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1.
Food Chem ; 313: 125956, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31923864

RESUMO

Crude enzymes were extracted from beef, pork and chicken and were employed to hydrolyze 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC) and oxidized PLPC, i.e. hydroperoxide of PLPC (PLPC-OOH) and hydroxide of PLPC (PLPC-OH). HPLC-ELSD and ESI-MS were used to characterize and determinate hydrolytic products. After hydrolysis at 37 °C for 180 min, 26.8 ~ 27.4%, 21.6 ~ 22.8% and 17.8 ~ 19.0% of substrates were hydrolyzed by crude enzymes from beef, pork and chicken, respectively. Phospholipase A2 (PLA2) was the major contributor to hydrolysis, which accounted for 47.8 ~ 49.6%, 45.8 ~ 48.7% and 46.6 ~ 46.8% of hydrolysis of PLPC, PLPC-OOH and PLPC-OH, respectively. Crude enzymes demonstrated almost same specificities towards PLPC, PLPC-OOH and PLPC-OH. Under actions of crude enzymes, hydroperoxyoctadecadienoic acids (HpODE) and hydroxyoctadecadienoic acids (HODE) were yielded as hydrolytic products of PLPC-OOH and PLPC-OH, respectively. These finding would be helpful to better understand the fate of hydroperoxides of phospholipids and formation of HODE during meat products manufacturing.


Assuntos
Músculos/enzimologia , Fosfatidilcolinas/metabolismo , Fosfolipases A2/metabolismo , Animais , Bovinos , Galinhas , Cromatografia Líquida de Alta Pressão , Ácidos Graxos Insaturados/análise , Ácidos Graxos Insaturados/metabolismo , Hidrólise , Fosfatidilcolinas/química , Suínos
2.
Aquat Toxicol ; 218: 105359, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31765944

RESUMO

Carbohydrate metabolism switches from aerobic to anaerobic (glycolysis) to supply energy in response to acute hypoxic stress. Acute hypoxic stress with dissolved oxygen (DO) levels of 1.2 ±â€¯0.1 mg/L for 24 h and 12 h re-oxygenation was used to investigate the response of the anaerobic glycolytic pathway in Micropterus salmoides muscle. The results showed that the glucose concentration was significantly lower in muscle, while the lactic acid and pyruvic acid concentrations tended to increase during hypoxic stress. No significant difference was observed in muscle glycogen, and ATP content fluctuated significantly. The activities of gluconeogenesis-related enzymes were slightly elevated, such as phosphoenolpyruvate carboxykinase (PEPCK). The activities of the glycolytic enzymes increased after the induction of hypoxia, such as hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH). Curiously, phosphofructokinase (PFK) activity was significantly down-regulated within 4 h during hypoxia, although these effects were transient, and most indices returned to control levels after 12 h of re-oxygenation. Upregulated hif-1α, ampkα, hk, glut1, and ldh mRNA expression suggested that carbohydrate metabolism was reprogrammed under hypoxia. Lactate transport was regulated by miR-124-5p according to quantitative polymerase chain reaction and dual luciferase reporter assays. Our findings provide new insight into the molecular regulatory mechanism of hypoxia in Micropterus salmoides muscle.


Assuntos
Aclimatação/fisiologia , Bass/metabolismo , Hipóxia/metabolismo , Ácido Láctico/metabolismo , MicroRNAs/genética , Transportadores de Ácidos Monocarboxílicos/genética , Músculos/metabolismo , Simportadores/genética , Aclimatação/genética , Animais , Bass/genética , Metabolismo dos Carboidratos/genética , Metabolismo dos Carboidratos/fisiologia , Regulação da Expressão Gênica , Hipóxia/genética , Músculos/enzimologia , Oxigênio/metabolismo
3.
Adv Neurobiol ; 23: 125-145, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31667807

RESUMO

Glycogen constitutes the main store of glucose in animal cells. Being present at much lower concentrations in the brain than in liver and muscles, brain glycogen has long been considered as an emergency source of glucose, mobilized under stress conditions (including hypoglyceamia). Nevertheless, over the past decade, multiple studies have shed a new light on the roles of brain glycogen, being notably an energy supply critical for high-cognitive processes such as learning and memory consolidation. Glycogen phosphorylase (GP) is the key enzyme regulating the mobilization of glycogen in cells. It is found in humans as three isozymes: muscle (mGP), liver (lGP) and brain GP (bGP). In the brain, astrocytes express both mGP and bGP while neurons only express the brain isoform. Although GP isozymes are very similar, their distinct regulatory features confer them distinct metabolic functions that are strongly related to the roles of glycogen in different tissues. Here, we provide an overview of the functions, the regulations and the structures of GPs in the brain and their relation to the specific roles of glycogen in astrocytes and neurons. We also discuss novel findings concerning the specific regulations of bGP by oxidative stress, and the potential of these enzymes as therapeutic targets in the brain.


Assuntos
Encéfalo/enzimologia , Glicogênio Fosforilase/química , Glicogênio Fosforilase/metabolismo , Glicogênio , Animais , Encéfalo/metabolismo , Glicogênio/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Músculos/enzimologia , Músculos/metabolismo
4.
Artigo em Inglês | MEDLINE | ID: mdl-31422160

RESUMO

In this paper, the total carbonic anhydrase (CA) enzyme was purified from horse mackerel (Trachurus trachurus) muscle with a specific activity of 23,063.93 EU/mg, purification fold of 551.08, total activity of 1522.22 EU/mL and a yield of 18.50% using sulfanilamide affinity column chromatography. For obtaining the subunit molecular mass and enzyme purity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for this part was performed and a single band was clearly recorded. The molecular mass of this enzyme was found approximately 35 kDa. The optimum temperature and pH values were obtained from Arrhenius plot. In addition, the inhibitory effects of different heavy metal ions (Fe2+, Cu2+, Co2+, Pb2+ Hg2+ and As3+) and some pesticides (thiram, clofentezine, propineb, deltamethrin, azoxystrobin and thiophanate) on horse mackerel (Trachurus trachurus) muscle tissue CA enzyme activities were investigated by utilizing esterase assay activity. The used metal ions and pesticides had IC50 values in the range of 0.21-13.84 mM and 3.78-70.58 mM, respectively.


Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/isolamento & purificação , Perciformes/metabolismo , Animais , Inibidores da Anidrase Carbônica/química , Cinética , Metais Pesados/química , Músculos/enzimologia , Praguicidas/química
5.
Biomed Res Int ; 2019: 6289380, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31275980

RESUMO

Early recovery from muscular injury is crucial for elite athletes. Hyperbaric oxygen therapy (HBOT) has been reported to be beneficial in terms of accelerating cell recovery and tissue repair, which are considered to be helpful for eliminating fatigue and recovering stamina. This study was performed to evaluate the efficacy of HBOT for exercise-related muscular injury. Forty-one athletes with exercise-related muscular injuries were recruited and randomized into an HBOT group and a control group. All participants received 10 sessions of either HBOT or placebo treatment. The brief pain inventory (BPI) was completed, and serum samples were analyzed. Data were collected before treatment (T1), at the end of the fifth treatment session (T2), at the end of the tenth treatment session (T3), and two weeks after T3 (T4). At T3, the HBOT group showed prominent reductions in the levels of creatine phosphokinase (CK), glutamic oxaloacetate transaminase (GOT), and myoglobin (MB), which lasted until T4. However, the control group did not present any statistical differences in levels from T1 to T4. In terms of pain intensity and interference, the HBOT group showed significant improvements at T3, while no improvements were observed in the control group. In conclusion, HBOT facilitates the early recovery of exercise-related muscular injury. This trial is registered with ISRCTN17817041.


Assuntos
Exercício/fisiologia , Oxigenação Hiperbárica , Músculos/lesões , Adulto , Enzimas/sangue , Humanos , Masculino , Músculos/enzimologia , Dor/patologia , Medição da Dor , Resultado do Tratamento , Adulto Jovem
6.
Food Chem ; 293: 537-544, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151645

RESUMO

To verify the effect of protein phosphorylation on glycolysis and elucidate the regulatory mechanism from the perspective of enzyme activity, ovine muscle was treated with a kinase inhibitor, dimethyl sulfoxide, or a phosphatase inhibitor and the activities of glycogen phosphorylase, pyruvate kinase and phosphofructokinase were determined. The protein phosphorylation level was significantly different after incubation of muscle with kinase or phosphatase inhibitors. The pH value and lactate content revealed that a high phosphorylation level was the reason for the fast glycolysis. The glycogen phosphorylase, pyruvate kinase and phosphofructokinase activities were significantly higher in the phosphatase inhibitor group than in the other two groups (p < 0.05). Therefore, protein phosphorylation is involved in activating these three enzymes. In summary, protein phosphorylation plays a role in post-mortem glycolysis through the regulation of enzyme activity in ovine muscle.


Assuntos
Glicogênio Fosforilase/metabolismo , Músculos/enzimologia , Fosfofrutoquinases/metabolismo , Piruvato Quinase/metabolismo , Animais , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Glicólise/efeitos dos fármacos , Fosfofrutoquinases/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piruvato Quinase/antagonistas & inibidores , Ovinos
7.
Artigo em Inglês | MEDLINE | ID: mdl-31059784

RESUMO

This study evaluated the efficacy of replacing dietary fish oil (FO) with vegetable oils (virgin coconut and corn oil) on enzyme activities (glycolytic, oxidative and lipid metabolites), mRNA expression of lipid metabolic genes and histomorphology of liver and intestine in O. niloticus. O. niloticus (6.07 ±â€¯0.07 g) was fed six experimental diets where fish oil (FO) served as the control diet, and then was supplemented by dietary oils; virgin coconut oil (VCO) {3%FO + 3%VCO; 3FVCO}, and corn oil (CO) {3%FO + 3%CO; 3FCO}, 6%VCO (VCO), 6%CO (CO) and 6%VO {3%VCO + 3%CO; VO}. Growth performances measured indicated fish fed diet 3FCO had higher weight gain (WG) and specific growth rate (SGR). Fish fed diet 3FCO recorded the highest activities in lactate dehydrogenase (LDH), pyruvate kinase (PK), citrate synthase (CS), cytochrome coxidase (COX), malic enzymes (ME) and lipoprotein lipase (LPL) respectively. Stearoyl-CoA desaturase (SCD1) was upregulated in groups fed diets 3FVCO and 3FCO. Also, groups fed diet VCO and CO expressed highly in LPL, whereas, elongase of very long-chain fatty acids (ELOVL-5) was not influenced by the lipid sources. Histological representations in the liver were highly impacted in vegetable diets where lipid accumulation was higher except those fed VCO. However, in the digestive tract from distal to middle and posterior, the same group (VCO) exhibited altered morphological structure as those fed diet 3FCO were similar to FO. The study shows that, corn oil in diets relates positively to growth and enzymatic activities which becomes evident in their depositions in liver and functional intestinal tracts. This study indicates dietary alternatives may cause alterations in lipid metabolic pathways (LPL and SCD1) involved in fatty acid transport. As such, polyunsaturated fatty acid (PUFA) rich diets (CO) based on this study results increases metabolic activities involving especially the production, distribution and consumption of adenosine triphosphate (ATP) in O. niloticus.


Assuntos
Ciclídeos/fisiologia , Gorduras Insaturadas na Dieta/farmacologia , Proteínas de Peixes/genética , Fígado/enzimologia , Músculos/enzimologia , Trifosfato de Adenosina/metabolismo , Ração Animal , Animais , Peso Corporal/efeitos dos fármacos , Ciclídeos/genética , Ciclídeos/crescimento & desenvolvimento , Óleo de Coco/farmacologia , Óleo de Milho/farmacologia , Enzimas/metabolismo , Óleos de Peixe/farmacologia , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Intestinos/citologia , Intestinos/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Fígado/citologia , Fígado/efeitos dos fármacos , Músculos/efeitos dos fármacos
8.
J Hum Genet ; 64(7): 637-645, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30948790

RESUMO

The genetic causes of Leigh syndrome are heterogeneous, with a poor genotype-phenotype correlation. To date, more than 50 nuclear genes cause nuclear gene-encoded Leigh syndrome. NDUFS6 encodes a 13 kiloDaltons subunit, which is part of the peripheral arm of complex I and is localized in the iron-sulfur fraction. Only a few patients were reported with proven NDUFS6 pathogenic variants and all presented with severe neonatal lactic acidemia and complex I deficiency, leading to death in the first days of life. Here, we present a patient harboring two NDUFS6 variants with a phenotype compatible with Leigh syndrome. Although most of previous reports suggested that NDUFS6 pathogenic variants invariably lead to early neonatal death, this report shows that the clinical spectrum could be larger. We found a severe decrease of NDUFS6 protein level in patient's fibroblasts associated with a complex I assembly defect in patient's muscle and fibroblasts. These data confirm the importance of NDUFS6 and the Zn-finger domain for a correct assembly of complex I.


Assuntos
Doença de Leigh/genética , NADH Desidrogenase/genética , Acidose Láctica/genética , Núcleo Celular/genética , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/genética , Fibroblastos/enzimologia , Estudos de Associação Genética , Humanos , Lactente , Doença de Leigh/diagnóstico por imagem , Doença de Leigh/enzimologia , Masculino , Mitocôndrias/genética , Músculos/enzimologia , NADH Desidrogenase/metabolismo , Domínios Proteicos/genética , Análise de Sequência de DNA
9.
Carbohydr Res ; 477: 58-65, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31005807

RESUMO

Glycogen phosphorylase enzymes (GP) catalyse reversible reactions; the glucose transfer from glycogen to inorganic phosphate (Pi, phosphorolysis) or the reverse glucose transfer from glucose-1-phosphate (G-1-P) to glycogen (synthesis). Rabbit muscle GPb (rmGPb) was used as a model enzyme to study the reversible enzyme reaction. To follow both directions of this reversible reaction, we have developed a novel isothermal titration calorimetry (ITC) method for the determination of the direct reaction rate. The preference of forward or reverse reaction was ensured by the 0.1 or 10 concentration ratios of G-1-P/Pi, respectively. Substrate specificity was studied using different maltooligosaccharides and glycogen. Based on the KM values, glycogen and 2-chloro-4-nitrophenyl maltoheptaoside (CNP-G7) were found to be analogous substrates, which allowed to optimize the method by taking advantage of the CNP chromophore being detectable in HPLC. In case of CNP-G7, substrate inhibition was observed and characterised by Ki of 23 ±â€¯7 mM. Inhibition of human GP is a promising strategy for the treatment of diabetes. Our ITC measurements have confirmed that caffeine and glucopyranosylidene-spiro-thiohydantoin (GTH), as known GPb inhibitors, inhibit the rmGPb-catalysed reversible reaction in both directions. Ki values obtained in the direction of synthesis (1.92 ±â€¯0.14 mM for caffeine and 11.5 ±â€¯2.0 µM for GTH) have been shown to be in good agreement with the Ki values obtained in the direction of phosphorolysis (4.05 ±â€¯0.26 mM for caffeine and 13.8 ±â€¯1.6 µM for GTH). The higher difference between the inhibition constants of caffeine was explained by the non-competitive mechanism. The described ITC method using the developed experimental design and reaction conditions is suitable for activity measurements of different phosphorylase enzymes on various substrates and is applicable for inhibition studies as well.


Assuntos
Calorimetria , Glicogênio Fosforilase Muscular/metabolismo , Músculos/enzimologia , Animais , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase Muscular/antagonistas & inibidores , Humanos , Cinética , Músculos/efeitos dos fármacos , Músculos/metabolismo , Coelhos , Relação Estrutura-Atividade
10.
Orphanet J Rare Dis ; 14(1): 62, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30832705

RESUMO

BACKGROUND: Late-onset Pompe disease (LOPD) is a recessive disease caused by α-glucosidase (GAA) deficiency, leading to progressive muscle weakness and/or respiratory failure in children and adults. Respiratory derangement can be the first indication of LOPD, but the diagnosis may be difficult for pneumologists. We hypothesize that assessing the GAA activity in suspected patients by a dried blood spot (DBS) may help the diagnosis of LOPD in the pneumological setting. POPULATION AND METHODS: We performed a multicenter DBS survey of patients with suspected LOPD according to a predefined clinical algorithm. From February 2015 to December 2017, 140 patients (57 ± 16 yrs., 80 males) were recruited in 19 Italian pneumological units. The DBS test was performed by a drop of blood collected on absorbent paper. Patients with GAA activity < 2.6 µmol/L/h were considered positive. A second DBS test was performed in the patients positive to the first assay. Patients testing positive at the re-test underwent a skeletal muscle biopsy to determine the GAA enzymatic activity. RESULTS: 75 recruited subjects had outpatient access, 65 subjects were admitted for an acute respiratory failure episode. Two patients tested positive in both the first and second DBS test (1.4% prevalence), and the LOPD diagnosis was confirmed through histology, with patients demonstrating a deficient GAA muscle activity (3.6 and 9.1 pmol/min/mg). A further five subjects were positive in the first DBS test but were not confirmed at re-test. The two positive cases were both diagnosed after hospitalization for acute respiratory failure and need of noninvasive ventilation. Most of the recruited patients had reduced maximal respiratory pressures (MIP 50 ± 27% and MEP 55 ± 27% predicted), restrictive pattern (FEV1/FVC 81.3 ± 13.6) and hypoxaemia (PaO2 70.9 ± 14.5 mmHg). Respiratory symptoms were present in all the patients, but only 48.6% of them showed muscle weakness in the pelvic girdle and/or in the scapular girdle (35.7%). CONCLUSIONS: DBS GAA activity test may be a powerful screening tool among pneumologists, particularly in the acute setting. A simple clinical algorithm may aid in the selection of patients on which to administer the DBS test.


Assuntos
Teste em Amostras de Sangue Seco/normas , Doença de Depósito de Glicogênio Tipo II/complicações , Doença de Depósito de Glicogênio Tipo II/diagnóstico , Transtornos de Início Tardio/diagnóstico , Pneumopatias/complicações , Pneumologia/métodos , Adulto , Idoso , Biópsia , Diagnóstico Precoce , Feminino , Doença de Depósito de Glicogênio Tipo II/sangue , Doença de Depósito de Glicogênio Tipo II/enzimologia , Humanos , Itália , Transtornos de Início Tardio/sangue , Transtornos de Início Tardio/enzimologia , Pneumopatias/sangue , Masculino , Pessoa de Meia-Idade , Músculos/enzimologia , Músculos/cirurgia , alfa-Glucosidases/metabolismo
11.
Ecotoxicol Environ Saf ; 173: 482-493, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30802737

RESUMO

Cholinesterases are frequent targets for toxic effects, namely by insecticides derived from phosphoric and carbamic acids. This effects allows the use of cholinesterase inhibition as a biomarker for contamination of aquatic environments by these specific chemical agents. However, cholinesterases are differently responsive to environmental contaminants, according to their different forms and locations. In addition, cholinesterases seem also to be inhibited by metals, so their use as an environmental criterion requires the prior characterization of their specific forms in each species and tissues, and the study of their sensitivity. The objective of this study was to characterize the cholinesterase isoenzymes present in the brain and dorsal muscle of three tropical fish species, namely Phalloceros harpagos (Lucinda, 2008), Pterygoplichthys pardalis (Castelnau, 1855) and Astyanax altiparanae (Garutti and Britski, 2000). In vitro assays were conducted to quantify the effect of pesticides (dimethoate and carbaryl) and metals (lead and copper) on cholinesterases activity. Although acetylcholinesterase seems to be the most prevalent and abundant form, as commonly described in vertebrates, the here-obtained results showed that three cholinesterase isoenzymes occur in tissues of the three fish species. In addition, the pesticide carbaryl caused a stronger inhibition than dimethoate. Copper caused a significantly higher cholinesterasic inhibition than lead, which is also in line with most results concerning the anticholinesterasic effects by these metals. The here obtained results allowed to conclude that acetylcholinesterase is the predominant form in all tissues from the three analyzed species. In addition, cholinesterases of these three fish were responsive to common environmental contaminants, namely metals and pesticides, similarly to what was already described for fish of temperate areas. This allows using the here proposed fish species in environmental studies for the assessment of the presence of neurotoxicants under neotropical conditions.


Assuntos
Peixes-Gato/metabolismo , Inibidores da Colinesterase/toxicidade , Cobre/toxicidade , Ciprinodontiformes/metabolismo , Chumbo/toxicidade , Praguicidas/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Carbaril/toxicidade , Colinesterases/metabolismo , Dimetoato/toxicidade , Feminino , Proteínas de Peixes/metabolismo , Masculino , Músculos/efeitos dos fármacos , Músculos/enzimologia
12.
Environ Toxicol ; 34(4): 457-468, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30604913

RESUMO

The toxicity of titanium dioxide nanoparticles (TiO2 -NP) in the blood, liver, muscle, and brain of a Neotropical detritivorous fish, Prochilodus lineatus, was tested. Juvenile fish were exposed to 0, 1, 5, 10, and 50 mg L-1 of TiO2 -NP for 48 hours (acute exposure) or 14 days (subchronic exposure) to evaluate changes in hematology, red blood cell (RBC) genotoxicity/mutagenicity, liver function (reactive oxygen species (ROS) production, antioxidant responses, detoxification, and histopathology), acetylcholinesterase (AChE) activity in muscles and brain, and Ti bioaccumulation. TiO2 -NP did not cause genetic damage to RBC, but acutely decreased white blood cells (WBC) and increased monocytes. Subchronically, RBC decreased, mean cell volume and hemoglobin increased, and WBC and lymphocytes decreased. Therefore, NP has the potential to affect immune system and increase energy expenditure, reducing the fish's ability to avoid predator and to resist pathogens. In the liver, acute exposure decreased ROS and increased glutathione (GSH) content, while subchronic exposure decreased superoxide dismutase activity and increased glutathione-S-transferase (GST) activity and GSH content. GSH and GST seem to play an essential role in metabolizing NP and ROS, likely increasing hepatocytes' metabolic rate, which may be the cause of observed cell hypertrophy, disarrangement of hepatic cords and degenerative morphological alterations. Although most studies indicate that the kidney is responsible for metabolizing and/or eliminating TiO2 -NP, this study shows that the liver also has a main role in these processes. Nevertheless, Ti still accumulated in the liver, muscle, and brain and decreased muscular AChE activity after acute exposure, showing neurotoxic potential. More studies are needed to better understand the biochemical pathways TiO2 -NP are metabolized and how its bioaccumulation may affect fish homeostasis and survival in the environment.


Assuntos
Encéfalo/efeitos dos fármacos , Caraciformes/sangue , Fígado/efeitos dos fármacos , Músculos/efeitos dos fármacos , Nanopartículas/toxicidade , Titânio/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Antioxidantes/metabolismo , Contagem de Células Sanguíneas , Encéfalo/enzimologia , Encéfalo/patologia , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Fígado/enzimologia , Fígado/patologia , Músculos/enzimologia , Músculos/patologia , Nanopartículas/metabolismo , Titânio/metabolismo , Poluentes Químicos da Água/metabolismo
13.
Artigo em Inglês | MEDLINE | ID: mdl-30594527

RESUMO

The cell cycle comprises a series of steps necessary for cell growth until cell division. The participation of proteins responsible for cell cycle regulation, known as cyclin dependent kinases or Cdks, is necessary for cycle progression. Cyclin dependent kinase 2 (Cdk-2) is one of the most studied Cdks. This kinase regulates the passage through the G1/S phase and is involved in DNA replication in the S phase. Cdks have been extensively studied in mammals, but there is little information about these proteins in crustaceans. In the present work, the nucleotide and amino acid sequence of Cdk-2 from the white shrimp (Cdk-2) and its expression during hypoxia and reoxygenation are reported. Cdk-2 is a highly conserved protein and contains the serine/threonine catalytic domain, an ATP binding site and the PSTAIRE sequence. The predicted Cdk-2 structure showed the two-lobed structure characteristic of kinases. Expression of Cdk-2 was detected in hepatopancreas, gills and muscle, with hepatopancreas having the highest expression during normoxic conditions. Cdk-2 expression was significantly induced after hypoxia for 24 h in muscle cells, but in hypoxia exposure for 24 followed by 1 h of reoxygenation, the expression levels returned to the levels found in normoxic conditions, suggesting induction of cell cycle progression in muscular cells during hypoxia. No significant changes in expression of Cdk-2 were detected in these conditions in hepatopancreas and gills.


Assuntos
Proteínas de Artrópodes/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Hipóxia/enzimologia , Oxigênio/metabolismo , Penaeidae/enzimologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Sequência de Bases , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/genética , Brânquias/enzimologia , Hepatopâncreas/enzimologia , Músculos/enzimologia , Penaeidae/metabolismo , Filogenia
14.
Drug Chem Toxicol ; 42(5): 463-470, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29258333

RESUMO

In the present study, the acute toxicity of chlorpyrifos (an organophosphate, OP) and cypermethrin (a pyrethroid) pesticides was estimated for 96 h in Heteropneustes fossilis. The LC50 for chlorpyrifos (CPF) and cypermethrin was found to be 1.90 mg/L and 0.085 mg/L, respectively. The acetylcholinesterase (AChE, EC 3.1.1.7) activity in Heteropneustes fossilis exposed to both the insecticides was assayed in brain, muscle and gills. In general, tissue specific as well as dose-dependent decrease in the AChE activity was exhibited by both pesticides. In response to the increasing concentrations of chlorpyrifos and cypermethrin as well, a significant decrease in the activity of AChE was found in brain while muscle and gills exhibited lesser inhibition. Thus, the brain was the main target organ for both insecticides, followed by muscle and gills. Between the two pesticides chlorpyrifos acted as more potent AChE inhibitor than cypermethrin since more intense changes in behavioral pattern was observed with the chlorpyrifos. These changes indicate that the effects of these pesticides are at neural as well as neuromuscular level.


Assuntos
Acetilcolinesterase/metabolismo , Peixes-Gato , Clorpirifos/toxicidade , Inibidores da Colinesterase/toxicidade , Piretrinas/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , Dose Letal Mediana , Músculos/efeitos dos fármacos , Músculos/enzimologia , Testes de Toxicidade Aguda
15.
Arthritis Res Ther ; 20(1): 134, 2018 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-29976235

RESUMO

BACKGROUND: Dermatomyositis and polymyositis are the best known idiopathic inflammatory myopathies (IIMs). Classic histopathologic findings include the infiltration of inflammatory cells into muscle tissues. Neutrophil serine proteinases (NSPs) are granule-associated enzymes and play roles in inflammatory cell migration by increasing the permeability of vascular endothelial cells. In this study, we aimed to find the roles of NSPs in pathogenesis of IIMs. METHODS: RNA and DNA were isolated to measure the relative expression of NSPs and their methylation levels. The expression of NSPs in serum and muscle tissues was tested by enzyme-linked immunosorbent assay, immunohistochemistry, and immunofluorescence, respectively. Serum from patients was used to culture the human dermal microvascular endothelial cells (HDMECs), and then we observed the influence of serum on expression of VE-cadherin, endothelial cell tube formation, and transendothelial migration of peripheral blood mononuclear cells (PBMCs). RESULTS: We found that the expression of NSPs was increased in PBMCs, serum, and muscle tissues of IIM patients; these NSPs were hypomethylated in the PBMCs of patients. Serum NSPs were positively correlated with clinical indicators of IIM patients, including lactic dehydrogenase, erythrocyte sedimentation rate, C-reactive protein, immunoglobulin G, immunoglobulin M, and immunoglobulin A. Patients with anti-Jo-1, with anti-Ro-52, or without interstitial lung disease had lower levels of proteinase 3. Serum NSPs degraded the VE-cadherin of HDMECs, and serum NSP application increased the permeability of HDMECs. CONCLUSIONS: Our studies indicate, for the first time, that NSPs play an important role in muscle inflammatory cell infiltration by increasing the permeability of vascular endothelial cells in IIM patients.


Assuntos
Músculos/enzimologia , Miosite/enzimologia , Neutrófilos/enzimologia , Serina Endopeptidases/metabolismo , Adulto , Antígenos CD/sangue , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/sangue , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Células Cultivadas , Células Endoteliais/enzimologia , Células Endoteliais/fisiologia , Feminino , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/enzimologia , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Músculos/metabolismo , Músculos/patologia , Miosite/sangue , Miosite/genética , Permeabilidade , Serina Endopeptidases/sangue , Serina Endopeptidases/genética , Migração Transendotelial e Transepitelial
16.
Artigo em Inglês | MEDLINE | ID: mdl-30017911

RESUMO

Glycogen, as an intracellular deposit of polysaccharide, takes important roles in energy balance of many animals. In fish, however, the role of glycogen during development is poorly understood. In the present study, we assessed changes in glycogen concentration and gene expression patterns of glycogen-metabolizing enzymes in developing masu salmon (Oncorhynchus masou masou), a salmonid species inhabiting west side of North Pacific Ocean. As we measured glycogen levels in the bodies and yolk sacs containing the liver separately, the glycogen concentration increased in both parts as the fish developed, whereas it transiently decreased in the yolk sac after hatching, implying glycogen synthesis and breakdown in these tissues. Immunofluorescence staining using anti-glycogen monoclonal antibody revealed localization of glycogen in the liver, muscle and yolk syncytial layer of the pre-hatching embryos and hatched larvae. In order to estimate glycogen metabolism in the fish, the genes encoding homologs of glycogen synthase (gys1 and gys2) and glycogen phosphorylase (pygma, pygmb and pygl) were cloned, and their expression patterns were assessed by quantitative PCR and in situ hybridization. In the fish, gys1 and gys2 were robustly expressed in the muscle and liver, respectively. Also, expression of pyg isoforms was found in muscle, liver and yolk syncytial layer during hatching. With changes in glycogen concentration and expression patterns of relevant genes, our results suggest, for the first time, possible involvement of glycogen in energy balance of fish embryos, especially during hatching.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Glicogênio/metabolismo , Fígado/enzimologia , Músculos/enzimologia , Salmão/metabolismo , Animais , Clonagem Molecular , Feminino , Imunofluorescência , Glicogênio Fosforilase/metabolismo , Fígado/crescimento & desenvolvimento , Masculino , Desenvolvimento Muscular , Filogenia , RNA Mensageiro/genética , Salmão/genética , Salmão/crescimento & desenvolvimento
17.
Fish Shellfish Immunol ; 80: 458-466, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29859312

RESUMO

The aim of this study was the assessment of preloaded feed pellets as a delivery system for plasmid DNA (pDNA), with the purpose of evaluating the potential administration of DNA vaccines orally in aquacultured fish. Pellets were made up by usual feed ingredients, which were mixed with chitosan nanoparticles entrapping a model plasmid (pCMVß) expressible in eukaryotic cells before being elaborated. The plasmid is characterized by the insertion of the reporter gene lacZ, encoding for the bacterial enzyme ß-galactosidase (ß-gal). The possible in vivo expression of the exogenous gene was measured in different fish tissues of gilthead sea bream (Sparus aurata) juveniles by two different procedures. On the one hand, the activity of the enzyme ß-gal was detected and quantified in muscle, liver and intestine; on the other, specific IgM against ß-gal antigen was titrated in blood samples. Intramuscular (i.m.) injection of equal amounts of plasmid was also carried out for the purpose of comparison with oral administration. The expression of the reporter gene was detected in fish tissues following both oral and i. m. administration of pDNA up to 60 days. However, organ distribution of the gene expression was more evident after oral (ß-gal activity measured in gut, liver and muscle) than after parenteral administration (restricted to adjacent muscle tissues). In agreement, specific IgM titration indicated that humoral immune response was more intense and sustained throughout the experimental period after oral than after i. m. delivery of equal amounts of pDNA. These results suggest that feed pellets containing chitosan nanoparticles might enable efficient oral delivery of pDNA, a fact that might imply valuable applications in terms of on-farm mass immunization purposes, especially with regard to DNA-based vaccines and small size fish, in which i. m. administration remains unfeasible.


Assuntos
Quitosana/administração & dosagem , DNA/administração & dosagem , Nanopartículas/administração & dosagem , Dourada/imunologia , Vacinas de DNA , Administração Oral , Ração Animal , Animais , Técnicas de Transferência de Genes , Genes Reporter , Imunoglobulina M/sangue , Intestinos/enzimologia , Fígado/enzimologia , Músculos/enzimologia , Plasmídeos , Dourada/metabolismo , beta-Galactosidase/imunologia , beta-Galactosidase/metabolismo
18.
Aquat Toxicol ; 200: 266-274, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29807214

RESUMO

Lipid regulators are among the most prescribed human pharmaceuticals worldwide. Gemfibrozil, which belongs to this class of pharmaceuticals, is one of the most frequently encountered in the aquatic environment. However, there is limited information concerning the mechanisms involved in gemfibrozil effects to aquatic organisms, particularly to marine organisms. Based on this knowledge gap, the current study aimed to assess biochemical and behavioral effects following a sublethal exposure to gemfibrozil (1.5, 15, 150, 1500 and 15,000 µg L-1) in the estuarine/marine fish Sparus aurata. After the exposure to 1.5 µg L-1 of gemfibrozil, fish had reduced ability to swim against a water flow and increased lipid peroxidation in the liver. At concentrations between 15-15,000 µg L-1, the activities of some enzymes involved in antioxidant defense were induced, appearing to be sufficient to prevent oxidative damage. Depending on the organ, different responses to gemfibrozil were displayed, with enzymes like catalase being more stimulated in gills, whereas glutathione peroxidase was more activated in liver. Although there were no obvious concentration-response relationships, the integrated biomarker response version 2 (IBRv2) analysis revealed that the highest concentrations of gemfibrozil (between 150-15,000 µg L-1) caused more alterations. All the tested concentrations of gemfibrozil induced effects in S. aurata, in terms of behavior and/or oxidative stress responses. Oxidative damage was found at a concentration that is considered environmentally relevant, suggesting a potential of this pharmaceutical to impact fish populations.


Assuntos
Biomarcadores/metabolismo , Genfibrozila/toxicidade , Dourada/metabolismo , Animais , Encéfalo/enzimologia , Catalase/metabolismo , Colinesterases/metabolismo , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Glutationa Peroxidase/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Músculos/enzimologia , Estresse Oxidativo/efeitos dos fármacos , Natação/fisiologia , Poluentes Químicos da Água/toxicidade
19.
Oxid Med Cell Longev ; 2018: 5763256, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29636849

RESUMO

The aim of this study was to determine whether oxidative stress markers are influenced by low-intensity laser therapy (LLLT) in rats subjected to a high-intensity resistive exercise session (RE). Female Wistar rats divided into three experimental groups (Ctr: control, 4J: LLLT, and RE) and subdivided based on the sampling times (instantly or 24 h postexercise) underwent irradiation with LLLT using three-point transcutaneous method on the hind legs, which was applied to the gastrocnemius muscle at the distal, medial, and proximal points. Laser (4J) or placebo (device off) were carried out 60 sec prior to RE that consisted of four climbs bearing the maximum load with a 2 min time interval between each climb. Lipoperoxidation levels and antioxidant capacity were obtained in muscle. Lipoperoxidation levels were increased (4-HNE and CL markers) instantly post-RE. LLLT prior to RE avoided the increase of the lipid peroxidation levels. Similar results were also notified for oxidation protein assays. The GPx and FRAP activities did not reduce instantly or 24 h after RE. SOD increased 24 h after RE, while CAT activity did not change with RE or LLLT. In conclusion, LLLT prior to RE reduced the oxidative stress markers, as well as, avoided reduction, and still increased the antioxidant capacity.


Assuntos
Terapia com Luz de Baixa Intensidade , Estresse Oxidativo/efeitos da radiação , Condicionamento Físico Animal , Animais , Feminino , Peroxidação de Lipídeos/efeitos da radiação , Músculos/enzimologia , Músculos/patologia , Músculos/efeitos da radiação , Ratos Wistar
20.
Ecotoxicol Environ Saf ; 155: 162-170, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-29522881

RESUMO

The use of multiple biomarkers has been shown to be an efficient method for evaluating environmental contamination. In this work, we evaluate neurotoxic effects and the antioxidant system responses of the R. branneri collected in two streams of lower Iguazu River basin, relating them with different percentage of vegetation coverture, presence of pesticides and fall and winter seasons. The biological samples were collected in March and August of 2015, from two streams that belong to the lower Iguazu River basin (Brazil): the Manoel Gomes River and the Arquimedes Stream. Soil analyses were performed, and the results showed the presence of the following organophosphates in the Manoel Gomes River and the Arquimedes Stream: disulfoton, methyl parathion, and ronnel. The present study detected inhibition of cholinesterase activity in the brain and muscle of fish samples during the fall from the Manoel Gomes River and the Arquimedes Stream. In the Manoel Gomes River, elevated lipoperoxidation was also observed during the fall. It was observed that the increase or decrease of biomarkers was related to temporal variation and, possibly, to the exposure of animals to agrochemicals. Although the Manoel Gomes River and the Arquimedes Stream are located in regions with large areas of vegetation, the soil analyses show that agrochemical residues are able to reach these locations, which suggests that the fauna are in contact with oxidant and anti-cholinesterase agents during the fall, in addition to respond differently during each season.


Assuntos
Peixes-Gato/metabolismo , Inibidores da Colinesterase/toxicidade , Organofosfatos/toxicidade , Praguicidas/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Biomarcadores/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Brasil , Colinesterases/metabolismo , Monitoramento Ambiental/métodos , Músculos/efeitos dos fármacos , Músculos/enzimologia , Rios , Estações do Ano
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