A co-localizes and interacts with mu- and m-calpains. Purified, active PP2A directly dephosphorylates mu- and m-calpains in vitro. Overexpression of the PP2A catalytic subunit (PP2A/C) suppresses nicotine-stimulated phosphorylation of mu- and m-calpains, which is associated with inhibition of calpain activity, wound healing, cell migration, and invasion. By contrast, depletion of PP2A/C by RNA interference enhances calpain phosphorylation, calpain activity, cell migration, and invasion. Importantly, C2-ceramide-induced suppression of calpain phosphorylation results in decreased secretion of mu- and m-calpains from lung cancer cells into culture medium, which may have potential clinic relevance in controlling metastasis of lung cancer. These findings reveal a novel role for PP2A as a physiological calpain phosphatase that not only directly dephosphorylates but also inactivates mu- and m-calpains, leading to suppression of migration and invasion of human lung cancer cells.

  2. Immunological detection of m- and µ-calpains in the skeletal muscle of Marchigiana cattle

    Directory of Open Access Journals (Sweden)

    E. Varricchio

    2013-01-01

    Full Text Available Calpains are Ca2+-dependent proteases able to cleave a large number of proteins involved in many biological functions. Particularly, in skeletal muscle they are involved in meat tenderizing during post mortem storage. In this report we analyzed the presence and expression of µ- and m-calpains in two skeletal muscles of the Marchigiana cattle soon after slaughter, using immunocytochemical and immunohistochemical techniques, Western blotting analysis and Casein Zymography. Therefore, the presence and the activity of these proteases was investigated until 15th day post-mortem during normal process of meat tenderizing. The results showed m- and µ-calpain immunosignals in the cytoplasm both along the Z disk/I band regions and in the form of intracellular stores. Moreover, the expression level of µ-calpain but not m-calpain decreased after 10 days of storage. Such a decrease in µ-calpain was accompanied by a gradual reduction of activity. On the contrary, m-calpain activity persisted up to 15 days of post-mortem storage. Such data indicate that expression and activity of both µ-calpain and m-calpain analyzed in the Marchigiana cattle persist longer than reported in literature for other bovines and may be related to both the type of muscle and breed examined.

  3. Calpains are required for invasive and metastatic potentials of human HCC cells.

    Science.gov (United States)

    Chen, Bo; Tang, Juan; Guo, Yun-Shan; Li, Yong; Chen, Zhi-Nan; Jiang, Jian-Li

    2013-07-01

    Calpains are a conserved family of calcium-dependent cysteine proteinases involved in various cellular functions. Two ubiquitous isoforms, µ- and m-calpain, are key members of the calpain family that play essential roles in regulating cell migration and invasion. However, it remains unclear whether they are involved in the progression of hepatocellular carcinoma (HCC). Here, we investigated the functions of µ- and m-calpain in the invasive and metastatic processes of human hepatoma cells. Our results indicated that the expression levels of calpains were elevated in HCC cells compared with those in normal hepatic cells. Our results indicated that small interfering RNA (siRNA)-mediated silencing of µ- and m-calpain expressions significantly suppressed the adhesive, migrative and invasive potentials of human hepatoma cells. The matrix metalloproteinases (MMPs) are key regulators of malignant tumour invasion and metastasis. siRNA-mediated down-regulation of µ- and m-calpain expressions also significantly attenuated MMP-2 and MMP-9 secretion. Thus µ- and m-calpain may play important roles in the invasion and metastasis of human hepatoma cells, and calpains may be drug targets for preventing HCC metastasis.

  4. 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.

  5. Immunological detection of m- and µ-calpains in the skeletal muscle of Marchigiana cattle.

    Science.gov (United States)

    Varricchio, E; Russolillo, M G; Maruccio, L; Velotto, S; Campanile, G; Paolucci, M; Russo, F

    2013-01-14

    Calpains are Ca(2+)-dependent proteases able to cleave a large number of proteins involved in many biological functions. Particularly, in skeletal muscle they are involved in meat tenderizing during post mortem storage. In this report we analyzed the presence and expression of µ- and m-calpains in two skeletal muscles of the Marchigiana cattle soon after slaughter, using immunocytochemical and immunohistochemical techniques, Western blotting analysis and Casein Zymography. Therefore, the presence and the activity of these proteases was investigated until 15th day post-mortem during normal process of meat tenderizing. The results showed m- and µ-calpain immunosignals in the cytoplasm both along the Z disk/I band regions and in the form of intracellular stores. Moreover, the expression level of µ-calpain but not m-calpain decreased after 10 days of storage. Such a decrease in µ-calpain was accompanied by a gradual reduction of activity. On the contrary, m-calpain activity persisted up to 15 days of post-mortem storage. Such data indicate that expression and activity of both µ-calpain and m-calpain analyzed in the Marchigiana cattle persist longer than reported in literature for other bovines and may be related to both the type of muscle and breed examined.

  6. Characterization of the definitive classical calpain family of vertebrates using phylogenetic, evolutionary and expression analyses.

    Science.gov (United States)

    Macqueen, Daniel J; Wilcox, Alexander H

    2014-04-09

    The calpains are a superfamily of proteases with extensive relevance to human health and welfare. Vast research attention is given to the vertebrate 'classical' subfamily, making it surprising that the evolutionary origins, distribution and relationships of these genes is poorly characterized. Consequently, there exists uncertainty about the conservation of gene family structure, function and expression that has been principally defined from work with mammals. Here, more than 200 vertebrate classical calpains were incorporated in phylogenetic analyses spanning an unprecedented range of taxa, including jawless and cartilaginous fish. We demonstrate that the common vertebrate ancestor had at least six classical calpains, including a single gene that gave rise to CAPN11, 1, 2 and 8 in the early jawed fish lineage, plus CAPN3, 9, 12, 13 and a novel calpain gene, hereafter named CAPN17. We reveal that while all vertebrate classical calpains have been subject to persistent purifying selection during evolution, the degree and nature of selective pressure has often been lineage-dependent. The tissue expression of the complete classic calpain family was assessed in representative teleost fish, amphibians, reptiles and mammals. This highlighted systematic divergence in expression across vertebrate taxa, with most classic calpain genes from fish and amphibians having more extensive tissue distribution than in amniotes. Our data suggest that classical calpain functions have frequently diverged during vertebrate evolution and challenge the ongoing value of the established system of classifying calpains by expression.

  7. calpain and cell apoptosis%calpain与细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    张巍; 官大威; 刘嵘; 周哲

    2007-01-01

    calpain是钙激活中性蛋白酶,属于半胱氨酸蛋白水解酶超家族成员.是由分子量为80KD的催化亚单位和分子量为30KD的调解亚单位组成的异二聚体.研究最多的是calpain1和2,它们又称μ-calpain和m-calpain.calpain被认为与细胞骨架重塑、信号转导途径、调控细胞周期、基因表达调控、某些凋亡途径以及长程增强效应等有关.calpain参与了细胞凋亡,而calpain抑制剂可以阻止细胞的凋亡.

  8. Calpains: attractive targets for the development of synthetic inhibitors.

    Science.gov (United States)

    Pietsch, Markus; Chua, Krystle C H; Abell, Andrew D

    2010-01-01

    The physiological roles of calpains are discussed, as are the associated pathological disorders that result from their over-activation. We also present practical information for establishing functional inhibition assays and an overview of X-ray crystal structures of calpain-inhibitor complexes to aid inhibitor design. These structures reveal the expected extended beta-strand conformation for the inhibitor backbone, a geometry that has been engineered into inhibitors with the introduction of either an N-terminal heterocycle or a macrocycle that links the P(1) and P(3) residues. The structure and function of all the main classes of inhibitors are reviewed, with most examples being classified according to the nature of the C-terminal reactive warhead group that reacts with the active site cysteine of calpains. These inhibitor classes include epoxysuccinate derivatives, aldehydes, aldehyde prodrugs (hemiacetals) and alpha-keto carbonyl compounds. Inhibitors derived from the endogenous inhibitor calpastatin and examples lacking a warhead, are now known and these are also discussed.

  9. Complement 3-deficient mice are not protected against MPTP-induced dopaminergic neurotoxicity.

    Science.gov (United States)

    Liang, Yajie; Li, Shurong; Guo, Qiang; Zhang, Yanling; Zhang, Yanliang; Wen, Can; Zou, Qiang; Su, Bingyin

    2007-10-31

    Recent studies have invoked inflammation as a major contributor to the pathogenesis of Parkinson's disease (PD). Emerging evidence indicated that components of complement system may be involved in such disorder and contribute to its development. We thus observed the influence of deficiency of complement 3 (C3), the key component of complement system, on the death of dopaminergic neurons in substantia nigra pars compacta (SNpc) and the loss of dopaminergic fibers in striatum induced by acute or chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Immunohistochemical staining of dopaminergic neurons in SNpc and neurochemical analysis of dopamine and its metabolites in striata revealed that there was no significant difference between the two genotypes. Longer survival time also indicated that C3 might not mediate the spontaneous recovery of dopaminergic fibers in mouse striatum acutely challenged by MPTP. We conclude that, despite growing evidence indicating the involvement of complement system in the pathogenesis of PD, our data do not support a role for C3 in this established model of PD, as indicated by results from HPLC analysis and immunohistochemical staining.

  10. The atypical calpains: evolutionary analyses and roles in Caenorhabditis elegans cellular degeneration.

    Directory of Open Access Journals (Sweden)

    Peter I Joyce

    Full Text Available The calpains are physiologically important Ca(2+-activated regulatory proteases, which are divided into typical or atypical sub-families based on constituent domains. Both sub-families are present in mammals, but our understanding of calpain function is based primarily on typical sub-family members. Here, we take advantage of the model organism Caenorhabditis elegans, which expresses only atypical calpains, to extend our knowledge of the phylogenetic evolution and function of calpains. We provide evidence that a typical human calpain protein with a penta EF hand, detected using custom profile hidden Markov models, is conserved in ancient metazoans and a divergent clade. These analyses also provide evidence for the lineage-specific loss of typical calpain genes in C. elegans and Ciona, and they reveal that many calpain-like genes lack an intact catalytic triad. Given the association between the dysregulation of typical calpains and human degenerative pathologies, we explored the phenotypes, expression profiles, and consequences of inappropriate reduction or activation of C. elegans atypical calpains. These studies show that the atypical calpain gene, clp-1, contributes to muscle degeneration and reveal that clp-1 activity is sensitive to genetic manipulation of [Ca(2+](i. We show that CLP-1 localizes to sarcomeric sub-structures, but is excluded from dense bodies (Z-disks. We find that the muscle degeneration observed in a C. elegans model of dystrophin-based muscular dystrophy can be suppressed by clp-1 inactivation and that nemadipine-A inhibition of the EGL-19 calcium channel reveals that Ca(2+ dysfunction underlies the C. elegans MyoD model of myopathy. Taken together, our analyses highlight the roles of calcium dysregulation and CLP-1 in muscle myopathies and suggest that the atypical calpains could retain conserved roles in myofilament turnover.

  11. The atypical calpains: evolutionary analyses and roles in Caenorhabditis elegans cellular degeneration.

    Science.gov (United States)

    Joyce, Peter I; Satija, Rahul; Chen, Maozi; Kuwabara, Patricia E

    2012-01-01

    The calpains are physiologically important Ca(2+)-activated regulatory proteases, which are divided into typical or atypical sub-families based on constituent domains. Both sub-families are present in mammals, but our understanding of calpain function is based primarily on typical sub-family members. Here, we take advantage of the model organism Caenorhabditis elegans, which expresses only atypical calpains, to extend our knowledge of the phylogenetic evolution and function of calpains. We provide evidence that a typical human calpain protein with a penta EF hand, detected using custom profile hidden Markov models, is conserved in ancient metazoans and a divergent clade. These analyses also provide evidence for the lineage-specific loss of typical calpain genes in C. elegans and Ciona, and they reveal that many calpain-like genes lack an intact catalytic triad. Given the association between the dysregulation of typical calpains and human degenerative pathologies, we explored the phenotypes, expression profiles, and consequences of inappropriate reduction or activation of C. elegans atypical calpains. These studies show that the atypical calpain gene, clp-1, contributes to muscle degeneration and reveal that clp-1 activity is sensitive to genetic manipulation of [Ca(2+)](i). We show that CLP-1 localizes to sarcomeric sub-structures, but is excluded from dense bodies (Z-disks). We find that the muscle degeneration observed in a C. elegans model of dystrophin-based muscular dystrophy can be suppressed by clp-1 inactivation and that nemadipine-A inhibition of the EGL-19 calcium channel reveals that Ca(2+) dysfunction underlies the C. elegans MyoD model of myopathy. Taken together, our analyses highlight the roles of calcium dysregulation and CLP-1 in muscle myopathies and suggest that the atypical calpains could retain conserved roles in myofilament turnover.

  12. Expression of Calpain 1 and 2 in Liver Cancer Cells with Different Migration Abilities%Calpain-1及Calpain-2在不同迁移能力肝癌细胞系中的表达

    Institute of Scientific and Technical Information of China (English)

    王宁; 陈腾祥; 刘振华; 王飞清; 潘娅

    2016-01-01

    目的:研究钙激活中性蛋白酶蛋白-1(Calpain-1)及Calpain-2在不同迁移能力肝癌细胞系中的表达.方法:常规细胞培养高迁移能力的MHCC97-H、低迁移能力的MHCC97-L、无迁移能力的HepG2的人肝癌细胞及正常人肝HL-7702细胞,采用western-bolt方法观察Calpain-1及Calpain-2在3株人肝癌细胞及正常肝细胞中的表达.结果:Calpain-1在正常肝细胞HL-7702、高迁移能力的MHCC97-H、低迁移能力的MHCC97-L及无迁移能力的HepG2细胞中均有表达,表达量差异无统计学意义,P>0.05;与正常肝细胞HL-7702比较,高、低、迁移能力肝癌细胞系中Calpain-2不同程度表达,随着细胞迁移能力增强而成递增趋势,P<0.01或P<0.05.结论:Calpain-2随肝癌细胞迁移能力的增强表达增加,表明Calpain-2与肝癌细胞的侵袭转移可能有密切关系.

  13. Myofibrillar protein turnover: the proteasome and the calpains.

    Science.gov (United States)

    Goll, D E; Neti, G; Mares, S W; Thompson, V F

    2008-04-01

    Metabolic turnover of myofibrillar proteins in skeletal muscle requires that, before being degraded to AA, myofibrillar proteins be removed from the myofibril without disrupting the ability of the myofibril to contract and develop tension. Skeletal muscle contains 4 proteolytic systems in amounts such that they could be involved in metabolic protein turnover: 1) the lysosomal system, 2) the caspase system, 3) the calpain system, and 4) the proteasome. The catheptic proteases in lysosomes are not active at the neutral pH of the cell cytoplasm, so myofibrillar proteins would have to be degraded inside lysosomes if the lysosomal system were involved. Lysosomes could not engulf a myofibril without destroying it, so the lysosomal system is not involved to a significant extent in metabolic turnover of myofibrillar proteins. The caspases are not activated until initiation of apoptosis, and, therefore, it is unlikely that the caspases are involved to a significant extent in myofibrillar protein turnover. The calpains do not degrade proteins to AA or even to small peptides and do not catalyze bulk degradation of the sarcoplasmic proteins, so they cannot be the only proteolytic system involved in myofibrillar protein turnover. Research during the past 20 yr has shown that the proteasome is responsible for 80 to 90% of total intracellular protein turnover, but the proteasome degrades peptide chains only after they have been unfolded, so that they can enter the catalytic chamber of the proteasome. Thus, although the proteasome can degrade sarcoplasmic proteins, it cannot degrade myofibrillar proteins until they have been removed from the myofibril. It remains unclear how this removal is done. The calpains degrade those proteins that are involved in keeping the myofibrillar proteins assembled in myofibrils, and it was proposed over 30 yr ago that the calpains initiated myofibrillar protein turnover by disassembling the outer layer of proteins from the myofibril and releasing

  14. Calcium-dependent proteolytic system and muscle dysfunctions: a possible role of calpains in sarcopenia.

    Science.gov (United States)

    Dargelos, E; Poussard, S; Brulé, C; Daury, L; Cottin, P

    2008-02-01

    The calcium-dependent proteolytic system is composed of cysteine proteases named calpains. They are ubiquitous or tissue-specific enzymes. The two best characterised isoforms are the ubiquitously expressed mu- and m-calpains. Besides its regulation by calcium, calpain activity is tightly controlled by calpastatin, the specific endogenous inhibitor, binding to phospholipids, autoproteolysis and phosphorylation. Calpains are responsible for limited proteolytic events. Among the multitude of substrates identified so far are cytoskeletal and membrane proteins, enzymes and transcription factors. Calpain activity is involved in a large number of physiological and pathological processes. In this review, we will particularly focus on the implication of the calcium-dependent proteolytic system in relation to muscle physiology. Because of their ability to remodel cytoskeletal anchorage complexes, calpains play a major role in the regulation of cell adhesion, migration and fusion, three key steps of myogenesis. Calcium-dependent proteolysis is also involved in the control of cell cycle. In muscle tissue, in particular, calpains intervene in the regeneration process. Another important class of calpain substrates belongs to apoptosis regulating factors. The proteases may thus play a role in muscle cell death, and as a consequence in muscle atrophy. The relationships between calcium-dependent proteolysis and muscle dysfunctions are being further developed in this review with a particular emphasis on sarcopenia.

  15. The activity of calpains in lymphocytes is glucose-dependent and is decreased in diabetic patients.

    Science.gov (United States)

    Díaz-Villaseñor, Andrea; Hiriart, Marcia; Cebrián, Mariano E; Zacarías-Castillo, Rogelio; Ostrosky-Wegman, Patricia

    2008-01-01

    Calpains are nonlysosomal calcium-dependent cysteine proteases that participate in insulin secretion and action. Polymorphisms in the calpain-10 gene have been shown to increase the risk for type 2 diabetes. Since white blood cells have been used to study glucose homeostasis, the present study was carried to find out if calpains have different activity and/or expression in accessible cells such as lymphocytes of individuals with or without type 2 diabetes. Fasting blood glucose concentration was significantly higher in diabetic subjects, whereas the difference in the activity of calpains evaluated in basal and stimulating extracellular glucose concentration was significantly higher in the lymphocytes from the control group. The mRNA expression of calpain-10 was similar in the lymphocytes of both patients and controls. The protein blots showed four bands that ranged between 75 and 50 kDa; however, no statistical differences were observed in the expression of the calpain-10 isoforms between controls and patients. Data obtained showed that human lymphocytes express calpain-10 mRNA and protein, showing a similar expression between diabetic and control subjects, nevertheless in the diabetic group calpain activity was less glucose-sensitive.

  16. Calpain inhibitors reduce retinal hypoxia in ischemic retinopathy by improving neovascular architecture and functional perfusion.

    Science.gov (United States)

    Hoang, Mien V; Smith, Lois E H; Senger, Donald R

    2011-04-01

    In ischemic retinopathies, underlying hypoxia drives abnormal neovascularization that damages retina and causes blindness. The abnormal neovasculature is tortuous and leaky and fails to alleviate hypoxia, resulting in more pathological neovascularization and retinal damage. With an established model of ischemic retinopathy we found that calpain inhibitors, when administered in moderation, reduced architectural abnormalities, reduced vascular leakage, and most importantly reduced retinal hypoxia. Mechanistically, these calpain inhibitors improved stability and organization of the actin cytoskeleton in retinal endothelial cells undergoing capillary morphogenesis in vitro, and they similarly improved organization of actin cables within new blood vessels in vivo. Hypoxia induced calpain activity in retinal endothelial cells and severely disrupted the actin cytoskeleton, whereas calpain inhibitors preserved actin cables under hypoxic conditions. Collectively, these findings support the hypothesis that hyper-activation of calpains by hypoxia contributes to disruption of the retinal endothelial cell cytoskeleton, resulting in formation of neovessels that are defective both architecturally and functionally. Modest suppression of calpain activity with calpain inhibitors restores cytoskeletal architecture and promotes formation of a functional neovasculature, thereby reducing underlying hypoxia. In sharp contrast to "anti-angiogenesis" strategies that cannot restore normoxia and may aggravate hypoxia, the therapeutic strategy described here does not inhibit neovascularization. Instead, by improving the function of neovascularization to reduce underlying hypoxia, moderate calpain inhibition offers a method for alleviating retinal ischemia, thereby suggesting a new treatment paradigm based on improvement rather than inhibition of new blood vessel growth.

  17. Calpain inhibition prevents amyloid-beta-induced neurodegeneration and associated behavioral dysfunction in rats

    NARCIS (Netherlands)

    Granic, Ivica; Nyakas, Csaba; Luiten, Paul G. M.; Eisel, Ulrich L. M.; Halmy, Laszlo G.; Gross, Gerhard; Schoemaker, Hans; Moeller, Achim; Nimmrich, Volker

    2010-01-01

    Amyloid-beta (A beta) is toxic to neurons and such toxicity is - at least in part - mediated via the NMDA receptor. Calpain, a calcium dependent cystein protease, is part of the NMDA receptor-induced neurodegeneration pathway, and we previously reported that inhibition of calpain prevents excitotoxi

  18. Calpains mediate epithelial-cell death during mammary gland involution: mitochondria and lysosomal destabilization.

    Science.gov (United States)

    Arnandis, T; Ferrer-Vicens, I; García-Trevijano, E R; Miralles, V J; García, C; Torres, L; Viña, J R; Zaragozá, R

    2012-09-01

    Our aim was to elucidate the physiological role of calpains (CAPN) in mammary gland involution. Both CAPN-1 and -2 were induced after weaning and its activity increased in isolated mitochondria and lysosomes. CAPN activation within the mitochondria could trigger the release of cytochrome c and other pro-apoptotic factors, whereas in lysosomes it might be essential for tissue remodeling by releasing cathepsins into the cytosol. Immunohistochemical analysis localized CAPNs mainly at the luminal side of alveoli. During weaning, CAPNs translocate to the lysosomes processing membrane proteins. To identify these substrates, lysosomal fractions were treated with recombinant CAPN and cleaved products were identified by 2D-DIGE. The subunit b(2) of the v-type H(+) ATPase is proteolyzed and so is the lysosomal-associated membrane protein 2a (LAMP2a). Both proteins are also cleaved in vivo. Furthermore, LAMP2a cleavage was confirmed in vitro by addition of CAPNs to isolated lysosomes and several CAPN inhibitors prevented it. Finally, in vivo inhibition of CAPN1 in 72-h-weaned mice decreased LAMP2a cleavage. Indeed, calpeptin-treated mice showed a substantial delay in tissue remodeling and involution of the mammary gland. These results suggest that CAPNs are responsible for mitochondrial and lysosomal membrane permeabilization, supporting the idea that lysosomal-mediated cell death is a new hallmark of mammary gland involution.

  19. Role of calpains in the injury-induced dysfunction and degeneration of the mammalian axon.

    Science.gov (United States)

    Ma, Marek

    2013-12-01

    Axonal injury and degeneration, whether primary or secondary, contribute to the morbidity and mortality seen in many acquired and inherited central nervous system (CNS) and peripheral nervous system (PNS) disorders, such as traumatic brain injury, spinal cord injury, cerebral ischemia, neurodegenerative diseases, and peripheral neuropathies. The calpain family of proteases has been mechanistically linked to the dysfunction and degeneration of axons. While the direct mechanisms by which transection, mechanical strain, ischemia, or complement activation trigger intra-axonal calpain activity are likely different, the downstream effects of unregulated calpain activity may be similar in seemingly disparate diseases. In this review, a brief examination of axonal structure is followed by a focused overview of the calpain family. Finally, the mechanisms by which calpains may disrupt the axonal cytoskeleton, transport, and specialized domains (axon initial segment, nodes, and terminals) are discussed.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation

    DEFF Research Database (Denmark)

    Grossi, Alberto; Karlsson, Anders H; Lawson, Moira Ann

    2008-01-01

    reorganization due to the activity of the ubiquitous proteolytic enzymes, calpains, has been reported. Whether there is a link between stretch- or load-induced signaling and calpain expression and activation is not known. Using a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have...... demonstrated that mechanical stimulation via laminin receptors leads to an increase in m-calpain expression, but no increase in the expression of other calpain isoforms. Our study revealed that after a short period of stimulation, m-calpain relocates into focal adhesion complexes and is followed by a breakdown...... of specific focal adhesion proteins previously identified as substrates for this enzyme. We show that stimulation also leads to an increase in calpain activity in these cells. These data support the pivotal role for m-calpain in the control of muscle precursor cell differentiation and thus strengthen the idea...

  5. Extracellular calpains increase tubular epithelial cell mobility. Implications for kidney repair after ischemia.

    Science.gov (United States)

    Frangié, Carlos; Zhang, Wenhui; Perez, Joëlle; Dubois, Yi-Chun Xu; Haymann, Jean-Philippe; Baud, Laurent

    2006-09-08

    Calpains are intracellular Ca2+-dependent cysteine proteases that are released in the extracellular milieu by tubular epithelial cells following renal ischemia. Here we show that externalized calpains increase epithelial cell mobility and thus are critical for tubule repair. In vitro, exposure of human tubular epithelial cells (HK-2 cells) to mu-calpain limited their adhesion to extracellular matrix and increased their mobility. Calpains acted primarily by promoting the cleavage of fibronectin, thus preventing fibronectin binding to the integrin alphavbeta3. Analyzing downstream integrin effects, we found that the cyclic AMP-dependent protein kinase A pathway was activated in response to alphavbeta3 disengagement and was essential for calpain-mediated increase in HK-2 cell mobility. In a murine model of ischemic acute renal failure, injection of a fragment of calpastatin, which specifically blocked calpain activity in extracellular milieu, markedly delayed tubule repair, increasing functional and histological lesions after 24 and 48 h of reperfusion. These findings suggest that externalized calpains are critical for tubule repair process in acute renal failure.

  6. Changes in calpains and calpastatin in the soleus muscle of Daurian ground squirrels during hibernation.

    Science.gov (United States)

    Yang, Chen-Xi; He, Yue; Gao, Yun-Fang; Wang, Hui-Ping; Goswami, Nandu

    2014-10-01

    We investigated changes in muscle mass, calpains, calpastatin and Z-disk ultrastructure in the soleus muscle (SOL) of Daurian ground squirrels (Spermophilus dauricus) after hibernation or hindlimb suspension to determine possible mechanisms by which muscle atrophy is prevented in hibernators. Squirrels (n=30) were divided into five groups: no hibernation group (PRE, n=6); hindlimb suspension group (HLS, n=6); two month hibernation group (HIB, n=6); two day group after 90±12 days of hibernation (POST, n=6); and forced exercise group (one time forced, moderate-intensity treadmill exercise) after arousal (FE, n=6). Activity and protein expression of calpains were determined by casein zymography and western blotting, and Z-disk ultrastructure was observed by transmission electron microscopy. The following results were found. Lower body mass and higher SOL muscle mass (mg) to total body mass (g) ratio were observed in HIB and POST; calpain-1 activity increased significantly by 176% (P=0.034) in HLS compared to the PRE group; no significant changes were observed in calpain-2 activity. Protein expression of calpain-1 and calpain-2 increased by 83% (P=0.041) and 208% (P=0.029) in HLS compared to the PRE group, respectively; calpastatin expression increased significantly by 180% (Pcalpain activity and consequently calpain-mediated protein degradation by highly elevated calpastatin protein expression levels may be an important mechanism for preventing muscle protein loss during hibernation and ensuring that Z-lines remained ultrastructurally intact.

  7. Calcium influx and calpain activation mediate preclinical retinal neurodegeneration in autoimmune optic neuritis.

    Science.gov (United States)

    Hoffmann, Dorit B; Williams, Sarah K; Bojcevski, Jovana; Müller, Andreas; Stadelmann, Christine; Naidoo, Vinogran; Bahr, Ben A; Diem, Ricarda; Fairless, Richard

    2013-08-01

    Optic neuritis is a common manifestation of multiple sclerosis, an inflammatory demyelinating disease of the CNS. Recently, the neurodegenerative component of multiple sclerosis has come under focus particularly because permanent disability in patients correlates well with neurodegeneration; and observations in both humans and multiple sclerosis animal models highlight neurodegeneration of retinal ganglion cells as an early event. After myelin oligodendrocyte glycoprotein immunization of Brown Norway rats, significant retinal ganglion cell loss precedes the onset of pathologically defined autoimmune optic neuritis. To study the role calcium and calpain activation may play in mediating early degeneration, manganese-enhanced magnetic resonance imaging was used to monitor preclinical calcium elevations in the retina and optic nerve of myelin oligodendrocyte glycoprotein-immunized Brown Norway rats. Calcium elevation correlated with an increase in calpain activation during the induction phase of optic neuritis, as revealed by increased calpain-specific cleavage of spectrin. The relevance of early calpain activation to neurodegeneration during disease induction was addressed by performing treatment studies with the calpain inhibitor calpeptin. Treatment not only reduced calpain activity but also protected retinal ganglion cells from preclinical degeneration. These data indicate that elevation of retinal calcium levels and calpain activation are early events in autoimmune optic neuritis, providing a potential therapeutic target for neuroprotection.

  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.

  9. Suppression of Calpain Expression by NSAIDs is Associated with Inhibition of Cell Migration in Rat Duodenum.

    Science.gov (United States)

    Silver, Kristopher; Littlejohn, A; Thomas, Laurel; Bawa, Bhupinder; Lillich, James D

    2017-03-22

    Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the alleviation of pain and inflammation, but these drugs are also associated with a suite of negative side effects. Gastrointestinal (GI) toxicity is particularly concerning since it affects an estimated 70% of individuals taking NSAIDs routinely, and evidence suggests the majority of toxicity is occurring in the small intestine. Traditionally, NSAID-induced GI toxicity has been associated with indiscriminate inhibition of cyclooxygenase isoforms, but other mechanisms, including inhibition of cell migration, intestinal restitution, and wound healing, are likely to contribute to toxicity. Previous efforts demonstrated that treatment of cultured intestinal epithelial cells (IEC) with NSAIDs inhibits expression and activity of calpain proteases, but the effects of specific inhibition of calpain expression in vitro or the effects of NSAIDs on intestinal cell migration in vivo remain to be determined. Accordingly, we examined the effect of suppression of calpain protease expression with siRNA on cell migration in cultured IECs and evaluated the effects of NSAID treatment on epithelial cell migration and calpain protease expression in rat duodenum. Our results show that calpain siRNA inhibits protease expression and slows migration in cultured IECs. Additionally, NSAID treatment of rats slowed migration up the villus axis and suppressed calpain expression in duodenal epithelial cells. Our results are supportive of the hypothesis that suppression of calpain expression leading to slowing of cell migration is a potential mechanism through which NSAIDs cause GI toxicity.

  10. Brain-derived neurotrophic factor and epidermal growth factor activate neuronal m-calpain via mitogen-activated protein kinase-dependent phosphorylation.

    Science.gov (United States)

    Zadran, Sohila; Jourdi, Hussam; Rostamiani, Karoline; Qin, Qingyu; Bi, Xiaoning; Baudry, Michel

    2010-01-20

    Calpain is a calcium-dependent protease that plays a significant role in synaptic plasticity, cell motility, and neurodegeneration. Two major calpain isoforms are present in brain, with mu-calpain (calpain1) requiring micromolar calcium concentrations for activation and m-calpain (calpain2) needing millimolar concentrations. Recent studies in fibroblasts indicate that epidermal growth factor (EGF) can activate m-calpain independently of calcium via mitogen-activated protein kinase (MAPK)-mediated phosphorylation. In neurons, MAPK is activated by both brain-derived neurotrophic factor (BDNF) and EGF. We therefore examined whether these growth factors could activate m-calpain by MAPK-dependent phosphorylation using cultured primary neurons and HEK-TrkB cells, both of which express BDNF and EGF receptors. Calpain activation was monitored by quantitative analysis of spectrin degradation and by a fluorescence resonance energy transfer (FRET)-based assay, which assessed the truncation of a calpain-specific peptide flanked by the FRET fluorophore pair DABCYL and EDANS. In both cell types, BDNF and EGF rapidly elicited calpain activation, which was completely blocked by MAPK and calpain inhibitors. BDNF stimulated m-calpain but not mu-calpain serine phosphorylation, an effect also blocked by MAPK inhibitors. Remarkably, BDNF- and EGF-induced calpain activation was preferentially localized in dendrites and dendritic spines of hippocampal neurons and was associated with actin polymerization, which was prevented by calpain inhibition. Our results indicate that, in cultured neurons, both BDNF and EGF activate m-calpain by MAPK-mediated phosphorylation. These results strongly support a role for calpain in synaptic plasticity and may explain why m-calpain, although widely expressed in CNS, requires nonphysiological calcium levels for activation.

  11. Calpains mediate the proteolytic modification of human cytomegalovirus UL112-113 proteins.

    Science.gov (United States)

    Wang, Shang-Kwei; Jiang, Meei Jyh; Lin, Shin-Rung; Chen, Mei-Yin; Wang, Hung-Hsueh; Duh, Chang-Yih

    2015-05-01

    The human cytomegalovirus (HCMV) UL112-113 gene is implicated in lytic viral replication. The UL112-113 proteins p34, p43, p50 and p84 are expressed via alternative splicing. However, the mechanism for the generation of three additional virus-associated proteins (p20, p26 and p28), which share the UL112 reading frame, remains unknown. Bioinformatic analyses indicated that p34, p43, p50 and p84 contain potential PEST-like degradation motifs. In this study, inhibitors of calpains, lysosomes and proteasomes reduced p20, p26 and p28 levels in virus-infected cells, suggesting the involvement of proteolytic modification. Moreover, maitotoxin, which increases intracellular calcium levels and activates calpain activity, induced the intracellular proteolysis of p34 into p20, p26 and p28 and the cleavage of p43, p50 and p84 into p38 and a novel protein, p34c. Proteolytic assays further indicated that p34, p43, p50 and p84 were substrates of calpain-1 and calpain-2 and that they generated proteolytic products that corresponded to those detected during the HCMV infectious period. Furthermore, substitution mutations in the putative calpain cleavage sites of p34 reduced accumulation of proteolytic products. The knockdown of endogenous calpain-1 and calpain-2 by RNA interference reduced accumulation of p20, p26 and p28 and concurrently increased levels of nascent p43, p50 and p84 during the infectious cycle. Intriguingly, calpain depletion enhanced viral genome synthesis. Moreover, HCMV-permissive cells that stably expressed p20, p26 or p28 exhibited reduced viral genome synthesis and mature virus production. Our findings suggest that cognate UL112-113 proteins derived from calpain-catalysed proteolysis are involved in the HCMV replication process.

  12. MDL28170, a calpain inhibitor, affects Trypanosoma cruzi metacyclogenesis, ultrastructure and attachment to Rhodnius prolixus midgut.

    Directory of Open Access Journals (Sweden)

    Vítor Ennes-Vidal

    Full Text Available BACKGROUND: Trypanosoma cruzi is the etiological agent of Chagas' disease. During the parasite life cycle, many molecules are involved in the differentiation process and infectivity. Peptidases are relevant for crucial steps of T. cruzi life cycle; as such, it is conceivable that they may participate in the metacyclogenesis and interaction with the invertebrate host. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we have investigated the effect of the calpain inhibitor MDL28170 on the attachment of T. cruzi epimastigotes to the luminal midgut surface of Rhodnius prolixus, as well as on the metacyclogenesis process and ultrastructure. MDL28170 treatment was capable of significantly reducing the number of bound epimastigotes to the luminal surface midgut of the insect. Once the cross-reactivity of the anti-Dm-calpain was assessed, it was possible to block calpain molecules by the antibody, leading to a significant reduction in the capacity of adhesion to the insect guts by T. cruzi. However, the antibodies were unable to interfere in metacyclogenesis, which was impaired by the calpain inhibitor presenting a significant reduction in the number of metacyclic trypomastigotes. The calpain inhibitor also promoted a direct effect against bloodstream trypomastigotes. Ultrastructural analysis of epimastigotes treated with the calpain inhibitor revealed disorganization in the reservosomes, Golgi and plasma membrane disruption. CONCLUSIONS/SIGNIFICANCE: The presence of calpain and calpain-like molecules in a wide range of organisms suggests that these proteins could be necessary for basic cellular functions. Herein, we demonstrated the effects of MDL28170 in crucial steps of the T. cruzi life cycle, such as attachment to the insect midgut and metacyclogenesis, as well as in parasite viability and morphology. Together with our previous findings, these results help to shed some light on the functions of T. cruzi calpains. Considering the potential roles of

  13. CLK2 inhibition ameliorates autistic features associated with SHANK3 deficiency.

    Science.gov (United States)

    Bidinosti, Michael; Botta, Paolo; Krüttner, Sebastian; Proenca, Catia C; Stoehr, Natacha; Bernhard, Mario; Fruh, Isabelle; Mueller, Matthias; Bonenfant, Debora; Voshol, Hans; Carbone, Walter; Neal, Sarah J; McTighe, Stephanie M; Roma, Guglielmo; Dolmetsch, Ricardo E; Porter, Jeffrey A; Caroni, Pico; Bouwmeester, Tewis; Lüthi, Andreas; Galimberti, Ivan

    2016-03-11

    SH3 and multiple ankyrin repeat domains 3 (SHANK3) haploinsufficiency is causative for the neurological features of Phelan-McDermid syndrome (PMDS), including a high risk of autism spectrum disorder (ASD). We used unbiased, quantitative proteomics to identify changes in the phosphoproteome of Shank3-deficient neurons. Down-regulation of protein kinase B (PKB/Akt)-mammalian target of rapamycin complex 1 (mTORC1) signaling resulted from enhanced phosphorylation and activation of serine/threonine protein phosphatase 2A (PP2A) regulatory subunit, B56β, due to increased steady-state levels of its kinase, Cdc2-like kinase 2 (CLK2). Pharmacological and genetic activation of Akt or inhibition of CLK2 relieved synaptic deficits in Shank3-deficient and PMDS patient-derived neurons. CLK2 inhibition also restored normal sociability in a Shank3-deficient mouse model. Our study thereby provides a novel mechanistic and potentially therapeutic understanding of deregulated signaling downstream of Shank3 deficiency.

  14. 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.

  15. Ex vivo measurement of calpain activation in human peripheral blood lymphocytes by detection of immunoreactive products of calpastatin degradation.

    Directory of Open Access Journals (Sweden)

    Jacek M Witkowski

    2008-01-01

    Full Text Available Limited proteolysis of multiple intracellular proteins by endogenous Ca-dependent cysteine proteases--calpains--is an important regulatory mechanism for cell proliferation, apoptosis etc. Its importance for cellular functions is stressed by existence of endogenous calpain inhibitors--calpastatins. The calpain-calpastatin system within living cells is in a fragile balance, which depends on both partners. The interdependence of calpain--a protease--and calpastatin--an endogenous inhibitor and at the same time a substrate for this enzyme makes any assessment of actual activity of this enzyme in the cells very difficult. In this work we made an attempt to estimate and compare the activity of calpain in human peripheral blood lymphocytes by assessing the levels of limited proteolysis of calpastatin in these cells by western blot, while at the same time the levels of calpain protein inside these cells was measured by flow cytometry. Our results indicate that it is possible to compare (semi-quantitatively the activities of calpain in peripheral blood CD4+ and CD19+ lymphocytes from various donors that way. Preliminary results showed that calpain activity is increased in the CD4+ T cells isolated from peripheral blood of rheumatoid arthritis patients as compared to control lymphocytes. Extremely high intrinsic activity of calpain was detected in chronic lymphocytic leukemia (CD19+ cells. All this confirms the detection of immunoreactive products of calpastatin as a good maker of endogenous calpain activity.

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

    Directory of Open Access Journals (Sweden)

    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

  17. Sarcomere length influences u-calpain mediated proteolysis of troponin-T

    Science.gov (United States)

    Muscle shortening and postmortem proteolysis are well established as mechanisms controlling beef tenderness. Inherent myofibril structure and the extent of overlap between myosin and actin filaments are hypothesized to affect the availability of substrates for degradation by calpains. The objective ...

  18. 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.

  19. 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

  20. Contribution of calpains to photoreceptor cell death in N-methyl-N-nitrosourea-treated rats.

    Science.gov (United States)

    Oka, Takayuki; Nakajima, Takeshi; Tamada, Yoshiyuki; Shearer, Thomas R; Azuma, Mitsuyoshi

    2007-03-01

    The purpose of the present study was to determine if proteolysis by the calcium-dependent enzyme calpains (EC 3.4.22.17) contributed to retinal cell death in a rat model of photoreceptor degeneration induced by intraperitoneal injection of N-methyl-N-nitrosourea (MNU). Retinal degeneration was evaluated by H&E staining, and cell death was determined by TUNEL assay. Total calcium in retina was measured by atomic absorption spectrophotometry. Activation of calpains was determined by casein zymography and immunoblotting. Proteolysis of alpha-spectrin and p35 (regulator of Cdk5) were evaluated by immunoblotting. Calpain inhibitor SNJ-1945 was orally administrated to MNU-treated rats to test drug efficacy. MNU decreased the thickness of photoreceptor cell layer, composed of the outer nuclear layer (ONL) and outer segment (OS). Numerous cells in the ONL showed positive TUNEL staining. Total calcium was increased in retina after MNU. Activation of calpains and calpain-specific proteolysis of alpha-spectrin were observed after MNU injection. Oral administration of SNJ-1945 to MNU-treated rats showed a significant protective effect against photoreceptor cell loss, confirming involvement of calpains in photoreceptor degeneration. Conversion of p35 to p25 was well correlated with calpain activation, suggesting prolonged activation of Cdk5/p25 as a possible downstream mechanism for MNU-induced photoreceptor cell death. SNJ-1945 reduced photoreceptor cells death, even though MNU is one of the most severe models of photoreceptor cell degeneration. Oral calpain inhibitor SNJ-1945 may be a candidate for testing as a medication against retinal degeneration in retinitis pigmentosa.

  1. Molecular determinants of survival motor neuron (SMN protein cleavage by the calcium-activated protease, calpain.

    Directory of Open Access Journals (Sweden)

    Jennifer L Fuentes

    Full Text Available Spinal muscular atrophy (SMA is a leading genetic cause of childhood mortality, caused by reduced levels of survival motor neuron (SMN protein. SMN functions as part of a large complex in the biogenesis of small nuclear ribonucleoproteins (snRNPs. It is not clear if defects in snRNP biogenesis cause SMA or if loss of some tissue-specific function causes disease. We recently demonstrated that the SMN complex localizes to the Z-discs of skeletal and cardiac muscle sarcomeres, and that SMN is a proteolytic target of calpain. Calpains are implicated in muscle and neurodegenerative disorders, although their relationship to SMA is unclear. Using mass spectrometry, we identified two adjacent calpain cleavage sites in SMN, S192 and F193. Deletion of small motifs in the region surrounding these sites inhibited cleavage. Patient-derived SMA mutations within SMN reduced calpain cleavage. SMN(D44V, reported to impair Gemin2 binding and amino-terminal SMN association, drastically inhibited cleavage, suggesting a role for these interactions in regulating calpain cleavage. Deletion of A188, a residue mutated in SMA type I (A188S, abrogated calpain cleavage, highlighting the importance of this region. Conversely, SMA mutations that interfere with self-oligomerization of SMN, Y272C and SMNΔ7, had no effect on cleavage. Removal of the recently-identified SMN degron (Δ268-294 resulted in increased calpain sensitivity, suggesting that the C-terminus of SMN is important in dictating availability of the cleavage site. Investigation into the spatial determinants of SMN cleavage revealed that endogenous calpains can cleave cytosolic, but not nuclear, SMN. Collectively, the results provide insight into a novel aspect of the post-translation regulation of SMN.

  2. Calpain-10 expression is elevated in pancreatic islets from patients with type 2 diabetes.

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    Charlotte Ling

    Full Text Available BACKGROUND: Calpain-10 was the first gene to be identified influencing the risk of type 2 diabetes (T2D by positioning cloning. Studies in beta-cell lines and rodent islets suggest that calpain-10 may act as a regulator of insulin secretion. However, its role in human pancreatic islets remains unclear. The aim of this study was to examine if calpain-10 expression is altered in islets from patients with T2D and if the transcript level correlates with insulin release. We also tested if polymorphisms in the CAPN10 gene are associated with gene expression and insulin secretion in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Calpain-10 mRNA expression was analysed in human pancreatic islets from 34 non-diabetic and 10 T2D multi-organ donors. CAPN10 SNP-43 and SNP-44 were genotyped and related to gene expression and insulin release in response to glucose, arginine and glibenclamide. The mRNA level of calpain-10 was elevated by 64% in pancreatic islets from patients with T2D compared with non-diabetic donors (P = 0.01. Moreover, the calpain-10 expression correlated positively with arginine-stimulated insulin release in islets from non-diabetic donors (r = 0.45, P = 0.015. However, this correlation was lost in islets from patients with T2D (r = 0.09; P = 0.8. The G/G variant of SNP-43 was associated with reduced insulin release in response to glucose (Pcalpain-10 expression correlates with insulin release in non-diabetic human islets, this correlation is lost in T2D suggesting that a stimulatory effect of calpain-10 could be lost in patients with T2D.

  3. 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.

  4. Calpains promote α2β1 integrin turnover in nonrecycling integrin pathway.

    Science.gov (United States)

    Rintanen, Nina; Karjalainen, Mikko; Alanko, Jonna; Paavolainen, Lassi; Mäki, Anita; Nissinen, Liisa; Lehkonen, Moona; Kallio, Katri; Cheng, R Holland; Upla, Paula; Ivaska, Johanna; Marjomäki, Varpu

    2012-02-01

    Collagen receptor integrins recycle between the plasma membrane and endosomes and facilitate formation and turnover of focal adhesions. In contrast, clustering of α2β1 integrin with antibodies or the human pathogen echovirus 1 (EV1) causes redistribution of α2 integrin to perinuclear multivesicular bodies, α2-MVBs. We show here that the internalized clustered α2 integrin remains in α2-MVBs and is not recycled back to the plasma membrane. Instead, receptor clustering and internalization lead to an accelerated down-regulation of α2β1 integrin compared to the slow turnover of unclustered α2 integrin. EV1 infection or integrin degradation is not associated with proteasomal or autophagosomal processes and shows no significant association with lysosomal pathway. In contrast, degradation is dependent on calpains, such that it is blocked by calpain inhibitors. We show that active calpain is present in α2-MVBs, internalized clustered α2β1 integrin coprecipitates with calpain-1, and calpain enzymes can degrade α2β1 integrin. In conclusion, we identified a novel virus- and clustering-specific pathway that diverts α2β1 integrin from its normal endo/exocytic traffic to a nonrecycling, calpain-dependent degradative endosomal route.

  5. 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.

  6. Expression and proteolytic activity of calpain in lens epithelial cells of oxidative cataract

    Institute of Scientific and Technical Information of China (English)

    徐雯; 姚克; 孙朝晖; 王凯军; 申屠形超

    2004-01-01

    Objective: To study the role of calpain in the mechanism of oxidative cataract through detecting the level of intracellular free Ca2+, the expression and proteolytic activity of calpain in the lens epithelial cells (LECs) of H2O2-induced cataract. Methods: Rat lenses were cultured in vitro and cataract was induced by H2O2. The level of intracellular free Ca2+ was measured by fluorescence determination with fura-2/AM. The expression of m-calpain protein in LECs was detected with immunohistochemical method. The proteolytic activity in LECs was measured using a fluorogenic synthetic substrate. Results: There were significant differences of the level of intracellular free Ca2+ (P=0.001, 0.000, 0.000), the expression of m-calpain (P=0.001, 0.000, 0.000) and the proteolytic activity of calpain (P=0.001, 0.000, 0.000) between H2O2-induced and control group at 6, 12 and 24 h, respectively. Conclusions: H2O2 can increase intracellular free Ca2+, then enhance the expression and proteolytic activity of calpain which may play a role in the mechanism of oxidative cataract of rat.

  7. 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.

  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.

  9. 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.

  10. m-calpain Activation Is Regulated by Its Membrane Localization and by Its Binding to Phosphatidylinositol 4,5-Bisphosphate*

    Science.gov (United States)

    Leloup, Ludovic; Shao, Hanshuang; Bae, Yong Ho; Deasy, Bridget; Stolz, Donna; Roy, Partha; Wells, Alan

    2010-01-01

    m-calpain plays a critical role in cell migration enabling rear de-adhesion of adherent cells by cleaving structural components of the adhesion plaques. Growth factors and chemokines regulate keratinocyte, fibroblast, and endothelial cell migration by modulating m-calpain activity. Growth factor receptors activate m-calpain secondary to phosphorylation on serine 50 by ERK. Concurrently, activated m-calpain is localized to its inner membrane milieu by binding to phosphatidylinositol 4,5-bisphosphate (PIP2). Opposing this, CXCR3 ligands inhibit cell migration by blocking m-calpain activity secondary to a PKA-mediated phosphorylation in the C2-like domain. The failure of m-calpain activation in the absence of PIP2 points to a key regulatory role, although whether this PIP2-mediated membrane localization is regulatory for m-calpain activity or merely serves as a docking site for ERK phosphorylation is uncertain. Herein, we report the effects of two CXCR3 ligands, CXCL11/IP-9/I-TAC and CXCL10/IP-10, on the EGF- and VEGF-induced redistribution of m-calpain in human fibroblasts and endothelial cells. The two chemokines block the tail retraction and, thus, the migration within minutes, preventing and reverting growth factor-induced relocalization of m-calpain to the plasma membrane of the cells. PKA phosphorylation of m-calpain blocks the binding of the protease to PIP2. Unexpectedly, we found that this was due to membrane anchorage itself and not merely serine 50 phosphorylation, as the farnesylation-induced anchorage of m-calpain triggers a strong activation of this protease, leading notably to an increased cell death. Moreover, the ERK and PKA phosphorylations have no effect on this membrane-anchored m-calpain. However, the presence of PIP2 is still required for the activation of the anchored m-calpain. In conclusion, we describe a novel mechanism of m-calpain activation by interaction with the plasma membrane and PIP2 specifically, this phosphoinositide acting as a

  11. Altered Ca2+ kinetics associated with α-actinin-3 deficiency may explain positive selection for ACTN3 null allele in human evolution.

    Directory of Open Access Journals (Sweden)

    Stewart I Head

    Full Text Available Over 1.5 billion people lack the skeletal muscle fast-twitch fibre protein α-actinin-3 due to homozygosity for a common null polymorphism (R577X in the ACTN3 gene. α-Actinin-3 deficiency is detrimental to sprint performance in elite athletes and beneficial to endurance activities. In the human genome, it is very difficult to find single-gene loss-of-function variants that bear signatures of positive selection, yet intriguingly, the ACTN3 null variant has undergone strong positive selection during recent evolution, appearing to provide a survival advantage where food resources are scarce and climate is cold. We have previously demonstrated that α-actinin-3 deficiency in the Actn3 KO mouse results in a shift in fast-twitch fibres towards oxidative metabolism, which would be more "energy efficient" in famine, and beneficial to endurance performance. Prolonged exposure to cold can also induce changes in skeletal muscle similar to those observed with endurance training, and changes in Ca2+ handling by the sarcoplasmic reticulum (SR are a key factor underlying these adaptations. On this basis, we explored the effects of α-actinin-3 deficiency on Ca2+ kinetics in single flexor digitorum brevis muscle fibres from Actn3 KO mice, using the Ca2+-sensitive dye fura-2. Compared to wild-type, fibres of Actn3 KO mice showed: (i an increased rate of decay of the twitch transient; (ii a fourfold increase in the rate of SR Ca2+ leak; (iii a threefold increase in the rate of SR Ca2+ pumping; and (iv enhanced maintenance of tetanic Ca2+ during fatigue. The SR Ca2+ pump, SERCA1, and the Ca2+-binding proteins, calsequestrin and sarcalumenin, showed markedly increased expression in muscles of KO mice. Together, these changes in Ca2+ handling in the absence of α-actinin-3 are consistent with cold acclimatisation and thermogenesis, and offer an additional explanation for the positive selection of the ACTN3 577X null allele in populations living in cold environments

  12. Altered Ca2+ kinetics associated with α-actinin-3 deficiency may explain positive selection for ACTN3 null allele in human evolution.

    Science.gov (United States)

    Head, Stewart I; Chan, Stephen; Houweling, Peter J; Quinlan, Kate G R; Murphy, Robyn; Wagner, Sören; Friedrich, Oliver; North, Kathryn N

    2015-01-01

    Over 1.5 billion people lack the skeletal muscle fast-twitch fibre protein α-actinin-3 due to homozygosity for a common null polymorphism (R577X) in the ACTN3 gene. α-Actinin-3 deficiency is detrimental to sprint performance in elite athletes and beneficial to endurance activities. In the human genome, it is very difficult to find single-gene loss-of-function variants that bear signatures of positive selection, yet intriguingly, the ACTN3 null variant has undergone strong positive selection during recent evolution, appearing to provide a survival advantage where food resources are scarce and climate is cold. We have previously demonstrated that α-actinin-3 deficiency in the Actn3 KO mouse results in a shift in fast-twitch fibres towards oxidative metabolism, which would be more "energy efficient" in famine, and beneficial to endurance performance. Prolonged exposure to cold can also induce changes in skeletal muscle similar to those observed with endurance training, and changes in Ca2+ handling by the sarcoplasmic reticulum (SR) are a key factor underlying these adaptations. On this basis, we explored the effects of α-actinin-3 deficiency on Ca2+ kinetics in single flexor digitorum brevis muscle fibres from Actn3 KO mice, using the Ca2+-sensitive dye fura-2. Compared to wild-type, fibres of Actn3 KO mice showed: (i) an increased rate of decay of the twitch transient; (ii) a fourfold increase in the rate of SR Ca2+ leak; (iii) a threefold increase in the rate of SR Ca2+ pumping; and (iv) enhanced maintenance of tetanic Ca2+ during fatigue. The SR Ca2+ pump, SERCA1, and the Ca2+-binding proteins, calsequestrin and sarcalumenin, showed markedly increased expression in muscles of KO mice. Together, these changes in Ca2+ handling in the absence of α-actinin-3 are consistent with cold acclimatisation and thermogenesis, and offer an additional explanation for the positive selection of the ACTN3 577X null allele in populations living in cold environments during recent

  13. Probing of primed and unprimed sites of calpains: Design, synthesis and evaluation of epoxysuccinyl-peptide derivatives as selective inhibitors.

    Science.gov (United States)

    Dókus, Levente E; Menyhárd, Dóra K; Tantos, Ágnes; Hudecz, Ferenc; Bánóczi, Zoltán

    2014-07-23

    Calpains are intracellular cysteine proteases with important physiological functions. Up- or downregulation of their expression can be responsible for several diseases, therefore specific calpain inhibitors may be considered as promising candidates for drug discovery. In this paper we describe the synthesis and characterization of a new class of inhibitors derived from the analysis of amino acid preferences in primed and unprimed sites of calpains by incorporation of l- or d-epoxysuccinyl group (Eps). Amino acids for replacement were chosen by considering the substrate preference of calpain 1 and 2 enzymes. The compounds were characterized by RP-HPLC, amino acid analysis and ESI-MS. Selectivity of the compounds was studied by using calpain 1 and 2; and cathepsin B. We have identified five calpain specific inhibitors with different extent of selectivity. Two of these also exhibited isoform selectivity. Compound NH2-Thr-Pro-Leu-(d-Eps)-Thr-Pro-Pro-Pro-Ser-NH2 proved to be a calpain 2 enzyme inhibitor with at least 11.8-fold selectivity, while compound NH2-Thr-Pro-Leu-(l-Eps)-Ser-Pro-Pro-Pro-Ser-NH2 possesses calpain 1 enzyme inhibition with at least 4-fold selectivity. The results of molecular modeling calculations suggest that the orientation of the bound inhibitor in the substrate binding cleft is markedly dependent on the stereochemistry of the epoxysuccinyl group.

  14. Effect of cortisol on calpains in the C2C12 and 3T3-L1 cells.

    Science.gov (United States)

    Muthuraman, Pandurangan; Ravikumar, Sambandam; Muthuviveganandavel, Veerappan; Kim, Jongpil

    2014-03-01

    The present study was carried out to understand the effect of cortisol on calpain system in the C2C12 and 3T3-L1 adipocyte cells under co-culture system. Cells were co-cultured by using transwell inserts with a 0.4 μm porous membrane to separate C2C12 and 3T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates. Ten microgram per milliliter of cortisol was added to the medium. Following treatment for 3 days, the cells in the lower well were harvested for analysis. Calpains such as μ-calpain, m-calpain, and calpastatin were selected for the analysis. RT-PCR results indicated the significant increase in the mRNA expression of μ-calpain, m-calpain, and calpastatin. In addition, the confocal microscopical investigation indicated the cortisol treatment increases calpain expression in the C2C12 and 3T3-L1 cells. Taking all these together, cortisol treatment with co-culture system shows most reliable status of calpains expression in the cells, which is quite distinct from one-dimensional monocultured cells.

  15. Calpain-Calcineurin-Nuclear Factor Signaling and the Development of Atrial Fibrillation in Patients with Valvular Heart Disease and Diabetes

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    Yong Zhao

    2016-01-01

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

  16. 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

    This study investigated the influence of post-mortem pH decline on calpain activity and myofibrillar degradation.From 80 pigs, 30 Longissimus dorsi (LD) muscles were selected on the basis of pH values at 3 h post-mortem and classified into groups of 10 as fast, intermediate and slow pH decline....... 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...... measured. A faster decrease in pH resulted in reduced level of l-calpain activity and increased autolysis of the enzyme, and hence an earlier loss of activity due to activation of l-calpain in muscles with a fast pH decline. Paralleling the l-calpain activation in muscles with a fast pH decline a higher...

  17. Calpains are involved in Entamoeba histolytica-induced death of HT-29 colonic epithelial cells.

    Science.gov (United States)

    Jang, Yun Soo; Song, Kyoung-Ju; Kim, Ju Young; Lee, Young Ah; Kim, Kyeong Ah; Lee, Sang Kyou; Shin, Myeong Heon

    2011-06-01

    Entamoeba histolytica is an enteric tissue-invading protozoan parasite that can cause amebic colitis and liver abscess in humans. E. histolytica has the capability to kill colon epithelial cells in vitro; however, information regarding the role of calpain in colon cell death induced by ameba is limited. In this study, we investigated whether calpains are involved in the E. histolytica-induced cell death of HT-29 colonic epithelial cells. When HT-29 cells were co-incubated with E. histolytica, the propidium iodide stained dead cells markedly increased compared to that in HT-29 cells incubated with medium alone. This pro-death effect induced by ameba was effectively blocked by pretreatment of HT-29 cells with the calpain inhibitor, calpeptin. Moreover, knockdown of m- and µ-calpain by siRNA significantly reduced E. histolytica-induced HT-29 cell death. These results suggest that m- and µ-calpain may be involved in colon epithelial cell death induced by E. histolytica.

  18. Expression and proteolytic activity of calpain in lens epithelial cells of oxidative cataract

    Institute of Scientific and Technical Information of China (English)

    徐雯; 姚克; 孙朝晖; 王凯军; 申屠形超

    2004-01-01

    Objective: To study the role of calpain in the mechanism of oxidative cataract through detecting the level of intracellular free Ca2+, the expression and proteolytic activity of calpain in the lens epithelial cells (LECs) of H2O2-induced cataract. Methods:Rat lenses were cultured in vitro and cataract was induced by H2O2. The level of intracellular free Ca2+ was measured by fluorescence determination with fura-2/AM. The expression of m-calpain protein in LECs was detected with immunohistochemical method. The proteolytic activity in LECs was measured using a fluorogenic synthetic substrate.Results: There were significant differences of the level of intracellular free Ca2+(P=0.001, 0.000, 0.000), the expression of m-calpain (P=0.001, 0.000, 0.000) and the proteolytic activity ofcalpain (P=O.O01,0.000, 0.000) between H2O2-induced and control group at 6, 12 and 24 h, respectively. Conclusions: H2O2 can increase intracellular free Ca2+, then enhance the expression and proteolytic activity of calpain which may play a role in the mechanism of oxidative cataract of rat.

  19. Calpain activation through galectin-3 inhibition sensitizes prostate cancer cells to cisplatin treatment

    Science.gov (United States)

    Wang, Y; Nangia-Makker, P; Balan, V; Hogan, V; Raz, A

    2010-01-01

    Prostate cancer will develop chemoresistance following a period of chemotherapy. This is due, in part, to the acquisition of antiapoptotic properties by the cancer cells and, therefore, development of novel strategies for treatment is of critical need. Here, we attempt to clarify the role of the antiapoptotic molecule galectin-3 in prostate cancer cells using siRNA and antagonist approaches. The data showed that Gal-3 inhibition by siRNA or its antagonist GCS-100/modified citrus pectin (MCP) increased cisplatin-induced apoptosis of PC3 cells. Recent studies have indicated that cisplatin-induced apoptosis may be mediated by calpain, a calcium-dependent protease, as its activation leads to cleavage of androgen receptor into an androgen-independent isoform in prostate cancer cells. Thus, we examined whether calpain activation is associated with the Gal-3 function of regulating apoptosis. Here, we report that Gal-3 inhibition by siRNA or GCS-100/MCP enhances calpain activation, whereas Gal-3 overexpression inhibits it. Inhibition of calpain using its inhibitor and/or siRNA attenuated the proapoptotic effect of Gal-3 inhibition, suggesting that calpain activation may be a novel mechanism for the proapoptotic effect of Gal-3 inhibition. Thus, a paradigm shift for treating prostate cancer is suggested whereby a combination of a non-toxic anti-Gal-3 drug together with a toxic chemotherapeutic agent could serve as a novel therapeutic modality for chemoresistant prostate cancers. PMID:21368866

  20. Effects of Calpain I,Calpain II on atrial fibrosis in human Rheumatic Atrial Fibrillation%卡配因参与风心病房颤患者心房纤维化过程

    Institute of Scientific and Technical Information of China (English)

    纪磊; 刘盈盈; 贡郡利; 李树岩

    2013-01-01

    目的 检测Calpain I、Calpain II、caspase-12在风湿性心脏病心房颤动患者心房组织中的表达,探讨其在心房纤维化发展中的作用及其对房颤发生、维持的作用.为临床慢性房颤患者心房纤维化及心房结构重构的防治提供新的靶点.方法 取32例风湿性心脏病行外科换瓣手术患者左心房组织,分为A组:永久性房颤组(Permanent AF组)16例,B组:阵发性或持续性房颤组(Paroxysmal or persistent AF组)10例,C组:窦性心律组(Sinus rhythm组)6例.采用免疫组化法测Calpain I、Calpain II、caspase-12在左心房组织中的表达并计算其灰度值,心肌胶原染色采用VG染色,光镜下观察组织纤维化程度,并计算胶原容积积分(CVF).结果 风湿性心脏病心房颤动患者calpain I、calpain II、caspase-12在左心房组织表达明显增多(P<0.05),calpain I、calpain II与CVF呈正相关.结论 calpain I、calpain II通过促进心肌细胞凋亡促进了风湿性心脏病心房颤动心房纤维化过程.

  1. 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.

  2. Mechanical stimuli activation of calpain is required for myoblast differentiation and occurs via an ERK/MAP kinase signaling pathway

    DEFF Research Database (Denmark)

    Grossi, Alberto; Karlsson, Anders H; Lawson, Moira Ann

    fusion, cell membrane and cytoskeleton component reorganization due to the activity of ubiquitous proteolytic enzymes known as calpains has been reported. Whether there is a link between stretch- or load induced signals, the MAPK pathway and calpain expression and activation is not known. Using......, with each individual myotube containing fewer nuclei. Mechanical stimulation increases not only the expression of m-calpain but also the overall activity of calpain in the cells through the MAPK signaling cascade. Our findings underline that the mechanical modulation of MAPK signaling cascade enhances...... the expression and activity of m-calpain, which play a pivotal role during myoblast fusion, strengthening the idea of its implication during the initial events of muscle development....

  3. Oxytocin improves behavioral and electrophysiological deficits in a novel Shank3-deficient rat

    Science.gov (United States)

    Harony-Nicolas, Hala; Kay, Maya; du Hoffmann, Johann; Klein, Matthew E; Bozdagi-Gunal, Ozlem; Riad, Mohammed; Daskalakis, Nikolaos P; Sonar, Sankalp; Castillo, Pablo E; Hof, Patrick R; Shapiro, Matthew L; Baxter, Mark G; Wagner, Shlomo; Buxbaum, Joseph D

    2017-01-01

    Mutations in the synaptic gene SHANK3 lead to a neurodevelopmental disorder known as Phelan-McDermid syndrome (PMS). PMS is a relatively common monogenic and highly penetrant cause of autism spectrum disorder (ASD) and intellectual disability (ID), and frequently presents with attention deficits. The underlying neurobiology of PMS is not fully known and pharmacological treatments for core symptoms do not exist. Here, we report the production and characterization of a Shank3-deficient rat model of PMS, with a genetic alteration similar to a human SHANK3 mutation. We show that Shank3-deficient rats exhibit impaired long-term social recognition memory and attention, and reduced synaptic plasticity in the hippocampal-medial prefrontal cortex pathway. These deficits were attenuated with oxytocin treatment. The effect of oxytocin on reversing non-social attention deficits is a particularly novel finding, and the results implicate an oxytocinergic contribution in this genetically defined subtype of ASD and ID, suggesting an individualized therapeutic approach for PMS. DOI: http://dx.doi.org/10.7554/eLife.18904.001 PMID:28139198

  4. 25-Hydroxyvitamin D3-deficiency enhances oxidative stress and corticosteroid resistance in severe asthma exacerbation.

    Directory of Open Access Journals (Sweden)

    Nan Lan

    Full Text Available Oxidative stress plays a significant role in exacerbation of asthma. The role of vitamin D in oxidative stress and asthma exacerbation remains unclear. We aimed to determine the relationship between vitamin D status and oxidative stress in asthma exacerbation. Severe asthma exacerbation patients with 25-hydroxyvitamin D3-deficiency (V-D deficiency or 25-hydroxyvitamin D-sufficiency (V-D sufficiency were enrolled. Severe asthma exacerbation with V-D-deficiency showed lower forced expiratory volume in one second (FEV1 compared to that with V-D-sufficiency. V-D-deficiency intensified ROS release and DNA damage and increased TNF-α, OGG1 and NFκB expression and NFκB phosphorylation in severe asthma exacerbation. Supplemental vitamin D3 significantly increased the rates of FEV1 change and decreased ROS and DNA damage in V-D-deficiency. Vitamin D3 inhibited LPS-induced ROS and DNA damage and were associated with a decline in TNF-α and NFκB in epithelial cells. H2O2 reduces nuclear translocation of glucocorticoid receptors in airway epithelial cell lines. V-D pretreatment enhanced the dexamethasone-induced nuclear translocation of glucocorticoid receptors in airway epithelial cell lines and monocytes from 25-hydroxyvitamin D3-deficiency asthma patients. These findings indicate that V-D deficiency aggravates oxidative stress and DNA damage, suggesting a possible mechanism for corticosteroid resistance in severe asthma exacerbation.

  5. 25-Hydroxyvitamin D3-deficiency enhances oxidative stress and corticosteroid resistance in severe asthma exacerbation.

    Science.gov (United States)

    Lan, Nan; Luo, Guangyan; Yang, Xiaoqiong; Cheng, Yuanyuan; Zhang, Yun; Wang, Xiaoyun; Wang, Xing; Xie, Tao; Li, Guoping; Liu, Zhigang; Zhong, Nanshan

    2014-01-01

    Oxidative stress plays a significant role in exacerbation of asthma. The role of vitamin D in oxidative stress and asthma exacerbation remains unclear. We aimed to determine the relationship between vitamin D status and oxidative stress in asthma exacerbation. Severe asthma exacerbation patients with 25-hydroxyvitamin D3-deficiency (V-D deficiency) or 25-hydroxyvitamin D-sufficiency (V-D sufficiency) were enrolled. Severe asthma exacerbation with V-D-deficiency showed lower forced expiratory volume in one second (FEV1) compared to that with V-D-sufficiency. V-D-deficiency intensified ROS release and DNA damage and increased TNF-α, OGG1 and NFκB expression and NFκB phosphorylation in severe asthma exacerbation. Supplemental vitamin D3 significantly increased the rates of FEV1 change and decreased ROS and DNA damage in V-D-deficiency. Vitamin D3 inhibited LPS-induced ROS and DNA damage and were associated with a decline in TNF-α and NFκB in epithelial cells. H2O2 reduces nuclear translocation of glucocorticoid receptors in airway epithelial cell lines. V-D pretreatment enhanced the dexamethasone-induced nuclear translocation of glucocorticoid receptors in airway epithelial cell lines and monocytes from 25-hydroxyvitamin D3-deficiency asthma patients. These findings indicate that V-D deficiency aggravates oxidative stress and DNA damage, suggesting a possible mechanism for corticosteroid resistance in severe asthma exacerbation.

  6. 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.

  7. RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury

    Science.gov (United States)

    Newton, K; Dugger, D L; Maltzman, A; Greve, J M; Hedehus, M; Martin-McNulty, B; Carano, R A D; Cao, T C; van Bruggen, N; Bernstein, L; Lee, W P; Wu, X; DeVoss, J; Zhang, J; Jeet, S; Peng, I; McKenzie, B S; Roose-Girma, M; Caplazi, P; Diehl, L; Webster, J D; Vucic, D

    2016-01-01

    Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia–reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia–reperfusion injury model, the high-dose TNF model, and in A20−/− mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia–reperfusion injury model and no benefit in A20−/− mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease. PMID:27177019

  8. [Calpains: general characteristics and role in various states of the organism].

    Science.gov (United States)

    Starodub, M F; Samokhina, L M; Koval', S M; Snigurs'ka, I O

    2014-01-01

    Calpains are a family of cytoplasmic calcium-dependent proteinases with papain-like activity. They participate in a variety of processes in the body: age changes, functioning of endothelium and pulmonary system, regulation of apoptosis and necrosis, development of various hypometabolic states, arterial hypertension, diabetes and chronic kidney disease, tumor growth. It is concluded that calpains, causing limited proteolysis of substrates, play an important role in a wide range of biological phenomena. Their activity is associated with the response to the calcium-dependent signaling and the effects of aging. Inhibition of calpains activity contributes to inhibition of endothelial dysfunction, cardiovascular disease, formation of structural and functional changes in the kidney tissue, has neuroprotective effect, preventing sarcopenia, reduces inflammatory reactions caused by hyperventilation of the lungs.

  9. 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.

  10. Dual Vulnerability of Tau to Calpains and Caspase-3 Proteolysis Under Neurotoxic and Neurodegenerative Conditions

    Directory of Open Access Journals (Sweden)

    Ming Cheng Liu

    2010-11-01

    Full Text Available Axonally specific microtubule-associated protein tau is an important component of neurofibrillary tangles found in AD (Alzheimer's disease and other tauopathy diseases such as CTE (chronic traumatic encephalopathy. Such tau aggregate is found to be hyperphosphorylated and often proteolytically fragmented. Similarly, tau is degraded following TBI (traumatic brain injury. In the present study, we examined the dual vulnerability of tau to calpain and caspase-3 under neurotoxic and neurodegenerative conditions. We first identified three novel calpain cleavage sites in rat tau (four-repeat isoform as Ser130 ↓ Lys131, Gly157 ↓ Ala158 and Arg380 ↓ Glu381. Fragment-specific antibodies to target the major calpain-mediated TauBDP-35K (35 kDa tau-breakdown product and the caspase-mediated TauBDP-45K respectively were developed. In rat cerebrocortical cultures treated with excitotoxin [NMDA (N-methyl-D-aspartate], tau is significantly degraded into multiple fragments, including a dominant signal of calpain-mediated TauBDP-35K with minimal caspase-mediated TauBDP-45K. Following apoptosis-inducing EDTA treatment, tau was truncated only to TauBDP-48K/45K-exclusively by caspase. Cultures treated with another apoptosis inducer STS (staurosporine, dual fragmentation by calpain (TauBDP-35K and caspase-3 (TauBDP-45K was observed. Tau was also fragmented in injured rat cortex following TBI in vivo to BDPs of 45-42 kDa (minor, 35 kDa and 15 kDa, followed by TauBDP-25K. Calpain-mediated TauBDP-35K-specific antibody confirmed robust signals in the injured cortex, while caspase-mediated TauBDP-45K-specific antibody only detected faint signals. Furthermore, intravenous administration of a calpain-specific inhibitor SNJ-1945 strongly suppressed the TauBDP-35K formation. Taken together, these results suggest that tau protein is dually vulnerable to calpain and caspase-3 proteolysis under different neurotoxic and injury conditions.

  11. [Calpains and their endo- and exogenous regulators in various neurodegeneration models].

    Science.gov (United States)

    Lysenko, L A; Kantserova, N P; Rendakov, N L; Nemova, N N

    2014-01-01

    On the basis of experimental series with murine models there was obtained the evidence on calcium-dependent protease activity changes in rat brain at induced neurodegeneration. The properties of the proteolytic and regulatory components of calpain system under the effect of neurotoxic stimuli--amyloid beta-peptide or glutamate--were characterized; the basic endogenous regulatory mechanisms of calcium-dependent proteolysis modulation were determined as well. Neuroprotective properties of exogenous calpain regulators differing in the mechanisms of action (sex steroids, calcium regulators) were tested on studied neurodegeneration models.

  12. 一种新的糖尿病易感基因--Calpain-10

    Institute of Scientific and Technical Information of China (English)

    杨莉丽; 刘德敏

    2005-01-01

    @@ 大量研究表明,2型糖尿病是一种异质性、多基因遗传病.其基因致病因素并不仅仅与一些主要的基因缺陷相关,而与多个小的基因突变积累有关.最近有研究报道,钙蛋白(Calpain)家族成员之一的Calpain-10基因(CAPN10)可能是新的糖尿病易感基因.

  13. Survival and differentiation defects contribute to neutropenia in glucose-6-phosphatase-β (G6PC3) deficiency in a model of mouse neutrophil granulocyte differentiation.

    Science.gov (United States)

    Gautam, S; Kirschnek, S; Gentle, I E; Kopiniok, C; Henneke, P; Häcker, H; Malleret, L; Belaaouaj, A; Häcker, G

    2013-08-01

    Differentiation of neutrophil granulocytes (neutrophils) occurs through several steps in the bone marrow and requires a coordinate regulation of factors determining survival and lineage-specific development. A number of genes are known whose deficiency disrupts neutrophil generation in humans and in mice. One of the proteins encoded by these genes, glucose-6-phosphatase-β (G6PC3), is involved in glucose metabolism. G6PC3 deficiency causes neutropenia in humans and in mice, linked to enhanced apoptosis and ER stress. We used a model of conditional Hoxb8 expression to test molecular and functional differentiation as well as survival defects in neutrophils from G6PC3(-/-) mice. Progenitor lines were established and differentiated into neutrophils when Hoxb8 was turned off. G6PC3(-/-) progenitor cells underwent substantial apoptosis when differentiation was started. Transgenic expression of Bcl-XL rescued survival; however, Bcl-XL-protected differentiated cells showed reduced proliferation, immaturity and functional deficiency such as altered MAP kinase signaling and reduced cytokine secretion. Impaired glucose utilization was found and was associated with ER stress and apoptosis, associated with the upregulation of Bim and Bax; downregulation of Bim protected against apoptosis during differentiation. ER-stress further caused a profound loss of expression and secretion of the main neutrophil product neutrophil elastase during differentiation. Transplantation of wild-type Hoxb8-progenitor cells into irradiated mice allowed differentiation into neutrophils in the bone marrow in vivo. Transplantation of G6PC3(-/-) cells yielded few mature neutrophils in bone marrow and peripheral blood. Transgenic Bcl-XL permitted differentiation of G6PC3(-/-) cells in vivo. However, functional deficiencies and differentiation abnormalities remained. Differentiation of macrophages from Hoxb8-dependent progenitors was only slightly disturbed. A combination of defects in differentiation

  14. Molecular cloning and localization of a calpain-like protease from the abdominal muscle of Norway lobster Nephrops norvegicus.

    Science.gov (United States)

    Gornik, S G; Westrop, G D; Coombs, G H; Neil, D M

    2010-04-01

    Calpains are ubiquitous cysteine-proteases found in many, if not all, living organisms and their roles within these organisms are diverse, ranging from the mediation of cytoskeletal remodeling to the regulation of gene expression. In crustaceans calpains have so far been shown to be important mainly during moulting and growth. In the present study we report the expression of a calpain in the abdominal muscle of Norway lobster (Nephrops norvegicus) using degenerate primer, rapid amplification of cDNA ends (5'-3'-RACE), reverse transcriptase-PCR and RNA in situ hybridization approaches. The full-length mRNA sequence (2,774 bp) was found to include an open reading frame (bp 225-1,940) encoding a 572 amino acid polypeptide with a predicted mass of 65.9 kDa and a predicted pI of 5.17. The calpain was found to be an arthropod M-class calpain homologue to Homarus americanus Calpain M (Ha-CalpM) and has thus been termed Nephrops norvegicus calpain M (Nn-CalpM). When its expression pattern in abdominal muscle of adult intermoult Nephrops norvegicus was investigated an exclusive expression in a thin layer of connective tissue cells surrounding muscle fibres was found. This localization suggests a role in tenderizing connective tissue networks during growth and moulting.

  15. The DEK1 Calpain Linker Functions in Three-Dimensional Body Patterning in Physcomitrella patens1[OPEN

    Science.gov (United States)

    Demko, Viktor; Mekhlif, Ahmed Khaleel

    2016-01-01

    The DEFECTIVE KERNEL1 (DEK1) calpain is a conserved 240-kD key regulator of three-dimensional body patterning in land plants acting via mitotic cell plane positioning. The activity of the cytosolic C-terminal calpain protease is regulated by the membrane-anchored DEK1 MEM, which is connected to the calpain via the 600-amino acid residue Linker. Similar to the calpain and MEM domains, the Linker is highly conserved in the land plant lineage, the similarity dropping sharply compared with orthologous charophyte sequences. Using site-directed mutagenesis, we studied the effect on Physcomitrella patens development by deleting the Linker and two conserved Linker motifs. The results show that removal of the Linker has nearly the same effect as removal of the entire DEK1 gene. In contrast, deletion of the conserved Laminin_G3 (LG3) domain had a milder effect, perturbing leafy gametophore patterning and archegonia development. The LG3 domain from Marchantia polymorpha is fully functional in P. patens, whereas angiosperm sequences are not functional. Deletion of a C-terminal Linker subsegment containing a potential calpain autolytic site severely disturbs gametophore development. Finally, changing one of the three calpain active-site amino acid residues results in the same phenotype as deleting the entire DEK1 gene. Based on the conserved nature of animal and DEK1 calpains, we propose that the DEK1 MEM-Linker complex inactivates the calpain by forcing apart the two calpain subunits carrying the three amino acids of the active site. PMID:27506240

  16. Evidence supporting the role of calpain in the α-processing of amyloid-β precursor protein.

    Science.gov (United States)

    Nguyen, Huey T; Sawmiller, Darrell R; Wu, Qi; Maleski, Jerome J; Chen, Ming

    2012-04-13

    Amyloid plaques are a hallmark of the aging and senile dementia brains, yet their mechanism of origins has remained elusive. A central issue is the regulatory mechanism and identity of α-secretase, a protease responsible for α-processing of amyloid-β precursor protein (APP). A remarkable feature of this enzyme is its high sensitivity to a wide range of cellular stimulators, many of which are agonists for Ca(2+) signaling. This feature, together with previous work in our laboratory, has suggested that calpain, a Ca(2+)-dependent protease, plays a key role in APP α-processing. In this study we report that overexpression of the μ-calpain gene in HEK293 cells resulted in a 2.7-fold increase of the protein levels. Measurements of intracellular calpain enzymatic activity revealed that the calpain overexpressing cells displayed a prominent elevation of the activity compared to wild-type cells. When the cells were stimulated by nicotine, glutamate or phorbol 12,13-dibutylester, the activity increase was even more remarkable and sensitive to calpeptin, a calpain inhibitor. Meanwhile, APP secretion from the calpain overexpressing cells was robustly increased under both resting and stimulated conditions over wild-type cells. Furthermore, cell surface biotinylation experiments showed that μ-calpain was clearly detected among the cell surface proteins. These data together support our view that calpain should be a reasonable candidate for α-secretase for further study. This model is discussed with an interesting fact that three other deposited proteins (tau, spectrin and crystalline) are also the known substrates of calpain. Finally we discuss some current misconceptions in senile dementia research.

  17. Activation of proteolysis by calpains and structural changes in human paroxysmal and persistent atrial fibrillation

    NARCIS (Netherlands)

    Brundel, BJJM; Ausma, J; van Gelder, IC; Van Der Want, JJL; van Gilst, WH; Crijns, HJGM; Henning, RH

    2002-01-01

    Objective: Atrial fibrillation (AF) is accompanied by electrical. structural and ion-channel protein remodeling. We tested if proteolysis by calpain and proteasome is activated during AF. and studied the relation with the remodeling processes. Methods: Right atrial appendages were obtained from pati

  18. Calpains: potential targets for alternative chemotherapeutic intervention against human pathogenic trypanosomatids.

    Science.gov (United States)

    Branquinha, M H; Marinho, F A; Sangenito, L S; Oliveira, S S C; Goncalves, K C; Ennes-Vidal, V; d'Avila-Levy, C M; Santos, A L S

    2013-01-01

    The treatment for both leishmaniasis and trypanosomiasis, which are severe human infections caused by trypanosomatids belonging to Leishmania and Trypanosoma genera, respectively, is extremely limited because of concerns of toxicity and efficacy with the available anti-protozoan drugs, as well as the emergence of drug resistance. Consequently, the urgency for the discovery of new trypanosomatid targets and novel bioactive compounds is particularly necessary. In this context, the investigation of changes in parasite gene expression between drug resistant/sensitive strains and in the up-regulation of virulence-related genes in infective forms has brought to the fore the involvement of calpain-like proteins in several crucial pathophysiological processes performed by trypanosomatids. These studies were encouraged by the publication of the complete genome sequences of three human pathogenic trypanosomatids, Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, which allowed in silico analyses that in turn directed the identification of numerous genes with interesting chemotherapeutic characteristics, including a large family of calpain-related proteins, in which to date 23 genes were assigned as calpains in T. brucei, 40 in T. cruzi and 33 in L. braziliensis. In the present review, we intend to add to these biochemical/biological reports the investigations performed upon the inhibitory capability of calpain inhibitors against human pathogenic trypanosomatids.

  19. 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.

  20. Calpain-mediated cleavage of collapsin response mediator protein-2 drives acute axonal degeneration

    Science.gov (United States)

    Zhang, Jian-Nan; Michel, Uwe; Lenz, Christof; Friedel, Caroline C.; Köster, Sarah; d’Hedouville, Zara; Tönges, Lars; Urlaub, Henning; Bähr, Mathias; Lingor, Paul; Koch, Jan C.

    2016-01-01

    Axonal degeneration is a key initiating event in many neurological diseases. Focal lesions to axons result in a rapid disintegration of the perilesional axon by acute axonal degeneration (AAD) within several hours. However, the underlying molecular mechanisms of AAD are only incompletely understood. Here, we studied AAD in vivo through live-imaging of the rat optic nerve and in vitro in primary rat cortical neurons in microfluidic chambers. We found that calpain is activated early during AAD of the optic nerve and that calpain inhibition completely inhibits axonal fragmentation on the proximal side of the crush while it attenuates AAD on the distal side. A screening of calpain targets revealed that collapsin response mediator protein-2 (CRMP2) is a main downstream target of calpain activation in AAD. CRMP2-overexpression delayed bulb formation and rescued impairment of axonal mitochondrial transport after axotomy in vitro. In vivo, CRMP2-overexpression effectively protected the proximal axon from fragmentation within 6 hours after crush. Finally, a proteomic analysis of the optic nerve was performed at 6 hours after crush, which identified further proteins regulated during AAD, including several interactors of CRMP2. These findings reveal CRMP2 as an important mediator of AAD and define it as a putative therapeutic target. PMID:27845394

  1. Calpains and neuronal damage in the ischemic brain: The swiss knife in synaptic injury.

    Science.gov (United States)

    Curcio, Michele; Salazar, Ivan L; Mele, Miranda; Canzoniero, Lorella M T; Duarte, Carlos B

    2016-08-01

    The excessive extracellular accumulation of glutamate in the ischemic brain leads to an overactivation of glutamate receptors with consequent excitotoxic neuronal death. Neuronal demise is largely due to a sustained activation of NMDA receptors for glutamate, with a consequent increase in the intracellular Ca(2+) concentration and activation of calcium- dependent mechanisms. Calpains are a group of Ca(2+)-dependent proteases that truncate specific proteins, and some of the cleavage products remain in the cell, although with a distinct function. Numerous studies have shown pre- and post-synaptic effects of calpains on glutamatergic and GABAergic synapses, targeting membrane- associated proteins as well as intracellular proteins. The resulting changes in the presynaptic proteome alter neurotransmitter release, while the cleavage of postsynaptic proteins affects directly or indirectly the activity of neurotransmitter receptors and downstream mechanisms. These alterations also disturb the balance between excitatory and inhibitory neurotransmission in the brain, with an impact in neuronal demise. In this review we discuss the evidence pointing to a role for calpains in the dysregulation of excitatory and inhibitory synapses in brain ischemia, at the pre- and post-synaptic levels, as well as the functional consequences. Although targeting calpain-dependent mechanisms may constitute a good therapeutic approach for stroke, specific strategies should be developed to avoid non-specific effects given the important regulatory role played by these proteases under normal physiological conditions.

  2. Epistasis between calpain 1 and its inhibitor calpastatin within breeds of cattle.

    Science.gov (United States)

    Barendse, W; Harrison, B E; Hawken, R J; Ferguson, D M; Thompson, J M; Thomas, M B; Bunch, R J

    2007-08-01

    The calpain gene family and its inhibitors have diverse effects, many related to protein turnover, which appear to affect a range of phenotypes such as diabetes, exercise-induced muscle injury, and pathological events associated with degenerative neural diseases in humans, fertility, longevity, and postmortem effects on meat tenderness in livestock species. The calpains are inhibited by calpastatin, which binds directly to calpain. Here we report the direct measurement of epistatic interactions of causative mutations for quantitative trait loci (QTL) at calpain 1 (CAPN1), located on chromosome 29, with causative mutations for QTL variation at calpastatin (CAST), located on chromosome 7, in cattle. First we identified potential causative mutations at CAST and then genotyped these along with putative causative mutations at CAPN1 in >1500 cattle of seven breeds. The maximum allele substitution effect on the phenotype of the CAPN1:c.947G>C single nucleotide polymorphism (SNP) was 0.14 sigma(p) (P = 0.0003) and of the CAST:c.155C>T SNP was also 0.14 sigma(p) (P = 0.0011) when measured across breeds. We found significant epistasis between SNPs at CAPN1 and CAST in both taurine and zebu derived breeds. There were more additive x dominance components of epistasis than additive x additive and dominance x dominance components combined. A minority of breed comparisons did not show epistasis, suggesting that genetic variation at other genes may influence the degree of epistasis found in this system.

  3. Association of the calpain-10 gene with type 2 diabetes in Europeans

    DEFF Research Database (Denmark)

    Tsuchiya, Takafumi; Schwarz, Peter E H; Bosque-Plata, Laura Del

    2006-01-01

    We conducted pooled and meta-analyses of the association of the calpain-10 gene (CAPN10) polymorphisms SNP-43, Indel-19 and SNP-63 individually and as haplotypes with type 2 diabetes (T2D) in 3237 patients and 2935 controls of European ancestry. In the pooled analyses, the common SNP-43*G allele ...

  4. Endurance training ameliorates complex 3 deficiency in a mouse model of Barth syndrome.

    Science.gov (United States)

    Soustek, Meghan S; Baligand, Celine; Falk, Darin J; Walter, Glenn A; Lewin, Alfred S; Byrne, Barry J

    2015-09-01

    Barth syndrome (BTHS) is an X-linked metabolic disorder that causes cardiomyopathy in infancy and is linked to mutations within the Tafazzin (TAZ) gene. The first mouse model, a TAZ knockdown model (TAZKD), has been generated to further understand the bioenergetics leading to cardiomyopathy. However, the TAZKD model does not show early signs of cardiomyopathy, and cardiac pathophysiology has not been documented until 7-8 months of age. Here we sought to determine the impact of endurance training on the cardiac and skeletal muscle phenotype in young TAZKD mice. TAZKD exercise trained (TAZKD-ET) and control exercise trained (CON-ET) mice underwent a 35-day swimming protocol. Non-trained aged matched TAZKD and CON mice were used as controls. At the end of the protocol, cardiac MRI was used to assess cardiac parameters. Cardiac MRI showed that training resulted in cardiac hypertrophy within both groups and did not result in a decline of ejection fraction. TAZKD mice exhibited a decrease in respiratory complex I, III, and IV enzymatic activity in cardiac tissue compared to control mice; however, training led to an increase in complex III activity in TAZKD-ET mice resulting in similar levels to those of CON-ET mice. (31)P magnetic resonance spectroscopy of the gastrocnemius showed a significantly lowered pH in TAZKD-ET mice post electrical-stimulation compared to CON-ET mice. Endurance training does not accelerate cardiac dysfunction in young TAZKD mice, but results in beneficial physiological effects. Furthermore, our results suggest that a significant drop in intracellular pH levels may contribute to oxidative phosphorylation defects during exercise.

  5. Relationship between Calpain-10 Gene Polymorphism and Insulin Resistance Phenotypes in Chinese

    Institute of Scientific and Technical Information of China (English)

    郑涓; 陈璐璐; 黎慧清

    2004-01-01

    In order to determine whether the variations in the calpain-10 gene constitutes risk of type 2 diabetes (T2DM) in Chinese, the frequency of UCSNP-43, 44 in 268 adults newly diagnosed with T2DM (according to the 1999 ADA criteria) and 153 non-diabetic control subjects was investigated. For all subjects, the height, weight, waist-to-hip ratio (W/H) and blood pressure, as well as following parameters were measured: (1) 75-g oral glucose tolerance test with insulin, C-peptide, HbA1c and blood lipid profiles; (2) Genomic DNA extracted from peripheral blood lymphocytes was genotyped for UCSNP-43 (calpain-10-g. 4852 G/A) and UCSNP-44 (calpain-10-g.4841T/C) by sequencing a polymerase chain reaction (PCR)-amplified fragment. PCR product was selected by single strand conformation polymorphism (SSCP) and then sequenced. The results showed that there was significant difference between T2DM group and normal control group in allele frequencies, haplotype frequencies, or haplotype combinations of UCSNP-43 and -44 either.But in newly diagnosed T2DM group, it was found that the individuals with the genotype UCSNP44 T/C+C/C had significantly increased fasting and post-challenge insulin levels (FIns and P2hIns), consistent with reduced insulin sensitivity. In the BMI>25 subgroup, the differences were even more significant. It was demonstrated that the Calpain-10 gene polymorphism UCSNP-44was associated with insulin sensitivity and FIns and P2hIns in newly diagnosed T2DM, although Calpain-10 doesn't appear as a major diabetes susceptible gene in this population.

  6. Calpains mediate integrin attachment complex maintenance of adult muscle in Caenorhabditis elegans.

    Science.gov (United States)

    Etheridge, Timothy; Oczypok, Elizabeth A; Lehmann, Susann; Fields, Brandon D; Shephard, Freya; Jacobson, Lewis A; Szewczyk, Nathaniel J

    2012-01-01

    Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line- or M-line-specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C. elegans lacks

  7. Calpains and proteasomes mediate degradation of ryanodine receptors in a model of cardiac ischemic reperfusion.

    Science.gov (United States)

    Pedrozo, Zully; Sánchez, Gina; Torrealba, Natalia; Valenzuela, Rodrigo; Fernández, Carolina; Hidalgo, Cecilia; Lavandero, Sergio; Donoso, Paulina

    2010-03-01

    Type-2 ryanodine receptors (RyR2)--the calcium release channels of cardiac sarcoplasmic reticulum--have a central role in cardiac excitation-contraction coupling. In the heart, ischemia/reperfusion causes a rapid and significant decrease in RyR2 content but the mechanisms responsible for this effect are not fully understood. We have studied the involvement of three proteolytic systems--calpains, the proteasome and autophagy--on the degradation of RyR2 in rat neonatal cardiomyocyte cultures subjected to simulated ischemia/reperfusion (sI/R). We found that 8h of ischemia followed by 16h of reperfusion decreased RyR2 content by 50% without any changes in RyR2 mRNA. Specific inhibitors of calpains and the proteasome prevented the decrease of RyR2 caused by sI/R, implicating both pathways in its degradation. Proteasome inhibitors also prevented the degradation of calpastatin, the endogenous calpain inhibitor, hindering the activation of calpain induced by calpastatin degradation. Autophagy was activated during sI/R as evidenced by the increase in LC3-II and beclin-1, two proteins involved in autophagosome generation, and in the emergence of GFP-LC3 containing vacuoles in adenovirus GFP-LC3 transduced cardiomyocytes. Selective autophagy inhibition, however, induced even further RyR2 degradation, making unlikely the participation of autophagy in sI/R-induced RyR2 degradation. Our results suggest that calpain activation as a result of proteasome-induced degradation of calpastatin initiates RyR2 proteolysis, which is followed by proteasome-dependent degradation of the resulting RyR2 fragments. The decrease in RyR2 content during ischemia/reperfusion may be relevant to the decrease of heart contractility after ischemia.

  8. Calpains mediate integrin attachment complex maintenance of adult muscle in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Timothy Etheridge

    2012-01-01

    Full Text Available Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line- or M-line-specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C

  9. Interaction of aspartic acid-104 and proline-287 with the active site of m-calpain.

    Science.gov (United States)

    Arthur, J S; Elce, J S

    1996-01-01

    In an ongoing study of the mechanisms of calpain catalysis and Ca(2+)-induced activation, the effects of Asp-104-->Ser and Pro-287-->Ser large subunit mutations on m-calpain activity, the pH-activity profile, Ca(2+)-sensitivity, and autolysis were measured. The importance of these positions was suggested by sequence comparisons between the calpain and papain families of cysteine proteinases. Asp-104 is adjacent to the active-site Cys-105, and Pro-287 is adjacent to the active-site Asn-286 and probably to the active-site His-262; both Asp-104 and Pro-287 are absolutely conserved in the known calpains, but are replaced by highly conserved serine residues in the papains. The single mutants had approx. 10-15% of wild-type activity, due mainly to a decrease in kcat, since Km was only slightly increased. The Pro-287-->Ser mutation appeared to cause a local perturbation of the catalytic Cys-105/His-262 catalytic ion pair, reducing its efficiency without major effect on the conformation and stability of the enzyme. The Asp-104-->Ser mutation caused a marked narrowing of the pH-activity curve, a 9-fold increase in Ca2+ requirement, and an acceleration of autolysis, when compared with the wild-type enzyme. The results indicated that Asp-104 alters the nature of its interaction with the catalytic ion pair during Ca(2+)-induced conformational change in calpain. This interaction may be direct or indirect, but is important in activation of the enzyme. PMID:8912692

  10. TLR3 deficiency renders astrocytes permissive to herpes simplex virus infection and facilitates establishment of CNS infection in mice

    DEFF Research Database (Denmark)

    Reinert, Line S; Harder, Louis; Holm, Christian K

    2012-01-01

    Herpes simplex viruses (HSVs) are highly prevalent neurotropic viruses. While they can replicate lytically in cells of the epithelial lineage, causing lesions on mucocutaneous surfaces, HSVs also establish latent infections in neurons, which act as reservoirs of virus for subsequent reactivation ...

  11. calpain1在卡那霉素致毒豚鼠耳蜗中的表达%Expression of calpain 1 in kanamycin-poisoned guinea pig cochlea

    Institute of Scientific and Technical Information of China (English)

    马德菊

    2011-01-01

    Aim To investigate the expression of calpain 1 in kanamycin( KM )-poisoned guinea pig cochlea. Methods Guinea pigs were randomly assigned to four groups : control group, KM 3 days group , 7 days group and 14 days group. Auditory brainstem response ( ABR )measurement was used to test the hearing of guinea pig. The expression of calpain I was detected by the SABC method of immunohistochemistry combined with imaging analysis technique. Auditory brainstem response( ABR )measurement was used to test the hearing of guinea pig. Results Immunoreactivity for calpain 1 was mainly found in hair cells , spiral ganglions, spiral ligament and stria vascularis. The results of imaging analysis indicated that immuncorectivity for calpain 1 gradually lowered with the days up. Conclusion Calpain 1 is found in Cochlea of control group and KM group. The results of imaging analysis indicate that immuncorectivity for calpain 1 gradually lower with the days up.%目的 探讨钙蛋白酶(calpain 1)在卡那霉素(kanamycin,KM)致毒豚鼠耳蜗中的定位与表达.方法 将40只豚鼠随机均分成4组:对照组、KM 3 d组、KM 7 d组和KM 14 d组,KM各组每天肌肉注射硫酸卡那霉素注射液200 mg·kg-1.应用电生理指标听性脑干反应(auditory brainstem response,ABR)观察用药前后豚鼠听力的变化;采用免疫组织化学SABC法结合显微图像分析技术,观察豚鼠耳蜗组织中calpain 1的表达.结果 calpain 1阳性免疫反应主要见于耳蜗毛细胞、螺旋神经节、螺旋韧带和血管纹,并随给药天数的增加,calpain 1在耳蜗上述部位的表达逐渐减弱.结论 calpain 1在正常豚鼠和卡那霉素致毒豚鼠耳蜗中都有表达,且随卡那霉素给药天数的增加表达逐渐减弱.

  12. FoxD3 deficiency promotes breast cancer progression by induction of epithelial–mesenchymal transition

    Energy Technology Data Exchange (ETDEWEB)

    Chu, Tian-Li [Department of General Surgery, The People’s Hospital of Wuqing, Tianjin (China); Zhao, Hong-Meng [Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Breast Surgery, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Li, Yue [Department of Respiration, Affiliated Hospital of Medical College of Chinese People’s Armed Police Force, Tianjin (China); Chen, Ao-Xiang; Sun, Xuan [Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Breast Surgery, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Ge, Jie, E-mail: gejie198003@163.com [Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China); Breast Surgery, Tianjin Medical University Cancer Institute and Hospital, Tianjin (China)

    2014-04-04

    Highlights: • FOXD3 is down-regulated in breast cancer tissues. • FOXD3 inhibits breast cancer cell proliferation and invasion. • FoxD3 deficiency induces epithelial–mesenchymal transition. - Abstract: The transcription factor forkhead box D3 (FOXD3) plays an important role in the development of neural crest and gastric cancer cells. However, the function and mechanisms of FOXD3 in the breast tumorigenesis and progression is still limited. Here, we report that FOXD3 is a tumor suppressor of breast cancer tumorigenicity and aggressiveness. We found that FOXD3 is down-regulated in breast cancer tissues. Patients with low FOXD3 expression have a poor outcome. Depletion of FOXD3 expression promotes breast cancer cell proliferation and invasion in vitro, whereas overexpression of FOXD3 inhibits breast cancer cell proliferation and invasion both in vitro and in vivo. In addition, depletion of FOXD3 is linked to epithelial–mesenchymal transition (EMT)-like phenotype. Our results indicate FOXD3 exhibits tumor suppressive activity and may be useful for breast therapy.

  13. The tyrosine phosphatase HD-PTP (PTPN23) is degraded by calpains in a calcium-dependent manner.

    Science.gov (United States)

    Castiglioni, Sara; Maier, Jeanette A M

    2012-05-04

    HD-PTP (PTPN23) is a non-transmembrane protein tyrosine phosphatase which contributes to the signal transduction pathways involved in the regulation of cell migration and invasion. We here demonstrate in T24 bladder carcinoma cells that HD-PTP undergoes calcium-dependent degradation which can be prevented by specific calpain inhibitors. In addition, treatment of the cells with the calpain inhibitor calpeptin results in the redistribution of endogenous HD-PTP to the periphery of the cells. Since (i) calpains are overexpressed in some tumors and (ii) the downregulation of HD-PTP enhances cell migration and invasion, we propose that HD-PTP degradation by calpains might result in the acquisition of a more aggressive phenotype in neoplastic cells.

  14. Sphingosine 1-Phosphate Receptor 3-Deficient Dendritic Cells Modulate Splenic Responses to Ischemia-Reperfusion Injury.

    Science.gov (United States)

    Bajwa, Amandeep; Huang, Liping; Kurmaeva, Elvira; Gigliotti, Joseph C; Ye, Hong; Miller, Jacqueline; Rosin, Diane L; Lobo, Peter I; Okusa, Mark D

    2016-04-01

    The plasticity of dendritic cells (DCs) permits phenotypic modulation ex vivo by gene expression or pharmacologic agents, and these modified DCs can exert therapeutic immunosuppressive effects in vivo through direct interactions with T cells, either inducing T regulatory cells (T(REG)s) or causing anergy. Sphingosine 1-phosphate (S1P) is a sphingolipid and the natural ligand for five G protein-coupled receptors (S1P1, S1P2, S1P3, S1P4, and S1P5), and S1PR agonists reduce kidney ischemia-reperfusion injury (IRI) in mice. S1pr3(-/-)mice are protected from kidney IRI, because DCs do not mature. We tested the therapeutic advantage of S1pr3(-/-) bone marrow-derived dendritic cell (BMDC) transfers in kidney IRI. IRI produced a rise in plasma creatinine (PCr) levels in mice receiving no cells (NCs) and mice pretreated with wild-type (WT) BMDCs. However, S1pr3(-/-) BMDC-pretreated mice were protected from kidney IRI. S1pr3(-/-) BMDC-pretreated mice had significantly higher numbers of splenic T(REG)s compared with NC and WT BMDC-pretreated mice. S1pr3(-/-) BMDCs did not attenuate IRI in splenectomized, Rag-1(-/-), or CD11c(+) DC-depleted mice. Additionally, S1pr3(-/-) BMDC-dependent protection required CD169(+)marginal zone macrophages and the macrophage-derived chemokine CCL22 to increase splenic CD4(+)Foxp3(+) T(REG)s. Pretreatment with S1pr3(-/-) BMDCs also induced T(REG)-dependent protection against IRI in an allogeneic mouse model. In summary, adoptively transferred S1pr3(-/-) BMDCs prevent kidney IRI through interactions within the spleen and expansion of splenic CD4(+)Foxp3(+) T(REG)s. We conclude that genetically induced deficiency of S1pr3 in allogenic BMDCs could serve as a therapeutic approach to prevent IRI-induced AKI.

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

    Directory of Open Access Journals (Sweden)

    Viviane Giese

    2008-09-01

    Full Text Available 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.

  16. 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.

  17. 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.

  18. Mechanical Stimulation of C2C12 Cells Increases m-Calpain Expression and Activity, Focal Adhesion Plaque Degradation and Cell Fusion

    DEFF Research Database (Denmark)

    Grossi, Alberto; Lawson, Moira Ann; Karlsson, Anders H

    Abstract Mechanical Stimulation of C2C12 Cells Increases m-calpain Expression and Activity, Focal Adhesion Plaque Degradation and Cell Fusion A. Grossi, A. H. Karlsson, M. A. Lawson; Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark...... to the activity of ubiquitous proteolytic enzymes known as calpains has been reported. Whether there is a link between stretch- or load induced signaling and calpain expression and activation is not known. Using a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have demonstrated...... that mechanical stimulation via laminin receptors leads to an increase in m-calpain expression, but no increase in the expression of other calpain isoforms. Our study revealed that after a short period of stimulation, m-calpain relocates into focal adhesion complexes and is followed by a breakdown of specific...

  19. Partial pyruvate kinase deficiency aggravates the phenotypic expression of band 3 deficiency in a family with hereditary spherocytosis

    NARCIS (Netherlands)

    van Zwieten, Rob; van Oirschot, Brigitte A; Veldthuis, Martijn; Dobbe, Johannes G; Streekstra, Geert J; van Solinge, Wouter W; Schutgens, Roger E G; van Wijk, Richard

    2015-01-01

    In a family with mild dominant spherocytosis, affected members showed partial band 3 deficiency. The index patient showed more severe clinical symptoms than his relatives, and his red blood cells displayed concomitant low pyruvate kinase activity. We investigated the contribution of partial PK defic

  20. Partial pyruvate kinase deficiency aggravates the phenotypic expression of band 3 deficiency in a family with hereditary spherocytosis.

    NARCIS (Netherlands)

    Zwieten, R. van; Oirschot, B.A.J.A. van; Veldthuis, M.; Dobbe, J.G.; Streekstra, G.J.; Solinge, W.W. van; Schutgens, R.E.; Wijk, R. Gerth van

    2015-01-01

    In a family with mild dominant spherocytosis, affected members showed partial band 3 deficiency. The index patient showed more severe clinical symptoms than his relatives, and his red blood cells displayed concomitant low pyruvate kinase activity. We investigated the contribution of partial PK defic

  1. 慢性阻塞性肺疾病模型大鼠骨骼肌中 Calpains 的表达%Expression of Calpains in skeletal muscle of rat COPD model

    Institute of Scientific and Technical Information of China (English)

    刘待见; 冯慧芬; 刘剑波

    2016-01-01

    Aim: To study the role of Calpains in rat skeletal muscle atrophy of chronic obstructive pulmonary disease (COPD).Methods: Forty healthy male Wistar rats were randomly allocated into COPD group and control group .COPD model was established by dripping lipopolysacharide (LPS) into trachea and exposing to cigarettes smoking .The pathological changes of lung tissue and skeletal muscle were observed by HE staining .Immunohistochemistry and RT-PCR were per-formed respectively to determine protein and mRNA expression of Calpain in skeletal muscle (μ-Calpain and m-Calpain). Results: Skeletal muscle atrophy was observed in COPD group instead of control group .Compared with control group,the expression of μ-Calpain and m-Calpain protein in extensor digitorum longus muscle and diaphragm of COPD group was en -hanced (P <0.05), as well as m-Calpain mRNA expression(P <0.05).Conclusion: The expression of Calpains in skel-etal muscle of rats COPD model upregulated .Calpains may be involved in COPD skeletal muscle atrophy .%目的:探讨 Calpains 在慢性阻塞性肺疾病(COPD)大鼠骨骼肌萎缩中的作用。方法:40只健康雄性Wistar 大鼠随机分为 COPD 模型组及对照组,模型组采用气管内注入脂多糖加香烟烟雾染毒法建立大鼠 COPD 模型,HE 染色法观察肺组织及骨骼肌(趾长伸肌、膈肌)组织的病理学改变;分别采用免疫组化和 RT-PCR 法测定骨骼肌组织中 Calpains(μ-Calpain 和 m-Calpain)蛋白及 mRNA 的表达。结果:在 COPD 模型组大鼠观察到骨骼肌萎缩,对照组大鼠未见骨骼肌萎缩。与对照组比较,COPD 模型组大鼠趾长伸肌及膈肌中μ-Calpain 和 m-Calpain 蛋白及 m-Calpain mRNA 表达增强(P <0.05),而μ-Calpain mRNA 的表达差异无统计学意义(P >0.05)。结论:Calpains在 COPD 模型大鼠骨骼肌中表达上调,可能参与 COPD 模型大鼠骨骼肌萎缩。

  2. Association of Smoking With Semen Quality and µ-Calpain Level in Normospermia: A Case-Control Study

    Directory of Open Access Journals (Sweden)

    Damoon Ashtary larky

    2016-05-01

    Full Text Available Objective: Calpains are a family of Ca2+ dependent proteases. There is some evidence that calpains involved in fusion process that occurs between spermatozoa and the oocyte. The current study aimed to investigate the association of smoking with semen quality and µ-calpain level.Materials and methods: This case-control study was conducted on 117 normospermia males  between June 2013 and march 2014 in Jahad Laboratory in ahvaz, Iran. The semen samples were collected from male smokers (n = 50 and non-smokers (n = 67. We divided these participants as light, moderate, or heavy smokers based on their cigarettes per day (CPD. ELISA assays were used to measure µ-calpain concentration. All semen samples were analyzed according to World Health Organization guidelines.Results: The analysis of semen showed the volume, concentration, motility and morphology of semen were significantly lower among the smoker men than the non-smoker men. Also this significant difference was observed based on the number (light, moderate and heavy smokers and duration (short term and long term smoker of smoking. Although, showed no significant difference between µ-calpain of smoker men and non-smoker men. CPD showed negatively correlation with semen volume, concentration, motility and morphology of sperm.Conclusion: Sperm quality was negatively correlated with CPD and duration of smoking. However, there is no significant correlation between smoking and µ-calpain concentration.

  3. Membrane-Permeable Calpain Inhibitors Promote Rat Oral Mucosal Epithelial Cell Proliferation by Inhibiting IL-1α Signaling.

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    Makoto Kondo

    Full Text Available To standardise regenerative medicine using cultured cells, the use of serum-free, chemically defined media will be necessary. We have reported that IL-1α inhibits the growth of epithelial cells in culture and that recombinant IL-1 receptor antagonist (IL-1RA significantly promotes epithelial cell growth in no feeder layer condition. In this study, we examined inhibitors of calpain, a cysteine proteinase that plays crucial roles in various cellular functions, including IL-1α maturation and secretion. The culturing of epithelial cells in serum-free media supplemented with a membrane-permeable calpain inhibitor significantly promoted growth while suppressing IL-1α maturation and secretion. By contrast, non-membrane-permeable calpain inhibitor treatment did not have these effects. Interestingly, immunoblotting analysis revealed that immature, untruncated, IL-1α expression was also downregulated by cell-permeable calpain inhibitor treatment, and the difference in IL-1α gene expression increased from day 2 to day 6. Although IL-1RA has been reported to promote epithelial cell growth, we detected no synergistic promotion of epithelial cell growth using a calpain inhibitor and IL-1RA. These findings indicate that calpain inhibitors promote epithelial cell proliferation by inhibiting IL-1α maturation at an early phase of epithelial cell culture and by suppressing the positive feedback-mediated amplification of IL-1α signalling.

  4. Calpain-2 expression is associated with response to platinum based chemotherapy, progression-free and overall survival in ovarian cancer

    Science.gov (United States)

    Storr, Sarah J; Safuan, Sabreena; Woolston, Caroline M; Abdel-Fatah, Tarek; Deen, Suha; Chan, Stephen Y; Martin, Stewart G

    2012-01-01

    Ovarian cancer is routinely treated with surgery and platinum-based chemotherapy. Resistance is a major obstacle in the efficacy of this chemotherapy regimen and the ability to identify those patients at risk of developing resistance is of considerable clinical importance. The expression of calpain-1, calpain-2 and calpastatin were determined using standard immunohistochemistry on a tissue microarray of 154 primary ovarian carcinomas from patients subsequently treated with platinum-based adjuvant chemotherapy. High levels of calpain-2 expression was significantly associated with platinum resistant tumours (P = 0.031). Furthermore, high expression of calpain-2 was significantly associated with progression-free (P = 0.049) and overall survival (P = 0.006) in this cohort. The association between calpain-2 expression and overall survival remained significant in multivariate analysis accounting for tumour grade, stage, optimal debulking and platinum sensitivity (hazard ratio = 2.174; 95% confidence interval = 1.144–4.130; P = 0.018). The results suggest that determining calpain-2 expression in ovarian carcinomas may allow prognostic stratification of patients treated with surgery and platinum-based chemotherapy. The findings of this study warrant validation in a larger clinical cohort. PMID:22435971

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

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    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.

  6. The Effects of Cisplatin on the Expression of Calpain in Mouse Cochlear Hair Cell in Vitro%顺铂对离体培养小鼠耳蜗毛细胞钙蛋白酶表达的影响

    Institute of Scientific and Technical Information of China (English)

    张立伟; 刘芳芳; 于利; 王爱梅

    2016-01-01

    Objective To investigate the effects of cisplatin on the expression of calpain in mouse cochlear hair cells in vitro ,and to explore the mechanism of cisplatin-induced apoptosis in mouse cochlear hair cells .Meth‐ods A total of 600 cochlear basilar membranes isolated from Kunming mice at postnatal day 3 were cultured for 24 hours ,then randomly divided into control group and 3 cisplatin groups (4 μg/ml ,8 μg/ml and 16 μg/ml) .Each group contained 150 basilar membranes .Four groups were continually cultured for another 24 hours .Hoechst 33258 staining was used to detect the apoptosis of cochlear hair cell .Immunofluorescent staining and Western blot were carried out for detecting the expressions of calpain 1 (μ-calpain) and calpain 2 (m-calpain) in mouse cochlear hair cells .Results The percent of apoptotic hair cells in the three cisplatin groups (15 .63% ± 0 .20% ,38 .40% ± 2 .64% and 64 .24% ± 0 .05% ,respectively) was greater than that of in the control group (5 .55% ± 0 .12% ) , showing a clear dose-response relationship (P<0 .01) .Furthermore ,the expressions of μ -calpain and m -cal‐pain in different cisplatin groups were increased ,and m -calpain expression was great remarkably with increased concentration of cisplatin (P<0 .01) .Conclusion Our results suggest that cisplatin can induce the apoptosis in mouse cochlear hair cells by regulating calpain pathway .This may play a role in the cisplatin-induced ototoxicity .%目的:观察顺铂对离体培养小鼠耳蜗毛细胞钙蛋白酶(calpain )表达的影响,探讨顺铂致耳蜗毛细胞凋亡的机制。方法取出生后3 d的昆明小鼠300只(600耳),分离出耳蜗基底膜600条,体外培养24 h后,随机分为对照组和4、8、16μg/ml顺铂组,每组150条;对照组加入2ml新鲜培养基,顺铂组分别加入2 ml含不同浓度顺铂(4、8、16μg/ml)的新鲜培养基,再继续培养24 h后,应用Hoechst 33258荧光染色观察耳蜗毛细胞

  7. 25-Hydroxyvitamin D3 Deficiency Independently Predicts Cognitive Impairment in Patients with Systemic Lupus Erythematosus.

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    Sen Hee Tay

    Full Text Available Cognitive dysfunction has been reported in 20-80% of SLE patients. Converging evidence has indicated the importance of vitamin D as a neuroimmunomodulator for cognitive function. In this study, we evaluated the relationship between vitamin D and cognitive dysfunction.Consecutive age- and gender-matched SLE patients and healthy controls (HCs were administered Automated Neuropsychological Assessment Metrics in this cross-sectional study. The primary outcome was the total throughput score (TTS. Anxiety and depression were measured using the Hospital Anxiety and Depression Scale (HADS. Levels of 25-hydroxyvitamin D [25(OHD3 and total 25(OHD] were measured using Liquid Chromatography-Tandem Mass Spectrometry.In total, 61 SLE patients and 61 HCs were studied. SLE patients scored significantly lower than HCs in the TTS (p = 0.004. There were no statistically significant differences in 25(OHD3 levels, total 25(OHD levels and total 25(OHD deficiency between SLE patients and HCs. However, more SLE patients had 25(OHD3 deficiency compared to HCs [12 (19.7% versus 2 (3.3%, p = 0.003]. Deficiency of 25(OHD3 (β = -63.667, SE = 27.456, p = 0.025, but not other vitamin D variables, independently predicted worse TTS after adjusting for age, education, gender, ethnicity, HADS-Total, duration of SLE, SELENA-SLEDAI, SLICC/ACR Damage Index and cumulative steroid dose in SLE patients. Age (β = -4.261, SE = 0.866, p < 0.001 was the only predictor of TTS after adjusting for education, gender, ethnicity, HADS-Total, vitamin D levels or status in HCs.Deficiency of 25(OHD3, a potentially modifiable risk factor, independently predicted cognitive impairment in SLE patients.

  8. Clinical significance and the expression level of AIF and Calpain-Ⅰ mRNA in gastric cancer%AIF与Calpain-Ⅰ在胃癌组织中的表达及其临床意义

    Institute of Scientific and Technical Information of China (English)

    赵悦; 彭杰; 李兴德; 朱中成; 张明云

    2013-01-01

    目的:检测胃癌组织中抑癌基因AIF与Calpain-Ⅰ mRNA水平的表达情况,探讨其在胃癌发生发展中的作用及其临床意义.方法:实时荧光定量PCR法检测64例胃癌组织及其对应的癌旁组织中AIF与Calpain-Ⅰ mRNA表达情况.结果:胃癌组织中AIF与Calpain-Ⅰ mRNA表达水平较癌旁组织上调,其阳性率分别为84%和75%,差异具有统计学意义(P<0.05).早期胃癌中AIF与Calpain-Ⅰ mRNA表达水平增高,随着肿瘤恶性程度的增高、临床分期的变晚,逐渐表现出下调趋势.结论:在胃癌组织中,AIF与Calpain-Ⅰ mR-NA表达水平较癌旁正常组织上调,且与胃癌的临床分期显著相关.%Objective; To detect the expression level of AIF and Calpain - I mRNA in gastric cancer and discuss the correlation with occurrence and development of gastric cancer and clinical significance. Methods: Real - time fluorescence quantitative RT - PCR was used to detect AIF and Calpain -I mRNA expression level in 64 cases of gastric cancer and para - cancer normal tissues. Results; The expression level of AIF(84% ) and Calpain -I(75% ) mRNA in gastric cancer was up -regulated obviously compared to para -cancer normal tissues(P <0.05). Furthermore,the expression level of AIF and Calpain - I mRNA was upper expressed in advanced gastric cancer compared to early gastric cancer(P<0.05). With the higher degree of malignant and clinical stage,it seems to decline gradually. Conclusion ; The expression level of AIF and Calpain - I mRNA was over expressed in gastric cancer tissues, and it had relationship with the clinical staging of gastric cancer.

  9. Effects of expression of calpain mRNA in rabbits exposed to vibration by hind legs%后肢接振对家兔脑和骨骼肌组织中calpain mRNA表达的影响

    Institute of Scientific and Technical Information of China (English)

    张兆强; 张春芝; 聂继池; 林立

    2016-01-01

    目的 探讨后肢接触振动(简称接振)对家兔脑和骨骼肌组织中calpain mRNA表达的影响.方法 依据4h等能量频率计权加速度有效值[a()(4)]将32只新西兰大白兔随机分为低强度(4.33 m/s2)、中强度(8.67 m/s2)、高强度(17.34 m/s2)组和对照组;后肢接振45 d后,取家兔脑和骨骼肌组织,实时荧光定量聚合酶链式反应(RT-qPCR)检测其calpain-1和calpain-2 mRNA的表达情况.结果 低、中、高强度组家兔脑组织中calpain-1 mRNA的相对含量分别为8.35±3.75、9.64±4.54、15.10±5.26,calpain-2 mRNA的相对含量分别为7.34±4.97、8.50±5.66、8.16±5.59,明显高于对照组(1.10±0.29、0.56±0.43),差异均有统计学意义(P<0.01);与对照组(0.98±0.59)比较,低、中、高强度组家兔骨骼肌组织中calpain-1 mRNA的相对含量(4.36±2.05、7.37±4.06、12.46±6.21)明显升高,差异均有统计学意义(P<0.05);各强度组与对照组家兔骨骼肌组织中calpain-2 mRNA的表达差异无统计学意义(P>0.05).结论 后肢接振能够上调家兔脑组织中calpain-1和calpain-2 mRNA以及骨骼肌组织calpain-1 mRNA的表达.%Objective To study the effects of expression of calpain mRNA in rabbits exposed to vibration by hind legs.Methods 32 New Zealand rabbits were randomly divided into a control group and 3 experimental groups according to 4-hour energy-equivalent frequency-weighted acceleration[a()(4)]:low (4.33 m/s2),moderate (8.67 m/s2) and high (17.34 m/s2) intensity group to accepted the vibration by hind legs.45 ds later,brain and skeletal muscle tissue of rabbits were taken to detect the expression of calpain-1 and calpain-2 mRNA by RT-qPCR technique.Results The relative content of calpain-1 mRNA in the brain tissues in rabbits of low,medium and high intensity group were 8.35±3.75,9.64±4.54,5.10±5.26.While the relative content of calpain-2 mRNA in the brain tissues in rabbits of low,medium and high intensity group were 7.34 ±4.97,8.50 ±5.66,8.16

  10. Amino acid sequence alignment of vertebrate CAPN3/calpain-3/p94

    Directory of Open Access Journals (Sweden)

    Yasuko Ono

    2015-12-01

    Full Text Available CAPN3 is a calpain superfamily member that is predominantly expressed in skeletal muscle. So far, clear CAPN3 orthologs were found only in vertebrates. CAPN3 is a unique protease in that it undergoes extremely rapid and exhaustive autolysis and that autolyzed fragments spontaneously associate each other to reconstitute the proteolytic activity. These unique properties of CAPN3 are dependent on IS1 and IS2, two CAPN3-characterizing sequences that do not exist in other calpains or any other proteases. To understand how IS1 and IS2 are conserved among vertebrates, this data article provides amino acid sequence alignment of representative vertebrate CAPN3s. For further analysis and discussion, see Ono et al. [1

  11. Effects of calcium ion, calpains, and calcium channel blockers on retinitis pigmentosa.

    Science.gov (United States)

    Nakazawa, Mitsuru

    2011-01-01

    Recent advances in molecular genetic studies have revealed many of the causative genes of retinitis pigmentosa (RP). These achievements have provided clues to the mechanisms of photoreceptor degeneration in RP. Apoptosis is known to be a final common pathway in RP and, therefore, a possible therapeutic target for photoreceptor rescue. However, apoptosis is not a single molecular cascade, but consists of many different reactions such as caspase-dependent and caspase-independent pathways commonly leading to DNA fractionation and cell death. The intracellular concentration of calcium ions is also known to increase in apoptosis. These findings suggest that calpains, one of the calcium-dependent proteinases, play some roles in the process of photoreceptor apoptosis and that calcium channel antagonists may potentially inhibit photoreceptor apoptosis. Herein, the effects of calpains and calcium channel antagonists on photoreceptor degeneration are reviewed.

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

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    Marton Siklos

    2015-11-01

    Full Text Available 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.

  13. Effects of Calcium Ion, Calpains, and Calcium Channel Blockers on Retinitis Pigmentosa

    Directory of Open Access Journals (Sweden)

    Mitsuru Nakazawa

    2011-01-01

    Full Text Available Recent advances in molecular genetic studies have revealed many of the causative genes of retinitis pigmentosa (RP. These achievements have provided clues to the mechanisms of photoreceptor degeneration in RP. Apoptosis is known to be a final common pathway in RP and, therefore, a possible therapeutic target for photoreceptor rescue. However, apoptosis is not a single molecular cascade, but consists of many different reactions such as caspase-dependent and caspase-independent pathways commonly leading to DNA fractionation and cell death. The intracellular concentration of calcium ions is also known to increase in apoptosis. These findings suggest that calpains, one of the calcium-dependent proteinases, play some roles in the process of photoreceptor apoptosis and that calcium channel antagonists may potentially inhibit photoreceptor apoptosis. Herein, the effects of calpains and calcium channel antagonists on photoreceptor degeneration are reviewed.

  14. Calpains participate in nerve terminal degeneration induced by spider and snake presynaptic neurotoxins.

    Science.gov (United States)

    Duregotti, Elisa; Tedesco, Erik; Montecucco, Cesare; Rigoni, Michela

    2013-03-15

    α-latrotoxin and snake presynaptic phospholipases A2 neurotoxins target the presynaptic membrane of axon terminals of the neuromuscular junction causing paralysis. These neurotoxins display different biochemical activities, but similarly alter the presynaptic membrane permeability causing Ca(2+) overload within the nerve terminals, which in turn induces nerve degeneration. Using different methods, here we show that the calcium-activated proteases calpains are involved in the cytoskeletal rearrangements that we have previously documented in neurons exposed to α-latrotoxin or to snake presynaptic phospholipases A2 neurotoxins. These results indicate that calpains, activated by the massive calcium influx from the extracellular medium, target fundamental components of neuronal cytoskeleton such as spectrin and neurofilaments, whose cleavage is functional to the ensuing nerve terminal fragmentation.

  15. 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.

  16. 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.

  17. Chronic hypobaric hypoxia mediated skeletal muscle atrophy: role of ubiquitin-proteasome pathway and calpains.

    Science.gov (United States)

    Chaudhary, Pooja; Suryakumar, Geetha; Prasad, Rajendra; Singh, Som Nath; Ali, Shakir; Ilavazhagan, Govindsamy

    2012-05-01

    The most frequently reported symptom of exposure to high altitude is loss of body mass and decreased performance which has been attributed to altered protein metabolism affecting skeletal muscles mass. The present study explores the mechanism of chronic hypobaric hypoxia mediated skeletal muscle wasting by evaluating changes in protein turnover and various proteolytic pathways. Male Sprague-Dawley rats weighing about 200 g were exposed to hypobaric hypoxia (7,620 m) for different durations of exposure. Physical performance of rats was measured by treadmill running experiments. Protein synthesis, protein degradation rates were determined by (14)C-Leucine incorporation and tyrosine release, respectively. Chymotrypsin-like enzyme activity of the ubiquitin-proteasome pathway and calpains were studied fluorimetrically as well as using western blots. Declined physical performance by more than 20%, in terms of time taken in exhaustion on treadmill, following chronic hypobaric hypoxia was observed. Compared to 1.5-fold increase in protein synthesis, the increase in protein degradation was much higher (five-folds), which consequently resulted in skeletal muscle mass loss. Myofibrillar protein level declined from 46.79 ± 1.49 mg/g tissue at sea level to 37.36 ± 1.153 (P calpains (three-fold) has been found to be important factors for the enhanced protein degradation rate. The study provided strong evidences suggesting that elevated protein turnover rate lead to skeletal muscle atrophy under chronic hypobaric hypoxia via ubiquitin-proteasome pathway and calpains.

  18. Calpain Inhibition Attenuates Apoptosis of Retinal Ganglion Cells in Acute Optic Neuritis

    Science.gov (United States)

    Smith, Amena W.; Das, Arabinda; Guyton, M. Kelly; Ray, Swapan K.; Rohrer, Baerbel

    2011-01-01

    Purpose. Optic neuritis (ON), inflammation of the optic nerve, is strongly associated with the pathogenesis of multiple sclerosis (MS) and is initiated by the attack of autoreactive T cells against self-myelin antigens, resulting in demyelination, degeneration of retinal ganglion cells (RGCs), and cumulative visual impairment. Methods. Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats on day 0, and animals received daily intraperitoneal injections of calpain inhibitor (calpeptin) or vehicle from day 1 until killed. Retinal cell death was analyzed by DNA fragmentation, and surviving ganglion cells were quantified after double labeling of retinal tissue with TUNEL and Brn3a. The expression of apoptotic and inflammatory proteins was determined by Western blotting. Results. It was demonstrated that calpain inhibition downregulates expression of proapoptotic proteins and the proinflammatory molecule nuclear factor-kappa B (NF-κB) in the retina of Lewis rats with acute EAE. Immunofluorescent labeling revealed that apoptotic cells in the RGC layer of vehicle-treated EAE animals were Brn3a positive, and a moderate dose of calpeptin dramatically reduced the frequency of apoptotic RGCs. Conclusions. These results suggest that calpain inhibition might be a useful supplement to immunomodulatory therapies such as corticosteroids in ON, due to its neuroprotective effect on RGCs. PMID:21613375

  19. 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).

  20. Calpain-Ⅰand Atrial Fibrillation%钙蛋白酶Ⅰ与心房颤动

    Institute of Scientific and Technical Information of China (English)

    冯曼; 杨群辉; 胡建民

    2014-01-01

    心房颤动是临床常见的心律失常,能致使血栓栓塞、心功能下降和心律失常性心肌疾病,给人类健康带来极大危害。Ca2+超载是导致心房颤动发病的重要机制之一,钙蛋白酶Ⅰ(calpain-Ⅰ)是一种由 Ca2+激活的蛋白水解酶,对心血管系统产生广泛的生物学效应。研究表明,在心房颤动发生过程中,calpain-Ⅰ活化后会降解心肌收缩蛋白、参与心房肌结构重构和促进细胞凋亡等。因此,calpain-Ⅰ有望成为治疗房颤的新靶点。论文简要阐述了 calpain-Ⅰ与心房颤动的关系。%Atrial fibrillation(AF)is the most prevalent cardiac arrhymia,which severely damages the health of pa-tients by causing stroke,decreasing heart function and developing myocardial disease.Ca2+ overloading is an im-portant mechanism in the pathogenesis of AF,calpain-Ⅰis Ca2+-activated protease,which play many kinds of bio-logical effects in the cardiovascular system.Recent studies have shown that activated calpain-Ⅰmay be a new target for the treatment of AF,involved in atrial structural remodeling,promote myocardial apoptosis in AF,and may be a new target for the treatment of AF.The relationship between calpain-Ⅰand AF were briefly reviewed in this arti-cle.

  1. Protein kinase Ciota promotes nicotine-induced migration and invasion of cancer cells via phosphorylation of micro- and m-calpains.

    Science.gov (United States)

    Xu, Lijun; Deng, Xingming

    2006-02-17

    Nicotine is a major component in cigarette smoke that activates the growth-promoting pathways to facilitate the development of lung cancer. However, it is not clear whether nicotine affects cell motility to facilitate tumor metastasis. Here we discovered that nicotine potently induces phosphorylation of both mu- and m-calpains via activation of protein kinase Ciota (PKCiota), which is associated with accelerated migration and invasion of human lung cancer cells. Purified PKCiota directly phosphorylates mu- and m-calpains in vitro. Overexpression of PKCiota results in increased phosphorylation of both mu- and m-calpains in vivo. Nicotine also induces activation of c-Src, which is a known PKCiota upstream kinase. Treatment of cells with the alpha(7) nicotinic acetylcholine receptor inhibitor alpha-bungarotoxin can block nicotine-induced calpain phosphorylation with suppression of calpain activity, wound healing, cell migration, and invasion, indicating that nicotine-induced calpain phosphorylation occurs, at least in part, through a signaling pathway involving the upstream alpha(7) nicotinic acetylcholine receptor. Intriguingly, depletion of PKCiota by RNA interference suppresses nicotine-induced calpain phosphorylation, calpain activity, cell migration, and invasion, indicating that PKCiota is a necessary component in nicotine-mediated cell motility signaling. Importantly, nicotine potently induces secretion of mu- and m-calpains from lung cancer cells into culture medium, which may have potential to cleave substrates in the extracellular matrix. These findings reveal a novel role for PKCiota as a nicotine-activated, physiological calpain kinase that directly phosphorylates and activates calpains, leading to enhanced migration and invasion of human lung cancer cells.

  2. 风湿性心脏病心房颤动的左心房中calpain-Ⅰ的表达和意义%Expression and significance of calpain-Ⅰ in left atria of patients with rheumatic heart disease

    Institute of Scientific and Technical Information of China (English)

    陈运清; 王琳; 苏晞; 陶凉; 陈绪发

    2007-01-01

    目的:检测calpain-Ⅰ在风湿性心脏病心房颤动(Af)患者左心房中的表达,研究其在Af发病机制中的作用.方法:选择接受外科换瓣手术的风湿性心脏病患者43例,其中窦性心律(RSR)组15例,阵发性Af(Paf)组8例,慢性Af(Caf)组20例.在外科手术中取左心房组织,应用免疫印迹方法测定calpain-Ⅰ和肌钙蛋白I(cTnⅠ)的蛋白含量.用逆转录-聚合酶链反应法测定calpain-Ⅰ在mRNA水平上的表达.结果:①与RSR组比较,Caf组calpain-I蛋白含量增加到(344±101.9)%(P<0.01);cTnI的蛋白含量则降低到(45.0±13.4)%(P<0.01).②Caf组calpain-Ⅰ的mRNA表达为2.49±0.86,比Paf组的(1.23±0.31)和RSR组(0.89±0.23)的明显增加(均P<0.01);calpain-I的蛋白含量与cTnI的蛋白含量也呈明显负相关(r=-0.898,P<0.01).而Paf组calpain-Ⅰ和cTnⅠ蛋白表达则无明显变化.③Caf组calpain-Ⅰ的蛋白含量和mRAN水平的表达分别与左心房内径和Af持续时间呈明显正相关(P<0.05或P<0.01).结论:Caf时左心房组织calpain-Ⅰ的蛋白含量明显升高,促进心房结构和功能的改变,参与Af的发病机制.

  3. 547 An Earlier, More Severe Presentation of G6pc3 Deficiency in a Male Infant From Mexico

    OpenAIRE

    Cruz, Alonso

    2012-01-01

    Background Severe congenital neutropenia is a bone marrow failure syndrome characterized by severe neutropenia present from birth. We present a case of G6PC3 deficiency presenting at an earlier age, with a more severe clinical picture than previously reported. Case report A 3-month-old boy, born to nonconsanguineous parents was delivered by C-section at 35 weeks gestation. He was admitted to neonatal intensive care unit for prematurity and poor respiratory effort requiring mechanical ventilat...

  4. A20 (Tnfaip3 deficiency in myeloid cells protects against influenza A virus infection.

    Directory of Open Access Journals (Sweden)

    Jonathan Maelfait

    Full Text Available The innate immune response provides the first line of defense against viruses and other pathogens by responding to specific microbial molecules. Influenza A virus (IAV produces double-stranded RNA as an intermediate during the replication life cycle, which activates the intracellular pathogen recognition receptor RIG-I and induces the production of proinflammatory cytokines and antiviral interferon. Understanding the mechanisms that regulate innate immune responses to IAV and other viruses is of key importance to develop novel therapeutic strategies. Here we used myeloid cell specific A20 knockout mice to examine the role of the ubiquitin-editing protein A20 in the response of myeloid cells to IAV infection. A20 deficient macrophages were hyperresponsive to double stranded RNA and IAV infection, as illustrated by enhanced NF-κB and IRF3 activation, concomitant with increased production of proinflammatory cytokines, chemokines and type I interferon. In vivo this was associated with an increased number of alveolar macrophages and neutrophils in the lungs of IAV infected mice. Surprisingly, myeloid cell specific A20 knockout mice are protected against lethal IAV infection. These results challenge the general belief that an excessive host proinflammatory response is associated with IAV-induced lethality, and suggest that under certain conditions inhibition of A20 might be of interest in the management of IAV infections.

  5. Partial pyruvate kinase deficiency aggravates the phenotypic expression of band 3 deficiency in a family with hereditary spherocytosis.

    Science.gov (United States)

    van Zwieten, Rob; van Oirschot, Brigitte A; Veldthuis, Martijn; Dobbe, Johannes G; Streekstra, Geert J; van Solinge, Wouter W; Schutgens, Roger E G; van Wijk, Richard

    2015-03-01

    In a family with mild dominant spherocytosis, affected members showed partial band 3 deficiency. The index patient showed more severe clinical symptoms than his relatives, and his red blood cells displayed concomitant low pyruvate kinase activity. We investigated the contribution of partial PK deficiency to the phenotypic expression of mutant band 3 in this family. Pyruvate kinase deficiency and band 3 deficiency were characterized by DNA analysis. Results of red cell osmotic fragility testing, the results of cell deformability obtained by the Automated Rheoscope and Cell Analyzer and the results obtained by Osmotic Gradient Ektacytometry, which is a combination of these tests, were related to the red cell ATP content. Spherocytosis in this family was due to a novel heterozygous mutation in SLC4A1, the gene for band 3. Reduced PK activity of the index patient was attributed to a novel mutation in PKLR inherited from his mother, who was without clinical symptoms. Partial PK deficiency was associated with decreased red cell ATP content and markedly increased osmotic fragility. This suggests an aggravating effect of low ATP levels on the phenotypic expression of band 3 deficiency.

  6. Calpain抑制剂对肝癌细胞MHCC97-H中Calpain-2蛋白表达的影响%Effect of Calpain Inhibitors of Different Concentration on Expression of Calpain-2 Protein in Hepatocellular Carcinoma Cell Line MHCC97-H

    Institute of Scientific and Technical Information of China (English)

    张敏; 王宁; 陈腾祥; 潘娅

    2016-01-01

    目的:观察4种Calpain抑制剂不同浓度及作用时间对肝癌细胞MHCC97-H中Calpain-2蛋白表达的影响.方法:实验分为对照组、DMSO组及4种抑制剂组,4种抑制剂组分别给予高、中、低浓度处理肝癌MH-CC97-H细胞24、48及72 h;用Western blot方法检测MHCC97-H中Calpain-2蛋白的表达,确定4种抑制剂的最适浓度和作用时间.结果:与对照组、DMSO组、其他时段各浓度组及同时段其他浓度组比较,高浓度ALM作用72 h后肝癌细胞MHCC97-H中Calpain-2蛋白的表达下降(P<0.05);与对照组、DMSO组、其他时段各浓度组及同时段其他浓度组比较,高浓度Calpain inhibitor Ⅳ、Calpain inhibitor Ⅵ、Calpastatin抑制剂72 h组肝癌细胞MH-CC97-H中Calpain-2蛋白的表达量显著下降(P<0.01).结论:4种高浓度Calpain抑制剂处理肝癌细胞MH-CC97-H 72 h后可抑制肝癌细胞MHCC97-H中Calpain-2蛋白的表达.

  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 inhibitor attenuated optic nerve damage in acute optic neuritis in rats

    Science.gov (United States)

    Das, Arabinda; Guyton, M. Kelly; Smith, Amena; Wallace, Gerald; McDowell, Misty L.; Matzelle, Denise D.; Ray, Swapan K.; Banik, Naren L.

    2012-01-01

    Optic neuritis (ON), which is an acute inflammatory autoimmune demyelinating disease of the central nervous system (CNS), often occurs in multiple sclerosis (MS). ON is an early diagnostic sign in most MS patients caused by damage to the optic nerve leading to visual dysfunction. Various features of both MS and ON can be studied following induction of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in Lewis rats. Inflammation and cell death in the optic nerve, with subsequent damage to the retinal ganglion cells in the retina, are thought to correlate with visual dysfunction. Thus, characterizing the pathophysiological changes that lead to visual dysfunction in EAE animals may help develop novel targets for therapeutic intervention. We treated EAE animals with and without the calpain inhibitor calpeptin (CP). Our studies demonstrated that the Ca2+-activated neutral protease calpain was upregulated in the optic nerve following induction of EAE at the onset of clinical signs (OCS) of the disease and these changes were attenuated following treatment with CP. These reductions correlated with decreases in inflammation (cytokines, iNOS, COX-2, NF-κB), and microgliosis (i.e. activated microglia). We observed that calpain inhibition reduced astrogliosis (reactive astroglia) and expression of aquaporin 4 (AQP4). The balance of Th1/Th2 cytokine production and also expression of the Th1-related CCR5 and CXCR3 chemokine receptors influence many pathological processes and play both causative and protective roles in neuron damage. Our data indicated that CP suppressed cytokine imbalances. Also, Bax:Bcl-2 ratio, production of tBid, PARP-1, expression and activities of calpain and caspases, and internucleosomal DNA fragmentation were attenuated after treatment with CP. Our results demonstrated that CP decreased demyelination [loss of myelin basic protein (MBP)] and axonal damage [increase in dephosphorylated neurofilament protein (de-NFP), and also

  9. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2016-01-01

    The neuroprotective property of quercetin is well reported against hypoxia and ischemia in past stud-ies. 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 isch-emic stroke. Hence, it may be considered as a potential drug target for the treatment of hypoxia induced neuronal injury. As the inhibitory property ofμ-calpain is yet to be explored in details, hence, in the present study, we investigated the interaction of quercetin withμ-calpain through a molecular dynamics simula-tion study as a tool through clarifying the molecular mechanism of such inhibition and determining the probable sites and modes of quercetin interaction with theμ-calpain catalytic domain. In addition, we also investigated the structure-activity relationship of quercetin withμ-calpain. Afifnity binding of quercetin withμ-calpain had a value of–28.73 kJ/mol and a Ki value of 35.87μM that may be a probable reason to lead to altered functioning ofμ-calpain. Hence, quercetin was found to be an inhibitor ofμ-calpain which might have a possible therapeutic role in hypoxic injury.

  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. Novel roles for ceramides, calpains and caspases in kidney proximal tubule cell apoptosis: lessons from in vitro cadmium toxicity studies.

    Science.gov (United States)

    Lee, Wing-Kee; Thévenod, Frank

    2008-12-01

    Apoptosis is a tightly regulated physiological process, which can be initiated by toxic stimuli, such as cadmium (Cd2+). Cd2+ (10-50 microM) induces a rapid increase in reactive oxygen species (ROS) (> or = 30 min) in a cell line derived from the S1 segment of rat kidney proximal tubule, without any apparent mitochondrial dysfunction. The sphingolipid ceramide is an important second messenger in apoptosis. Short exposure to Cd2+ (3h) causes an increase in ceramides, which occurs downstream of ROS formation, and may interact with cellular components, such as endoplasmic reticulum and mitochondria. Following apoptosis initiation, execution must take place. The classical executioners of apoptosis are caspases, a family of cysteine proteases. However, increasing studies report caspase-independent apoptosis, which questions the essentiality of caspases for apoptosis implementation. With low micromolar Cd2+ concentrations (calpains, has emerged. Calpain activation by Cd2+ (3-6h) seems to be regulated by ceramide levels, in order to induce apoptosis. Calpain and caspase substrates overlap but yield different fragments, which may explain their diverse downstream targets. Furthermore, calpains and caspases may interact with one another to enhance, as seen by Cd2+, or diminish apoptosis. In this review, we discuss novel roles for ceramides, calpains and caspases as part of Cd2+-induced apoptotic signalling pathways in the kidney proximal tubule and their in vivo relevance to Cd2+-induced nephrotoxicity.

  13. Inhibition of caspases but not of calpains temporarily protect against C2-ceramide-induced death of CAD cells.

    Science.gov (United States)

    Arboleda, Gonzalo; Waters, Catherine; Gibson, Rosemary

    2007-06-29

    Evidence has implicated apoptosis as a mechanism underlying cell death in diverse neurodegenerative diseases including Parkinson's disease (PD). Endogenous agents such as TNF-alpha, INF-gamma, IL-1beta and others stress signals activate the sphingomyelin pathway increasing ceramide levels. Ceramide triggers apoptotic pathways while inhibiting survival signalling, and is involved in the regulation of intracellular Ca(2+) homeostasis and compartmentalisation. The contribution of caspases in neuronal apoptosis has been highlighted by the increased survival exerted by caspase inhibition, but the involvement of calpains during neuronal apoptosis and the potential benefit of their inhibition is still controversial. In the present paper, we have analysed the contribution of caspases and calpains to cell death of CAD cells, a catecholaminergic cell line of mesencephalic origin, following C2-ceramide exposure. Ceramide caused CAD cell death by a dose and time dependant mechanism. 25microM of C2-ceramide caused apoptosis. Analysis of activation of caspases and calpains by differential cleavage of alpha-fodrin showed that although calpains are activated before caspases following C2-ceramide exposure, only caspase inhibition increased cell survival. These results demonstrate the activation of caspases and calpains in C2-ceramide-induced cell death, and support the role of caspase inhibition as a neuroprotective strategy and a plausible therapeutic approach to decrease catecholaminergic cell death.

  14. 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.

  15. The repetitive cytoskeletal protein H49 of Trypanosoma cruzi is a calpain-like protein located at the flagellum attachment zone.

    Directory of Open Access Journals (Sweden)

    Alexandra Galetović

    Full Text Available BACKGROUND: Trypanosoma cruzi has a single flagellum attached to the cell body by a network of specialized cytoskeletal and membranous connections called the flagellum attachment zone. Previously, we isolated a DNA fragment (clone H49 which encodes tandemly arranged repeats of 68 amino acids associated with a high molecular weight cytoskeletal protein. In the current study, the genomic complexity of H49 and its relationships to the T. cruzi calpain-like cysteine peptidase family, comprising active calpains and calpain-like proteins, is addressed. Immunofluorescence analysis and biochemical fractionation were used to demonstrate the cellular location of H49 proteins. METHODS AND FINDINGS: All of H49 repeats are associated with calpain-like sequences. Sequence analysis demonstrated that this protein, now termed H49/calpain, consists of an amino-terminal catalytic cysteine protease domain II, followed by a large region of 68-amino acid repeats tandemly arranged and a carboxy-terminal segment carrying the protease domains II and III. The H49/calpains can be classified as calpain-like proteins as the cysteine protease catalytic triad has been partially conserved in these proteins. The H49/calpains repeats share less than 60% identity with other calpain-like proteins in Leishmania and T. brucei, and there is no immunological cross reaction among them. It is suggested that the expansion of H49/calpain repeats only occurred in T. cruzi after separation of a T. cruzi ancestor from other trypanosomatid lineages. Immunofluorescence and immunoblotting experiments demonstrated that H49/calpain is located along the flagellum attachment zone adjacent to the cell body. CONCLUSIONS: H49/calpain contains large central region composed of 68-amino acid repeats tandemly arranged. They can be classified as calpain-like proteins as the cysteine protease catalytic triad is partially conserved in these proteins. H49/calpains could have a structural role, namely that of

  16. 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

    2016-08-09

    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.

  17. Bid and calpains cooperate to trigger oxaliplatin-induced apoptosis of cervical carcinoma HeLa cells.

    Science.gov (United States)

    Anguissola, Sergio; Köhler, Barbara; O'Byrne, Robert; Düssmann, Heiko; Cannon, Mary D; Murray, Frank E; Concannon, Caoimhin G; Rehm, Markus; Kögel, Donat; Prehn, Jochen H M

    2009-11-01

    The Bcl-2 homology 3-only protein Bid is an important mediator of death receptor-induced apoptosis. Recent reports and this study suggest that Bid may also mediate genotoxic drug-induced apoptosis of various human cancer cells. Here, we characterized the role of Bid and the mechanism of Bid activation during oxaliplatin-induced apoptosis of HeLa cervical cancer cells. Small hairpin RNA-mediated silencing of Bid protected HeLa cells against both death receptor- and oxaliplatin-induced apoptosis. Expression of a Bid mutant in which caspase-8 cleavage site was mutated (D59A) reactivated oxaliplatin-induced apoptosis in Bid-deficient cells but failed to reactivate death receptor-induced apoptosis, suggesting that caspase-8-mediated Bid cleavage did not contribute to oxaliplatin-induced apoptosis. Overexpression of bcl-2 or treatment with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone abolished caspase-2, -8, -9, and -3 activation as well as Bid cleavage in response to oxaliplatin, suggesting that Bid cleavage occurred downstream of mitochondrial permeabilization and was predominantly mediated by caspases. We also detected an early activation of calpains in response to oxaliplatin. Calpain inhibition reduced Bid cleavage, mitochondrial depolarization, and activation of caspase-9, -3, -2, and -8 in response to oxaliplatin. Further experiments, however, suggested that Bid cleavage by calpains was not a prerequisite for oxaliplatin-induced apoptosis: single-cell imaging experiments using a yellow fluorescent protein-Bid-cyan fluorescent protein probe demonstrated translocation of full-length Bid to mitochondria that was insensitive to calpain or caspase inhibition. Moreover, calpain inhibition showed a potent protective effect in Bid-silenced cells. In conclusion, our data suggest that calpains and Bid act in a cooperative, but mutually independent, manner to mediate oxaliplatin-induced apoptosis of HeLa cells.

  18. Prostacyclin analog-suppressed ischemia-reperfusion injury of the rat liver: evaluation by calpain mu activation.

    Science.gov (United States)

    Wang, M; Sakon, M; Miyoshi, H; Umeshita, K; Kishimoto, S; Taniguchi, K; Gotoh, M; Imajoh-Ohmi, S; Monden, M

    1997-12-01

    Prostaglandin I2 has a protective effect on hepatic ischemia-reperfusion injury. However, the exact intracellular mechanisms of this effect have not been elucidated. Calpain micro, a Ca2+-dependent protease, has been found to play a role in the ischemia-reperfusion injury of various organs. The hilar area of the left lateral and median lobes of rat livers was clamped for 60 min. A prostaglandin I2 analog (OP2507, C35H41NO4) was intravenously administered at 0.1, 0.32, or 1.0 microg/kg/min from 20 min before the ischemia. In addition to biochemical and microscopic analyses, the activation of calpain mu was investigated using specific antibodies against the intermediate (activated) and preactivated forms of calpain mu. The degradation of talin was also studied by Western blotting. When OP2507 was infused at 0.32 and 1.0 microg/kg/min, bile flow significantly increased after reperfusion compared with the control group, consistent with the decrease in serum transaminase levels. Membrane bleb formation and the appearance of the intermediate form of calpain mu were observed at 60 min of ischemia in the control and OP2507 (0.1 microg/kg/min) groups and remained present until 120 min after reperfusion. OP2507 (1.0 microg/kg/min) markedly suppressed not only membrane bleb formation but also calpain mu activation and the degradation of talin. In conclusion, OP2507 suppresses ischemia-reperfusion injury of the rat liver, and its cytoprotective effect is closely associated with the inhibition of calpain mu activation.

  19. The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants.

    Science.gov (United States)

    Liang, Zhe; Demko, Viktor; Wilson, Robert C; Johnson, Kenneth A; Ahmad, Rafi; Perroud, Pierre-François; Quatrano, Ralph; Zhao, Sen; Shalchian-Tabrizi, Kamran; Otegui, Marisa S; Olsen, Odd-Arne; Johansen, Wenche

    2013-09-01

    DEK1, the single calpain of land plants, is a member of the ancient membrane bound TML-CysPc-C2L calpain family that dates back 1.5 billion years. Here we show that the CysPc-C2L domains of land plant calpains form a separate sub-clade in the DEK1 clade of the phylogenetic tree of plants. The charophycean alga Mesostigma viride DEK1-like gene is clearly divergent from those in land plants, suggesting that a major evolutionary shift in DEK1 occurred during the transition to land plants. Based on genetic complementation of the Arabidopsis thaliana dek1-3 mutant using CysPc-C2L domains of various origins, we show that these two domains have been functionally conserved within land plants for at least 450 million years. This conclusion is based on the observation that the CysPc-C2L domains of DEK1 from the moss Physcomitrella patens complements the A. thaliana dek1-3 mutant phenotype. In contrast, neither the CysPc-C2L domains from M. viride nor chimeric animal-plant calpains complement this mutant. Co-evolution analysis identified differences in the interactions between the CysPc-C2L residues of DEK1 and classical calpains, supporting the view that the two enzymes are regulated by fundamentally different mechanisms. Using the A. thaliana dek1-3 complementation assay, we show that four conserved amino acid residues of two Ca²⁺-binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1.

  20. 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.

  1. Local structural preferences of calpastatin, the intrinsically unstructured protein inhibitor of calpain.

    Science.gov (United States)

    Kiss, Robert; Kovács, Dénes; Tompa, Péter; Perczel, András

    2008-07-01

    Calpain, the calcium-activated intracellular cysteine protease, is under the tight control of its intrinsically unstructured inhibitor, calpastatin. Understanding how potent inhibition by calpastatin can be reconciled with its unstructured nature provides deeper insight into calpain function and a more general understanding of how proteins devoid of a well-defined structure carry out their function. To this end, we performed a full NMR assignment of hCSD1 to characterize it in its solution state. Secondary chemical shift values and NMR relaxation data, R 1, R 2, and hetero-NOE, as well as spectral density function analysis have shown that conserved regions of calpastatin, subdomains A and C, which are responsible for calcium-dependent anchoring of the inhibitor to the enzyme, preferentially sample partially helical backbone conformations of a reduced flexibility. Moreover, the linker regions between subdomains are more flexible with no structural preference. The primary determinant of calpain inhibition, subdomain B, also has a non-fully random conformational preference, resembling a beta-turn structure also ascertained by prior studies of a 27-residue peptide encompassing the inhibitory region. This local structural preference is also confirmed by a deviation in chemical shift values between full-length calpastatin domain 1 and a truncated construct cut in the middle of subdomain B. At the C-terminal end of the molecule, a nascent helical region was found, which in contrast to the overall structural properties of the molecule may indicate a previously unknown functional region. Overall, these observations provide further evidence that supports previous suggestions that intrinsically unstructured proteins use preformed structural elements in efficient partner recognition.

  2. 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.

  3. Calpain-10 gene polymorphisms and risk of type 2 diabetes mellitus in Mexican mestizos.

    Science.gov (United States)

    Picos-Cárdenas, V J; Sáinz-González, E; Miliar-García, A; Romero-Zazueta, A; Quintero-Osuna, R; Leal-Ugarte, E; Peralta-Leal, V; Meza-Espinoza, J P

    2015-03-27

    The calpain-10 gene is expressed primarily in tissues important in glucose metabolism; thus, some of its polymorphisms have been associated with type 2 diabetes. In this study, we examined the association between the calpain-10 single-nucleotide polymorphism (SNP)-43, SNP-19, and SNP-63 and type 2 diabetes in Mexican mestizos. We included 211 patients and 152 non-diabetic subjects. Polymerase chain reaction was used to identify alleles. We compared allele, genotype, haplotype, and diplotype frequencies between both groups and used the chi-square test to calculate the risk. The allele frequency of SNP-43 allele 1 was 70% in controls and 72% in patients; the GG, GA, and AA genotype frequencies were 48.7, 42.8, and 8.5% in controls and 51.2, 41.7, and 7.1% in patients, respectively. For SNP- 19, the prevalence of allele 1 (2R) was 32% in controls and 39% in patients. In controls, homozygosity (2R/2R) was 10.5%, heterozygosity was 42.8%, and 3R/3R was 46.7%; in cases, these values were 13.3, 50.7, and 36.0%, respectively. For SNP-63, the frequency of allele 1 was 87% in controls and 83% in patients; genotype frequencies in controls were 75.7% (CC), 23% (CT), and 1.3% (TT), and were 69.7, 27.5, and 2.8%, respectively for the cases. Genotype distributions were consistent with Hardy-Weinberg equilibrium. No significant intergroup differences for allele, genotype, haplotype, or diplotype frequencies were observed. We found no association between these polymorphisms and diabetes. However, our sample size was small, so the role of calpain-10 risk alleles should be further examined.

  4. 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.

  5. Activation of Both the Calpain and Ubiquitin-Proteasome Systems Contributes to Septic Cardiomyopathy through Dystrophin Loss/Disruption and mTOR Inhibition

    Science.gov (United States)

    Freitas, Ana Caroline Silva; Figueiredo, Maria Jose; Campos, Erica Carolina; Soave, Danilo Figueiredo; Ramos, Simone Gusmao; Tanowitz, Herbert B.

    2016-01-01

    Cardiac dysfunction caused by the impairment of myocardial contractility has been recognized as an important factor contributing to the high mortality in sepsis. Calpain activation in the heart takes place in response to increased intracellular calcium influx resulting in proteolysis of structural and contractile proteins with subsequent myocardial dysfunction. The purpose of the present study was to test the hypothesis that increased levels of calpain in the septic heart leads to disruption of structural and contractile proteins and that administration of calpain inhibitor-1 (N-acetyl-leucinyl-leucinyl-norleucinal (ALLN)) after sepsis induced by cecal ligation and puncture prevents cardiac protein degradation. We also tested the hypothesis that calpain plays a role in the modulation of protein synthesis/degradation through the activation of proteasome-dependent proteolysis and inhibition of the mTOR pathway. Severe sepsis significantly increased heart calpain-1 levels and promoted ubiquitin and Pa28β over-expression with a reduction in the mTOR levels. In addition, sepsis reduced the expression of structural proteins dystrophin and β-dystroglycan as well as the contractile proteins actin and myosin. ALLN administration prevented sepsis-induced increases in calpain and ubiquitin levels in the heart, which resulted in decreased of structural and contractile proteins degradation and basal mTOR expression levels were re-established. Our results support the concept that increased calpain concentrations may be part of an important mechanism of sepsis-induced cardiac muscle proteolysis. PMID:27880847

  6. Effects of an n-3-deficient diet on brain, retina, and liver fatty acyl composition in artificially reared rats.

    Science.gov (United States)

    Moriguchi, Toru; Lim, Sun-Young; Greiner, Rebecca; Lefkowitz, William; Loewke, James; Hoshiba, Junji; Salem, Norman

    2004-08-01

    Rat pups born to dams fed a diet with 3.1% of total fatty acids as alpha-linolenic acid (LNA) were fed, using an artificial rearing system, either an n-3-deficient (n-3-Def) or an n-3-adequate (n-3-Adq) diet. Both diets contained 17.1% linoleic acid, but the n-3-Adq diet also contained 3.1% LNA. The percentage of brain docosahexaenoic acid (DHA) continuously decreased (71%) with time over the 29 days of the experiment, with concomitant increases in docosapentaenoic acid (DPAn-6). In the retina, the percentage of DHA rose in the n-3-Adq group, with an apparent increased rate around the time of eye opening. However, there was a flat curve for the percentage of DHA in the n-3-Def group and a rising DPAn-6 with time. Liver DHA was highest at the time of birth in the n-3-Adq group but fell off somewhat over the course of 29 days. This decrease was more pronounced in the n-3-Def group, and the DPAn-6 rose considerably during the second half of the experiment. This method presents a first-generation model for n-3 deficiency that is more similar to the case of human nutrition than is the commonly employed two-generation model.

  7. Foxi3 deficiency compromises hair follicle stem cell specification and activation

    Science.gov (United States)

    Shirokova, Vera; Biggs, Leah C.; Jussila, Maria; Ohyama, Takahiro; Groves, Andrew K.; Mikkola, Marja L.

    2017-01-01

    The hair follicle is an ideal system to study stem cell specification and homeostasis due to its well characterized morphogenesis and stereotypic cycles of stem cell activation upon each hair cycle to produce a new hair shaft. The adult hair follicle stem cell niche consists of two distinct populations, the bulge and the more activation-prone secondary hair germ. Hair follicle stem cells are set aside during early stages of morphogenesis. This process is known to depend on the Sox9 transcription factor, but otherwise the establishment of the hair follicle stem cell niche is poorly understood. Here we show that that mutation of Foxi3, a Forkhead family transcription factor mutated in several hairless dog breeds, compromises stem cell specification. Further, loss of Foxi3 impedes hair follicle downgrowth and progression of the hair cycle. Genome-wide profiling revealed a number of downstream effectors of Foxi3 including transcription factors with a recognized function in hair follicle stem cells such as Lhx2, Runx1, and Nfatc1, suggesting that the Foxi3 mutant phenotype results from simultaneous downregulation of several stem cell signature genes. We show that Foxi3 displays a highly dynamic expression pattern during hair morphogenesis and cycling, and identify Foxi3 as a novel secondary hair germ marker. Absence of Foxi3 results in poor hair regeneration upon hair plucking, and a sparse fur phenotype in unperturbed mice that exacerbates with age, caused by impaired secondary hair germ activation leading to progressive depletion of stem cells. Thus, Foxi3 regulates multiple aspects of hair follicle development and homeostasis. PMID:26992132

  8. 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.

  9. 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.

  10. Inhibition of Calpain Prevents N-Methyl-D-aspartate-Induced Degeneration of the Nucleus Basalis and Associated Behavioral Dysfunction

    NARCIS (Netherlands)

    Nimmrich, Volker; Szabo, Robert; Nyakas, Csaba; Granic, Ivica; Reymann, Klaus G.; Schroeder, Ulrich H.; Gross, Gerhard; Schoemaker, Hans; Wicke, Karsten; Moeller, Achim; Luiten, Paul

    2008-01-01

    N-Methyl-D-aspartate( NMDA) receptor-mediated excitotoxicity is thought to underlie a variety of neurological disorders, and inhibition of either the NMDA receptor itself, or molecules of the intracellular cascade, may attenuate neurodegeneration in these diseases. Calpain, a calcium-dependent cyste

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

    Science.gov (United States)

    Lian, Ting; Wang, Linjie; Liu, Yiping

    2013-03-01

    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 calpastatin (CAST) have the closest relationship with tenderness in livestock. They are involved in a wide range of physiological processes including muscle growth and differentiation, pathological conditions and post-mortem meat aging. Whereas, Calpain3 (CAPN3) has been established as an important activating enzyme specifically expressed in livestock's skeletal muscle, but its role in domestic animals meat tenderization remains controversial. In this review, we summarize the role of CAPN1, calpain II (CAPN2) and CAST in post-mortem meat tenderization, and analyse the relationship between CAPN3 and tenderness in domestic animals. Besides, the possible mechanism affecting post-mortem meat aging and improving meat tenderization, and current possible causes responsible for divergence (whether CAPN3 contributes to animal meat tenderization or not) are inferred. Only the possible mechanism of CAPN3 in meat tenderization has been confirmed, while its exact role still needs to be studied further.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Cloning and biological characteristic of calpain-like gene of Toxoplasma gondii%弓形虫依钙蛋白激酶Calpain-like的克隆及生物学特性

    Institute of Scientific and Technical Information of China (English)

    陈雪艳; 付玉才; 李璐; 耿艺介; 黄达娜; 高世同; 张仁利

    2008-01-01

    目的 克隆弓形虫RH株Calpain(依钙蛋白酶)-like基因,分析免疫生物学功能,筛选预防弓形虫感染的疫苗候选分子. 方法 利用人类、鼠类和其它寄生虫的Calpain基因同源性比较的保守区域合成混合引物,以弓形虫的RNA为模板.应用RT-PCR扩增弓形虫Calpain-like 基因片段,扩增的片段通过TA克隆插入克隆载体pET32A,筛选阳性克隆进行双酶切鉴定和DNA序列分析,用克隆的基因片段制备特异性基因探针,经Northern blot技术证实弓形虫Calpain-like基因mRNA. 结果 RT-PCR扩增了316bp弓形虫Calpmn-like基因,其DNA序列与日本血吸虫Calpain基因同一区域的同源性为70%,与计算机推测的弓形虫Calpain-like基因的同源性为100%.Northern blot显示了弓形虫Calpmn-like基因mRNA的大小约6300bp. 结论 弓形虫RH株基因组中存在Calpmn-like基因,Calpain-like基因在弓形虫RH株中高度表达.

  14. Effect of Calpain on The Degradation of Tau in Rat Brain Cortex Extracts%Calpain对细胞骨架蛋白tau降解作用的研究

    Institute of Scientific and Technical Information of China (English)

    方征宇; 刘世杰; 王小川; 刘蓉; 王群; 陈正跃; 王建枝

    2003-01-01

    Calpain is a calcium-activated protease and there are two ubiquitously distributed mammalian calpains, namely calpain 1 (μ-calpain and CAPN1) and calpain 2 (m-calpain and CAPN2). Calpains regulate the function of many proteins by limited proteolysis. To determine the nature of different subtypes of calpain on degradation of microtubule-associated protein tau, the rat brain cortex extracts were incubated with 0.2 mmol/L, 1 mmol/L, 3 mmol/L and 5 mmol/L of CaCl2 for 15 min at 37℃. The findings were that Ca2+ treatment at concentration 1~5 mmol/L led to significant proteolysis of tau protein and this degradation was blocked by calpain inhibitor, calpeptin. In addition, when the extracts containing 1 mmol/L CaCl2 were treated with μ-calpain inhibitor (0.05 μmol/L of calpastatin) or m-calpain inhibitor (100 μmol/L calpain inhibitor Ⅳ) or both, the Ca2+-induced degradation of tau protein was decreased to 8.6%,92.5% and 97.8%, respectively. These data suggest that both μ-calpain and m-calpain in brain cortex extracts are activated by Ca2+ and both of them degrade tau protein, although, m-calpain plays a more important role in proteolysis of tau.%Calpain是钙依赖性中性蛋白酶,根据其对钙敏感性的不同,可分为m-和μ-calpain两型.分别用不同浓度CaCl2溶液孵育Wistar大鼠脑皮质匀浆液,并用蛋白质印迹和定量图像分析技术检测不同亚型calpain对tau蛋白的降解作用.研究发现:在37℃用1 mmol/L Ca2+孵育底物15 min,可见tau蛋白明显降解,并在分子质量为29 ku处出现tau蛋白降解片段;当Ca2+浓度为5 mmol/L时,tau蛋白几乎全部被降解;这种tau蛋白降解可被calpain特异性抑制剂完全逆转.进一步的研究发现,分别用μ-calpain抑制剂(0.05 μmol/L calpastatin),m-calpain抑制剂(100 μmol/L calpain inhibitor Ⅳ)或总calpain抑制剂(552 μmol/L calpeptin)与1 mmol/L Ca2+共同孵育Wistar大鼠脑皮质匀浆液,Ca2+激活的tau蛋白降解分别被抑制8.6%,92.5%

  15. 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.

  16. 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.

  17. The effect of acetyl-L-carnitine on lenticular calpain activity in prevention of selenite-induced cataractogenesis.

    Science.gov (United States)

    Elanchezhian, R; Sakthivel, M; Geraldine, P; Thomas, P A

    2009-05-01

    The present study sought to determine whether acetyl-L-carnitine (ALCAR) prevents selenite cataractogenesis by mechanisms involving lenticular calpain activity, Wistar rat pups were divided into 3 groups of 15 each. Group I (normal) rats received an intraperitoneal (i.p.) injection of normal saline on postpartum day 10; Group II (cataract-untreated) rats received a single subcutaneous (s.c.) injection of sodium selenite (19micromol/kg body weight) on postpartum day 10; Group III (cataract-treated) pups received a single s.c. injection of sodium selenite on postpartum day 10 and intraperitoneal injections of acetyl-L-carnitine (200mg/kg body weight) on postpartum days 9-14. At the end of the study period (postpartum day 16), both eyes of each rat pup were examined by slit-lamp biomicroscopy. There was dense lenticular opacification in all Group II rats, minimal lenticular opacification in 33% of Group III rats, and no lenticular opacification in 67% of Group III and in all Group I rats. Group II lenses exhibited significantly lower mean values of calpain activity and Lp82 (lens-specific calpain) protein expression, decreases in relative transcript level of m-calpain mRNA and significantly higher mean Ca(2+) concentrations than Group I or Group III lenses; the values of these parameters in Group III rat lenses (ALCAR-treated) approximated those in Group I rat lenses. The results suggest that, in addition to its already-described antioxidant potential, ALCAR prevents selenite cataractogenesis by maintaining calpain activity at near normal levels. These findings may stimulate further efforts to develop ALCAR as a novel drug for prevention of cataract.

  18. 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...... documented and has been shown to affect transcription of specific gene sequences, protein synthesis, the immune system and increase in Ca2+ influx. The past 10 years has seen a dramatic increase in the understanding of how proteolytic enzymes such as calpains can affect the growth of muscle. In vivo studies...... have shown that m-calpain is necessary for myoblast fusion leading to the formation of muscle fibers and that inhibition of this enzyme restricts myotube formation. Whether there is a link between stretchor load induced signaling and m-calpain expression and activation is not known. Using a magnetic...

  19. 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 K; Lametsch, René; Elce, John S;

    2008-01-01

    Dynamic regulation of the actin cytoskeleton is important for cell motility, spreading and the formation of membrane surface extensions such as lamellipodia, ruffles and blebs. The ubiquitous calpains contribute to integrin-mediated cytoskeletal remodelling during cell migration and spreading......, by cleavage of focal adhesion components and signalling molecules. In the present study, the live-cell morphology of calpain-knockout and wild-type cells was examined by time-lapse fluorescence microscopy, and a role of calpain in mediating the formation of sporadic membrane blebs was established. Membrane...... 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...

  20. Morphological and behavioral characterization of adult mice deficient for SrGAP3.

    Science.gov (United States)

    Bertram, Jonathan; Koschützke, Leif; Pfannmöller, Jörg P; Esche, Jennifer; van Diepen, Laura; Kuss, Andreas W; Hartmann, Bianca; Bartsch, Dusan; Lotze, Martin; von Bohlen Und Halbach, Oliver

    2016-10-01

    SrGAP3 belongs to the family of Rho GTPase proteins. These proteins are thought to play essential roles in development and in the plasticity of the nervous system. SrGAP3-deficient mice have recently been created and approximately 10 % of these mice developed a hydrocephalus and died shortly after birth. The others survived into adulthood, but displayed neuroanatomical alteration, including increased ventricular size. We now show that SrGAP3-deficient mice display increased brain weight together with increased hippocampal volume. This increase was accompanied by an increase of the thickness of the stratum oriens of area CA1 as well as of the thickness of the molecular layer of the dentate gyrus (DG). Concerning hippocampal adult neurogenesis, we observed no significant change in the number of proliferating cells. The density of doublecortin-positive cells also did not vary between SrGAP3-deficient mice and controls. By analyzing Golgi-impregnated material, we found that, in SrGAP3-deficient mice, the morphology and number of dendritic spines was not altered in the DG. Likewise, a Sholl-analysis revealed no significant changes concerning dendritic complexity as compared to controls. Despite the distinct morphological alterations in the hippocampus, SrGAP3-deficient mice were relatively inconspicuous in their behavior, not only in the open-field, nest building but also in the Morris water-maze. However, the SrGAP3-deficient mice showed little to no interest in burying marbles; a behavior that is seen in some animal models related to autism, supporting the view that SrGAP3 plays a role in neurodevelopmental disorders.

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

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    Cristina Salmerón

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

  2. Acetylcholine Attenuated TNF-α-Induced Apoptosis in H9c2 Cells: Role of Calpain and the p38-MAPK Pathway

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    Ming Zhao

    2015-07-01

    Full Text Available Background: Previous studies have shown that inflammation is associated with excessive activation of calpains. Acetylcholine (ACh has been reported to inhibit pro-inflammatory cytokine release and protect against cardiomyocyte injury. However, there is no direct evidence regarding whether ACh can regulate calpains to exert cardioprotection. To this end, we investigated the effect of ACh on tumour necrosis factor alpha (TNF-α-induced cardiomyocyte injury and further explored the underlying mechanism. Methods: Flow cytometry and transmission electron microscopy were performed to evaluate apoptosis and cellular ultrastructure. Western blotting was performed to assess changes in protein expression. siRNA was employed to silence specific proteins. Results: TNF-α treatment increased the expression of cleaved caspase-3, calpain-1 and p38-mitogen-activated protein kinase (p38-MAPK. The calpain inhibitor PD150606 and the p38-MAPK inhibitor SB203580 inhibited apoptosis induced by TNF-α. Moreover, SB203580 decreased the expression and activity of calpain-1, possibly related to the up-regulation of calpastatin. ACh significantly inhibited TNF-α-induced cell apoptosis, as evidenced by decreases in caspase-3 cleavage, p38-MAPK phosphorylation, and calpain-1 expression and activity as well as increases in calpastatin expression. These beneficial effects of ACh were abolished by atropine or M2AChR siRNA. Conclusion: Our results suggest that ACh ameliorated TNF-α-induced calpain activation by decreasing p38-MAPK phosphorylation and enhancing calpastatin expression, indicating that calpain may be an important link between inflammatory factors and myocardial cell apoptosis.

  3. 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.

  4. 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.

  5. Expression of calpains in the cochlea in kanamycin-poisoned guinea pig%钙蛋白酶在卡那霉素致毒豚鼠耳蜗中的表达

    Institute of Scientific and Technical Information of China (English)

    马德菊; 王爱梅

    2011-01-01

    目的 探讨钙蛋白酶(calpain)在卡那霉素(kanamycin,KM)致耳中毒豚鼠耳蜗的表达.方法 将豚鼠随机分成对照组、KM 3 d组、KM 7 d组和KM 14 d组,应用免疫组织化学SABC(streptavidin-biotin peroxidae complex,链霉亲合素-生物素过氧化物酶复合物)法和显微图像分析技术检测耳蜗中钙蛋白酶的表达,用药前后给予短纯音刺激检测听性脑干反应阈值,观察豚鼠听力的变化.结果 对照组calpain 1阳性免疫反应主要见于耳蜗毛细胞、螺旋神经节、血管纹和螺旋韧带,以螺旋神经节的染色较深,而其它部位均呈阴性.肌肉注射KM后,calpain 1在耳蜗中的阳性反应部位与对照组大致相同,显微图像分析结果表明,随着给药天数的增加,calpain 1在耳蜗上述部位的阳性反应逐渐减弱.Calpain 2在各组豚鼠耳蜗中的表达部位与calpain 1的相同,显微图像分析结果提示,随着给药天数的增加,calpain 2在耳蜗上述部位的阳性反应逐渐增强.结论 正常豚鼠耳蜗中有calpain1和calpain 2的表达.注射KM后,随着给药天数的增加,calpain 1在耳蜗的表达逐渐减弱,而calpain 2的表达则逐渐增强,提示calpain 2可能参与了卡那霉素致耳中毒的过程.%Objective To investigate expression of calpains in the cochlea in kanamycin (KM) -poisoned guinea pigs. Methods Guinea pigs were randomly assigned to serve as controls or to receive KM for 3, 7 or 14 days. SABC im-munohistochemistry and imaging analysis were used to examine expression of calpain 1 and calpain 2. Auditory brainstem responses (ABRs) were used to determine auditory thresholds and their changes. Results Immunoreactivity for clapin 1 was mainly seen in the hair cell, spiral ganglion, stria vascularis and spiral ligament in the control group, with greater staining in the spiral ganglion. The location of calpain 1 expression in the cochlea was similar in the control and KM treatment groups. Imaging analysis indicated that

  6. 钙蛋白酶系统与烧伤后骨骼肌消耗的关系研究进展%Advances in the research of the relationship between calpains and post-burn skeletal muscle wasting

    Institute of Scientific and Technical Information of China (English)

    马丽; 柴家科

    2013-01-01

    Calpains are intracellular nonlysosomal Ca2+-regulated cysteine proteases,widely located in the tissues of most mammals.Skeletal muscle tissue mainly expresses m-calpain,μ-caplain,n-calpain,and their endogenous inhibitor calpastatin.They are closely related to the cell apoptosis,cytoskeleton formation,cell cycles,etc.Calpains are also considered to be participating in the protein degradation process.Severe burns are typically followed by hypermetabolic responses that are characterized by hyperdynamic circulatory responses with increased proteolysis and cell apoptosis.Recently,overloading of Ca2+ in skeletal muscle cells,which activates the calpains is observed after a serious burn.This paper aims to review the current research of the relationship between calpains and post-burn skeletal muscle wasting from the perspectives of structure,function,and physiological activities.

  7. Calpastatin-mediated inhibition of calpains in the mouse brain prevents mutant ataxin 3 proteolysis, nuclear localization and aggregation, relieving Machado-Joseph disease.

    Science.gov (United States)

    Simões, Ana T; Gonçalves, Nélio; Koeppen, Arnulf; Déglon, Nicole; Kügler, Sebastian; Duarte, Carlos Bandeira; Pereira de Almeida, Luís

    2012-08-01

    Machado-Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-repetition of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within the ataxin 3 protein, which upon proteolysis may trigger Machado-Joseph disease. We investigated the role of calpains in the generation of toxic ataxin 3 fragments and pathogenesis of Machado-Joseph disease. For this purpose, we inhibited calpain activity in mouse models of Machado-Joseph disease by overexpressing the endogenous calpain-inhibitor calpastatin. Calpain blockage reduced the size and number of mutant ataxin 3 inclusions, neuronal dysfunction and neurodegeneration. By reducing fragmentation of ataxin 3, calpastatin overexpression modified the subcellular localization of mutant ataxin 3 restraining the protein in the cytoplasm, reducing aggregation and nuclear toxicity and overcoming calpastatin depletion observed upon mutant ataxin 3 expression. Our findings are the first in vivo proof that mutant ataxin 3 proteolysis by calpains mediates its translocation to the nucleus, aggregation and toxicity and that inhibition of calpains may provide an effective therapy for Machado-Joseph disease.

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Study on purification of Calpain system in beef by DEAE-sephacel and DEAE-sepharose-FF%DEAE-Sephacel和DEAE-Sepharose-FF 对牛肉中Calpains分离纯化的比较研究

    Institute of Scientific and Technical Information of China (English)

    许永平; 罗欣; 孙清亮; 胡鹏; 毛衍伟

    2007-01-01

    实验通过DEAE离子交换层析对牛背长肌中钙激活酶(Calpains)的分离研究,比较了DEAE-Sepharose-FF和DEAE-Sephacel对钙激活酶(Calpains)的分离效果,确定了Calpains分离纯化的条件.Sephacel不耐压,分离时间长,因此选择Sepharose-FF分离纯化Calpains.

  11. Inhibition of calpains fails to improve regeneration through a peripheral nerve conduit.

    Science.gov (United States)

    Hausner, Thomas; Marvaldi, Letizia; Márton, Gábor; Pajer, Krisztián; Hopf, Rudolf; Schmidhammer, Robert; Hausott, Barbara; Redl, Heinz; Nógrádi, Antal; Klimaschewski, Lars

    2014-04-30

    Intramuscular injection of the calpain inhibitor leupeptin promotes peripheral nerve regeneration in primates (Badalamente et al., 1989 [13]), and direct positive effects of leupeptin on axon outgrowth were observed in vitro (Hausott et al., 2012 [12]). In this study, we applied leupeptin (2mg/ml) directly to collagen-filled nerve conduits in the rat sciatic nerve transection model. Analysis of myelinated axons and retrogradely labeled motoneurons as well as functional 'CatWalk' video analysis did not reveal significant differences between vehicle controls and leupeptin treated animals. Therefore, leupeptin does not improve nerve regeneration via protease inhibition in regrowing axons or in surrounding Schwann cells following a single application to a peripheral nerve conduit suggesting indirect effects on motor endplate integrity if applied systemically.

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

    Directory of Open Access Journals (Sweden)

    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. 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.

  14. 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.

  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. p-Benzoquinone, a reactive metabolite of benzene, prevents the processing of pre-interleukins-1{alpha} and -1{beta} to active cytokines by inhibition of the processing enzymes, calpain, and interleukin-1{beta} converting enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Kalf, G.F.; Renz, J.F.; Niculescu, R. [Thomas Jefferson Univ., Philadelphia, PA (United States)

    1996-12-01

    Chronic exposure of humans to benzene affects hematopoietic stem and progenitor cells and leads to aplastic anemia. The stromal macrophage, a target of benzene toxicity, secretes interieukin-1 (IL-1), which induces the stromal fibroblast to synthesize hematopoietic colony-stimulating factors. In a mouse model, benzene causes an acute marrow hypocellularity that can be prevented by the concomitant administration of IL-1{alpha}. The ability of benzene to interfere with the production and secretion of IL-1{alpha} was tested. Stromal macrophages from benzene-treated mice were capable of the transcription of the IL-1{alpha} gene and the translation of the message but showed an inability to process the 34-kDa pre-IL-1{alpha} precursor to the 17-kDa biologically active cytokine. Treatment of normal murine stromal macrophages in culture with hydroquinone (HQ) also showed an inhibition in processing of pre-IL-1{alpha}. Hydroquinone is oxidized by a peroxidase-mediated reaction in the stromal macrophage to p-benzoquinone, which interacts with the sulfhydryl (SH) groups of proteins and was shown to completely inhibit the activity of calpain, the SH-dependent protease that cleaves pre-IL-1{alpha}. In a similar manner, HQ, via peroxidase oxidation to p-benzoquinone, was capable of preventing the IL-1{beta} autocrine stimulation of growth of human B1 myeloid tumor cells by preventing the processing of pre-IL-1{beta} to mature cytokine. Benzoquinone was also shown to completely inhibit the ability of the SH-dependent IL-1{beta} converting enzyme. Thus benzene-induced bone marrow hypocellularity may result from apoptosis of hematopoietic progenitor cells brought about by lack of essential cylokines and deficient IL-1{alpha} production subsequent to the inhibition of calpain by p-benzoquinone and the prevention of pre-IL-1 processing. 34 refs., 8 figs.

  17. Effects of concentric and repeated eccentric exercise on muscle damage and calpain-calpastatin gene expression in human skeletal muscle

    DEFF Research Database (Denmark)

    Vissing, K.; Overgaard, K.; Nedergaard, A.

    2008-01-01

    , and was compared to a control-group (n = 6). Muscle strength and soreness and plasma creatine kinase and myoglobin were measured before and during 7 days following exercise bouts. Muscle biopsies were collected from m. vastus lateralis of both legs prior to and at 3, 24 h and 7 days after exercise and quantified...... for muscle Ca2+-content and mRNA levels for calpain isoforms and calpastatin. Exercise reduced muscle strength and increased muscle soreness predominantly in the eccentric leg (P ... eccentric exercise bout (P muscle Ca2+-content did not differ between interventions. mRNA levels for calpain 2 and calpastatin were upregulated exclusively by eccentric exercise 24 h post-exercise (P

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

    Directory of Open Access Journals (Sweden)

    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. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Cleavage of Tau by calpain in Alzheimer's disease: the quest for the toxic 17 kD fragment.

    Science.gov (United States)

    Garg, Sarika; Timm, Thomas; Mandelkow, Eva-Maria; Mandelkow, Eckhard; Wang, Yipeng

    2011-01-01

    The amyloid cascade hypothesis of Alzheimer's disease (AD) posits that the generation of β-amyloid (Aβ) triggers Tau neurofibrillary pathology. Recently a "17 kD" calpain-induced Tau fragment, comprising residues 45-230 (molecular weight [MW], 18.7 kD), was proposed to mediate Aβ-induced toxicity. Here, we demonstrate that the "17 kD" fragment is actually much smaller, containing residues 125-230 (molecular weight, 10.7 kD). Inducing Tau phosphorylation by okadaic acid or mimicking phosphorylation by Glu mutations at the epitopes of Alzheimer-diagnostic antibodies AT100/AT8/PHF1 could not prevent the generation of this fragment. The fragment can be induced not only by Aβ oligomers, but also by other cell stressors, e.g., thapsigargin (a Ca(2+)-ATPase inhibitor) or glutamate (an excitatory neurotransmitter). However, overexpression of neither Tau(45-230) nor Tau(125-230) fragment is toxic to Chinese hamster ovary (CHO) cells, neuroblastoma cells (N2a) or primary hippocampal neurons. Finally, the calpain-induced fragment can be observed both in Alzheimer's disease brains and in control normal human brains. We conclude that the 17 kD Tau fragment is not a mediator of Aβ-induced toxicity, leaving open the possibility that upstream calpain activation might cause both Tau fragmentation and toxicity.

  4. Role of mitochondrial calpains in apoptosis%线粒体calpains与细胞凋亡关系的研究进展

    Institute of Scientific and Technical Information of China (English)

    宋必卫; 樊俏玫; 何治宇

    2014-01-01

    Calpains are the family of Ca2+-activated cysteine pro-teases. Although calpains are considered to be cytoplasmic en-zymes, recent research has demonstrated that μ-calpain, m-cal-pain, calpain 10 and their endogenous inhibitor calpastatin are present in the mitochondria and play important roles both in caspase -dependent and-independent pathways in cell death phe-nomena. Calpains exert direct and indirect effects on the caspases and regulators of apoptosis pathway such as Bcl-2, Bax, Bid, promoting the release of Cyt-C, AIF, then result in cellular ap-optosis. To allow pharmacological targeting of these enzymes, thorough knowledge of their patterns of activation and further in-teractions with already known apoptotic pathways is necessary.%calpains是一类由 Ca2+激活的内源性半胱氨酸蛋白酶。尽管先前研究认为calpains是一种胞质酶,最近研究发现μ-calpain、m-calpain、calpain10以及内源性抑制剂 cal-pastatin也同时存在于线粒体中,在caspase依赖型和非依赖型细胞凋亡通路中均发挥着重要的作用。 calpain 除了与caspase相互作用外,还可剪切凋亡相关蛋白如 Bcl-2、Bax、Bid等,促进Cyt-C、AIF的释放,从而参与调控细胞凋亡的病理过程。 calpains作为潜在的治疗靶点,研究线粒体calpains与细胞凋亡通路的关系具有重大意义。

  5. 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.

  6. 心房颤动犬心房肌 Calpains mRNA 与蛋白表达改变的研究%A Study on the Changes of Atrial Calpains mRNA and Protein Expression in Atrial Fibrillation Dogs

    Institute of Scientific and Technical Information of China (English)

    初杉; 薛红杰; 李悦

    2008-01-01

    目的 观察长期心房快速起搏诱发房颤犬心房肌 Calpain Ⅰ、Calpain Ⅱ mRNA 和蛋白表达情况.方法 RT-PCR 和 Western-blot 方法 检测对照组及房颤组犬心房肌 Calpain Ⅰ和 Calpain Ⅱ mRNA 及蛋白表达情况;光镜、电镜下,观察心房肌病理组织学和超微结构改变.结果 房颤组犬心房肌 Calpain Ⅰ mRNA 和蛋白表达较对照组犬显著上调 (P<0.01,P<0.05);房颤组犬心房肌肌溶解明显.结论 房颤组犬心房肌Calpain Ⅰ mRNA 和蛋白表达显著增加,可能通过引起心房肌肌溶解参与导致房颤心房结构重构.

  7. Calpain-5 gene variants are associated with diastolic blood pressure and cholesterol levels

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    Morón Francisco J

    2007-01-01

    Full Text Available Abstract Background Genes implicated in common complex disorders such as obesity, type 2 diabetes mellitus (T2DM or cardiovascular diseases are not disease specific, since clinically related disorders also share genetic components. Cysteine protease Calpain 10 (CAPN10 has been associated with T2DM, hypertension, hypercholesterolemia, increased body mass index (BMI and polycystic ovary syndrome (PCOS, a reproductive disorder of women in which isunlin resistance seems to play a pathogenic role. The calpain 5 gene (CAPN5 encodes a protein homologue of CAPN10. CAPN5 has been previously associated with PCOS by our group. In this new study, we have analysed the association of four CAPN5 gene variants(rs948976A>G, rs4945140G>A, rs2233546C>T and rs2233549G>A with several cardiovascular risk factors related to metabolic syndrome in general population. Methods Anthropometric measurements, blood pressure, insulin, glucose and lipid profiles were determined in 606 individuals randomly chosen from a cross-sectional population-based epidemiological survey in the province of Segovia in Central Spain (Castille, recruited to investigate the prevalence of anthropometric and physiological parameters related to obesity and other components of the metabolic syndrome. Genotypes at the four polymorphic loci in CAPN5 gene were detected by polymerase chain reaction (PCR. Results Genotype association analysis was significant for BMI (p ≤ 0.041, diastolic blood pressure (p = 0.015 and HDL-cholesterol levels (p = 0.025. Different CAPN5 haplotypes were also associated with diastolic blood pressure (DBP (0.0005 ≤ p ≤ 0.006 and total cholesterol levels (0.001 ≤ p ≤ 0.029. In addition, the AACA haplotype, over-represented in obese individuals, is also more frequent in individuals with metabolic syndrome defined by ATPIII criteria (p = 0.029. Conclusion As its homologue CAPN10, CAPN5 seems to influence traits related to increased risk for cardiovascular diseases. Our

  8. Susceptibility of Phytomonas serpens to calpain inhibitors in vitro: interference on the proliferation, ultrastructure, cysteine peptidase expression and interaction with the invertebrate host

    Science.gov (United States)

    de Oliveira, Simone Santiago Carvalho; Gonçalves, Diego de Souza; Garcia-Gomes, Aline dos Santos; Gonçalves, Inês Correa; Seabra, Sergio Henrique; Menna-Barreto, Rubem Figueiredo; Lopes, Angela Hampshire de Carvalho Santos; D’Avila-Levy, Claudia Masini; dos Santos, André Luis Souza; Branquinha, Marta Helena

    2016-01-01

    A pleiotropic response to the calpain inhibitor MDL28170 was detected in the tomato parasite Phytomonas serpens. Ultrastructural studies revealed that MDL28170 caused mitochondrial swelling, shortening of flagellum and disruption of trans Golgi network. This effect was correlated to the inhibition in processing of cruzipain-like molecules, which presented an increase in expression paralleled by decreased proteolytic activity. Concomitantly, a calcium-dependent cysteine peptidase was detected in the parasite extract, the activity of which was repressed by pre-incubation of parasites with MDL28170. Flow cytometry and Western blotting analyses revealed the differential expression of calpain-like proteins (CALPs) in response to the pre-incubation of parasites with the MDL28170, and confocal fluorescence microscopy confirmed their surface location. The interaction of promastigotes with explanted salivary glands of the insect Oncopeltus fasciatus was reduced when parasites were pre-treated with MDL28170, which was correlated to reduced levels of surface cruzipain-like and gp63-like molecules. Treatment of parasites with anti-Drosophila melanogaster (Dm) calpain antibody also decreased the adhesion process. Additionally, parasites recovered from the interaction process presented higher levels of surface cruzipain-like and gp63-like molecules, with similar levels of CALPs cross-reactive to anti-Dm-calpain antibody. The results confirm the importance of exploring the use of calpain inhibitors in studying parasites’ physiology. PMID:27925020

  9. Increased mitochondrial emission of reactive oxygen species and calpain activation are required for doxorubicin-induced cardiac and skeletal muscle myopathy.

    Science.gov (United States)

    Min, Kisuk; Kwon, Oh-Sung; Smuder, Ashley J; Wiggs, Michael P; Sollanek, Kurt J; Christou, Demetra D; Yoo, Jeung-Ki; Hwang, Moon-Hyon; Szeto, Hazel H; Kavazis, Andreas N; Powers, Scott K

    2015-04-15

    Although doxorubicin (DOX) is a highly effective anti-tumour agent used to treat a variety of cancers, DOX administration is associated with significant side effects, including myopathy of both cardiac and skeletal muscles. The mechanisms responsible for DOX-mediated myopathy remain a topic of debate. We tested the hypothesis that both increased mitochondrial reactive oxygen species (ROS) emission and activation of the cysteine protease calpain are required for DOX-induced myopathy in rat cardiac and skeletal muscle. Cause and effect was determined by administering a novel mitochondrial-targeted anti-oxidant to prevent DOX-induced increases in mitochondrial ROS emission, whereas a highly-selective pharmacological inhibitor was exploited to inhibit calpain activity. Our findings reveal that mitochondria are a major site of DOX-mediated ROS production in both cardiac and skeletal muscle fibres and the prevention of DOX-induced increases in mitochondrial ROS emission protects against fibre atrophy and contractile dysfunction in both cardiac and skeletal muscles. Furthermore, our results indicate that DOX-induced increases in mitochondrial ROS emission are required to activate calpain in heart and skeletal muscles and, importantly, calpain activation is a major contributor to DOX-induced myopathy. Taken together, these findings show that increased mitochondrial ROS production and calpain activation are significant contributors to the development of DOX-induced myopathy in both cardiac and skeletal muscle fibres.

  10. KERAGAMAN GEN CALPASTATIN, CALPAIN 3 DAN MYOSTATIN PADA DOMBA DI UP3 JONGGOL

    Directory of Open Access Journals (Sweden)

    Cece Sumantri

    2012-04-01

    Full Text Available The aim of this study was to identify the genetic polymorphisms of calpastatin (CAST, calpain 3 (CAPN3 and myostatin (MSTN on local sheep at Jonggol Animal Science Teaching and Research Unit (JASTRU. A total number of 294 blood samples were collected from JASTRU. The identification of polymorhism in CAST and CAPN3 genes performed by using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP while MSTN gene by using PCR-SSCP methods. The results showed that CAST|MspI, CAST|NcoI and CAPN3|MaeII loci were polymorphic, whereas The MSTN locus was monomorphic for G (1.0. The frequency of allele M (0.87 on the locus (CAST|MspI higher than the N allele (0.13. At locus CAST|NcoI, the frequency of allele M (0.96 higher than the N allele (0.04. At the CAPN3|MaeII, allele G (0.85 and allele T (0.15. Locus CAST|NcoI has higher observed heterozygosity (Ho = 0.92 compared to CAPN3|MaeII and CAST|MspI (Ho = 0.74-0.77, however has lower compared to CAPN3|MaeII and CAST|MspI in expected of heterozygosity (He = 0.08 vs 0.23-0.26 and in index fixation (Fis = -0.04 vs 0.03-0.12.

  11. 急性病毒性心肌炎小鼠心肌中Calpain mRNA表达与心肌细胞凋亡的关系研究%A Study on the Correlation between Calpain mrna Expression and cardiomyocyte apoptosis in Acute murine coxsackievirus B3 myocarditis

    Institute of Scientific and Technical Information of China (English)

    李明辉; 汪云开; 虞勇; 陈瑞珍; 杨英珍

    2009-01-01

    目的 检测Calpain mRNA在柯萨奇病毒B3(Coxsackievirus B3,CVB3)急性心肌炎小鼠心肌组织中的表达,并分析其与心肌细胞凋亡的关系.方法 以CVB3单次感染Balb/c小鼠建立急性病毒性心肌炎(n=10)模型,同时设立药物干预组(n=12)及药物对照组(n=10);同期小鼠腹腔无菌注射等剂量不含病毒的DMEM液作为正常对照组(n=8).以缺口末端标记法(TUNEL)检测各组心肌细胞凋亡情况,计数凋亡率.用逆转录聚合酶链反应(RT-PCR)方法检测小鼠心肌组织中Calpain-1及Calpain-2 mRNA的表达.结果 急性病毒性心肌炎组较正常对照组及药物对照组心肌组织内Calpain-1(P=0.02及P=0.04)和Calpain-2(P<0.01及P=0.02)mRNA表达升高,心肌细胞凋亡率亦增高(P值均<0.01);药物对照组与正常对照组心肌组织内Calpain-1(P>0.05)和Calpain-2(P>0.05)mRNA及心肌细胞凋亡率均没有统计学差异(P>0.05);药物干预组较正常组心肌组织内Calpain-1(P<0.01)及Calpain-2(P<0.01)mRNA表达升高,心肌细胞凋亡率亦升高(P<0.01);药物干预组较急性病毒性心肌炎组心肌组织内Calpain-1(P=0.04)及Calpain-2(P<0.01)mRNA表达升高,心肌细胞凋亡率亦升高(P<0.01).各实验组小鼠心肌组织中Calpain-1及calpain-2 mRNA的表达水平分别与心肌细胞的凋亡呈正相关.结论 柯萨奇B3病毒通过激活心肌组织内Calpain,导致心肌细胞的凋亡,从而参与病毒性心肌炎的发生.

  12. 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.

  13. Striatal inhibition of calpains prevents levodopa-induced neurochemical changes and abnormal involuntary movements in the hemiparkinsonian rat model.

    Science.gov (United States)

    Chagniel, Laure; Robitaille, Christine; Lebel, Manon; Cyr, Michel

    2012-01-01

    Pharmacological dopamine replacement with l-3,4-dihydroxyphenylalanine (L-DOPA) remains the most effective approach to treat the motor symptoms of Parkinson's disease (PD). However, as the disease progresses, the therapeutic response to L-DOPA gradually becomes erratic and is associated with the emergence of dyskinesia in the majority of patients. The pathogenesis of L-DOPA-induced dyskinesia (LID) is still unknown. In the current study, using the 6-hydroxydopamine (6-OHDA)-lesioned rat model of PD, we demonstrated that the calcium-dependent proteins calpains and cdk5 of the striatum play a critical role in the behavioral and molecular changes evoked by L-DOPA therapy. We first confirmed that L-DOPA reversed PD symptoms, assessed by the cylinder, stepping and vibrissae-elicited reaching tests in this animal model, and elicited robust abnormal involuntary movements (AIMs) reminiscent of LID. Interestingly, intrastriatal infusion of the calpains inhibitor MDL28170, and to a lower extent the cdk5 inhibitor roscovitine, reduced the severity and amplitude of AIMs without affecting L-DOPA's antiparkinsonian effects. Notably, the calpains and cdk5 inhibitors totally reversed the striatal molecular changes attributed to L-DOPA therapy, such as ERK1/2 and dynamin phosphorylation. Another fascinating observation was that L-DOPA therapy, in combination with intrastriatal infusion of MDL28170, augmented tyrosine hydroxylase levels in the striatum of lesioned rats without affecting the number of dopaminergic cells in the substantia nigra. These findings disclose a novel mechanism underlying the maladaptive alterations induced by L-DOPA therapy in the 6-OHDA rat model of PD.

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

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    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.

  15. Lrit3 deficient mouse (nob6): a novel model of complete congenital stationary night blindness (cCSNB).

    Science.gov (United States)

    Neuillé, Marion; El Shamieh, Said; Orhan, Elise; Michiels, Christelle; Antonio, Aline; Lancelot, Marie-Elise; Condroyer, Christel; Bujakowska, Kinga; Poch, Olivier; Sahel, José-Alain; Audo, Isabelle; Zeitz, Christina

    2014-01-01

    Mutations in LRIT3, coding for a Leucine-Rich Repeat, immunoglobulin-like and transmembrane domains 3 protein lead to autosomal recessive complete congenital stationary night blindness (cCSNB). The role of the corresponding protein in the ON-bipolar cell signaling cascade remains to be elucidated. Here we genetically and functionally characterize a commercially available Lrit3 knock-out mouse, a model to study the function and the pathogenic mechanism of LRIT3. We confirm that the insertion of a Bgeo/Puro cassette in the knock-out allele introduces a premature stop codon, which presumably codes for a non-functional protein. The mouse line does not harbor other mutations present in common laboratory mouse strains or in other known cCSNB genes. Lrit3 mutant mice exhibit a so-called no b-wave (nob) phenotype with lacking or severely reduced b-wave amplitudes in the scotopic and photopic electroretinogram (ERG), respectively. Optomotor tests reveal strongly decreased optomotor responses in scotopic conditions. No obvious fundus auto-fluorescence or histological retinal structure abnormalities are observed. However, spectral domain optical coherence tomography (SD-OCT) reveals thinned inner nuclear layer and part of the retina containing inner plexiform layer, ganglion cell layer and nerve fiber layer in these mice. To our knowledge, this is the first time that SD-OCT technology is used to characterize an animal model for CSNB. This phenotype is noted at 6 weeks and at 6 months. The stationary nob phenotype of mice lacking Lrit3, which we named nob6, confirms the findings previously reported in patients carrying LRIT3 mutations and is similar to other cCSNB mouse models. This novel mouse model will be useful for investigating the pathogenic mechanism(s) associated with LRIT3 mutations and clarifying the role of LRIT3 in the ON-bipolar cell signaling cascade.

  16. Lrit3 deficient mouse (nob6: a novel model of complete congenital stationary night blindness (cCSNB.

    Directory of Open Access Journals (Sweden)

    Marion Neuillé

    Full Text Available Mutations in LRIT3, coding for a Leucine-Rich Repeat, immunoglobulin-like and transmembrane domains 3 protein lead to autosomal recessive complete congenital stationary night blindness (cCSNB. The role of the corresponding protein in the ON-bipolar cell signaling cascade remains to be elucidated. Here we genetically and functionally characterize a commercially available Lrit3 knock-out mouse, a model to study the function and the pathogenic mechanism of LRIT3. We confirm that the insertion of a Bgeo/Puro cassette in the knock-out allele introduces a premature stop codon, which presumably codes for a non-functional protein. The mouse line does not harbor other mutations present in common laboratory mouse strains or in other known cCSNB genes. Lrit3 mutant mice exhibit a so-called no b-wave (nob phenotype with lacking or severely reduced b-wave amplitudes in the scotopic and photopic electroretinogram (ERG, respectively. Optomotor tests reveal strongly decreased optomotor responses in scotopic conditions. No obvious fundus auto-fluorescence or histological retinal structure abnormalities are observed. However, spectral domain optical coherence tomography (SD-OCT reveals thinned inner nuclear layer and part of the retina containing inner plexiform layer, ganglion cell layer and nerve fiber layer in these mice. To our knowledge, this is the first time that SD-OCT technology is used to characterize an animal model for CSNB. This phenotype is noted at 6 weeks and at 6 months. The stationary nob phenotype of mice lacking Lrit3, which we named nob6, confirms the findings previously reported in patients carrying LRIT3 mutations and is similar to other cCSNB mouse models. This novel mouse model will be useful for investigating the pathogenic mechanism(s associated with LRIT3 mutations and clarifying the role of LRIT3 in the ON-bipolar cell signaling cascade.

  17. Deletion of FGFR3 in Osteoclast Lineage Cells Results in Increased Bone Mass in Mice by Inhibiting Osteoclastic Bone Resorption.

    Science.gov (United States)

    Su, Nan; Li, Xiaogang; Tang, Yubin; Yang, Jing; Wen, Xuan; Guo, Jingyuan; Tang, Junzhou; Du, Xiaolan; Chen, Lin

    2016-09-01

    Fibroblast growth factor receptor 3 (FGFR3) participates in bone remodeling. Both Fgfr3 global knockout and activated mice showed decreased bone mass with increased osteoclast formation or bone resorption activity. To clarify the direct effect of FGFR3 on osteoclasts, we specifically deleted Fgfr3 in osteoclast lineage cells. Adult mice with Fgfr3 deficiency in osteoclast lineage cells (mutant [MUT]) showed increased bone mass. In a drilled-hole defect model, the bone remodeling of the holed area in cortical bone was also impaired with delayed resorption of residual woven bone in MUT mice. In vitro assay demonstrated that there was no significant difference between the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts derived from wild-type and Fgfr3-deficient bone marrow monocytes, suggesting that FGFR3 had no remarkable effect on osteoclast formation. The bone resorption activity of Fgfr3-deficient osteoclasts was markedly decreased accompanying with downregulated expressions of Trap, Ctsk, and Mmp 9. The upregulated activity of osteoclastic bone resorption by FGF2 in vitro was also impaired in Fgfr3-deficient osteoclasts, indicating that FGFR3 may participate in the regulation of bone resorption activity of osteoclasts by FGF2. Reduced adhesion but not migration in osteoclasts with Fgfr3 deficiency may be responsible for the impaired bone resorption activity. Our study for the first time genetically shows the direct positive regulation of FGFR3 on osteoclastic bone resorption. © 2016 American Society for Bone and Mineral Research.

  18. Altered cognitive performance and synaptic function in the hippocampus of mice lacking C3.

    Science.gov (United States)

    Perez-Alcazar, Marta; Daborg, Jonny; Stokowska, Anna; Wasling, Pontus; Björefeldt, Andreas; Kalm, Marie; Zetterberg, Henrik; Carlström, Karl E; Blomgren, Klas; Ekdahl, Christine T; Hanse, Eric; Pekna, Marcela

    2014-03-01

    Previous work implicated the complement system in adult neurogenesis as well as elimination of synapses in the developing and injured CNS. In the present study, we used mice lacking the third complement component (C3) to elucidate the role the complement system plays in hippocampus-dependent learning and synaptic function. We found that the constitutive absence of C3 is associated with enhanced place and reversal learning in adult mice. Our findings of lower release probability at CA3-CA1 glutamatergic synapses in combination with unaltered overall efficacy of these synapses in C3 deficient mice implicate C3 as a negative regulator of the number of functional glutamatergic synapses in the hippocampus. The C3 deficient mice showed no signs of spontaneous epileptiform activity in the hippocampus. We conclude that C3 plays a role in the regulation of the number and function of glutamatergic synapses in the hippocampus and exerts negative effects on hippocampus-dependent cognitive performance.

  19. Calpains, cleaved mini-dysferlinC72, and L-type channels underpin calcium-dependent muscle membrane repair.

    Science.gov (United States)

    Lek, Angela; Evesson, Frances J; Lemckert, Frances A; Redpath, Gregory M I; Lueders, Ann-Katrin; Turnbull, Lynne; Whitchurch, Cynthia B; North, Kathryn N; Cooper, Sandra T

    2013-03-20

    Dysferlin is proposed as a key mediator of calcium-dependent muscle membrane repair, although its precise role has remained elusive. Dysferlin interacts with a new membrane repair protein, mitsugumin 53 (MG53), an E3 ubiquitin ligase that shows rapid recruitment to injury sites. Using a novel ballistics assay in primary human myotubes, we show it is not full-length dysferlin recruited to sites of membrane injury but an injury-specific calpain-cleavage product, mini-dysferlinC72. Mini-dysferlinC72-rich vesicles are rapidly recruited to injury sites and fuse with plasma membrane compartments decorated by MG53 in a process coordinated by L-type calcium channels. Collective interplay between activated calpains, dysferlin, and L-type channels explains how muscle cells sense a membrane injury and mount a specialized response in the unique local environment of a membrane injury. Mini-dysferlinC72 and MG53 form an intricate lattice that intensely labels exposed phospholipids of injury sites, then infiltrates and stabilizes the membrane lesion during repair. Our results extend functional parallels between ferlins and synaptotagmins. Whereas otoferlin exists as long and short splice isoforms, dysferlin is subject to enzymatic cleavage releasing a synaptotagmin-like fragment with a specialized protein- or phospholipid-binding role for muscle membrane repair.

  20. Osmostress-induced apoptosis in Xenopus oocytes: role of stress protein kinases, calpains and Smac/DIABLO.

    Directory of Open Access Journals (Sweden)

    Nabil Ben Messaoud

    Full Text Available Hyperosmotic shock induces cytochrome c release and caspase-3 activation in Xenopus oocytes, but the regulators and signaling pathways involved are not well characterized. Here we show that hyperosmotic shock induces rapid calpain activation and high levels of Smac/DIABLO release from the mitochondria before significant amounts of cytochrome c are released to promote caspase-3 activation. Calpain inhibitors or EGTA microinjection delays osmostress-induced apoptosis, and blockage of Smac/DIABLO with antibodies markedly reduces cytochrome c release and caspase-3 activation. Hyperosmotic shock also activates the p38 and JNK signaling pathways very quickly. Simultaneous inhibition of both p38 and JNK pathways reduces osmostress-induced apoptosis, while sustained activation of these kinases accelerates the release of cytochrome c and caspase-3 activation. Therefore, at least four different pathways early induced by osmostress converge on the mitochondria to trigger apoptosis. Deciphering the mechanisms of hyperosmotic shock-induced apoptosis gives insight for potential treatments of human diseases that are caused by perturbations in fluid osmolarity.

  1. Osmostress-induced apoptosis in Xenopus oocytes: role of stress protein kinases, calpains and Smac/DIABLO.

    Science.gov (United States)

    Ben Messaoud, Nabil; Yue, Jicheng; Valent, Daniel; Katzarova, Ilina; López, José M

    2015-01-01

    Hyperosmotic shock induces cytochrome c release and caspase-3 activation in Xenopus oocytes, but the regulators and signaling pathways involved are not well characterized. Here we show that hyperosmotic shock induces rapid calpain activation and high levels of Smac/DIABLO release from the mitochondria before significant amounts of cytochrome c are released to promote caspase-3 activation. Calpain inhibitors or EGTA microinjection delays osmostress-induced apoptosis, and blockage of Smac/DIABLO with antibodies markedly reduces cytochrome c release and caspase-3 activation. Hyperosmotic shock also activates the p38 and JNK signaling pathways very quickly. Simultaneous inhibition of both p38 and JNK pathways reduces osmostress-induced apoptosis, while sustained activation of these kinases accelerates the release of cytochrome c and caspase-3 activation. Therefore, at least four different pathways early induced by osmostress converge on the mitochondria to trigger apoptosis. Deciphering the mechanisms of hyperosmotic shock-induced apoptosis gives insight for potential treatments of human diseases that are caused by perturbations in fluid osmolarity.

  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 (Pcalpain 1 was increased in the adult and the aged groups with AF (Pcalpain 1. The general pathophysiological alterations in normal aged atria may therefore produce a substrate that is conducive to AF.

  3. 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.

  4. Citicoline protects brain against closed head injury in rats through suppressing oxidative stress and calpain over-activation.

    Science.gov (United States)

    Qian, Ke; Gu, Yi; Zhao, Yumei; Li, Zhenzong; Sun, Ming

    2014-07-01

    Citicoline, a natural compound that functions as an intermediate in the biosynthesis of cell membrane phospholipids, is essential for membrane integrity and repair. It has been reported to protect brain against trauma. This study was designed to investigate the protective effects of citicoline on closed head injury (CHI) in rats. Citicoline (250 mg/kg i.v. 30 min and 4 h after CHI) lessened body weight loss, and improved neurological functions significantly at 7 days after CHI. It markedly lowered brain edema and blood-brain barrier permeability, enhanced the activities of superoxide dismutase and the levels of glutathione, reduced the levels of malondialdehyde and lactic acid. Moreover, citicoline suppressed the activities of calpain, and enhanced the levels of calpastatin, myelin basic protein and αII-spectrin in traumatic tissue 24 h after CHI. Also, it attenuated the axonal and myelin sheath damage in corpus callosum and the neuronal cell death in hippocampal CA1 and CA3 subfields 7 days after CHI. These data demonstrate the protection of citicoline against white matter and grey matter damage due to CHI through suppressing oxidative stress and calpain over-activation, providing additional support to the application of citicoline for the treatment of traumatic brain injury.

  5. 脊髓损伤中钙蛋白酶的作用%The function of calpain in spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    袁景(综述); 甄平(审校)

    2014-01-01

    脊髓损伤后可发生不同程度的神经坏死和组织变性,导致脊髓神经功能障碍。脊髓继发性损伤过程中钙蛋白酶( calpain)在脊髓损伤中发挥重要作用。 Calpain为一Ca2+依赖性半胱氨酸蛋白酶,该酶包括2种同分异构体μ-calpain 和 m-calpain,分别由微摩尔和毫摩尔浓度的Ca2+激活。细胞内 calpain 以非活性酶原形式存在,细胞内游离Ca2+浓度升高后,calpain酶原被活化,引起calpain活化后一系列分子生物化学的改变,在脊髓引起细胞凋亡或损伤。应用calpain的特异性抑制剂进行干预后,脊髓calpain的表达和活性受到抑制,脊髓的病理改变和细胞凋亡得到缓解,神经功能得以保护。该文将calpain在脊髓损伤发展过程中的作用作一综述。

  6. Human S100A7 Induces Mature Interleukin1α Expression by RAGE-p38 MAPK-Calpain1 Pathway in Psoriasis

    Science.gov (United States)

    Lei, Hu; Li, Xiangyun; Jing, Bo; Xu, Hanzhang; Wu, Yingli

    2017-01-01

    Psoriatic keratinocytes express exaggerated levels of inflammatory cytokines, and show aberrant hyperproliferation and terminal differentiation in the pathogenesis of psoriasis. The antimicrobial protein hS100A7 (psoriasin) has been found highly expressed in psoriatic skin, but the mechanism and physiological function remain largely unknown. We observed that hS100A7 induces mature interleukin 1α (17kDa) expression in normal human epidermal keratinocytes, which is dependent on RAGE-p38 MAPK and calpain-1 as the inhibitors or knockdown of them completely decreased the expression of mature interleukin1α. Then, we proved mS100a7a15, mature IL-1α and calpain-1 were highly expressed in imquimod-induced psoriasis model and mouse IL-17a-neutralizing antibody treatment attenuated mS100a7a15 expression. At last, PD 151746 (calpain-1 inhibitor) treatment decreased epidermal thickness in imquimod-induced psoriasis model. Taken together, our results suggest that mature IL-1α induced by hS100A7 is via RAGE-p38 MAPK and calpain-1 pathway in keratinocyte and this mechanism may play an important role during psoriasis. PMID:28060905

  7. 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.

  8. 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.

  9. Lipoxin A4 induces apoptosis of renal interstitial fibroblasts via calcium-dependent up-regulation of calpain 10 and Smac expressions

    Institute of Scientific and Technical Information of China (English)

    Shenghua Wu; Chao Lu; Ling Dong; Guoping Zhou; Ziqing Chen

    2005-01-01

    Objective: To examine whether lipoxin A4 (LXA4) induces apoptosis of renal interstitial fibroblasts and explore the mechanisms of signal pathway of LXA4. Methods: Rat renal interstitial fibroblasts (NRK-49F cells) were exposed to LXA4 at different concentrations. Prior to the experiment, the cells were transfected with Smac or calpain 10 antisense oligodeoxynucleotide (ODN), or treated with calcium channel inhibitor SK&F96365. Apoptosis of cells was recognized by double staining using acridine orange and ethidium bromide, observed in laser scanning confocal microscope, and counted by a flow cytometer. Caspase-3 activities were measured by colorimetric assay. The levels of free cytosolic calcium ([Ca2+ ]i) were analyzed in fura-2-loaded cells by laser scanning confocal microscopy. Expression of calpain 10 mRNA was determined by RT-PCR. Expressions of Smac protein and threonine phosphorylated Akt1 proteins at 308 site were determined by a Western blotting analysis. Activity of signal transducers and activators of transcription-3 (STAT3) was determined by electrophoretic mobility shift assay. Results: LXA4 at the concentrations of 0.1 and 1μmol/L induced 9.83% and 33.82% apoptosis of NRK-49F cells respectively, reduced at S and G2-M phase and increased the cells at G0-G1 phase in a dose-dependent manner. Treatment of the cells with LXA4 increased the expressions of calpain 10 and Smac, the levels of [Ca2+ ]i and activity of caspase-3. It also down-regulated the DNA-binding activity of STAT3 and expression of threonine phosphorylated Akt1. Transfection of the cells with calpain 10 antisense ODN inhibited the LXA4-induced apoptosis, activity of caspase-3 and expression of calpain 10, and ameliorated the decreased activity of STAT3. Transfection of the cells with Smac antisense ODN inhibited the LXA4-induced apoptosis, activity of caspase-3 and expression of Smac. Pretreatment of the cells with SK & F96365 inhibited the LXA4-induced apoptosis, levels of [Ca2+ ]i

  10. Vitamin D3 deficiency increases DNA damage and modify the expression of genes associated with hypertension in normotensive and hypertensive rats

    Directory of Open Access Journals (Sweden)

    Carla Silva Machado

    2015-05-01

    Full Text Available Vitamin D3 is a lipophilic micronutrient obtained from the diet (salmon, sardines, mackerel and cod liver oil or by the conversion of 7-dehydrocholesterol on skin after exposure to UVB radiation. This vitamin participates in several cellular processes, contributes to the maintenance of calcium concentrations, acts on phosphorus absorption, and is also related to the development and progression of chronic diseases. In hypertension, it is known that vitamin D3 act on renin-angiotensin-aldosterone system, regulates the gene expression and can induce or attenuate oxidative DNA damage. Vitamin D3 deficiency is present in 30-50% of human population (Pilz et al., 2009, and has been associated with increase of chromosomal instability and DNA damage (Nair-Shalliker; Armstrong; Fenech, 2012. Since experimental and clinical studies have suggested a relationship between vitamin D3 and blood pressure, the aim of this study was to evaluate whether vitamin D3 deficiency or supplementation lead to an increase or decrease in DNA damage, regulates the expression of genes associated with hypertension and changes the systolic blood pressure. Spontaneously hypertensive rats (SHR, used as a model of human essential hypertension, and their normotensive controls (Wistar Kyoto – WKY were fed a control diet (vitamin D3 at 1.000 UI/kg, a deficient diet (vitamin D3 at 0 UI/kg or a supplemented diet (vitamin D3 at 10.000 UI/kg for 12 weeks. DNA damage was assessed by comet assay in cardiac muscle tissue and blood tissue, following the methodology proposed by Singh et al. (1988 and Tice et al. (2000; gene expression of 84 genes was assessed by RT2ProfilerTM PCR Array in cardiac muscle tissue; and systolic blood pressure was measured weekly by a noninvasive method using tail plethysmography. In SHR and WKY rats, vitamin D3 deficiency increased DNA damage in the blood tissue and did not change the DNA damage in cardiac muscle tissue; vitamin D3 supplementation maintained the

  11. Calpain regulates thymidylate synthase-5-fluoro-dUMP complex levels associated with response to 5-fluorouracil in gastric cancer cells.

    Science.gov (United States)

    Nabeya, Yoshihiro; Suzuki, Takao; Furuya, Aki; Koide, Naoki; Ohkoshi, Motohiro; Takiguchi, Masaki; Ochiai, Takenori; Matsubara, Hisahiro; Hiwasa, Takaki

    2011-08-01

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

  12. 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.

  13. Selective cognitive deficits and reduced hippocampal brain-derived neurotrophic factor mRNA expression in small-conductance calcium-activated K+ channel deficient mice

    DEFF Research Database (Denmark)

    Jacobsen, J P R; Redrobe, J P; Hansen, H H;

    2009-01-01

    and the question is difficult to address pharmacologically due to the lack of subtype-selective SK-channel modulators. In this study, we used doxycycline-induced conditional SK3-deficient (T/T) mice to address the cognitive consequences of selective SK3 deficiency. In T/T mice SK3 protein is near-eliminated from...... the brain following doxycycline treatment. We tested T/T and wild type (WT) littermate mice in five distinct learning and memory paradigms. In Y-maze spontaneous alternations and five-trial inhibitory avoidance the performance of T/T mice was markedly inferior to WT mice. In contrast, T/T and WT mice...

  14. 动物钙蛋白酶系统基因与肉质嫩度关联研究进展%Research Progress in Calpain System Gene and Its Association with Meat Tenderness

    Institute of Scientific and Technical Information of China (English)

    马丽娜; 李颖康

    2011-01-01

    钙蛋白酶系统由钙激活中性蛋白酶I(μ-calpain,calpain l,CAPN1)、钙激活中性蛋白酶Ⅱ(m-calpain,calpain 2,CAPN2)及其内源性抑制剂钙蛋白酶抑制蛋白(calpastatin,CAST)3个部分组成,钙蛋白酶的活性会影响畜禽肌肉增长和肉的嫩度.对钙蛋白酶系统的特性、作用机理及其对肉质的影响进行了综述,并对其应用前景进行讨论.

  15. Altered spatiotemporal expression of collagen types I, III, IV, and VI in Lpar3-deficient peri-implantation mouse uterus.

    Science.gov (United States)

    Diao, Honglu; Aplin, John D; Xiao, Shuo; Chun, Jerold; Li, Zuguo; Chen, Shiyou; Ye, Xiaoqin

    2011-02-01

    Lpar3 is upregulated in the preimplantation uterus, and deletion of Lpar3 leads to delayed uterine receptivity in mice. Microarray analysis revealed that there was higher expression of Col3a1 and Col6a3 in the Preimplantation Day 3.5 Lpar3(-/-) uterus compared to Day 3.5 wild-type (WT) uterus. Since extracellular matrix (ECM) remodeling is indispensable during embryo implantation, and dynamic spatiotemporal alteration of specific collagen types is part of this process, this study aimed to characterize the expression of four main uterine collagen types: fibril-forming collagen (COL) I and COL III, basement membrane COL IV, and microfibrillar COL VI in the peri-implantation WT and Lpar3(-/-) uterus. An observed delay of COL III and COL VI clearance in the Lpar3(-/-) uterus may be associated with higher preimplantation expression of Col3a1 and Col6a3. There was also delayed clearance of COL I and delayed deposition of COL IV in the decidual zone in the Lpar3(-/-) uterus. These changes were different from the effects of 17beta-estradiol and progesterone on uterine collagen expression in ovariectomized WT uterus, indicating that the altered collagen expression in Lpar3(-/-) uterus is unlikely to be a result of alterations in ovarian hormones. Decreased expression of several genes encoding matrix-degrading metallo- and serine proteinases was observed in the Lpar3(-/-) uterus. These results demonstrate that pathways downstream of LPA3 are involved in the dynamic remodeling of ECM in the peri-implantation uterus.

  16. 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

  17. Th2 Regulation of Viral Myocarditis in Mice: Different Roles for TLR3 versus TRIF in Progression to Chronic Disease

    Directory of Open Access Journals (Sweden)

    Eric D. Abston

    2012-01-01

    Full Text Available Viral infections are able to induce autoimmune inflammation in the heart. Here, we investigated the role of virus-activated Toll-like receptor (TLR3 and its adaptor TRIF on the development of autoimmune coxsackievirus B3 (CVB3 myocarditis in mice. Although TLR3- or TRIF-deficient mice developed similarly worse acute CVB3 myocarditis and viral replication compared to control mice, disease was significantly worse in TRIF compared to TLR3-deficient mice. Interestingly, TLR3-deficient mice developed an interleukin (IL-4-dominant T helper (Th2 response during acute CVB3 myocarditis with elevated markers of alternative activation, while TRIF-deficient mice elevated the Th2-associated cytokine IL-33. Treatment of TLR3-deficient mice with recombinant IL-33 improved heart function indicating that elevated IL-33 in the context of a classic Th2-driven response protects against autoimmune heart disease. We show for the first time that TLR3 versus TRIF deficiency results in different Th2 responses that uniquely influence the progression to chronic myocarditis.

  18. Co-culture of C2C12 and 3T3-L1 preadipocyte cells alters the gene expression of calpains, caspases and heat shock proteins.

    Science.gov (United States)

    Pandurangan, Muthuraman; Jeong, Dawoon; Amna, Touseef; Van Ba, Hoa; Hwang, Inho

    2012-10-01

    The present study was carried out to understand the co-culture effect of C2C12 and 3T3-L1 preadipocyte cells on calpain, caspase, and heat shock protein (Hsp) systems. Calpains, caspases, and heat shock proteins play critical roles in the growth and development of mammalian cells. Cells were co-cultured using transwell inserts with a 0.4-μm porous membrane to separate C2C12 and 3T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates and inserts containing C2C12 transferred to 3T3-L1 plates. Following co-culture for 24 and 48 h, the cells in the lower well were harvested for analysis. Calpains include μ-calpain, m-calpain, and their specific inhibitor calpastatin. The expression pattern of μ-calpain did not change in the co-cultured C2C12 and 3T3-L1 cells, whereas m-capain mRNA expression significantly reduced in the 48-h co-cultured 3T3-L1 cells. Calpastatin mRNA expression significantly increased in the 48-h co-cultured C2C12 cells. Caspase-7 mRNA expression did not change in the 24- and 48-h co-cultured C2C12 and 3T3-L1 cells. Caspase-3 mRNA expression significantly reduced in the 24- and 48-h co-cultured 3T3-L1 cells; caspase-9 mRNA had a significant reduction only at 48 h, whereas caspase-9 mRNA expression significantly increased in the 48-h co-cultured C2C12 cells. Hsp27 and Hsp90 mRNA expressions are significantly reduced in the 24- and 48-h co-cultured C2C12 and 3T3-L1 cells, whereas Hsp70 mRNA expression significantly increased in the 48-h co-cultured 3T3-L1 cells. The co-culture reflects three-dimensional views of C2C12 and 3T3-L1 cell types as in vivo, which is quite distinct from the one-dimensional monocultured C2C12 and 3T3-L1 cells.

  19. Calpain Inhibition Reduces Amplitude and Accelerates Decay of the Late Sodium Current in Ventricular Myocytes from Dogs with Chronic Heart Failure

    Science.gov (United States)

    Undrovinas, Albertas; Maltsev, Victor A.; Sabbah, Hani N.

    2013-01-01

    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 (1μM) 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. Conclusions Calpain inhibition reverses INaL changes in failing dog ventricular cardiomyocytes in the

  20. Calpain and reactive oxygen species targets Bax for mitochondrial permeabilisation and caspase activation in zerumbone induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Praveen K Sobhan

    Full Text Available Fluorescent protein based signaling probes are emerging as valuable tools to study cell signaling because of their ability to provide spatio- temporal information in non invasive live cell mode. Previously, multiple fluorescent protein probes were employed to characterize key events of apoptosis in diverse experimental systems. We have employed a live cell image based approach to visualize the key events of apoptosis signaling induced by zerumbone, the active principle from ginger Zingiber zerumbet, in cancer cells that enabled us to analyze prominent apoptotic changes in a hierarchical manner with temporal resolution. Our studies substantiate that mitochondrial permeabilisation and cytochrome c dependent caspase activation dominate in zerumbone induced cell death. Bax activation, the essential and early event of cell death, is independently activated by reactive oxygen species as well as calpains. Zerumbone failed to induce apoptosis or mitochondrial permeabilisation in Bax knockout cells and over-expression of Bax enhanced cell death induced by zerumbone confirming the essential role of Bax for mitochondrial permeabilsation. Simultaneous inhibition of reactive oxygen species and calpain is required for preventing Bax activation and cell death. However, apoptosis induced by zerumbone was prevented in Bcl 2 and Bcl-XL over-expressing cells, whereas more protection was afforded by Bcl 2 specifically targeted to endoplasmic reticulum. Even though zerumbone treatment down-regulated survival proteins such as XIAP, Survivin and Akt, it failed to affect the pro-apoptotic proteins such as PUMA and BIM. Multiple normal diploid cell lines were employed to address cytotoxic activity of zerumbone and, in general, mammary epithelial cells, endothelial progenitor cells and smooth muscle cells were relatively resistant to zerumbone induced cell death with lesser ROS accumulation than cancer cells.

  1. 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.

  2. THE PARKINSONIAN NEUROTOXIN ROTENONE ACTIVATES CALPAIN AND CASPASE-3 LEADING TO MOTONEURON DEGENERATION IN SPINAL CORD OF LEWIS RATS

    Science.gov (United States)

    SAMANTARAY, S.; KNARYAN, V. H.; GUYTON, M. K.; MATZELLE, D. D.; RAY, S. K.; BANIK, N. L.

    2007-01-01

    Exposure to environmental toxins increases the risk of neurodegenerative diseases including Parkinson’s disease (PD). Rotenone is a neurotoxin that has been used to induce experimental parkinsonism in rats. We used the rotenone model of experimental parkinsonism to explore a novel aspect of extra-nigral degeneration, the neurodegeneration of spinal cord (SC), in PD. Rotenone administration to male Lewis rats caused significant neuronal cell death in cervical and lumbar SC as compared to control animals. Dying neurons were motoneurons as identified by double immunofluorescent labeling for TUNEL+ cells and ChAT-immunoreactivity. Neuronal death was accompanied by abundant astrogliosis and microgliosis as evidenced from GFAP-immunoreactivity and OX-42-immunoreactivity, respectively, implicating an inflammatory component during neurodegeneration in SC. However, the integrity of the white matter in SC was not affected by rotenone administration as evidenced from the non co-localization of any TUNEL+ cells with GFAP-immunoreactivity and MBP-immunoreactivity, the selective markers for astrocytes and oligodendrocytes, respectively. Increased activities of 76 kD active m-calpain and 17/19 kD active caspase-3 further demonstrated involvement of these enzymes in cell death in SC. The finding of ChAT+ cell death also suggested degeneration of SC motoneurons in rotenone-induced experimental parkinsonism. Thus, this is the first report of its kind in which the selective vulnerability of a putative parkinsonian target outside of nigrostriatal system has been tested using an environmental toxin to understand the pathophysiology of PD. Moreover, rotenone-induced degeneration of SC motoneuron in this model of experimental parkinsonism progressed with upregulation of calpain and caspase-3. PMID:17367952

  3. Calpain-Mediated Positional Information Directs Cell Wall Orientation to Sustain Plant Stem Cell Activity, Growth and Development.

    Science.gov (United States)

    Liang, Zhe; Brown, Roy C; Fletcher, Jennifer C; Opsahl-Sorteberg, Hilde-Gunn

    2015-09-01

    Eukaryotic development and stem cell control depend on the integration of cell positional sensing with cell cycle control and cell wall positioning, yet few factors that directly link these events are known. The DEFECTIVE KERNEL1 (DEK1) gene encoding the unique plant calpain protein is fundamental for development and growth, being essential to confer and maintain epidermal cell identity that allows development beyond the globular embryo stage. We show that DEK1 expression is highest in the actively dividing cells of seeds, meristems and vasculature. We further show that eliminating Arabidopsis DEK1 function leads to changes in developmental cues from the first zygotic division onward, altered microtubule patterns and misshapen cells, resulting in early embryo abortion. Expression of the embryonic marker genes WOX2, ATML1, PIN4, WUS and STM, related to axis organization, cell identity and meristem functions, is also altered in dek1 embryos. By monitoring cell layer-specific DEK1 down-regulation, we show that L1- and 35S-induced down-regulation mainly affects stem cell functions, causing severe shoot apical meristem phenotypes. These results are consistent with a requirement for DEK1 to direct layer-specific cellular activities and set downstream developmental cues. Our data suggest that DEK1 may anchor cell wall positions and control cell division and differentiation, thereby balancing the plant's requirement to maintain totipotent stem cell reservoirs while simultaneously directing growth and organ formation. A role for DEK1 in regulating microtubule-orchestrated cell wall orientation during cell division can explain its effects on embryonic development, and suggests a more general function for calpains in microtubule organization in eukaryotic cells.

  4. Glucose deprivation induces reticulum stress by the PERK pathway and caspase-7- and calpain-mediated caspase-12 activation.

    Science.gov (United States)

    de la Cadena, Selene García; Hernández-Fonseca, Karla; Camacho-Arroyo, Ignacio; Massieu, Lourdes

    2014-03-01

    Glucose is the main energy source in brain and it is critical for correct brain functioning. Type 1 diabetic patients might suffer from severe hypoglycemia if exceeding insulin administration, which can lead to acute brain injury if not opportunely corrected. The mechanisms leading to hypoglycemic brain damage are not completely understood and the role of endoplasmic reticulum (ER) stress has not been studied. ER stress resulting from the accumulation of unfolded or misfolded proteins in the ER is counteracted by the unfolded protein response (UPR). When the UPR is sustained, apoptotic death might take place. We have examined UPR activation during glucose deprivation (GD) in hippocampal cultured neurons and its role in the induction of apoptosis. Activation of the PERK pathway of the UPR was observed, as increased phosphorylation of eIF2α and elevated levels of the transcription factor ATF4, occurred 30 min after GD and the levels of the chaperone protein, GRP78 and the transcription factor CHOP, increased after 2 h of GD. In addition, we observed an early activation of caspase-7 and 12 during GD, while caspase-3 activity increased only transiently during glucose reintroduction. Inhibition of caspase-3/7 and the calcium-dependent protease, calpain, significantly decreased caspase-12 activity. The ER stress inhibitor, salubrinal prevented neuronal death and caspase-12 activity. Results suggest that the PERK pathway of the UPR is involved in GD-induced apoptotic neuronal death through the activation of caspase-12, rather than the mitochondrial-dependent caspase pathway. In addition, we show that calpain and caspase-7 are soon activated after GD and mediate caspase-12 activation and neuronal death.

  5. MET receptor variant R970C favors calpain-dependent generation of a fragment promoting epithelial cell scattering.

    Science.gov (United States)

    Montagne, Rémi; Baranzelli, Anne; Muharram, Ghaffar; Catherine, Leroy; Lesaffre, Marie; Vinchent, Audrey; Kherrouche, Zoulika; Werkmeister, Elisabeth; Cortot, Alexis B; Tulasne, David

    2017-01-04

    The receptor tyrosine kinase MET and its ligand, the hepatocyte growth factor, are essential to embryonic development, whereas deregulation of MET signaling is associated with tumorigenesis leading to various cancers, including lung carcinoma. Mutations in the MET kinase domain lead to constitutive kinase activity and are associated with tumorigenesis. In lung cancer, however, some mutations are found in the juxtamembrane domain, and their functional consequences are unknown. Because the juxtamembrane domain of MET is targeted by several proteolytic cleavages, involved in its degradation during cell death or under steady-state conditions, we evaluated the influence of these mutations on the MET proteolytic cleavages. In stably transfected epithelial cells expressing MET, the juxtamembrane mutations R970C, P991S, and T992I were found not to modify the known caspase or presenilin-dependent regulated intramembrane proteolysis. Yet when overexpressed, the R970C variant caused generation of an as yet undescribed 45-kDa fragment (p45 MET). This fragment was found in the confluent lung cancer cell line NCI-H1437 carrying the R970C mutation and at a lesser extent in cell lines expressing WT MET, suggesting that R970C mutation favors this cleavage. Generation of p45 MET required the activity of the calpain proteases, confirming the involvement of proteolysis. Ectopic expression of reconstituted p45 MET in epithelial cell lines favored cell scattering and invasion indicating active role of this fragment in HGF/SF induced responses. Hence, although the juxtamembrane mutations of MET do not affect its known proteolytic cleavages, the R970C MET variant favors calpain dependent proteolytic cleavage in lung cancer cells.

  6. Effect of leptin on expression of calpain-1 and Bcl-2 and apoptosis in myocardial tissue of neonatal rats after asphyxia%瘦素对窒息新生大鼠心肌组织calpain-1和Bcl-2的表达及细胞凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    吴丹丹; 吴星恒; 张丽娜

    2016-01-01

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

  7. Time course changes of calpain activity following FPI to rats' neurons and the effect of mild hypothermia on calpain activity%大鼠神经元液压伤后依钙蛋白酶的时程变化及亚低温的影响

    Institute of Scientific and Technical Information of China (English)

    隋立森; 刘恩重; 姜晓丹; 徐如祥; 韩富; 张相彤; 戴钦舜

    2007-01-01

    目的 研究常温时液压冲击伤后神经元内依钙蛋白酶活性变化及亚低温的干预效果. 方法 体外培养大鼠神经元并制作液压冲击伤模型,在不同时间点进行亚低温干预,以紫外分光光度法检测液压冲击伤后的神经元内Calpain的活性变化及在不同时段亚低温对Calpain活性的干预效应. 结果 常温时,在细胞损伤后Calpain的活性发生显著变化.亚低温组与常温组Calpain的活性不同,且亚低温对伤后Calpain活性的干预效应与作用起始时间密切相关. 结论 液压冲击伤后神经元内Calpain活性发生的改变参与细胞创伤的病理过程,亚低温可能通过这一机制具有一定保护创伤性脑损伤的作用.亚低温干预的效果与开始干预时间相关.%Objective To investigate time course changes of intraneuronal calpain activity following fluid percussion injury(FPI)under normothermia(37℃)and mild hypothermia(32+0.5)℃.Methods Rats' neurons produced by external culture and FPI models prepared, mild hypothermia was applied for intervention at different time points and the changes in intraneuronal calpain activity following FPI and the intervention effect of mild hypothermia on calpain activity during different time courses were detected by UV-spectrophotometry. Results Remarkable changes occurred to calpain activity following FPI at 37℃. While the intervention effect of mild hypothermia on calpain activity was different, closely related to the starting time point of intervention. Conclusion The changes of intraneuronal calpain activity following FPI join the pathological process of cellular trauma; mild hypothermia might protect patients with traumatic brain injury(TBI)to some extent through the regulation of calpain activity. And, the intervention effect of mild hypothermia has some correlation with the starting time point of intervention.

  8. 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.

  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....... This was done by investigating the effect of the irreversible cysteine protease inhibitor zFA-fmk, the cathepsin B inhibitor CA-074-Me and the calpain inhibitor ALLN on the viability of UVB-irradiated human keratinocytes and HeLa cells. At concentrations of 10 microM and above zVAD-fmk conferred partial dose...

  10. 高钙日粮对农安籽鹅不同部位肌肉μ-Calpain mRNA表达量的影响%Effects of high dietary calcium on mRNA muscular expression of μ-calpain at different parts of geese

    Institute of Scientific and Technical Information of China (English)

    王剑; 孙会

    2011-01-01

    选用8周龄农安籽鹅120只,随机分成4组,每组3个重复,每个重复10只.其中对照组饲喂基础日粮(Ca 0.6%),其余为试验1至3组,钙含量分别为0.9%、1.2%和1.5%.预饲期为7 d,试验期为28 d.分别于试验期的第7、14、21、28 d进行屠宰.试验结果表明:日粮中添加不同水平的丙酸钙对鹅胸肌和腿肌μ-Calpain mRNA表达量无显著影响(P>0.05).不同屠宰时间对胸肌μ-Calpain mRNA表达量影响差异极显著(P<0.01),而对腿肌μ-Calpain mRNA表达量无显著影响(P>0.05).在不同屠宰时间和添加不同钙水平的条件下,胸肌的μ-Calpain mRNA表达量均高于腿肌μ-Calpain mRNA表达量.

  11. Calpain- and caspase-mediated alphaII-spectrin and tau proteolysis in rat cerebrocortical neuronal cultures after ecstasy or methamphetamine exposure.

    Science.gov (United States)

    Warren, Matthew W; Zheng, Wenrong; Kobeissy, Firas H; Cheng Liu, Ming; Hayes, Ronald L; Gold, Mark S; Larner, Stephen F; Wang, Kevin K W

    2007-08-01

    Abuse of 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) and methamphetamine (Meth or Speed) is a growing international problem with an estimated 250 million users of psychoactive drugs worldwide. It is important to demonstrate and understand the mechanism of neurotoxicity so potential prevention and treatment therapies can be designed. In this study rat primary cerebrocortical neuron cultures were challenged with MDMA and Meth (1 or 2 mM) for 24 and 48 h and compared to the excitotoxin N-methyl-D-aspartate (NMDA). The neurotoxicity of these drugs, as assessed by microscopy, lactate dehydrogenase release and immunoblot, was shown to be both dose- and time-dependent. Immunoblot analysis using biomarkers of cell death showed significant proteolysis of both alphaII-spectrin and tau proteins. Breakdown products of alphaII-spectrin (SBDPs) of 150, 145, and 120 kDa and tau breakdown products (TBDPs) of 45, 32, 26, and 14 kDa were observed. The use of the protease inhibitors calpain inhibitor SJA6017 and caspase inhibitors z-VAD-fmk and Z-D-DCB, attenuated drug-induced alphaII-spectrin and tau proteolysis. The calpain inhibitor reduced the calpain-induced breakdown products SBDP145 and TBDP14, but there was an offset increase in the caspase-mediated breakdown products SBDP120 and TBDP45. The caspase inhibitors, on the other hand, decreased SBDP120 and TBDP45. These data suggest that both MDMA and Meth trigger concerted proteolytic attacks of the structural proteins by both calpain and caspase family of proteases. The ability of the protease inhibitors to reduce the damage caused by these drugs suggests that the treatment arsenal could include similar drugs as possible tools to combat the drug-induced neurotoxicity in vivo.

  12. 风湿性心房颤动患者左心房组织中钙蛋白酶-2的表达%Expression of calpain-2 in human left atrium in rheumatic atrial fibrillation

    Institute of Scientific and Technical Information of China (English)

    韩波; 王建春; 张涛; 朱小龙; 李丛; 王正军; 赵勇; 邹承伟

    2014-01-01

    目的:观察风湿性心房颤动(AF)患者左心房中钙蛋白酶-2(calpain-2)的表达,探讨其在AF发病机制中的作用。方法选取外科换瓣手术的风湿性心脏病患者39例,分为窦性心律组( n=16例)和心房颤动组( n=23例)。应用RT-PCR技术和免疫组化LSAB法半定量检测calpain-2的表达。结果心房颤动组calpain-2 mRNA的deltaCt值高于窦性心律组[(0.091±0.035) vs (0.066±0.022),P=0.017];两组左心房心肌细胞中均有棕黄色颗粒沉着,心外膜和心内膜细胞均未见棕黄色颗粒;心房颤动组中calpain-2表达含量明显高于窦性心律组( P<0.001),两者平均光密度值分别为0.92±0.17和0.65±0.01;calpain-2的含量与左心房收缩末期直径呈正相关(r=0.929, P<0.001)。结论 calpain-2在人类左心房心肌细胞中表达;AF时calpain-2的表达明显增加。%Objective To observe the expression of calpain-2 in the left atrum of patients with rheumatic atrial fibrilla-tion, and to explore its roles in the pathogenesis of atrial fibrillation.Methods A total of 39 patients with rheumatic heart disease who received surgical valve replacement were divided into two groups: the atrial fibrillation group ( n=23) and sinus rhythm group (n=16).The expression of calpain-2 was detected with LSAB immunohistochemical and the Real-time PCR technology.Results The deltaCt of calpain-2 mRNA of the atrial fibrillation group ( 0.091 ± 0.035) was higher than that of the sinus rhythm group (0.066 ±0.022, P=0.017).Brown particle deposits were ob-served in the left atrial myocytes in both groups, but the epicardial and endocardial cells showed no brown particles.The average optical density values of calpain-2 of the atrial fibrillation group and sinus rhythm group were (0.92 ±0.17) and (0.65 ±0.01)(P<0.001).Calpain-2 levels and left atrial systolic diameter were correlated (r=0.929, P<0.01).Conclusion

  13. Calpain 1 regulates TGF-β1-induced epithelial-mesenchymal transition in human lung epithelial cells via PI3K/Akt signaling pathway

    Science.gov (United States)

    Tan, Wei-Jun; Tan, Qiu-Yue; Wang, Ting; Lian, Min; Zhang, Li; Cheng, Zhen-Shun

    2017-01-01

    Cell proliferation, transformation, and epithelial-mesenchymal transition (EMT) are key processes involved in the development of idiopathic pulmonary fibrosis (IPF). This study investigated the regulatory factors and signaling pathways that mediate EMT in the human type II alveolar epithelial A549 cell line. A549 cells were cultured in RPMI-1640 medium and allocated to the following four groups: blank control group or treated with transforming growth factor-β1 (TGF-β1), TGF-β1 + PD 150606 (a calpain 1 inhibitor), or PD 150606. We examined E-cadherin (E-cad), α-smooth muscle actin (α-SMA), and calpain 1 mRNA transcript and protein expression levels in these four groups by performing RT-PCR and western blot analyses. The results indicated that TGF-β1 treatment significantly downregulated E-cad and upregulated α-SMA expression compared with that of the blank control group (Pcells. However, TGF-β1-induced ETM was not correlated with the ERK and JNK signaling pathways. These combined results indicate that calpain 1 could regulate EMT in TGF-β1-treated A549 epithelial cells via the PI3K/Akt signaling pathway.

  14. 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.

  15. Cloning and Expression of Caipain-like Gene in Toxoplasma gondii%弓形虫依钙蛋白激酶Calpain-like基因的克隆及表达

    Institute of Scientific and Technical Information of China (English)

    陈雪艳; 付玉才; 李璐; 耿艺介; 黄达娜; 高世同; 张仁利

    2008-01-01

    目的 克隆弓形虫RH株Calpain-like基因片段,构建原核表达载体,诱导表达Calpain-like基因重组蛋白.方法 收集、纯化弓形虫RH株速殖子,提取总RNA,在设计合成的引物中引入Sal I和EcoR I酶切位点.应用RT-PCR扩增弓形虫RH株Calpain-like基因片段,插入pGEM-T载体,提取重组质粒,双酶切获得目的基因,亚克隆到原核表达质粒pET32a,重组子经双酶切、PCR和DNA序列分析鉴定,转化大肠杆菌BL21并以IPTG诱导表达.结果 从弓形虫RH株速殖子cDNA中扩增出316 bp的Calpain-like基因片段;含pET32a/Calpain-like的重组质粒在宿主菌经诱导后,获得与预期分子量相符的表达产物.结论 成功地克隆和表达弓形虫RH株Calpain-like基因,弓形虫Calpain-like基因的克隆表达为进一步筛选弓形虫疫苗候选分子和治疗药物的靶位提供了研究基础.

  16. Amiloride slows down calpain-mediated ABCA1 degradation through in-hibition of hypoxia-induced NHE1 expression%Amiloride 通过抑制缺氧诱导的 NHE1表达而延缓 calpain 介导的 ABCA1降解

    Institute of Scientific and Technical Information of China (English)

    莫显刚; 张莉; 张洛超; 王龙; 向凝; 杨涓; 宋翔

    2015-01-01

    AIM:To examine the effects of hypoxia on sodium-hydrogen exchange 1 (NHE1) expression, in-tracellular Ca2+concentration ( [ Ca2+] i ) and calpain activity, and to explore the effect of amiloride on adenosine triphos-phate-binding cassette transporter A1 (ABCA1) degradation and its calpain-related mechanism.METHODS:RAW264.7 cells were exposed to hypoxia for 0 h, 12 h, 24 h and 48 h.The cell viability was measured by MTT assay and the expres-sion of NHE1 at mRNA and protein levels was detected by real-time PCR and Western blot.[ Ca2+] i was analyzed by flow cytometry.Calpain activity was assessed by the method of Suc-LLVY-aminoluciferin.Furthermore, the protein levels of ABCA1 in the RAW264.7 cells exposed to hypoxia for 24 h were determined after 6 h or 12 h treatment with NHE1 inhibi-tor amiloride in the presence of cycloheximide.ABCA1 protein levels and calpain activity were detected after 12 h incuba-tion with calpain inhibitor ALLN or intracellular calcium-chelating agent BAPTA.RESULTS: Hypoxia inhibited the cell viability in a time-dependent manner.Hypoxia up-regulated the mRNA and protein expression of NHE1, and increased [ Ca2+] i and calpain activity.Hypoxia increased the degradation of ABCA1 and amiloride slowed down the ABCA1 degra-dation.ALLN or BAPTA increased ABCA1 protein level and decreased calpain activity.CONCLUSION:NHE1 inhibitor amiloride attenuates the calpain-mediated degradation of ABCA1, indicating that hypoxia-induced NHE1 might, at least in part, participate in the ABCA1 degradation.%目的:研究缺氧对钠氢交换体1(NHE1)表达、细胞内钙离子浓度([Ca2+]i)和钙蛋白酶(calpain)活性的影响,探讨NHE1抑制剂阿米洛利( amiloride )对 ABCA1降解的影响以及与calpain 相关的机制。方法:RAW264.7细胞缺氧0、12、24和48 h。 MTT法检测细胞活力,real-time PCR及Western blot检测NHE1的表达。流式细胞术检测[ Ca2+] i ,荧光素法检测细胞

  17. 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

  18. Dismantling the autophagic arsenal when it is time to die: concerted AMBRA1 degradation by caspases and calpains.

    Science.gov (United States)

    Corazzari, Marco; Fimia, Gian Maria; Piacentini, Mauro

    2012-08-01

    Under stress conditions cells activate different response pathways which result in cell survival or apoptosis depending on: (1) the nature of the insults, (2) the type, if acute or chronic stress, and (3) how long the stress persists. Generally, autophagy is induced early to sustain cell survival and inhibit cell death. However, adverse conditions are able to overcome autophagy to promote cell death. Increasing evidence suggests that the inhibition of autophagy by the apoptotic machinery has been proposed as one of the crucial events responsible for the irreversible switch from survival to death. The mechanism seems to be related to the selective apoptotic protease-mediated degradation of key autophagic proteins. We recently found that AMBRA1, an important regulator of the autophagic process mediating the initial steps of autophagosome formation, is also irreversibly degraded by the combined activity of caspases and calpains. This phenomenon is not merely a consequence of apoptosis execution but represents a key step required to efficiently promote the autophagic vs apoptosis switch.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Complement deficiency promotes cutaneous wound healing in mice.

    Science.gov (United States)

    Rafail, Stavros; Kourtzelis, Ioannis; Foukas, Periklis G; Markiewski, Maciej M; DeAngelis, Robert A; Guariento, Mara; Ricklin, Daniel; Grice, Elizabeth A; Lambris, John D

    2015-02-01

    Wound healing is a complex homeostatic response to injury that engages numerous cellular activities, processes, and cell-to-cell interactions. The complement system, an intricate network of proteins with important roles in immune surveillance and homeostasis, has been implicated in many physiological processes; however, its role in wound healing remains largely unexplored. In this study, we employ a murine model of excisional cutaneous wound healing and show that C3(-/-) mice exhibit accelerated early stages of wound healing. Reconstitution of C3(-/-) mice with serum from C3(+/+) mice or purified human C3 abrogated the accelerated wound-healing phenotype. Wound histology of C3(-/-) mice revealed a reduction in inflammatory infiltrate compared with C3(+/+) mice. C3 deficiency also resulted in increased accumulation of mast cells and advanced angiogenesis. We further show that mice deficient in the downstream complement effector C5 exhibit a similar wound-healing phenotype, which is recapitulated in C5aR1(-/-) mice, but not C3aR(-/-) or C5aR2(-/-) mice. Taken together, these data suggest that C5a signaling through C5aR may in part play a pivotal role in recruitment and activation of inflammatory cells to the wound environment, which in turn could delay the early stages of cutaneous wound healing. These findings also suggest a previously underappreciated role for complement in wound healing, and may have therapeutic implications for conditions of delayed wound healing.

  1. 钙蛋白酶介导模拟失重大鼠心肌肌钙蛋白抑制亚基的降解%Calpain mediates cardiac troponin Ⅰ degradation in tail-suspended rats

    Institute of Scientific and Technical Information of China (English)

    徐彭涛; 宋振; 李全; 张琳; 王云英; 余志斌

    2010-01-01

    本文旨在观察尾部悬吊模拟失重大鼠心肌钙蛋白酶(calpain)与钙蛋白酶抑素(calpastatin)表达的变化,以探讨心肌肌钙蛋白抑制业基(cardiac troponin Ⅰ,cTnⅠ)降解的可能机制.采用尾部悬吊模拟失重大鼠模型,Western blotting技术观测心肌calpain-1、calpain-2与calpastatin的表达;PD150606抑制calpain活性,分析cTnⅠ降解程度的变化.结果显示:与同步对照组相比,悬吊2周与4周组大鼠心肌calpastatin表达呈显著性降低(P<0.05),calpain-1表达未改变,calpain-2表达略有降低:但是,心肌calpain-1/calpastatin及calpain-2/calpastatin的比值在悬吊2周与4周组明显增高(P<0.05,P<0.01).悬吊4周组cTnⅠ降解显著高于对照组(P<0.01);然而,用calpain非特异件抑制剂PD150606处理后,对照组及悬吊组cTnⅠ的降解均被显著抑制(P<0.01).这些结果提示模拟失重大鼠心肌calpain活性增高可能增加cTnⅠ的降解.

  2. 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.

  3. Tissue inhibitor of metalloproteinase-3 knockout mice exhibit enhanced energy expenditure through thermogenesis.

    Directory of Open Access Journals (Sweden)

    Yohsuke Hanaoka

    Full Text Available Tissue inhibitors of metalloproteinases (TIMPs regulate matrix metalloproteinase activity and maintain extracellular matrix homeostasis. Although TIMP-3 has multiple functions (e.g., apoptosis, inhibition of VEGF binding to VEGF receptor, and inhibition of TNFα converting enzyme, its roles in thermogenesis and metabolism, which influence energy expenditure and can lead to the development of metabolic disorders when dysregulated, are poorly understood. This study aimed to determine whether TIMP-3 is implicated in metabolism by analyzing TIMP-3 knockout (KO mice. TIMP-3 KO mice had higher body temperature, oxygen consumption, and carbon dioxide production than wild-type (WT mice, although there were no differences in food intake and locomotor activity. These results suggest that metabolism is enhanced in TIMP-3 KO mice. Real-time PCR analysis showed that the expression of PPAR-δ, UCP-2, NRF-1 and NRF-2 in soleus muscle, and PGC-1α and UCP-2 in gastrocnemius muscle, was higher in TIMP-3 KO mice than in WT mice, suggesting that TIMP-3 deficiency may increase mitochondrial activity. When exposed to cold for 8 hours to induce thermogenesis, TIMP-3 KO mice had a higher body temperature than WT mice. In the treadmill test, oxygen consumption and carbon dioxide production were higher in TIMP-3 KO mice both before and after starting exercise, and the difference was more pronounced after starting exercise. Our findings suggest that TIMP-3 KO mice exhibit enhanced metabolism, as reflected by a higher body temperature than WT mice, possibly due to increased mitochondrial activity. Given that TIMP-3 deficiency increases energy expenditure, TIMP-3 may present a novel therapeutic target for preventing metabolic disorders.

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

    Directory of Open Access Journals (Sweden)

    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

  5. Hippocampal neurons exposed to the environmental contaminants methylmercury and polychlorinated biphenyls undergo cell death via parallel activation of calpains and lysosomal proteases.

    Science.gov (United States)

    Tofighi, Roshan; Johansson, Carolina; Goldoni, Matteo; Ibrahim, Wan Norhamidah Wan; Gogvadze, Vladimir; Mutti, Antonio; Ceccatelli, Sandra

    2011-01-01

    Methylmercury (MeHg) and polychlorinated biphenyls (PCBs) are widespread environmental pollutants commonly found as contaminants in the same food sources. Even though their neurotoxic effects are established, the mechanisms of action are not fully understood. In the present study, we have used the mouse hippocampal neuronal cell line HT22 to investigate the mechanisms of neuronal death induced by MeHg, PCB 153, and PCB 126, alone or in combination. All chemicals induced cell death with morphological changes compatible with either apoptosis or necrosis. Mitochondrial functions were impaired as shown by the significant decrease in mitochondrial Ca²+ uptake capacity and ATP levels. MeHg, but not the PCBs, induced loss of mitochondrial membrane potential and release of cytochrome c into the cytosol. Also, pre-treatment with the antioxidant MnTBAP was protective only against cell death induced by MeHg. While caspase activation was absent, the Ca²+-dependent proteases calpains were activated after exposure to MeHg or the selected PCBs. Furthermore, lysosomal disruption was observed in the exposed cells. Accordingly, pre-treatment with the calpain specific inhibitor PD150606 and/or the cathepsin D inhibitor Pepstatin protected against the cytotoxicity of MeHg and PCBs, and the protection was significantly enhanced when the two inhibitors were combined. Simultaneous exposures to lower doses of MeHg and PCBs suggested mostly antagonistic interactions. Taken together, these data indicate that MeHg and PCBs induce caspase-independent cell death via parallel activation of calpains and lysosomal proteases, and that in this model oxidative stress does not play a major role in PCB toxicity.

  6. Location and characteristic of Calpain,candidate molecule of Schistosoma japonicumvaccine%日本血吸虫疫苗候选分子Calpain的原位表达

    Institute of Scientific and Technical Information of China (English)

    段立华; 张仁利; 吴少庭; 高世同; 林敏

    2002-01-01

    目的探讨日本血吸虫疫苗候选分子--钙离子激活的中性蛋白激酶(Calpain)在日本血吸虫体内的表达部位及其特征.方法用重组纯化的Calpain抗原免疫大鼠,制备抗Calpain血清,从感染鼠灌流分离的日本血吸虫成虫被切片,用羊抗大鼠IgG抗体作为二抗进行免疫组织化学分析,观察Calpain蛋白在日本血吸虫成虫体内的表达部位.结果重组Calpain抗原免疫大鼠后,产生了一个极高的抗Calpain特异性抗体,并在日本血吸虫成虫切片上识别自然的日本血吸虫Calpain,且大多数表达在成虫真皮层.结论日本血吸虫钙离子激活的中性蛋白激酶(Calpain)主要分布在成虫肌肉和真皮层,提示Calpain可能是日本血吸虫的一个疫苗候选分子,并提示这个分子的重组抗原有可能应用于血吸虫病诊断.

  7. Persistent oxygen-glucose deprivation induces astrocytic death through two different pathways and calpain-mediated proteolysis of cytoskeletal proteins during astrocytic oncosis.

    Science.gov (United States)

    Cao, Xu; Zhang, Ying; Zou, Liangyu; Xiao, Haibing; Chu, Yinghao; Chu, Xiaofan

    2010-07-26

    Astrocytes are thought to play a role in the maintenance of homeostasis and the provision of metabolic substrates for neurons as well as the coupling of cerebral blood flow to neuronal activity. Accordingly, astrocytic death due to various types of injury can critically influence neuronal survival. The exact pathway of cell death after brain ischemia is under debate. In the present study, we used astrocytes from rat primary culture treated with persistent oxygen-glucose-deprivation (OGD) as a model of ischemia to examine the pathway of cell death and the relevant mechanisms. We observed changes in the cellular morphology, the energy metabolism of astrocytes, and the percentage of apoptosis or oncosis of the astrocytes induced by OGD. Electron microscopy revealed the co-existence of ultrastructural features in both apoptosis and oncosis in individual cells. The cellular ATP content was gradually decreased and the percentages of apoptotic and oncotic cells were increased during OGD. After 4h of OGD, ATP depletion to less than 35% of the control was observed, and oncosis became the primary pathway for astrocytic death. Increased plasma membrane permeability due to oncosis was associated with increased calpain-mediated degradation of several cytoskeletal proteins, including paxillin, vinculin, vimentin and GFAP. Pre-treatment with the calpain inhibitor 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) could delay the OGD-induced astrocytic oncosis. These results suggest that there is a narrow range of ATP that determines astrocytic oncotic death induced by persistent OGD and that calpain-mediated hydrolysis of the cytoskeletal-associated proteins may contribute to astrocytes oncosis.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. Calpain-1在缺氧缺血性脑损伤新生大鼠心肌中的表达及意义%Expressions of calpain-1mRNA and protein in myocardium of rats with hypoxic-ischemic brain damage

    Institute of Scientific and Technical Information of China (English)

    赵红; 徐梅; 初桂兰

    2011-01-01

    Objective: To detect the expressions of calpain-lmRNA and protein in myocardium of rats with hypoxic-ischemic brain damage (HIBD).Methods: HIBD model was made by modified rice of 7-day-old Wistar rats, 64 rats were randomly divided into eight groups: control group and groups with 2 h、 12 h、24 h、2 d、3 d、5 d and 7 d of exposure to HIBD.Apoptosis index (AI) was detected by TUNEL.RT-PCR and Western blot were applied to detect the gene and protein expressions of calpain-1.Results:Apoptosis cells were found sporadically in control group and significantly higher in HIBD groups (P<0.01).AI was increased at 2 h and reached peak at 3 d,and it was decreased at 7 d but still more than that in control group (P<0.01).The expression of Calpain-1 mRNA began to increase at 12 h after HIBD and reached peak at 2 d (0.5400±0.0848, P<0.01), and then decreased but remained in a higher level than that of the con trol group until 5 d after HIBD (P<0.01); calpain-1 protein activation began to increase at 2 h after HIBD(P<0.05) and reached peak at 3 d(P<0.001), and it was still in higher level at 5 d (P<0.001) and same as the control at 7 d (P>0.05).The expressions of Calpain-1 mRNA and protein activation were positively correlated with AI (r=0.786,P<0.01; r=0.853,P<0.01).Conclusion: Apoptosis cells and the expressions of calpain-1 are increased in myocardium of neonatal rats with HIBD, showing positive correlation with AI.%目的:了解缺氧缺血性脑损伤(HIBD)新生大鼠心肌细胞凋亡和Calpain-l表达的变化.方法:采用改良的Rice法构建新生大鼠HIBD模型,64只大鼠随机分为对照组和HIBD后2、12、24 h及2、3、5、7 d组.应用TUNEL法检测心肌细胞凋亡情况,并计数凋亡指数(AI),RT-PCR法和Westem blot法检测各组大鼠心肌中Calpain-1mRNA及蛋白活性的变化.结果:对照组偶见凋亡细胞,HIBD组凋亡细胞均明显增多(P<0.01),3 d达凋亡高峰(P<0.001),此后开始降低,7 d

  13. 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.

  14. Preserved recovery of cardiac function following ischemia-reperfusion in mice lacking SIRT3.

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    Koentges, Christoph; Pfeil, Katharina; Meyer-Steenbuck, Maximilian; Lother, Achim; Hoffmann, Michael M; Odening, Katja E; Hein, Lutz; Bode, Christoph; Bugger, Heiko

    2016-01-01

    Lack of the mitochondrial deacetylase sirtuin 3 (SIRT3) impairs mitochondrial function and increases the susceptibility to induction of the mitochondrial permeability transition pore. Because these alterations contribute to myocardial ischemia-reperfusion (IR) injury, we hypothesized that SIRT3 deficiency may increase cardiac injury following myocardial IR. Hearts of 10-week-old mice were perfused in the isolated working mode and subjected to 17.5 min of global no-flow ischemia, followed by 30 min of reperfusion. Measurements before ischemia revealed a decrease in cardiac power (-20%) and rate pressure product (-15%) in SIRT3(-/-) mice. Mitochondrial state 3 respiration (-15%), ATP synthesis (-39%), and ATP/O ratios (-29%) were decreased in hearts of SIRT3(-/-) mice. However, percent recovery of cardiac power (WT 94% ± 9%; SIRT3(-/-) 89% ± 9%) and rate pressure product (WT 89% ± 16%; SIRT3(-/-) 96% ± 3%) following IR was similar in both groups. Myocardial infarct size was not increased in SIRT3(-/-) mice following permanent ligation of the left anterior descending coronary artery (LAD). Left ventricular pressure and dP/dtmax, and mitochondrial respiration and ATP synthesis were not different between groups following LAD ligation. Thus, despite pre-existing defects in cardiac function and mitochondrial respiratory capacity in SIRT3(-/-) mice, SIRT3 deficiency does not additionally impair cardiac function following IR or following myocardial infarction.

  15. Elk3 deficiency causes transient impairment in post-natal retinal vascular development and formation of tortuous arteries in adult murine retinae.

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    Christine Weinl

    Full Text Available Serum Response Factor (SRF fulfills essential roles in post-natal retinal angiogenesis and adult neovascularization. These functions have been attributed to the recruitment by SRF of the cofactors Myocardin-Related Transcription Factors MRTF-A and -B, but not the Ternary Complex Factors (TCFs Elk1 and Elk4. The role of the third TCF, Elk3, remained unknown. We generated a new Elk3 knockout mouse line and showed that Elk3 had specific, non-redundant functions in the retinal vasculature. In Elk3(-/- mice, post-natal retinal angiogenesis was transiently delayed until P8, after which it proceeded normally. Interestingly, tortuous arteries developed in Elk3(-/- mice from the age of four weeks, and persisted into late adulthood. Tortuous vessels have been observed in human pathologies, e.g. in ROP and FEVR. These human disorders were linked to altered activities of vascular endothelial growth factor (VEGF in the affected eyes. However, in Elk3(-/- mice, we did not observe any changes in VEGF or several other potential confounding factors, including mural cell coverage and blood pressure. Instead, concurrent with the post-natal transient delay of radial outgrowth and the formation of adult tortuous arteries, Elk3-dependent effects on the expression of Angiopoietin/Tie-signalling components were observed. Moreover, in vitro microvessel sprouting and microtube formation from P10 and adult aortic ring explants were reduced. Collectively, these results indicate that Elk3 has distinct roles in maintaining retinal artery integrity. The Elk3 knockout mouse is presented as a new animal model to study retinal artery tortuousity in mice and human patients.

  16. 钙激活蛋白酶系统与慢性房颤犬心房重构关系的研究%Effects of the calpain system expression on atrial structural remodeling in canine with atrial fibrillation

    Institute of Scientific and Technical Information of China (English)

    马骁; 张薇; 钟明; 黎莉; 苗雅; 孙慧; 张运

    2007-01-01

    目的:探讨慢性房颤犬心房肌钙激活蛋白酶(calpain)系统表达水平的改变及其与心房重构的相关性.方法:17只杂种犬随机分为心房颤动组(11只)和对照组(6只),于起搏前后均进行经胸超声心动图检查,测量舒张期左房内径.房颤组经颈外静脉将电极置入右心耳快速起搏(400 beats/min)8周复制房颤模型,开胸取心房组织,测定心房肌Ca2+浓度,用荧光实时定量PCR和Western blotting技术检测calpain及其抑制剂calpastatin mRNA和蛋白的表达量.结果:房颤组心房肌Ca2+ 浓度升高,左房内径显著大于起搏前及对照组(P<0.05),房颤组和对照组比较犬心房肌calpain Ⅰ、calpain ⅡmRNA表达无显著差异(P>0.05),房颤组calpastatin mRNA表达明显高于对照组(P<0.05);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、calpainⅡ蛋白表达水平与左房内径呈显著正相关(r=0.53,r=0.67,P<0.05),calpastatin蛋白表达水平与左房内径呈显著负相关(r=-0.74,P<0.05).结论:房颤所引起calpain系统的蛋白表达改变,使calpain/calpastatin系统相互间作用失衡,造成多种蛋白被降解可能是心房重构的重要机制.

  17. 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

  18. 延迟冷却对牛背最长肌中μ-calpain活性及嫩度的影响%Effect of Delay Chilling on μ-calpain Activities and the Tenderness of Bovine M.Iongissimus

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    胡鹏; 丁玉; 梁荣蓉; 罗欣

    2010-01-01

    μ-calpain activities and shear force values of bovine M.Iongissimus from Chinese Yellow crossbred bulls were analyzed,and the effect of delay chilling on μ-calpain activities and the tenderness of beef during postmortem aging were studied.The results showed that delay chilling significantly improved μ-calpain activities (P<0.05) and enhanced the tenderness of bovine M.Iongissimus during earlier aging periods compared with conventional chilling.But in later aging periods,delay chilling weakened the effect on the tenderness of beef because of premature consumption of μ-calpain.The experiment results confirmed that delay chilling improved the rate of postmortem aging of beef and remarkably enhanced the tenderness of beef through the effect of delay chilling on μ-calpain activities.%[目的]研究延迟冷却对牛背最长肌中μ-calpain活性及嫩度的影响,为进一步改善牛肉食用品质提供依据.[方法]测定中国杂交黄牛(鲁西黄牛x西门塔尔)牛背最长肌中μ-calpain活性及剪切力值,分析延迟冷却对牛背最长肌宰后成熟过程中μ-calpain活性及牛肉嫩度的影响.[结果]在宰后成熟早期,延迟冷却显著提高了μ-calpain的活性,提高了牛背最长肌的嫩度;成熟后期,由于μ-calpain被过早消耗,对牛肉的嫩化作用减弱.[结论]延迟冷却通过对μ-calpain活性的影响,改变了肉的成熟速度,显著改善了牛肉的嫩度.

  19. Calpain10基因多态性与维吾尔族2型糖尿病患者亲级关系的研究%Relationship between Calpain10 gene polymorphisms and diabetes in familial type 2 diabetes mellitus of Uygur in Xinjiang

    Institute of Scientific and Technical Information of China (English)

    樊勇; 鲜木斯娅·肉孜; 谢自敬; 肖艳; 王塞刚

    2009-01-01

    目的 分析Calpain10基因UCSNP43和UCSNP44位点多态性与新疆维吾尔族2型糖尿病家系中小同亲级的相关性.方法 以新疆62个维吾尔族2型糖尿病核心家系为样本,采用PPAP(population and pedigree analysis programs)统计软件和Penrose法来研究其遗传方式;将75个新疆维吾尔族2型糖尿病家系一级亲和二级亲成员481人做两组比较分析,采用基因芯片方法 榆测UCSNP43和UCSNP44位点多态性,应用SPSS 13.0统计软件分析Calpain10基凶多态性是否在2型糖球病家系不同亲级关系存在差异.结果 (1)2型糖尿病家系一级亲的遗传度为0.578,二级关的遗传度为0.032;(2)一级和二级亲属之间Calpain10基因UCSNP43、UCSNP44基因型和等位基因频率分布差异均无统汁学意义(P<0.05);(3)Calpain10其因UCSNP43位点AA基因型携带者体重指数、空腹胰岛素水平较GG和GA基因型携带者高(P<0.05或P<0.01).结论 新疆维吾尔族2型精尿病的遗传度随着亲缘系数的降低析减小;UCSNP43位点AA基因携带者胰岛素抵抗程度较GG和AG型携带者高.%Objective To analyse the relationship between Calpain10 gene UCSNP43 and UCSNP44 poly morphisms and diabetes in familial type 2 diabetes mellitus of Uygur in Xinjiang.Methods Sixty-two Uygur type 2 diabetic families were enrolled.The polymorphisms of Calpain10 gene UCSNP43 and UCSNP44 were measured by SNP-stream of gene chip.Results (1) The heritability of the first degree relative and the second degree relative in Uygur familial type 2 diabetes were 0.578 and 0.032.(2)There was no difference between UCSNP43 and UCSNP44 polymorphisms of the first and second degree relatives in familial type 2 diabetes mellitus.(3)The carriers of USCNP43 AA genotype had higher body mass index and fasting insulin than GG and GA genotypes carriers.Conclu sion With the downgrading of the familial relationship,the heritability of type 2 diabetes mellitus was decreasing. The carriers of UCSNP43 AA genotype

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

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    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

  1. Increased motor drive and sleep loss in mice lacking Kv3-type potassium channels.

    Science.gov (United States)

    Espinosa, F; Marks, G; Heintz, N; Joho, R H

    2004-04-01

    The voltage-gated potassium channels Kv3.1 and Kv3.3 are widely expressed in the brain, including areas implicated in the control of motor activity and in areas thought to regulate arousal states. Although Kv3.1 and Kv3.3-single mutants show some physiological changes, previous studies revealed relatively subtle behavioral alterations suggesting that Kv3.1 and Kv3.3 channel subunits may be encoded by a pair of redundant genes. In agreement with this hypothesis, Kv3.1/Kv3.3-deficient mice display a 'strong' mutant phenotype that includes motor dysfunction (ataxia, myoclonus, tremor) and hyperactivity when exposed to a novel environment. In this paper we report that Kv3.1/Kv3.3-deficient mice are also constitutively hyperactive. Compared to wildtype mice, double mutants display 'restlessness' that is particularly prominent during the light period, when mice are normally at rest, characterized by more than a doubling of ambulatory and stereotypic activity, and accompanied by a 40% sleep reduction. When we reinvestigated both single mutants, we observed constitutive increases of ambulatory and stereotypic activity in conjunction with sleep loss in Kv3.1-single mutants but not in Kv3.3-single mutants. These findings indicate that the absence of Kv3.1-channel subunits is primarily responsible for the increased motor drive and the reduction in sleep time.

  2. Novel roles for MLH3 deficiency and TLE6-like amplification in DNA mismatch repair-deficient gastrointestinal tumorigenesis and progression.

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    Peng-Chieh Chen

    2008-06-01

    Full Text Available DNA mismatch repair suppresses gastrointestinal tumorgenesis. Four mammalian E. coli MutL homologues heterodimerize to form three distinct complexes: MLH1/PMS2, MLH1/MLH3, and MLH1/PMS1. To understand the mechanistic contributions of MLH3 and PMS2 in gastrointestinal tumor suppression, we generated Mlh3(-/-;Apc(1638N and Mlh3(-/-;Pms2(-/-;Apc(1638N (MPA mice. Mlh3 nullizygosity significantly increased Apc frameshift mutations and tumor multiplicity. Combined Mlh3;Pms2 nullizygosity further increased Apc base-substitution mutations. The spectrum of MPA tumor mutations was distinct from that observed in Mlh1(-/-;Apc(1638N mice, implicating the first potential role for MLH1/PMS1 in tumor suppression. Because Mlh3;Pms2 deficiency also increased gastrointestinal tumor progression, we used array-CGH to identify a recurrent tumor amplicon. This amplicon contained a previously uncharacterized Transducin enhancer of Split (Tle family gene, Tle6-like. Expression of Tle6-like, or the similar human TLE6D splice isoform in colon cancer cells increased cell proliferation, colony-formation, cell migration, and xenograft tumorgenicity. Tle6-like;TLE6D directly interact with the gastrointestinal tumor suppressor RUNX3 and antagonize RUNX3 target transactivation. TLE6D is recurrently overexpressed in human colorectal cancers and TLE6D expression correlates with RUNX3 expression. Collectively, these findings provide important insights into the molecular mechanisms of individual MutL homologue tumor suppression and demonstrate an association between TLE mediated antagonism of RUNX3 and accelerated human colorectal cancer progression.

  3. Galectin-3 Deletion Enhances Visceral Adipose Tissue Inflammation and Dysregulates Glucose Metabolism in Mice on a High-Fat Diet

    Directory of Open Access Journals (Sweden)

    Jeftic Ilija

    2016-09-01

    Full Text Available Obesity and type 2 diabetes mellitus (T2DM constitute major health problems worldwide. Increased visceral adiposity enhances the risk of insulin resistance and type 2 diabetes. The mechanisms involved in obesity-associated chronic inflammation in metabolic tissues (metaflammation that lead to insulin resistance and dysregulated glucose metabolism are incompletely defined. Galectin-3 (Gal-3, a β-galactoside-binding lectin, modulates immune/inflammatory responses and specifically binds to metabolic danger molecules. To dissect the role of Gal-3 in obesity and diabetes, Gal-3-deficient (LGALS3-/- and wild-type (WT C57Bl/6 male mice were placed on a high-fat diet (HFD, 60% kcal fat or a standard chow diet (10% kcal fat for 6 months and metabolic, histological and immunophenotypical analyses of the visceral adipose tissue were performed. HFD-fed LGALS3-/- mice had higher body weights and more body weight gain, visceral adipose tissue (VAT, hyperglycaemia, hyperinsulinemia, insulin resistance and hyperlipidemia than diet-matched WT mice. Compared to WT mice, the enlarged VAT in obese LGALS3-/- mice contained larger adipocytes. Additionally, we demonstrate enhanced inflammation in the VAT of LGALS3-/- mice compared with diet-matched WT mice. The VAT of LGALS3-/- mice fed a HFD contained more numerous dendritic cells and proinflammatory F4/80+CD11c+CD11b+ and F4/80high macrophages. In contrast to WT mice, the numbers of CXCR3+ and CD8+ T cells were increased in the VAT of Gal-3-deficient mice after 6 months of high-fat feeding. We provide evidence that Gal-3 ablation results in enhanced HFD-induced adiposity, inflammation in the adipose tissue, insulin resistance and hyperglycaemia. Thus, Gal-3 represents an important regulator of obesity-associated immunometabolic alterations.

  4. Calpain expression changes in response to hypothermia after traumatic brain injury%颅脑外伤后亚低温治疗对Calpain表达的影响

    Institute of Scientific and Technical Information of China (English)

    万杰清; 贾锋; 毛青; 江基尧

    2009-01-01

    Objective To determine the effect of hypothermia on gene transcription and protein expression of calpain after traumatic brain injury (TBI). Methods Twenty-seven rats were randomly divided into three groups, ie, normal control group, normothermia TBI group and hypothermia TBI group. All rats with TBI were suffered from a lateral fluid percussion injury (FPI) at the right parietal lobe. Hy-pothermia intervention [rectal temperature for (32 ± 0.5) ℃] was performed for four hours immediately after TBI in hypothermia TBI group. Fluorescence PCR and Western blot were utilized to semi-quantify gene transcription and protein expression of ealpain and immunofluorescence used to observe protein dis-tribution of Calpain. Results Compared with normothermia TBi group and normal control group, hypo-thermia TBI group showed increased calpain gene transcription at 12 and 24 hours respectively after FPI (P <0.05). However, the increase of ealpain protein expression in hypothermia TBI group was inhibited more significantly by hypothermia at 6,12,24 and 72 hours after TBI, compared with normothermia TBI group (P < 0.05). Conclusion Neuroproteetion of hypothermia after TBI may somewhat be related to the decrease of calpain protein expression after its gene transcription.%目的 探讨亚低温治疗对颅脑外伤后Calpain基因和蛋白表达的影响.方法 将27只SD大鼠按随机数字表法分为正常对照组、常温脑损伤组和亚低温脑损伤组.脑损伤组分别接受右顶部侧方中度液压损伤打击,其中亚低温组在伤后即刻接受持续4 h的亚低温治疗[肛温(32±0.5)℃].在伤后6,12,24和72 h四个时间点分别通过荧光PCR、Western blot定量检测各组打击伤灶周围皮层钙蛋白酶Calpain基因转录和蛋白分子表达的水平,同时运用免疫荧光观察Calpain蛋白分布情况.结果 与常温组比较,亚低温使颅脑外伤后12 h及24 h Calpain基因的转录较正常对照和同时间点常

  5. 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.

  6. Effects of the calpain system expression on atrial structural remodeling in chronic atrial fibrillation%心房纤颤患者心房肌钙激活蛋白酶系统表达变化及意义

    Institute of Scientific and Technical Information of China (English)

    马骁; 宋平; 张学义; 关宏; 赵宏兵; 王慧; 黄琳; 杜平

    2008-01-01

    目的 探讨慢性心房纤颤患者心房肌钙激活蛋白酶(calpain)系统表达水平改变及其与心房重构的相关性.方法 选取进行人工瓣膜置换术的风湿性心脏病房颤患者28例为研究对象,风湿性心脏病窦性心律患者14例为对照组.两组患者术前均进行经胸超声心动图检查,于手术时取左心耳心肌标本,采用荧光实时定量PCR和Western blot技术检测calpain及其抑制剂calpastatin的mRNA和蛋白的表达量.结果 房颤组左心房内径和右心房内径均显著大于对照组(P0.05),房颤组calpastatin的mRNA表达明显高于对照组(P<0.05):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、calpain Ⅱ蛋白表达水平与左房内径呈显著正相关(r=0.67,r=0.55,P<0.05),calpastatin蛋白表达水平与左房内径呈显著负相关(r=-0.73,P<0.05).结论 房颤所引起calpain系统的蛋白表达改变,使calpain/calpastatin系统相互间作用失衡,造成多种蛋白被降解可能是心房重构的重要机制.

  7. Disruption and molecular characterization of calpains-related (MoCAPN1, MoCAPN3 and MoCAPN4) genes in Magnaporthe oryzae.

    Science.gov (United States)

    Khan, Irshad Ali; Wang, Yao; Li, Hai-Jiao; Lu, Jian-Ping; Liu, Xiao-Hong; Lin, Fu-Cheng

    2014-11-01

    Calpains are intracellular, cysteine proteases found in plants, animals and fungi functioning as signal transduction components in different cellular pathways including sporulation and alkaline adaptation in fungi. Calpains-related MoCAPN1 (MGG_14872), MoCAPN3 (MGG_15810) and MoCAPN4 (MGG_04818) genes from Magnaporthe oryzae genome which are 2604, 3513 and 771-bp in length and encoding identical proteins of 867, 1170 and 256 amino acids were functionally characterized for different phenotypes through gene disruption method. All the mutants except those for MoCAPN1 showed normal phenotypes. In pathogenicity test, the mutants did not lead to any visible changes in phenotypes causing similar blast lesions on blast susceptible rice and barley leaves as those of the Guy-11 strain suggesting no major role in pathogenicity. Germ tubes formation, appressorium formation, mycelium radial growth and mating with 2539 strain were indistinguishable among the mutants and Guy-11 strains. Cell wall integrity (congo red) test, stress response under chemical pressure (ZnSO4, CuSO4 and CdCl2), osmotic and oxidative (NaCl and H2O2) stress response, growth response on glucose and nitrogen deficient media resulted in similar results in the mutants and Guy-11 strains. However, mutants for ΔMoCAPN1 gene produced reduced (0.57±0.15B and 0.54±0.05B) conidia compared to that (1.69±0.13A) of the Guy-11 strain showing its involvement in conidiation.

  8. 鸡胸肉中钙激活酶的纯化研究%The purification of calpains from chicken breast meat

    Institute of Scientific and Technical Information of China (English)

    慕妮; 王恺; 王璐莎; 黄明; 巴吐尔·阿不力克木

    2013-01-01

    对宰后10 min的鸡胸肉经匀浆后利用超高速冷冻离心、硫酸铵分级沉淀、离子交换层析和疏水层析等技术,从中分离纯化出μ-钙激活酶(μ-calpain)和μ/m-钙激活酶(μ/m-calpain).对分离纯化过程各主要步骤中的酶总活性、比活性等参数进行测定,同时采用活性电泳对纯化过程的样品进行分析.结果表明:样品通过Hitrap Capto DEAE层析柱,可以将μ-calpain和μ/m-calpain分别洗脱下来,μ-calpain比活性为1.02 U·mg-1,μ/m-calpain的比活性为3.04 U-mg-1;所得μ-calpain及μ/m-calpain样品分别依次经过Phenyl Sepharose 6 FF层析柱和Mono Q层析柱,最后得到的μ-calpain和μ/m-calpain比活性分别为26.89和34.96 U· mg-1.结果表明,随着纯化步骤的增加,鸡胸肉中的钙激活酶纯度逐步增加,并得到了较有效的纯化.

  9. EspC, an Autotransporter Protein Secreted by Enteropathogenic Escherichia coli, Causes Apoptosis and Necrosis through Caspase and Calpain Activation, Including Direct Procaspase-3 Cleavage

    Directory of Open Access Journals (Sweden)

    Antonio Serapio-Palacios

    2016-06-01

    Full Text Available Enteropathogenic Escherichia coli (EPEC has the ability to antagonize host apoptosis during infection through promotion and inhibition of effectors injected by the type III secretion system (T3SS, but the total number of these effectors and the overall functional relationships between these effectors during infection are poorly understood. EspC produced by EPEC cleaves fodrin, paxillin, and focal adhesion kinase (FAK, which are also cleaved by caspases and calpains during apoptosis. Here we show the role of EspC in cell death induced by EPEC. EspC is involved in EPEC-mediated cell death and induces both apoptosis and necrosis in epithelial cells. EspC induces apoptosis through the mitochondrial apoptotic pathway by provoking (i a decrease in the expression levels of antiapoptotic protein Bcl-2, (ii translocation of the proapoptotic protein Bax from cytosol to mitochondria, (iii cytochrome c release from mitochondria to the cytoplasm, (iv loss of mitochondrial membrane potential, (v caspase-9 activation, (vi cleavage of procaspase-3 and (vii an increase in caspase-3 activity, (viii PARP proteolysis, and (ix nuclear fragmentation and an increase in the sub-G1 population. Interestingly, EspC-induced apoptosis was triggered through a dual mechanism involving both independent and dependent functions of its EspC serine protease motif, the direct cleavage of procaspase-3 being dependent on this motif. This is the first report showing a shortcut for induction of apoptosis by the catalytic activity of an EPEC protein. Furthermore, this atypical intrinsic apoptosis appeared to induce necrosis through the activation of calpain and through the increase of intracellular calcium induced by EspC. Our data indicate that EspC plays a relevant role in cell death induced by EPEC.

  10. Remarkable reduction of MAP2 in the brains of scrapie-infected rodents and human prion disease possibly correlated with the increase of calpain.

    Directory of Open Access Journals (Sweden)

    Yan Guo

    Full Text Available Microtubule-associated protein 2 (MAP2 belongs to the family of heat stable MAPs, which takes part in neuronal morphogenesis, maintenance of cellular architecture and internal organization, cell division and cellular processes. To obtain insight into the possible alteration and the role of MAP2 in transmissible spongiform encephalopathies (TSEs, the MAP2 levels in the brain tissues of agent 263K-infected hamsters and human prion diseases were evaluated. Western blots and IHC revealed that at the terminal stages of the diseases, MAP2 levels in the brain tissues of scrapie infected hamsters, a patient with genetic Creutzfeldt-Jakob disease (G114V gCJD and a patient with fatal familial insomnia (FFI were almost undetectable. The decline of MAP2 was closely related with prolonged incubation time. Exposure of SK-N-SH neuroblastoma cell line to cytotoxic PrP106-126 peptide significantly down-regulated the cellular MAP2 level and remarkably disrupted the microtubule structure, but did not alter the level of tubulin. Moreover, the levels of calpain, which mediated the degradation of a broad of cytoskeletal proteins, were significantly increased in both PrP106-126 treated SK-N-SH cells and brain tissues of 263K prion-infected hamsters. Our data indicate that the decline of MAP2 is a common phenomenon in TSEs, which seems to occur at an early stage of incubation period. Markedly increased calpain level might contribute to the reduction of MAP2.

  11. µ-Calpain conversion of antiapoptotic Bfl-1 (BCL2A1 into a prodeath factor reveals two distinct alpha-helices inducing mitochondria-mediated apoptosis.

    Directory of Open Access Journals (Sweden)

    Juan García Valero

    Full Text Available Anti-apoptotic Bfl-1 and pro-apoptotic Bax, two members of the Bcl-2 family sharing a similar structural fold, are classically viewed as antagonist regulators of apoptosis. However, both proteins were reported to be death inducers following cleavage by the cysteine protease µ-calpain. Here we demonstrate that calpain-mediated cleavage of full-length Bfl-1 induces the release of C-terminal membrane active α-helices that are responsible for its conversion into a pro-apoptotic factor. A careful comparison of the different membrane-active regions present in the Bfl-1 truncated fragments with homologous domains of Bax show that helix α5, but not α6, of Bfl-1 induces cell death and cytochrome c release from purified mitochondria through a Bax/Bak-dependent mechanism. In contrast, both helices α5 and α6 of Bax permeabilize mitochondria regardless of the presence of Bax or Bak. Moreover, we provide evidence that the α9 helix of Bfl-1 promotes cytochrome c release and apoptosis through a unique membrane-destabilizing action whereas Bax-α9 does not display such activities. Hence, despite a common 3D-structure, C-terminal toxic domains present on Bfl-1 and Bax function in a dissimilar manner to permeabilize mitochondria and induce apoptosis. These findings provide insights for designing therapeutic approaches that could exploit the cleavage of endogenous Bcl-2 family proteins or the use of Bfl-1/Bax-derived peptides to promote tumor cell clearance.

  12. X-ray diffraction, solution structure, and computational studies on derivatives of (3-sec-butyl-2,3-dihydro-1H-isoquinolin-4-ylidene)acetic acid: compounds with activity as calpain inhibitors.

    Science.gov (United States)

    Alonso, Mercedes; Chicharro, Roberto; Miranda, Carlos; Arán, Vicente J; Maestro, Miguel A; Herradón, Bernardo

    2010-01-15

    A thorough experimental and computational study of derivatives of (3-sec-butyl-2,3-dihydroisoquinolin-4-ylidene)acetic acid was performed. Some of these compounds are calpain inhibitors and could be useful as therapeutic agents, since this enzyme is a Ca(2+)-dependent cysteine protease involved in a wide variety of metabolic and physiological processes, whose over-activation is associated to several pathological conditions. To gain a better understanding of the structure-activity relationships, a structural analysis was carried out with (1)H and (13)C NMR spectroscopy and DFT calculations together with the X-ray diffraction data of three compounds. The solid state structures showed that the crystal packing as well as the intermolecular interactions depend on the substituent nature of the COOR group. Also, the reactivity of the exocyclic double bond was theoretically evaluated, finding that the more reactive compound is the most potent inhibitor of calpain (IC(50) = 25 nM).

  13. Variants within the calpain-10 gene on chromosome 2q37 (NIDDM1) and relationships to type 2 diabetes, insulin resistance, and impaired acute insulin secretion among Scandinavian Caucasians

    DEFF Research Database (Denmark)

    Rasmussen, Søren K; Urhammer, Søren A; Berglund, Lars Erik;

    2002-01-01

    Variations in the calpain-10 gene (CAPN10) have been identified among Mexican-Americans, and an at-risk haplotype combination (112/121) defined by three polymorphisms, UCSNP-43, -19, and -63, confers increased risk of type 2 diabetes. Here we examine the three polymorphisms in 1,594 Scandinavian ...... the CAPN10 variants and type 2 diabetes, insulin resistance, or impaired insulin secretion....

  14. 钙激活中性蛋白酶-2对整合素β4水解的影响%Research on the Effect of Calpain-2 on the Proteolysis of Integrin β4 in MCF7 Cell

    Institute of Scientific and Technical Information of China (English)

    胡晓霞; 霍建云; 张金娟; 潘娅; 王晋星一; 陈妮; 张祥令; 陈腾祥

    2015-01-01

    目的:观察乳腺癌细胞系MCF7细胞中,钙激活中性蛋白酶2(calpain-2)对整合素(integrin)β4水解的影响.方法:利用“短发夹”RNA(shRNA)和微小RNA (mirRNA)技术结合的calpain-2基因沉默(gene silen-cing)慢病毒质粒(GIPZ lentiviral shRNAmir)转染乳腺癌细胞株MCF7,嘌呤霉素筛选稳定沉默calpain-2基因的细胞株,细胞株传4代,用荧光显微镜观察转染效率,用蛋白质印迹(Westernblot)实验检测calpain-2基因沉默效率及integrin β4水解的情况.结果:嘌呤霉素筛选、细胞传4代,荧光显微镜下观察显示转染和筛选后,EGFP表达阳性的MCF7细胞的比例分别达到(95.6±2.6)%(空shRNAmir载体)、(97.1±1.7)%(Calpain-2 shRNAmir1)和(97.7±0.2)%(Calpain-2 shRNAmir 2),差异无统计学意义(P>0.05);Western blot结果显示,基因沉默的MCF7细胞中calpain-2的表达被明显抑制,相对于空shRNAmir载体转染的MCF7细胞,差异有统计学意义(P<0.01),calpain-2的表达下调到21.5%(Calpain-2 shRNAmir 1)和18.8% (Calpain-2 shRNAmir 2),空shRNAmir载体转染的MCF7细胞中calpain-2的表达与未转染的MCF7细胞比较,差异无统计学意义(P>0.05);比较cal-pain-2基因沉默和空shRNAmir载体转染的MCF7细胞,发现integrin β4均被水解,水解片段的分子量主要有200 kD、130和95 kD; calpain-2基因沉默后,calpain-2基因沉默MCF7细胞中200 kD的水解片段比空shRNAmir载体转染的MCF7细胞减少(P<0.01).结论:在乳腺癌细胞MCF7中,calpain-2可能参与integrin β4的200 kD片段的形成,从而参与调整integrin β4的构象变化.

  15. Quercetin glycosides and chlorogenic acid in highbush blueberry leaf decoction prevent cataractogenesis in vivo and in vitro: Investigation of the effect on calpains, antioxidant and metal chelating properties.

    Science.gov (United States)

    Ferlemi, Anastasia-Varvara; Makri, Olga E; Mermigki, Penelope G; Lamari, Fotini N; Georgakopoulos, Constantinos D

    2016-04-01

    The present study investigates whether highbush blueberry leaf polyphenols prevent cataractogenesis and the underlying mechanisms. Chlorogenic acid, quercetin, rutin, isoquercetin and hyperoside were quantified in Vaccinium corymbosum leaf decoction (BBL) using HPLC-DAD. Wistar rats were injected subcutaneously with 20 μmol selenite (Na2SeO3)/kg body weight on postnatal (PN) day 10 (Se, n = 8-10/group) only or also intraperitoneally with 100 mg dry BBL/kg body weight on PN days 11 and 12 (SeBBL group, n = 10). Control group received only normal saline (C). Cataract evaluation revealed that BBL significantly prevented lens opacification. It, also, protected lens from selenite oxidative attack and prevented calpain activation, as well as protein loss and aggregation. In vitro studies showed that quercetin attenuated porcine lens turbidity caused by [Formula: see text] or Ca(2+) and interacted efficiently with those ions according to UV-Vis titration experiments. Finally, rutin, isoquercetin and hyperoside moderately inhibited pure human μ-calpain. Conclusively, blueberry leaf extract, a rich source of bioactive polyphenols, prevents cataractogenesis by their strong antioxidant, chelating properties and through direct/indirect inhibition of lens calpains.

  16. Localization and Expression of Calpain in the Cochlea of Normal Guinea Pig%钙蛋白酶在正常豚鼠耳蜗中的定位表达

    Institute of Scientific and Technical Information of China (English)

    马德菊; 王爱梅; 包翠芬; 宝东艳; 常志杰

    2008-01-01

    目的 研究钙蛋白酶(calpain)在正常豚鼠耳蜗中的定位与表达.方法 应用免疫组织化学SABC法结合显微图像分析技术,观察耳蜗组织中calpain的表达;同时采用电生理指标听性脑干反应(auditory brainstem response,ABR)检测豚鼠的听力.结果 calpain免疫反应活性主要见于耳蜗血管纹、螺旋韧带、螺旋神经节及毛细胞,螺旋神经节的染色较深;其中,calpain 1表达多见于螺旋神经节细胞的胞浆中,而calpain 2免疫染色几乎出现在整个螺旋神经节细胞中.结论 calpain在正常豚鼠耳蜗的多种细胞中都有表达.

  17. Sulindac metabolites decrease cerebrovascular malformations in CCM3-knockout mice.

    Science.gov (United States)

    Bravi, Luca; Rudini, Noemi; Cuttano, Roberto; Giampietro, Costanza; Maddaluno, Luigi; Ferrarini, Luca; Adams, Ralf H; Corada, Monica; Boulday, Gwenola; Tournier-Lasserve, Elizabeth; Dejana, Elisabetta; Lampugnani, Maria Grazia

    2015-07-07

    Cerebral cavernous malformation (CCM) is a disease of the central nervous system causing hemorrhage-prone multiple lumen vascular malformations and very severe neurological consequences. At present, the only recommended treatment of CCM is surgical. Because surgery is often not applicable, pharmacological treatment would be highly desirable. We describe here a murine model of the disease that develops after endothelial-cell-selective ablation of the CCM3 gene. We report an early, cell-autonomous, Wnt-receptor-independent stimulation of β-catenin transcription activity in CCM3-deficient endothelial cells both in vitro and in vivo and a triggering of a β-catenin-driven transcription program that leads to endothelial-to-mesenchymal transition. TGF-β/BMP signaling is then required for the progression of the disease. We also found that the anti-inflammatory drugs sulindac sulfide and sulindac sulfone, which attenuate β-catenin transcription activity, reduce vascular malformations in endothelial CCM3-deficient mice. This study opens previously unidentified perspectives for an effective pharmacological therapy of intracranial vascular cavernomas.

  18. VAV2 and VAV3 as candidate disease genes for spontaneous glaucoma in mice and humans.

    Directory of Open Access Journals (Sweden)

    Keiko Fujikawa

    Full Text Available BACKGROUND: Glaucoma is a leading cause of blindness worldwide. Nonetheless, the mechanism of its pathogenesis has not been well-elucidated, particularly at the molecular level, because of insufficient availability of experimental genetic animal models. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that deficiency of Vav2 and Vav3, guanine nucleotides exchange factors for Rho guanosine triphosphatases, leads to an ocular phenotype similar to human glaucoma. Vav2/Vav3-deficient mice, and to a lesser degree Vav2-deficient mice, show early onset of iridocorneal angle changes and elevated intraocular pressure, with subsequent selective loss of retinal ganglion cells and optic nerve head cupping, which are the hallmarks of glaucoma. The expression of Vav2 and Vav3 tissues was demonstrated in the iridocorneal angle and retina in both mouse and human eyes. In addition, a genome-wide association study screening glaucoma susceptibility loci using single nucleotide polymorphisms analysis identified VAV2 and VAV3 as candidates for associated genes in Japanese open-angle glaucoma patients. CONCLUSIONS/SIGNIFICANCE: Vav2/Vav3-deficient mice should serve not only as a useful murine model of spontaneous glaucoma, but may also provide a valuable tool in understanding of the pathogenesis of glaucoma in humans, particularly the determinants of altered aqueous outflow and subsequent elevated intraocular pressure.

  19. 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.

  20. 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

  1. Novel Bioinformatics–Based Approach for Proteomic Biomarkers Prediction of Calpain-2 & Caspase-3 Protease Fragmentation: Application to βII-Spectrin Protein

    Science.gov (United States)

    El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges; Kobeissy, Firas

    2017-01-01

    The crucial biological role of proteases has been visible with the development of degradomics discipline involved in the determination of the proteases/substrates resulting in breakdown-products (BDPs) that can be utilized as putative biomarkers associated with different biological-clinical significance. In the field of cancer biology, matrix metalloproteinases (MMPs) have shown to result in MMPs-generated protein BDPs that are indicative of malignant growth in cancer, while in the field of neural injury, calpain-2 and caspase-3 proteases generate BDPs fragments that are indicative of different neural cell death mechanisms in different injury scenarios. Advanced proteomic techniques have shown a remarkable progress in identifying these BDPs experimentally. In this work, we present a bioinformatics-based prediction method that identifies protease-associated BDPs with high precision and efficiency. The method utilizes state-of-the-art sequence matching and alignment algorithms. It starts by locating consensus sequence occurrences and their variants in any set of protein substrates, generating all fragments resulting from cleavage. The complexity exists in space O(mn) as well as in O(Nmn) time, where N, m, and n are the number of protein sequences, length of the consensus sequence, and length per protein sequence, respectively. Finally, the proposed methodology is validated against βII-spectrin protein, a brain injury validated biomarker.

  2. Calpain activator induced platelet apoptosis%活化钙离子依赖性蛋白酶诱导血小板凋亡的研究

    Institute of Scientific and Technical Information of China (English)

    王志成; 张晓峰

    2011-01-01

    目的:探讨钙离子依赖性蛋白酶(calpain)在血小板凋亡中的作用.方法:Calpain活化剂dibucaine处理血小板后,采用流式细胞仪检测血小板线粒体跨膜电位(△ψm)去极化,磷脂酰丝氨酸(PS)外翻;western blot检测caspase-3活化以及calpain底物talin酶切.结果:Dibucaine能诱导血小板△ψm去极化,PS外翻,caspase-3活化,以及calpain底物talin的酶切;calpain抑制剂能完全抑制dibucaine诱导的Aψm去极化、PS外翻、caspase-3活化,以及talin的酶切.结论:Calpain在血小板凋亡中发挥重要作用,活化calpain能诱导血小板凋亡.

  3. Novel Bioinformatics–Based Approach for Proteomic Biomarkers Prediction of Calpain-2 & Caspase-3 Protease Fragmentation: Application to βII-Spectrin Protein

    Science.gov (United States)

    El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges; Kobeissy, Firas

    2017-01-01

    The crucial biological role of proteases has been visible with the development of degradomics discipline involved in the determination of the proteases/substrates resulting in breakdown-products (BDPs) that can be utilized as putative biomarkers associated with different biological-clinical significance. In the field of cancer biology, matrix metalloproteinases (MMPs) have shown to result in MMPs-generated protein BDPs that are indicative of malignant growth in cancer, while in the field of neural injury, calpain-2 and caspase-3 proteases generate BDPs fragments that are indicative of different neural cell death mechanisms in different injury scenarios. Advanced proteomic techniques have shown a remarkable progress in identifying these BDPs experimentally. In this work, we present a bioinformatics-based prediction method that identifies protease-associated BDPs with high precision and efficiency. The method utilizes state-of-the-art sequence matching and alignment algorithms. It starts by locating consensus sequence occurrences and their variants in any set of protein substrates, generating all fragments resulting from cleavage. The complexity exists in space O(mn) as well as in O(Nmn) time, where N, m, and n are the number of protein sequences, length of the consensus sequence, and length per protein sequence, respectively. Finally, the proposed methodology is validated against βII-spectrin protein, a brain injury validated biomarker. PMID:28112201

  4. Limb-girdle muscular dystrophy type 2A resulting from homozygous G2338C transversion mutation in the calpain-3 gene.

    Science.gov (United States)

    Peddareddygari, Leema Reddy; Surgan, Victoria; Grewal, Raji P

    2010-12-01

    Limb-girdle muscular dystrophy represents a clinically and genetically heterogeneous group of myopathies. Limb-girdle muscular dystrophy Type 2A, which is transmitted in an autosomal-recessive pattern, is caused by mutations in the calpain-3 (CAPN3) gene. A number of mutations have been reported in patients from throughout the world but not in the Asian-Indian population. We describe a genotype/phenotype analysis of an Asian-Indian patient with a history, neurologic examination, and investigations consistent with muscular dystrophy. Genetic analysis of this patient showed a homozygous G2338C transversion resulting in an amino acid change from aspartic acid 780 histidine in the CAPN3 gene confirming Limb-girdle muscular dystrophy Type 2A. Subsequent testing of the patient's family revealed that his parents and sister were heterozygous unaffected carriers. The G2338C transversion was detected as a compound heterozygous mutation in one patient in Germany. We report a homozygous case and expand the clinical spectrum of limb-girdle muscular dystrophy Type 2A to include Asian-Indians.

  5. Selective cognitive deficits and reduced hippocampal brain-derived neurotrophic factor mRNA expression in small-conductance calcium-activated K+ channel deficient mice.

    Science.gov (United States)

    Jacobsen, J P R; Redrobe, J P; Hansen, H H; Petersen, S; Bond, C T; Adelman, J P; Mikkelsen, J D; Mirza, N R

    2009-09-29

    Small-conductance calcium-activated K(+) channels 1-3 (SK1-3) are important for neuronal firing regulation and are considered putative CNS drug targets. For instance non-selective SK blockers improve performance in animal models of cognition. The SK subtype(s) involved herein awaits identification and the question is difficult to address pharmacologically due to the lack of subtype-selective SK-channel modulators. In this study, we used doxycycline-induced conditional SK3-deficient (T/T) mice to address the cognitive consequences of selective SK3 deficiency. In T/T mice SK3 protein is near-eliminated from the brain following doxycycline treatment. We tested T/T and wild type (WT) littermate mice in five distinct learning and memory paradigms. In Y-maze spontaneous alternations and five-trial inhibitory avoidance the performance of T/T mice was markedly inferior to WT mice. In contrast, T/T and WT mice performed equally well in passive avoidance, object recognition and the Morris water maze. Thus, some aspects of working/short-term memory are disrupted in T/T mice. Using in situ hybridization, we further found the cognitive deficits in T/T mice to be paralleled by reduced brain-derived neurotrophic factor (BDNF) mRNA expression in the dentate gyrus and CA3 of the hippocampus. BDNF mRNA levels in the frontal cortex were not affected. BDNF has been crucially implicated in many cognitive processes. Hence, the biological substrate for the cognitive impairments in T/T mice could conceivably entail reduced trophic support of the hippocampus.

  6. Molecular signatures of neurodegeneration in the cortex of PS1/PS2 double knockout mice

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    Choi Se

    2008-10-01

    Full Text Available Abstract Background Familial Alzheimer's disease-linked variants of presenilin (PSEN1 and PSEN2 contribute to the pathophysiology of disease by both gain-of-function and loss-of-function mechanisms. Deletions of PSEN1 and PSEN2 in the mouse forebrain result in a strong and progressive neurodegenerative phenotype which is characterized by both anatomical and behavioral changes. Results To better understand the molecular changes associated with these morphological and behavioral phenotypes, we performed a DNA microarray transcriptome profiling of the hippocampus and the frontal cortex of the PSEN1/PSEN2 double knock-out mice and littermate controls at five different ages ranging from 2–8 months. Our data suggest that combined deficiencies of PSEN1 and PSEN2 results in a progressive, age-dependent transcriptome signature related to neurodegeneration and neuroinflammation. While these events may progress differently in the hippocampus and frontal cortex, the most critical expression signatures are common across the two brain regions, and involve a strong upregulation of cathepsin and complement system transcripts. Conclusion The observed neuroinflammatory expression changes are likely to be causally linked to the neurodegenerative phenotype observed in mice with compound deletions of PSEN1 and PSEN2. Furthermore, our results suggest that the evaluation of inhibitors of PS/γ-secretase activity for treatment of Alzheimer's Disease must include close monitoring for signs of calpain-cathepsin system activation.

  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. 慢性心房颤动右心房肌细胞内钙蛋白酶含量及细胞凋亡水平的变化%Changes in the Content of Calpain and the Level of Apoptosis in Right Atrium Myocytes in Patients with Chronic Atrial Fibrillation

    Institute of Scientific and Technical Information of China (English)

    吴敏; 陈鑫

    2007-01-01

    [目的]探讨风湿性二尖瓣病变伴心房颤动患者右心房心肌细胞中钙蛋白酶(Calpain)的含量变化及细胞凋亡水平的变化及相关关系.[方法]选择风湿性二尖瓣病变行二尖瓣置换手术的心脏病患者共30例,其中窦性心律(对照组)15例,慢性房颤(观察组)15例.在手术中取右心房组织,应用蛋白免疫印迹法测定各病人心肌细胞内Calpain I、Calpain Ⅱ的含量;应用TUNEL法检测各病人心房肌细胞凋亡指数(AI),并分析比较它们之间的关系.[结果]与对照组比较,观察组Calpain I含量明显上升(1.585±0.260 vs 1.191±0.099,P<0.01);Calpain Ⅱ含量无明显变化(O.845±0.071 vs 0.823士0.078,P=0.22);凋亡水平上升(34.00%±10.87%vs 15.53%±6.52%,P<0.01).慢性房颤组的Calpain I含量AI呈明显正相关(r=0.894,P<0.01).Calpain Ⅱ含量与AI无明显相关(r=0.24,P=0.39).[结论]Calpain I和凋亡可能在风湿性房颤的发展及维持中起重要作用,且二者关系密切.

  9. Embryo spacing and implantation timing are differentially regulated by LPA3-mediated lysophosphatidic acid signaling in mice.

    Science.gov (United States)

    Hama, Kotaro; Aoki, Junken; Inoue, Asuka; Endo, Tomoko; Amano, Tomokazu; Motoki, Rie; Kanai, Motomu; Ye, Xiaoqin; Chun, Jerold; Matsuki, Norio; Suzuki, Hiroshi; Shibasaki, Masakatsu; Arai, Hiroyuki

    2007-12-01

    In polytocous animals, blastocysts are evenly distributed along each uterine horn and implant. The molecular mechanisms underlying these precise events remain elusive. We recently showed that lysophosphatidic acid (LPA) has critical roles in the establishment of early pregnancy by affecting embryo spacing and subsequent implantation through its receptor, LPA3. Targeted deletion of Lpa3 in mice resulted in delayed implantation and embryo crowding, which is associated with a dramatic decrease in the prostaglandins and prostaglandin-endoperoxide synthase 2 expression levels. Exogenous administration of prostaglandins rescued the delayed implantation but did not rescue the defects in embryo spacing, suggesting the role of prostaglandins in implantation downstream of LPA3 signaling. In the present study, to know how LPA3 signaling regulates the embryo spacing, we determined the time course distribution of blastocysts during the preimplantation period. In wild-type (WT) uteri, blastocysts were distributed evenly along the uterine horns at Embryonic Day 3.8 (E3.8), whereas in the Lpa3-deficient uteri, they were clustered in the vicinity of the cervix, suggesting that the mislocalization and resulting crowding of the embryos are the cause of the delayed implantation. However, embryos transferred singly into E2.5 pseudopregnant Lpa3-deficient uterine horns still showed delayed implantation but on-time implantation in WT uteri, indicating that embryo spacing and implantation timing are two segregated events. We also found that an LPA3-specific agonist induced rapid uterine contraction in WT mice but not in Lpa3-deficient mice. Because the uterine contraction is critical for embryo spacing, our results suggest that LPA3 signaling controls embryo spacing via uterine contraction around E3.5.

  10. 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.

  11. Fetal origins of adult vascular dysfunction in mice lacking endothelial nitric oxide synthase.

    Science.gov (United States)

    Longo, Monica; Jain, Venu; Vedernikov, Yuri P; Bukowski, Radek; Garfield, Robert E; Hankins, Gary D; Anderson, Garland D; Saade, George R

    2005-05-01

    Epidemiological studies have shown increased incidence of hypertension and coronary artery disease in growth-restricted fetuses during their adult life. A novel animal model was used to test the hypothesis regarding the role of an abnormal uterine environment in fetal programming of adult vascular dysfunction. Mice lacking a functional endothelial nitric oxide synthase (NOS3-/-KO, where KO is knockout) and wild-type (WT) mice (NOS3+/+WT) were crossbred to produce homozygous NOS3-/-KO, maternally derived heterozygous (NOS3+/-mat, mother with NOS3 deficiency), paternally derived heterozygous (NOS3+/-pat, normal mother), and NOS3+/+WT litters. Number of fetuses per litter was smaller in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Adult female mice from these litters (7-8 wk old) were killed, and ring preparations of carotid and mesenteric arteries were mounted in a wire myograph to evaluate the passive and reactive vascular characteristics. Slope of the length-tension plot (a measure of vascular compliance) was increased, and optimal diameter (as calculated by Laplace equation) was decreased in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Acetylcholine caused vasorelaxation in NOS3+/-pat and NOS3+/+WT and contraction in NOS3-/-KO and NOS3+/-mat mice. Responses to phenylephrine and Ca2+ were increased in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Relaxation to isoproterenol was decreased in NOS3-/-KO and NOS3+/-mat vs. NOS3+/-pat and NOS3+/+WT mice. Abnormalities in the passive and reactive in vitro vascular properties seen in NOS+/-mat that developed in a NOS3-deficient maternal/uterine environment compared with the genetically identical NOS3+/-pat mice that developed in a normal environment are the first direct evidence in support of a role for uterine environment in determining vascular function in later life.

  12. 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

  13. Effects of soybean isoflavones on calpain expression and neuronal apoptosis in ischemic and reperfused brain tissues of ovariectomized female rats%大豆异黄酮对去势雌大鼠缺血再灌注脑组织细胞凋亡及calpain表达的影响

    Institute of Scientific and Technical Information of China (English)

    么晓轶; 于纪珠; 李颖

    2011-01-01

    目的:研究大豆异黄酮对大鼠脑缺血再灌注后缺血脑组织神经细胞凋亡和calpain表达的影响,探讨大豆异黄酮的神经保护作用.方法:成年雌性大鼠36只,随机分为药物组和对照组,药物组再随机分为小剂量和大剂量组.所有动物切除双侧卵巢,1周后各组每日给予大豆异黄酮和生理盐水,1个月后建立大脑缺血再灌注模型,原位末端标记检测神经细胞凋亡,采用原位杂交技术观察calpain mRNA表达情况,同时比较各组calpain活性变化.结果:大豆异黄酮组凋亡细胞数明显减少(P<0.05),神经细胞凋亡指数降低,calpain mRNA表达明显减少,calpain活性下降.药物大剂量组与小剂量组比较无明显差异.结论:大豆异黄酮使脑缺血再灌注后神经细胞凋亡减少,可能通过下调calpain mRNA表达,降低calpain的活性发挥神经保护作用.%AIM: To study the effects of soybean isoflavones on neuronal apoptosis and mRNA expression of calpain in transient focal cerebral ischemia and reperfusion ( FCIR ) rats. METHODS: Thirty - six adult healthy female SD rats were ovariectomized and randomly divided into soybean isoflavone - treated group and control group. The rats in soybean isoflavone - treated group were subdivided into low and high doses of soybean isoflavone groups. After 1 month of treatment, a FCIR model was established by intraluminal middle cerebral artery occlusion ( MCAO ) with a nylon monofila-ment suture. In situ hybridization was performed to examine the mRNA expression of calpain. Apoptosis was observed with terminal deoxynucleotidyl transferase - mediated dUTP nick - end labeling ( TUNEL ). The activity of calpain was also examined. RESULTS: The mRNA expression of calpain and TUNEL positive cells were observed in the cortex and striatum of ischemic hemisphere. Calpain mRNA was present at lower levels in soybean isoflavone - treated groups than that in control group ( P <0. 05 ). The number of apoptotic cells

  14. Effect of valsartan and carnitine on cardiomyocyte Calpain-1 and Bcl-xl expressions of dogs with chronic alcohol intake-induced cardiomyopathy%钙激活中性蛋白酶Calpain-1在犬酒精性心肌病心肌细胞凋亡中的表达及不同药物对其的干预作用

    Institute of Scientific and Technical Information of China (English)

    桑颖; 周立君; 井玲; 原琳; 鲁立新; 张庆辉

    2011-01-01

    目的 观察钙激活中性蛋白酶(Calpain-1)和凋亡相关蛋白在酒精性心肌病犬心肌细胞凋亡中的表达及缬沙坦和肉毒碱对其的干预作用.方法 实验犬28只,随机分为酒精组、缬沙坦+酒精组、肉毒碱+酒精组和正常对照组,每组7只动物,4组给予相同饮食,前3组通过采取逐渐增加饮用酒精浓度并长期定量摄入的方法建立酒精性心肌病模型,缬沙坦+酒精组、肉毒碱+酒精组分别给予缬沙坦及肉毒碱进行干预.6个月后病理观察各组犬心肌纤维化程度,电镜观察肌丝排列、润盘连接、细胞核、染色质、细胞器等超微结构、检测心功能和心肌细胞凋亡情况,Western blot法测定Calpain-1蛋白的表达,免疫组织化学法测定促凋亡蛋白(Bad)和抗凋亡蛋白(Bcl-xl)的表达.结果 酒精组、缬沙坦+酒精组和肉毒碱+酒精组左心窜射血分数和左心室短轴缩短率均低于正常对照组(P均<0.01),左心室舒张末期内径和左心室收缩末期内径则高于正常对照组(P均<0.01).缬沙坦+酒精组左心室射血分数和左心室短轴缩短率高于酒精组(P均<0.01),左心室舒张末期内径和左心室收缩末期内径则低于正常对照组(P均<0.01).肉毒碱+酒精组只有左心室射血分数和左心室短轴缩短率高于酒精组(P均<0.01).酒精组心肌细胞凋亡数、Bad和Calpain-1蛋白表达均明显高于正常对照组,而Bcl-xl蛋白明显低于正常对照组(P均<0.05).缬沙坦+酒精组和肉毒碱+酒精组心肌细胞凋亡数、Bad和Calpain-1蛋白表达明显低于酒精组,而Bcl-xl蛋白表达则明显高于酒精组(P均<0.05).结论 酒精加剧心肌细胞凋亡、心脏结构和功能恶化,缬沙坦和肉毒碱通过不同机制下调Calpain-1和Bad的蛋白表达,卜调Bcl-xl的蛋白表达,缬沙坦的干预效果优于肉毒碱.%Objective To evaluate the effects of valsartan and carnitine on cardiomyocyte Calpain-1and Bcl-xl expressions of

  15. Common polymorphisms of calpain-10 and the risk of Type 2 Diabetes in a Tunisian Arab population: a case-control study

    Directory of Open Access Journals (Sweden)

    Mahjoub Touhami

    2010-05-01

    Full Text Available Abstract Background Genetic variations in the calpain-10 gene (CAPN10, in particular the at-risk diplotype (112/121, were previously implicated with increased risk of type 2 diabetes (T2D. Methods We examined the association of CAPN10 UCSNP-43 (rs3792267, UCSNP-19 (rs3842570, and UCSNP-63 (rs5030952 SNPs with T2D in 917 Tunisian T2D patients and 748 non-diabetic controls. CAPN10 genotyping was done by PCR-RFLP. Results Enrichment of UCSNP-19 2R (minor allele and 2R/2R genotype was found in T2D patients; the allele and genotype distribution of UCSNP-43 and UCSNP-63 alleles and genotypes were not significantly different between patient groups and non-diabetic control subjects. Regression analysis demonstrated progressive increases in T2D risk in 3R/2R [OR (95% CI = 1.35 (1.08 - 1.68] and 2R/2R [OR (95% CI = 1.61 (1.20 - 2.18] genotypes. Of the six haplotypes detected, enrichment of haplotype 111 (UCSNP-43/UCSNP-19/UCSNP-63 was seen in patients (Pc = 0.034; the distribution of the other haplotypes was comparable between patients and control subjects; neither haplotype 211 nor haplotype 212 was observed. Furthermore, the frequency of all CAPN10 diplotypes identified, including the "high-risk diplotype (112/121 reported for Mexican-Americans and Northern Europeans, were comparable between patients and controls. Conclusions CAPN10 UCSNP-19 variant, and the 111 haplotype contribute to the risk of T2D in Tunisian subjects; no significant associations between CAPN10 diplotypes and T2D were demonstrated for Tunisians.

  16. 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

  17. 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.

  18. 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.

  19. 急性离心运动后骨骼肌超微结构、钙依赖性蛋白酶和泛素的动态变化%Dynamic Changes in Ultrastructure, Calpains and Ubiquitin in Skeletal Muscle after Acute Eccentric Exercise

    Institute of Scientific and Technical Information of China (English)

    金其贯; 刘霞; 李淑艳; 刘瑜

    2011-01-01

    Objective To investigate the dynamic changes in ultrastructure, calpains and ubiquitin in skeletal muscle after single bout of eccentric exercise. Methods Thirty male SD rats were randomly divided into control group, immediate post-exercise group, 24-hour post-exercise group and 7-day post-exercise group. Rats in exercise groups performed single bout of downhill running (16 m/ min, -16 degree) for 200 minutes. Quadriceps were drawn immediately 24 hours and 7 days after the eccentric exercise, respectively, and the changes in ultrastructure, serum LDH and CK activity, and calpain-1, calpain-2, and ubiquitin concentration quadriceps were observed. Results ① In quadriceps, mixed arrangement and curled filaments appeared immediately after eccentric exercise; mild dissolution, fracture, Z-line irregular, and partial disappearance of Z lines occurred 24 hours after eccentric exercise; there were no significant changes in control group 7 days aftereccentric exercise; the activities of serum CK and LDH changed consistently with the alteration of ultrastructures. ② As compared with the control group, the concentrations of calpain-1, calpain-2 and ubiquitin in quadriceps decreased immediately after exercise without significant statistical difference. The concentrations of calpain-1, calpain-2 and ubiquitin in quadriceps 24 hours post-exercise were significantly higher than that in control group and immediate post-exercise group. As compared with immediate post-exercise and 24-hour post-exercise, the contents of calpain-1, calpain-2 and ubiquitin in quadriceps significantly decreased 7 days post-exercise, whereas no significant statistical difference was found as compared with the control group. Conclusion ① The skeletal muscle damage induced by eccentric exercise appeared most seriously 24 hours after exercise and restored basically 7 days after exercise; ② The changes in calpain and ubiquitin contents in skeletal muscle were basically the same as the dynamic changes

  20. Studies on μ-Calpain Localization in Beef Muscle Fiber and Influence on Microstructure during Postmortem Aging%μ-Calpain在牛肉肌纤维中的定位及后熟过程中对肌纤维显微结构的影响

    I