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1.
Food Chem ; 306: 125616, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31622832

RESUMO

This research aimed to explore the role of protein S-nitrosylation in regulating the tenderness of postmortem beef, from the perspective of µ-calpain autolysis and protein proteolysis. Five bovine semimembranosus muscles were incubated with three treatments including S-nitrosoglutathione (GSNO, nitric oxide donor), normal saline and Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME, nitric oxide synthase inhibitor). The results showed that the level of protein S-nitrosylation was improved by GSNO treatment and reduced by L-NAME treatment (p < 0.05). Compared to the control, GSNO treatment had higher shear force while L-NAME treatment presented lower shear force at 7 d postmortem (p < 0.05). In addition, µ-calpain autolysis, myofibrillar protein and desmin degradation were reduced by GSNO treatment and accelerated by L-NAME treatment (p < 0.05). Therefore, it can be speculated that protein S-nitrosylation could affect beef tenderization by regulating the autolysis of µ-calpain and the degradation of myofibrillar proteins.


Assuntos
Proteína S/metabolismo , Carne Vermelha , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Calpaína/metabolismo , Bovinos , Desmina/metabolismo , Proteína S/química , Proteólise
2.
Adv Exp Med Biol ; 1185: 311-316, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884630

RESUMO

Autosomal dominant retinitis pigmentosa (adRP) is mainly caused by mutations responsible for rhodopsin (RHO) misfolding. Although it was previously proved that unfolded RHO is retained into the endoplasmatic reticulum (ER) eliciting ER-stress, consequent mechanisms underlying photoreceptor degeneration need to be further clarified. Several animal models of RHO mutants have been developed for this purpose and for development of neuroprotective treatments. Here, we compared two of the most used models of adRP, the P23H mutant RHO transgenic and knock-in mouse models, in order to define which are their limits and potentials. Although they were largely used, the differences on the activation of the cell death pathways occurring in these two models still remain to be fully characterized. We present data proving that activation of calpains is a mechanism of cell death shared by both models and that molecules targeting calpains are neuroprotective. Conversely, the role of ER-stress contribution to cell death appears to be divergent and remains controversial.


Assuntos
Calpaína/metabolismo , Morte Celular , Estresse do Retículo Endoplasmático , Retinite Pigmentosa/patologia , Rodopsina/metabolismo , Animais , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Camundongos , Camundongos Transgênicos , Dobramento de Proteína , Degeneração Retiniana , Retinite Pigmentosa/enzimologia , Rodopsina/genética
3.
Mem Inst Oswaldo Cruz ; 114: e190147, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31553371

RESUMO

BACKGROUND: Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. OBJECTIVES: Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. METHODS/FINDINGS: In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63. CONCLUSION: This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.


Assuntos
Calpaína/genética , Genoma de Protozoário/genética , Leishmania braziliensis/química , Macrófagos Peritoneais/metabolismo , Animais , Western Blotting , Calpaína/efeitos dos fármacos , Calpaína/metabolismo , Calpaína/ultraestrutura , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Citometria de Fluxo , Regulação da Expressão Gênica , Imuno-Histoquímica , Leishmania braziliensis/genética , Leishmania braziliensis/metabolismo , Leishmania braziliensis/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Virulência
4.
Adv Exp Med Biol ; 1155: 451-462, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468422

RESUMO

Objective To determine whether taurine has protective effects on chicken myocardial apoptosis induced by hypoxic condition through inhibiting calpain-1 derived mitochondrial apoptotic pathway. Methods Chicken primary embryonic myocardial cells were isolated and cultured at 37 °C under a 5% CO2 atmosphere. Firstly the optimum concentration of taurine or PD150606 was chosen by detecting the cell viability. Chicken cardiomyocytes were cultured in 95% N2-5% CO2 atmosphere for 12 h to produce hypoxic conditions. Before hypoxic treatment, 10 mM taurine and 10 uM PD150606 (a specific calpains inhibitor) were added separately or together. The cell apoptosis was detected by acridine orange/ethidium bromide (AO/EB) double staining. Western blotting was used to determine the protein expressions of calpain-1, cytochrome c, Bcl-2, procaspase-9 and procaspase-3 in the cardiomyocytes. Results Taurine administration effectively attenuated the myocardial apoptosis under hypoxic condition, reduced the calpain-1 protein level. In addition, pre-treated taurine could up-regulate the protein expressions of Bcl-2 and procaspase-3 in hypoxic myocardial cells, down-regulate protein expression levels of cytochrome c and procaspase-9. Moreover, taurine exhibited same inhibition effect as PD150606 on the cell apoptosis and proteins express under hypoxic condition. Conclusions Taurine could attenuate the chicken cardiomyocyte apoptosis impaired by hypoxia through inhibiting calpian-1-derived mitochondrial apoptotic pathway in vitro.


Assuntos
Apoptose , Calpaína/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Taurina/farmacologia , Acrilatos/farmacologia , Animais , Hipóxia Celular , Células Cultivadas , Galinhas , Mitocôndrias
5.
Parasit Vectors ; 12(1): 383, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31362766

RESUMO

BACKGROUND: Schistosoma mekongi, which causes schistosomiasis in humans, is an important public health issue in Southeast Asia. Treatment with praziquantel is the primary method of control but emergence of praziquantel resistance requires the development of alternative drugs and vaccines. Calcium-dependent cysteine protease (calpain) is a novel vaccine candidate that has been studied in S. mansoni, S. japonicum, and protozoans including malaria, leishmania and trypanosomes. However, limited information is available on the properties and functions of calpain in other Schistosoma spp., including S. mekongi. In this study, we functionally characterized calpain 1 of S. mekongi (SmeCalp1). RESULTS: Calpain 1 of S. mekongi was obtained from transcriptomic analysis of S. mekongi; it had the highest expression level of all isoforms tested and was predominantly expressed in the adult male. SmeCalp1 cDNA is 2274 bp long and encodes 758 amino acids, with 85% to 90% homology with calpains in other Schistosoma species. Recombinant SmeCalp1 (rSmeCalp1), with a molecular weight of approximately 86.7 kDa, was expressed in bacteria and stimulated a marked antibody response in mice. Native SmeCalp1 was detected in crude worm extract and excretory-secretory product, and it was mainly localized in the tegument of the adult male; less signal was detected in the adult female worm. Thus, SmeCalp1 may play a role in surface membrane synthesis or host-parasite interaction. We assessed the protease activity of rSmeCalp1 and demonstrated that rSmeCalp1 could cleave the calpain substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, that was inhibited by calpain inhibitors (MDL28170 and E64c). Additionally, rSmeCalp1 could degrade the biological substrates fibronectin (blood clotting protein) and human complement C3, indicating important roles in the intravascular system and in host immune evasion. CONCLUSIONS: SmeCalp1 is expressed on the tegumental surface of the parasite and can cleave host defense molecules; thus, it might participate in growth, development and survival during the entire life-cycle of S. mekongi. Information on the properties and functions of SmeCalp1 reported herein will be advantageous in the development of effective drugs and vaccines against S. mekongi and other schistosomes.


Assuntos
Antígenos de Helmintos/imunologia , Calpaína/genética , Calpaína/metabolismo , Schistosoma/enzimologia , Animais , Antígenos de Helmintos/genética , Cumarínicos/metabolismo , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Feminino , Imunização , Masculino , Camundongos , Camundongos Endogâmicos ICR , Oligopeptídeos/metabolismo , Schistosoma/genética , Esquistossomose/imunologia , Esquistossomose/parasitologia , Análise de Sequência de DNA
6.
Life Sci ; 232: 116662, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31323271

RESUMO

AIMS: Vascular endothelial cells act as a selective barrier between circulating blood and vessel wall and play an important role in the occurrence and development of cardiovascular diseases. Astragaloside IV (As-IV) has a protective effect on vascular endothelial cells, but its underlying mechanism remains unclear. This study is aimed at investigating the effect of As-IV on endothelial dysfunction (ED). METHODS: Male Sprague-Dawley (SD) were injected intraperitoneally with 65 mg/kg streptozotocin (STZ) to induce diabetes and then administered orally with As-IV (40, 80 mg/kg) for 8 weeks. Vascular function was evaluated by vascular reactivity in vivo and in vitro. The expression of calpain-1 and eNOS in the aorta of diabetic rats was examined by western blot. NO production was measured using nitrate reductase method. Oxidative stress was determined by measuring SOD, GSH-px and ROS. RESULTS: Our results showed that As-IV administration significantly improved diabetes associated ED in vivo, and both NAC (an antioxidant) and MDL-28170 (calpain-1 inhibitor) significantly attenuated hyperglycemia-induced ED in vitro. Meanwhile, pretreatment with the inhibitor l-NAME nearly abolished vasodilation to ACh in all groups of rats. Furthermore, As-IV increased NO production and the expression of eNOS in the thoracic aorta of diabetic rats. In addition, the levels of ROS were significantly increased, and the activity of SOD and GSH-px were decreased in diabetic rats, while As-IV administration reversed this change in a concentration-dependent manner. CONCLUSION: These results suggest that As-IV improves endothelial dysfunction in thoracic aortas from diabetic rats by reducing oxidative stress and calpain-1.


Assuntos
Calpaína/metabolismo , Endotélio Vascular/efeitos dos fármacos , Hiperglicemia/patologia , Estresse Oxidativo/efeitos dos fármacos , Saponinas/farmacologia , Triterpenos/farmacologia , Acetilcisteína/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Biomarcadores/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Diabetes Mellitus Experimental/metabolismo , Dipeptídeos/farmacologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Hiperglicemia/metabolismo , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Sprague-Dawley , Estreptozocina , Vasodilatação/efeitos dos fármacos
7.
Med Sci Monit ; 25: 5137-5142, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31292430

RESUMO

BACKGROUND This study aimed to investigate the association between the rs2975760 and rs3792267 single nucleotide polymorphisms (SNPs) of the calpain 10 (CAPN10) gene and gestational diabetes mellitus. MATERIAL AND METHODS The study included 138 patients with gestational diabetes mellitus and 152 healthy pregnant women. Venous blood was separated, and the DNA was extracted. The rs2975760 and rs3792267SNP polymorphisms of CAPN10 were detected using polymerase chain reaction (PCR). The frequencies of different genotypes in patients with gestational diabetes mellitus and healthy pregnant women were determined, and the relationship between different SNP genotypes and the risk of gestational diabetes mellitus was analyzed. RESULTS There were no significant differences in the frequencies of the TT, CT and CC genotypes of rs2975760 and the frequencies of the GG, AG and AA genotypes of rs3792267 between the women with gestational diabetes and the controls. Expression of rs2975760 and rs3792267 were not associated with the risk of gestational diabetes in the dominant model, recessive model, and additive model. However, grade B and grade D diabetes in the CC and TC genotypes of rs2975760 were significantly different from those in the TT genotype (P<0.05). Grade B and grade D diabetes in the AA and AG genotypes of rs3792267 were significantly different compared with those in the GG genotype (P<0.05), and allele A was significantly increased compared with allele G (P<0.05). CONCLUSIONS The rs2975760 and rs3792267 SNP polymorphisms of CAPN10 showed no significant association with the incidence of gestational diabetes mellitus and only a mild association with the severity.


Assuntos
Calpaína/genética , Diabetes Gestacional/genética , Adulto , Alelos , Grupo com Ancestrais do Continente Asiático/genética , Calpaína/metabolismo , Estudos de Casos e Controles , China , Diabetes Mellitus Tipo 2/genética , Feminino , Frequência do Gene/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Haplótipos , Humanos , Polimorfismo de Nucleotídeo Único/genética , Gravidez , Fatores de Risco
8.
Pak J Pharm Sci ; 32(3): 937-946, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31278703

RESUMO

Calpain 10 plays a role in insulin secretion, action and susceptibility to type 2 diabetes. The mechanism through which it influences the insulin secretion and action is not completely defined. A structural bioinformatics approach is applied to envision its mechanism of action using available tools on NCBI (blastp and blastn), EMBL-EBI, Ensembl, Swiss Model Repository websites, I-TASSER, PROCHECK program and Discovery Studio software. Homology of domain I and II of calpain10 (isoform a) was established with super family cysteine proteinase domains (II a and II b, e=1.30e-77, 1.00e-20). Remaining sequences of domain III and T from (isoform a and c) indicated some similarity (Avg. e=1.94e-37) to calpain large subunit domain III (PF01067), the isoform g (139 AA) showed similarity with a part of catalytic domain of cysteine protease super family (e-value 1.00e-20). Swiss-model repository for 3D structures of protein, showed structural resemblance of 29% with 1QXP template of mu-calpain, 27% with 1KFX of m-calpain and 32% with 2P0R of calpain 9 in complex with leupeptin. Models prepared through I-TASSER confirmed through Ramachandran (RC) plots. The calpain 10 isoforms a, c and g show partial structural and functional resemblance to m, mu and calpain 9. This information is useful to find new drugs for disease management.


Assuntos
Calpaína/química , Calpaína/metabolismo , Modelos Moleculares , Calpaína/genética , Biologia Computacional/métodos , Humanos , Conformação Proteica , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência
9.
Life Sci ; 233: 116631, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31278945

RESUMO

AIMS: Prior to reperfusion, Calpains remain inactive due to the acidic pH and elevated ionic strength in the ischemic myocardium; but Calpain is activated during myocardial reperfusion. The underlying mechanism of Calpain activation in the ischemia-reperfusion (I/R) is yet to be determined. Therefore, the present study aims to investigate the mechanism of Calpain in I/R-induced mice. MAIN METHODS: In order to detect the function of Calpain and the NLRP3/ASC/Caspase-1 axis in cardiomyocyte pyroptosis, endoplasmic reticulum (ER) stress and myocardial function, the cardiomyocytes were treated with hypoxia-reoxygenation (H/R), and NLRP3 were silenced, Calpain was overexpressed and Caspase-1 inhibitors were used to determine cardiomyocyte pyroptosis. The results obtained from the cell experiments were then verified with an animal experiment in I/R mice. KEY FINDINGS: There was an overexpression in Calpain, ASC, NLRP3, GRP78 and C/EBP homologous protein (CHOP) in cardiomyocytes following H/R. A significant increase was witnessed in lactic acid dehydrogenase (LDH) activity, cardiomyocyte pyroptosis rate, Calpain activity, reactive oxygen species (ROS) concentration, as well as activation of ER stress in cardiomyocytes after H/R. However, opposing results were observed in H/R cardiomyocytes that received siRNA Calpain, siRNA NLRP3 or Caspase-1 inhibitor treatment. Overall, the results obtained from the animal experiment were consistent with the results from the cell experiment. SIGNIFICANCE: The silencing of Calpain suppresses the activation of the NLRP3/ASC/Caspase-1 axis, thus inhibiting ER stress in mice and improving myocardial dysfunction induced by I/R, providing a novel therapeutic pathway for I/R.


Assuntos
Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Calpaína/antagonistas & inibidores , Caspase 1/química , Estresse do Retículo Endoplasmático , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Calpaína/genética , Calpaína/metabolismo , Caspase 1/genética , Caspase 1/metabolismo , Células Cultivadas , Inflamassomos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , RNA Interferente Pequeno/genética
10.
J Anim Sci ; 97(8): 3213-3227, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31212312

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) virus is one of the most economically significant pig pathogens worldwide. However, the metabolic explanation for reductions in tissue accretion observed in growing pigs remains poorly defined. Additionally, PRRS virus challenge is often accompanied by reduced feed intake, making it difficult to discern which effects are virus vs. feed intake driven. To account for this, a pair-fed model was employed to examine the effects of PRRS challenge and nutrient restriction on skeletal muscle and liver metabolism. Forty-eight pigs were randomly selected (13.1 ± 1.97 kg BW) and allotted to 1 of 3 treatments (n = 16 pigs/treatment): 1) PRRS naïve, ad libitum fed (Ad), 2) PRRS-inoculated, ad libitum fed (PRRS+), and 3) PRRS naïve, pair-fed to the PRRS-inoculated pigs' daily feed intake (PF). At days postinoculation (dpi) 10 and 17, 8 pigs per treatment were euthanized and tissues collected. Tissues were assayed for markers of proteolysis (LM only), protein synthesis (LM only), oxidative stress (LM only), gluconeogenesis (liver), and glycogen concentrations (LM and liver). Growth performance, feed intake, and feed efficiency were all reduced in both PRRS+ and PF pigs compared with Ad pigs (P < 0.001). Furthermore, growth performance and feed efficiency were additionally reduced in PRRS+ pigs compared with PF pigs (P < 0.05). Activity of most markers of LM proteolysis (µ-calpain, 20S proteasome, and caspase 3/7) was not increased (P > 0.10) in PRRS+ pigs compared with Ad pigs, although activity of m-calpain was increased in PRRS+ pigs compared with Ad pigs (P = 0.025) at dpi 17. Muscle reactive oxygen species production was not increased (P > 0.10) in PRRS+ pigs compared with Ad pigs. However, phosphorylation of protein synthesis markers was decreased in PRRS+ pigs compared with both Ad (P < 0.05) and PF (P < 0.05) pigs. Liver gluconeogenesis was not increased as a result of PRRS; however, liver glycogen was decreased (P < 0.01) in PRRS+ pigs compared with Ad and PF pigs at both time points. Taken together, this work demonstrates the differential impact a viral challenge and nutrient restriction have on metabolism of growing pigs. Although markers of skeletal muscle proteolysis showed limited evidence of increase, markers of skeletal muscle synthesis were reduced during PRRS viral challenge. Furthermore, liver glycogenolysis seems to provide PRRS+ pigs with glucose needed to fuel the immune response during viral challenge.


Assuntos
Gluconeogênese , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Proteólise , Animais , Biomarcadores/metabolismo , Calpaína/metabolismo , Ingestão de Alimentos , Feminino , Fígado/metabolismo , Músculo Esquelético/metabolismo , Estresse Oxidativo , Síndrome Respiratória e Reprodutiva Suína/virologia , Distribuição Aleatória , Suínos
11.
J Food Sci ; 84(5): 1054-1059, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31042817

RESUMO

This study was designed to determine the effects of µ/m-calpain on the degradation of cytoskeletal proteins in pectoralis major. Four chickens were slaughtered and the breasts were removed and stored for 12 hr at 4 °C. Each sample was divided into three groups and respectively immersed in control reagent, calpain inhibitor, and caspase inhibitor at 4 °C. The samples were used to evaluate troponin-T and desmin degradation, calpain activity, and myofibril ultrastructure at 12 hr, day 1, day 3, and day 7. Casein zymography revealed that µ-calpain could not be detected in all samples after 12 hr postmortem. The calpain inhibitor inhibited µ/m-calpain activity and reduced troponin-T and desmin degradation during 7 day postmortem. The caspase inhibitor inhibited µ/m-calpain activity and, troponin-T and desmin degradation before day 3 postmortem. The findings suggest that, µ/m-calpain had an effect on cytoskeletal protein degradation after 12 hr postmortem.


Assuntos
Calpaína , Carne/análise , Proteínas Musculares , Animais , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Galinhas , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Músculo Esquelético/química , Músculo Esquelético/metabolismo
12.
Meat Sci ; 155: 50-60, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31075739

RESUMO

The aim of this study was to determine the extent to which calpastatin (CASN) variants (based on two chromatographic peaks; CASN-P1 and CASN-P2) explain variation in µ-calpain autolysis, protein degradation, and changes in the sarcoplasmic proteome observed during postmortem aging of beef. The Longissimus lumborum (LL) and Triceps brachii (TB) muscles were obtained from six crossbred steers and samples prepared from day 0, 1 and 7 postmortem (pm). The decline of CASN activity during aging was due to decrease of CASN-P2 in both muscles. The CASN-P2:µ-calpain ratio at day 0 was greater for TB, which presented lesser calpain autolysis, myofibrillar protein degradation, and fewer sarcoplasmic proteome changes during aging. Changes in abundance of Heat shock protein 70 family in the sarcoplasmic fraction were positively associated to proteolysis during aging, with greater differences in LL.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/metabolismo , Músculo Esquelético/química , Carne Vermelha/análise , Animais , Bovinos , Proteínas de Choque Térmico HSP70/análise , Masculino , Miofibrilas , Mudanças Depois da Morte , Proteólise , Proteoma
13.
Poult Sci ; 98(11): 6131-6137, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31111925

RESUMO

Meat tenderization can be affected by a variety of factors including animal age. However, results obtained from goose, chicken, turkey, and ostrich studies have shown that the effects of age on meat tenderization were insignificant, which may be due to a very limited age difference in birds used in those studies. Therefore, the purpose of this study was to investigate the effects of animal age on postmortem proteolysis and tenderization of breast muscle from developing and mature White Roman geese. Goose carcasses from mature (50 mo old, n = 10) and young (3 mo old, n = 10) geese were vacuum-packaged and stored at 5°C within 10 min postmortem. Breast (pectoralis major) samples were taken at 0 (∼10 min postmortem), 1, 3, and 7 D of storage. Our results showed that the decrease in pH, calpain-1 and -11 activities, desmin content and shear force, as well as the increase in myofibrillar fragmentation index were more rapid (P < 0.05) in young than in mature goose breast. These results suggest that postmortem proteolysis and tenderization of goose muscle are extensively affected by age.


Assuntos
Calpaína/metabolismo , Gansos/fisiologia , Músculo Esquelético/fisiologia , Fatores Etários , Animais , Masculino , Carne/análise , Mudanças Depois da Morte , Proteólise
14.
Poult Sci ; 98(10): 4327-4337, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31111951

RESUMO

Consumer preference for slow-growing broiler chickens is rising because of increased demand for high-quality poultry products. Korat chicken (KRC) is a slow-growing chicken generated in Thailand. A goal of the KRC breeding program is to produce meat with a low purine content to benefit an aging population, without interfering with growth performance. Thus, this study aimed to investigate the effects of genes encoding melanocortin 4 receptor (MC4R), calpain 1 (CAPN1), and adenylosuccinate lyase (ADSL) on body weight, muscle fiber, and content of purine and its derivatives (i.e., adenine, guanine, hypoxanthine, and xanthine), to develop molecular markers for breeding programs. Genotypes of MC4R, CAPN1, and ADSL were obtained from 583 KRCs by PCR-single-strand conformation polymorphism. The body weight and purine contents of the KRCs were measured every 2 wk until the KRCs reached market weight at 10 wk of age. A significant association between the MC4R genotype and body weight at 2, 4, and 10 wk of age was detected. KRC possessing the BB genotype of CAPN1 showed significantly heavier body weight at 6 wk of age and guanine content at 4 wk of age, and a smaller muscle fiber diameter in the breast muscle at 10 wk of age, compared with those of the other genotypes. In addition, high expression levels of the CAPN1 and ADSL genes were detected in the breast muscle at 2 wk of age. Although higher purine contents were detected at a young age, no significant associations with the MC4R, CAPN1, and ADSL genes were detected. Our results indicate that MC4R and CAPN1 could be used as genetic markers for growth and meat quality in the slow-growing chicken breeding program.


Assuntos
Proteínas Aviárias/genética , Peso Corporal/genética , Galinhas/fisiologia , Purinas/metabolismo , Adenilossuccinato Liase/genética , Adenilossuccinato Liase/metabolismo , Animais , Proteínas Aviárias/metabolismo , Calpaína/genética , Calpaína/metabolismo , Galinhas/genética , Galinhas/crescimento & desenvolvimento , Feminino , Marcadores Genéticos , Genótipo , Masculino , Carne/análise , Polimorfismo Conformacional de Fita Simples , Receptor Tipo 4 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/metabolismo
15.
Biol Pharm Bull ; 42(8): 1282-1294, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31105116

RESUMO

The purpose of the paper is to study the differences in cell death mechanism of MGC-803 induced by "dextran-magnetic layered double hydroxide-fluorouracil" (DMF) drug delivery system and 5-Fluorouracil (5-Fu), respectively. The inhibitory effect on the proliferation was detected via CCK-8. The morphology of cell death was detected by transmission electron microscopy (TEM). Intracellular ATP, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and Cytosolic Free Ca (Ca2+) level were detected via some methods. The result showed that DMF had more obvious effect in suppressing proliferation compared with 5-Fu, and changed cell death pattern of 5-Fu from apoptosis to oncosis. The ATP decrease, MMP loss, Ca2+ increase, the activation of uncoupling protein-2 (UCP-2) and calpain-1 were significant after DMF exposure. However, DMF did not result in ROS accumulation. DMF could involve in activation of porimin, and the cascade reaction of caspases-3, -7, -9, and -12 and poly ADP-ribose polymerase (PARP) through Western blot. DMF showed a stronger injury on nuclear membrane in the cascade reaction of caspases-6, -8 and lamin-A. DMF triggered rapid depletion of ATP, which was consistent with the phenotype of oncosis. Endogenous mitochondrial apoptosis might not be the main cause of cell swelling. DMF could induce strong endoplasmic reticulum stress (ERS) effect, there might be some signaling pathways related with ERS during the process of oncosis. The calpain system might not be a key factor for structural damage in oncosis induced by DMF. DMF could induce the caspases cascade reactions similar to apoptosis, but inflicted a more strong damage on nuclear membrane and PARP.


Assuntos
Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dextranos/farmacologia , Fluoruracila/farmacologia , Hidróxidos/farmacologia , Apoptose/efeitos dos fármacos , Calpaína/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Humanos , Lamina Tipo A/metabolismo , Mitocôndrias/metabolismo , Fenótipo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas
16.
Anim Sci J ; 90(6): 728-736, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31006927

RESUMO

This study evaluated the effects of rice whole crop silage (RWCS) on growth, plasma levels of vitamin A, ß-carotene, vitamin E and IGF-1, and expression of genes involved in muscle protein degradation and synthesis in Japanese Black calves. Eleven calves were divided into RWCS (fed RWCS ad libitum and concentrate, n = 5) and control groups (fed hay ad libitum and concentrate, n = 6). Final body weight and dairy gain were significantly larger in the RWCS group compared with the control group. Plasma ß-carotene and vitamin E concentrations were significantly higher in the RWCS group compared with control group. Although plasma vitamin E concentration in the RWCS group significantly increased from 4 to 9 months of age, it did not increase in the control group. At 6 months of age in the RWCS group, ubiquitin B (p < 0.05) and calpain 1 (p = 0.097) mRNA expression were lower than control group, but they were not different between groups at 9 months of age. These results indicate that RWCS increases plasma ß-carotene level and promotes muscle growth because of a decrease in the rate of protein degradation, but the effect is lost with the increase in plasma vitamin E level.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal/fisiologia , Bovinos/crescimento & desenvolvimento , Bovinos/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Oryza , Proteólise , Silagem , Vitamina A/metabolismo , Vitamina E/metabolismo , beta Caroteno/metabolismo , Fenômenos Fisiológicos da Nutrição Animal/genética , Animais , Calpaína/genética , Calpaína/metabolismo , Expressão Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo
17.
Biochim Biophys Acta Mol Cell Res ; 1866(8): 1260-1271, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30959065

RESUMO

Several human acute and chronic diseases involve calpain over-activation. However, the mechanistic linkages between the etiology and the progression of cell damages are not yet completely understood. Here we show that different human cells and tissues, including brain tumor specimens, cell lines of nerve origin, breast tumor samples and peripheral blood mononuclear cells from healthy donors, express a calpastatin form that lacks all the exons coding for the domains responsible of calpain inhibition. The open reading frame of this new form of calpastatin, named hcast 3-25, starts inside the L-domain (exons 2 and 3) and continues with the exons from 25 to 29 that code for the conserved C-terminal tail shared by all the full-length calpastatins. We have here observed that unlike the other calpastatins forms, that are predominantly Δ3 splice variants, hcast 3-25 is endowed with exon 3. At a functional level, recombinant hcast 3-25 operates as a positive modulator of calpain-1 in vitro by preventing 1) calpain-1-mediated proteolytic degradation of the activated enzyme and 2) binding to calpain-1 of inhibitory calpastatins that contain the L-domain. Thus hcast 3-25 can be considered as a novel member and possible modulator of the calpain/calpastatin system acting by a mechanism alternative to inhibition.


Assuntos
Neoplasias Encefálicas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/metabolismo , Proteínas de Neoplasias/metabolismo , Proteólise , Neoplasias Encefálicas/genética , Proteínas de Ligação ao Cálcio/genética , Calpaína/genética , Linhagem Celular Tumoral , Humanos , Proteínas de Neoplasias/genética , Domínios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
18.
Meat Sci ; 153: 144-151, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30946977

RESUMO

A study was conducted to elucidate the impact of pulsed electric field (PEF) on the activity of calpains, proteolytic activity of desmin and troponin-T and physicochemical properties of beef Biceps femoris during ageing. The meat samples (meat blocks) were subjected to two PEF treatments; T1 (5 kV, 90 Hz, 0.38 kV/cm) and T2 (10 kV, 20 Hz, 0.61 kV/cm) and a non-treated control was run in parallel. The samples were vacuum packaged and aged for 1, 7 and 14 days at 4 ±â€¯1 °C. This study reports for the first time the impact of PEF-processing on the calpain activity in beef. Early post-mortem activation of calpain 2 was observed in PEF-treated samples. An increase in the calpain activity and proteolysis of desmin and troponin-T was observed. No significant effect of PEF was observed on the shear force of tough muscles from culled dairy animals during the entire ageing period.


Assuntos
Calpaína/metabolismo , Eletricidade , Manipulação de Alimentos/métodos , Carne Vermelha/análise , Animais , Bovinos , Desmina/metabolismo , Músculo Esquelético/metabolismo , Proteólise , Resistência ao Cisalhamento , Troponina T/metabolismo
19.
Blood ; 133(20): 2222-2232, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30819925

RESUMO

l-Asparaginase (l-ASNase) is a strategic component of treatment protocols for acute lymphoblastic leukemia (ALL). It causes asparagine deficit, resulting in protein synthesis inhibition and subsequent leukemic cell death and ALL remission. However, patients often relapse because of the development of resistance, but the underlying mechanism of ALL cell resistance to l-asparaginase remains unknown. Through unbiased genome-wide RNA interference screening, we identified huntingtin associated protein 1 (HAP1) as an ALL biomarker for l-asparaginase resistance. Knocking down HAP1 induces l-asparaginase resistance. HAP1 interacts with huntingtin and the intracellular Ca2+ channel, inositol 1,4,5-triphosphate receptor to form a ternary complex that mediates endoplasmic reticulum (ER) Ca2+ release upon stimulation with inositol 1,4,5-triphosphate3 Loss of HAP1 prevents the formation of the ternary complex and thus l-asparaginase-mediated ER Ca2+ release. HAP1 loss also inhibits external Ca2+ entry, blocking an excessive rise in [Ca2+]i, and reduces activation of the Ca2+-dependent calpain-1, Bid, and caspase-3 and caspase-12, leading to reduced number of apoptotic cells. These findings indicate that HAP1 loss prevents l-asparaginase-induced apoptosis through downregulation of the Ca2+-mediated calpain-1-Bid-caspase-3/12 apoptotic pathway. Treatment with BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)] reverses the l-asparaginase apoptotic effect in control cells, supporting a link between l-asparaginase-induced [Ca2+]i increase and apoptotic cell death. Consistent with these findings, ALL patient leukemic cells with lower HAP1 levels showed resistance to l-asparaginase, indicating the clinical relevance of HAP1 loss in the development of l-asparaginase resistance, and pointing to HAP1 as a functional l-asparaginase resistance biomarker that may be used for the design of effective treatment of l-asparaginase-resistant ALL.


Assuntos
Antineoplásicos/uso terapêutico , Asparaginase/uso terapêutico , Proteínas do Tecido Nervoso/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Adulto , Calpaína/metabolismo , Caspases/metabolismo , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Adulto Jovem
20.
Basic Res Cardiol ; 114(3): 17, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874894

RESUMO

We and others have reported that calpain-1 was increased in myocardial mitochondria from various animal models of heart disease. This study investigated whether constitutive up-regulation of calpain-1 restricted to mitochondria induced myocardial injury and heart failure and, if so, whether these phenotypes could be rescued by selective inhibition of mitochondrial superoxide production. Transgenic mice with human CAPN1 up-regulation restricted to mitochondria in cardiomyocytes (Tg-mtCapn1/tTA) were generated and characterized with low and high over-expression of transgenic human CAPN1 restricted to mitochondria, respectively. Transgenic up-regulation of mitochondria-targeted CAPN1 dose-dependently induced cardiac cell death, adverse myocardial remodeling, heart failure, and early death in mice, the changes of which were associated with mitochondrial dysfunction and mitochondrial superoxide generation. Importantly, a daily injection of mitochondria-targeted superoxide dismutase mimetics mito-TEMPO for 1 month starting from age 2 months attenuated cardiac cell death, adverse myocardial remodeling and heart failure, and reduced mortality in Tg-mtCapn1/tTA mice. In contrast, administration of TEMPO did not achieve similar cardiac protection in transgenic mice. Furthermore, transgenic up-regulation of mitochondria-targeted CAPN1 induced a reduction of ATP5A1 protein and ATP synthase activity in hearts. In cultured cardiomyocytes, increased calpain-1 in mitochondria promoted mitochondrial permeability transition pore (mPTP) opening and induced cell death, which were prevented by over-expression of ATP5A1, mito-TEMPO or cyclosporin A, an inhibitor of mPTP opening. In conclusion, this study has provided direct evidence demonstrating that increased mitochondrial calpain-1 is an important mechanism contributing to myocardial injury and heart failure by disrupting ATP synthase, and promoting mitochondrial superoxide generation and mPTP opening.


Assuntos
Calpaína/metabolismo , Cardiomiopatia Dilatada/etiologia , Insuficiência Cardíaca/etiologia , Mitocôndrias/metabolismo , Animais , Cardiomiopatia Dilatada/metabolismo , Morte Celular , Óxidos N-Cíclicos , Modelos Animais de Doenças , Insuficiência Cardíaca/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Miócitos Cardíacos/metabolismo , Superóxidos/metabolismo
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