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Pyruvate kinase (PK) activators have potential therapeutic applications in diseases such as sickle cell anemia. In this study, N-Substituted sulfonamide derivatives of 1,4-dihydropyridines were synthesized and evaluated as PK activators in vitro and using molecular docking studies. The compounds were synthesized by reacting dicarbonyl compounds with ammonium acetate, 5-nitrobenzaldehyde, and alumina sulfuric acid (ASA), followed by reduction and sulfonylation. The structures of the compounds were analyzed using spectroscopic techniques. DFT calculations provided insights into the electronic properties. Molecular docking of the compounds into the active site of PK showed favorable binding interactions. ADME evaluation indicated suitable solubility, BBB permeation, and lack of CYP450 inhibition. Overall, this study demonstrates the potential of new hybrid 1,4-dihydropyridine substituted sulfonamides as PK activators for further development. According to AC 50 values, the compound (DTSF7, 0.97μM) is about 100-fold higher affective than the clinically used sulfonamide compound (AC 50  = 90μM) for PK., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial/commercial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Discharges to the aquatic environment of pharmaceuticals represent a hazard to the aquatic organisms. Subchronic assay with 17-alpha-ethinylestradiol (EE 2 ) and in vitro essays with pharmaceuticals of environmental concern were conducted to examine the sensitivity of tissue acetylcholinesterase (AChE) and carboxylesterase (CbE) activities of Tinca tinca to them. Subchronic exposure to 17-alpha-EE 2 caused significant effects on brain, liver, and muscle CbE, but no on AChE activities. Most of the pharmaceuticals tested in vitro were considered as weak inhibitors of tissular AChE activity. Depending on the tissues, some compounds were classified as moderate inhibitors of CbE activity while other were categorized as weak enzymatic inhibitors. An opposite trend was observed depending on the tissue, while brain and liver CbE activities were inhibited, the muscle CbE activity was induced. Changes experienced on enzymatic activities after exposure to pharmaceuticals might affect the physiological functions in which these enzymes are involved. In vitro exposure to 17-alpha-EE 2 in tench could be an informative, but not a surrogate model to know the effect of this synthetic estrogen on AChE and CbE activities., (© 2024 The Authors. Environmental Toxicology published by Wiley Periodicals LLC.)

Muscle attachment sites (MASs, apodemes) in insects and other arthropods involve specialized epithelial cells, called tendon cells or tenocytes, that adhere to apical extracellular matrices containing chitin. Here, we have uncovered a function for chitin deacetylases (CDAs) in arthropod locomotion and muscle attachment using a double-stranded RNA-mediated gene-silencing approach targeted toward specific CDA isoforms in the red flour beetle, Tribolium castaneum ( Tc ). Depletion of TcCDA1 or the alternatively spliced TcCDA2 isoform, TcCDA2a, resulted in internal tendon cuticle breakage at the femur-tibia joint, muscle detachment from both internal and external tendon cells, and defective locomotion. TcCDA deficiency did not affect early muscle development and myofiber growth toward the cuticular MASs but instead resulted in aborted microtubule development, loss of hemiadherens junctions, and abnormal morphology of tendon cells, all features consistent with a loss of tension within and between cells. Moreover, simultaneous depletion of TcCDA1 or TcCDA2a and the zona pellucida domain protein, TcDumpy, prevented the internal tendon cuticle break, further supporting a role for force-dependent interactions between muscle and tendon cells. We propose that in T. castaneum , the absence of N -acetylglucosamine deacetylation within chitin leads to a loss of microtubule organization and reduced membrane contacts at MASs in the femur, which adversely affect musculoskeletal connectivity, force transmission, and physical mobility.

The widespread use of nanoparticles raises many serious concerns about the safety and environmental impact of nanoparticles. Therefore, risk assessments of specific nanoparticles in occupational and environmental exposure are essential before their large-scale production and applications, especially in medicine and for usage in household items. In this study, the effects of five different metal nanoparticles on the structure, stability, and function of four metabolic enzymes were evaluated using various biophysical techniques. Our results show that Cu nanoparticles exhibited the most significant adverse effects on the structures, stability, and activities of all the metabolic enzymes. Zn nanoparticles caused moderate adverse effects on these enzymes. The rest of the metal (Al, Fe, and Ni) nanoparticles had a relatively lower impact on the metabolic enzymes. Our data indicated that Cu nanoparticles promote metal-catalyzed disulfide bond formation in these proteins. In summary, some metal nanoparticles can cause adverse effects on the structure, function, and stability of metabolic enzymes. In addition, metal nanoparticles may affect protein homeostasis in the cytosol or extracellular fluids., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The crystal structures of free T-state and R-state glycogen phosphorylase (GP) and of R-state GP in complex with the allosteric activators IMP and AMP are reported at improved resolution. GP is a validated pharmaceutical target for the development of antihyperglycaemic agents, and the reported structures may have a significant impact on structure-based drug-design efforts. Comparisons with previously reported structures at lower resolution reveal the detailed conformation of important structural features in the allosteric transition of GP from the T-state to the R-state. The conformation of the N-terminal segment (residues 7-17), the position of which was not located in previous T-state structures, was revealed to form an α-helix (now termed α0). The conformation of this segment (which contains Ser14, phosphorylation of which leads to the activation of GP) is significantly different between the T-state and the R-state, pointing in opposite directions. In the T-state it is packed between helices α4 and α16 (residues 104-115 and 497-508, respectively), while in the R-state it is packed against helix α1 (residues 22'-38') and towards the loop connecting helices α4' and α5' of the neighbouring subunit. The allosteric binding site where AMP and IMP bind is formed by the ordering of a loop (residues 313-326) which is disordered in the free structure, and adopts a conformation dictated mainly by the type of nucleotide that binds at this site.

Linear ubiquitination is an atypic ubiquitination process that directly connects the N- and C-termini of ubiquitin and is catalyzed by HOIL-1-interacting protein (HOIP). It is involved in the immune response or apoptosis by activating the nuclear factor-κB pathway and is associated with polyglucosan body myopathy 1, an autosomal recessive disorder with progressive muscle weakness and cardiomyopathy. However, little is currently known regarding the function of linear ubiquitination in muscles. Here, we investigated the role of linear ubiquitin E3 ligase (LUBEL), a DrosophilaHOIP ortholog, in the development and aging of muscles. The muscles of the flies with down-regulation of LUBEL or its downstream factors, kenny and Relish, developed normally, and there were no obvious abnormalities in function in young flies. However, the locomotor activity of the LUBEL RNAi flies was reduced compared to age-matched control, while LUBEL RNAi did not affect the increased mitochondrial fusion or myofiber disorganization during aging. Interestingly, the accumulation of polyubiquitinated protein aggregation during aging decreased in muscles by silencing LUBEL, kenny, or Relish. Meanwhile, the levels of autophagy and global translation, which are implicated in the maintenance of proteostasis, did not change due to LUBEL down-regulation. In conclusion, we propose a new role of linear ubiquitination in proteostasis in the muscle aging., (Copyright © 2021 Elsevier Inc. All rights reserved.)

We investigated the effect of endogenous cathepsin L on surimi gel produced from olive flounder ( Paralichthys olivaceus ). The amino acid sequences of six proteins predicted or identified as cathepsin L were obtained from the olive flounder genome database, and a phylogenetic analysis was conducted. Next, cathepsin L activity toward N -α-benzyloxycarbonyl-l-phenylalanyl-l-arginine-(7-amino-4-methylcoumarin) (Z-F-R-AMC) was detected in crude olive flounder extract and a crude enzyme preparation. A considerable decrease in the level of myosin heavy chain (MHC) in surimi occurred during autolysis at 60 °C. In contrast, the levels of actin, troponin-T, and tropomyosin decreased only slightly. To prevent protein degradation by cathepsin L, a protease inhibitor was added to surimi. In the presence of 1.0% protease inhibitor, the autolysis of olive flounder surimi at 60 °C was inhibited by 12.2%; the degree of inhibition increased to 44.2% as the inhibitor concentration increased to 3.0%. In addition, the deformation and hardness of modori gel increased as the inhibitor concentration increased to 2.0%. Therefore, cathepsin L plays an important role in protein degradation in surimi, and the quality of surimi gel could be enhanced by inhibiting its activity.

Proteomics profiling disclosed the molecular mechanism underlying beef poor meat quality. This study aimed to identify protein markers indicating the quality of beef during postmortem storage at 4°C. Beef longissimus dorsi samples were stored at 4°C. The meat water holding capacity (WHC), pH value and moisture content were determined at different time points during the storage period. The iTRAQ MS/MS approach was used to determine the proteomics profiling at 0, 3.5 and 7 d during storage at 4°C. Bioinformatics analysis was performed to investigate the potential correlated proteins associated with meat quality. Storage at 4°C gradually decreased the pH value, WHC, and hence the moisture content. The iTRAQ proteomic analysis revealed that a cluster of glycolytic enzymes including malate dehydrogenase, cytoplasmic, L-lactate dehydrogenase, phosphoglycerate mutase and pyruvate kinase, and another cluster of proteins involved in oxygen transport and binding (myoglobin) and hemoglobin complex (including Globin A1 and hemoglobin subunit alpha) were decreased during the postmortem storage. These results suggest that the decreased glycolysis, oxygen, and heme-binding activities might be associated with the beef muscle low quality and the decline of tenderness during postmortem storage at 4°C., Competing Interests: The authors have declared that no competing interests exist.

Muscle fructose-1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose-1,6-bisphosphate (F1,6BP) to fructose-6-phosphate (F6P) and inorganic phosphate, regulates glucose homeostasis by controlling the glyconeogenic pathway. FBPase requires divalent cations, such as Mg2+, Mn2+, or Zn2+, for its catalytic activity; however, calcium ions inhibit the muscle isoform of FBPase by interrupting the movement of the catalytic loop. It has been shown that residue E69 in this loop plays a key role in the sensitivity of muscle FBPase towards calcium ions. The study presented here is based on five crystal structures of wild-type human muscle FBPase and its E69Q mutant in complexes with the substrate and product of the enzymatic reaction, namely F1,6BP and F6P. The ligands are bound in the active site of the studied proteins in the same manner and have excellent definition in the electron density maps. In all studied crystals, the homotetrameric enzyme assumes the same cruciform quaternary structure, with the κ angle, which describes the orientation of the upper dimer with respect to the lower dimer, of -85o. This unusual quaternary arrangement of the subunits, characteristic of the R-state of muscle FBPase, is also observed in solution by small-angle X-ray scattering (SAXS).

Good health depends on the maintenance of metabolic flexibility, which in turn is dependent on the maintenance of regulatory flexibility of a large number of regulatory enzymes, but especially the pyruvate dehydrogenase complex (PDC), because of its central role in carbohydrate metabolism. Flexibility in regulation of PDC is dependent on rapid changes in the phosphorylation state of PDC determined by the relative activities of the pyruvate dehydrogenase kinases (PDKs) and the pyruvate dehydrogenase phosphatases. Inactivation of the PDC by overexpression of PDK4 contributes to hyperglycemia, and therefore the serious health problems associated with diabetes. Loss of regulatory flexibility of PDC occurs in other disease states and pathological conditions that have received less attention than diabetes. These include cancers, non-alcoholic fatty liver disease, cancer-induced cachexia, diabetes-induced nephropathy, sepsis and amyotrophic lateral sclerosis. Overexpression of PDK4, and in some situations, the other PDKs, as well as under expression of the pyruvate dehydrogenase phosphatases, leads to inactivation of the PDC, mitochondrial dysfunction and deleterious effects with health consequences. The possible basis for this phenomenon, along with evidence that overexpression of PDK4 results in phosphorylation of "off-target" proteins and promotes excessive transport of Ca 2+ into mitochondria through mitochondria-associated endoplasmic reticulum membranes are discussed. Recent efforts to find small molecule PDK inhibitors with therapeutic potential are also reviewed., (© 2020 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

We explore the principles of pressure tolerance in enzymes of deep-sea fishes using lactate dehydrogenases (LDH) as a case study. We compared the effects of pressure on the activities of LDH from hadal snailfishes Notoliparis kermadecensis and Pseudoliparis swirei with those from a shallow-adapted Liparis florae and an abyssal grenadier Coryphaenoides armatus. We then quantified the LDH content in muscle homogenates using mass-spectrometric determination of the LDH-specific conserved peptide LNLVQR. Existing theory suggests that adaptation to high pressure requires a decrease in volume changes in enzymatic catalysis. Accordingly, evolved pressure tolerance must be accompanied with an important reduction in the volume change associated with pressure-promoted alteration of enzymatic activity ( Δ V PP ∘ ). Our results suggest an important revision to this paradigm. Here, we describe an opposite effect of pressure adaptation-a substantial increase in the absolute value of Δ V PP ∘ in deep-living species compared to shallow-water counterparts. With this change, the enzyme activities in abyssal and hadal species do not substantially decrease their activity with pressure increasing up to 1-2 kbar, well beyond full-ocean depth pressures. In contrast, the activity of the enzyme from the tidepool snailfish, L. florae, decreases nearly linearly from 1 to 2500 bar. The increased tolerance of LDH activity to pressure comes at the expense of decreased catalytic efficiency, which is compensated with increased enzyme contents in high-pressure-adapted species. The newly discovered strategy is presumably used when the enzyme mechanism involves the formation of potentially unstable excited transient states associated with substantial changes in enzyme-solvent interactions., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Branched-chain amino acids (BCAAs; valine, leucine, and isoleucine) are increased in starvation and diabetes mellitus. However, the pathogenesis has not been explained. It has been shown that BCAA catabolism occurs mostly in muscles due to high activity of BCAA aminotransferase, which converts BCAA and α-ketoglutarate (α-KG) to branched-chain keto acids (BCKAs) and glutamate. The loss of α-KG from the citric cycle (cataplerosis) is attenuated by glutamate conversion to α-KG in alanine aminotransferase and aspartate aminotransferase reactions, in which glycolysis is the main source of amino group acceptors, pyruvate and oxaloacetate. Irreversible oxidation of BCKA by BCKA dehydrogenase is sensitive to BCKA supply, and ratios of NADH to NAD + and acyl-CoA to CoA-SH. It is hypothesized that decreased glycolysis and increased fatty acid oxidation, characteristic features of starvation and diabetes, cause in muscles alterations resulting in increased BCAA levels. The main alterations include (i) impaired BCAA transamination due to decreased supply of amino groups acceptors (α-KG, pyruvate, and oxaloacetate) and (ii) inhibitory influence of NADH and acyl-CoAs produced in fatty acid oxidation on citric cycle and BCKA dehydrogenase. The studies supporting the hypothesis and pros and cons of elevated BCAA concentrations are discussed in the article.

A lag in the increase in oxygen consumption (MO 2 ) occurs at the start of sustainable exercise in trout. Waterborne dichloroacetate (0.58 and 3.49 mmol l -1 ), a compound which activates pyruvate dehydrogenase (PDH) by inhibiting PDH kinase in muscle, accelerates the increase in MO 2 during the first 10 min of sustainable exercise when velocity is elevated to 75% critical swimming speed in a swim tunnel. There are no effects on MO 2 thereafter or at rest. This indicates that a delay in PDH activation ("metabolic inertia") contributes to the lag phenomenon., (© 2020 Fisheries Society of the British Isles.)

Current study aims to determine difference in metal accumulation pattern in muscle of Liza parsia (pelagic, omnivore), Amblypharyngodon mola (surface feeder, herbivore) and Mystus gulio (benthic, carnivore) depending on their niche and feeding habit and how it affects the endogenous antioxidants and glucose metabolism in fish muscle. Fishes were collected from Malancha, Diamond Harbour and Chandanpiri, West Bengal, India. Concentrations of lead, zinc, cadmium, chromium were measured in water, sediment and fish muscle. Metal pollution index (MPI) and bioconcentration factor (BCF) was calculated to evaluate the ability of fish to accumulate specific metals in muscle tissue from the aquatic environment. Metal concentrations were found significantly higher (P < 0.05) in water, sediment, fish muscles from Malancha than Chandanpiri and Diamond Harbour. L. parsia (MPI: 0.4-1.6) showed highest metal deposition in their muscle followed by A. mola (MPI: 0.37-1.38) and M. gulio (MPI: 0.2-1.2). Malondealdehyde, superoxide dismutase, catalase, glutathione S transferase, glutathione reductase and cortisol levels increased in case of L. parsia from Malancha and Chandanpiri. Succinate dehydrogenase, lactate dehydrogenase, Ca +2 ATPase and cytochrome C oxidase levels were significantly (P < 0.05) lower at Malancha and Chandanpiri than Diamond Harbour. Heat shock protein (HSP70) expression was significantly (P < 0.05) higher in all fish species at Malancha followed by Chandanpiri and Diamond Harbour. Glucose, glycogen, hexokinase, phosphofructokinase and glycogen phosphorylase levels varied between sites and selected fish species. Serum cortisol level was measured and found to be the highest in L. parsia from Malancha (2.94 ± 0.12 ng/ml) and the lowest in M. gulio from Diamond Harbour (0.7 ± 0.05 ng/ml). The results indicate that metal toxicity alters antioxidant levels, oxidative status and energy production in fish in species specific manner. Our results also indicate that Mystus has the highest degree of adaptability in response to metal toxicity possibly due to its specific food habit and niche position. Therefore, it can be concluded that maintenance of oxidative and metabolic status to combat metal-induced oxidative load will be helpful for the fishes to acquire better resistance under such eco-physiological stress. Alteration of niche and interactive segregation in aquatic organism may be one of the key modulator of resistance against such stress., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The intertidal marine snail, Littorina littorea, has evolved to survive bouts of anoxia and extracellular freezing brought about by changing tides and subsequent exposure to harsh environmental conditions. Survival in these anoxic conditions depends on the animals entering a state of metabolic rate depression in order to maintain an appropriate energy production-consumption balance during periods of limited oxygen availability. This study investigated the kinetic, physical, and regulatory properties of pyruvate kinase (PK), which catalyzes the final reaction of aerobic glycolysis, from foot muscle of L. littorea to determine if the enzyme is differentially regulated in response to anoxia and freezing exposure. PK purified from foot muscle of anoxic animals exhibited a lower affinity for its substrate phosphoenolpyruvate than PK from control and frozen animals. PK from anoxic animals was also more sensitive to a number of allosteric regulators, including alanine and aspartate, which are key anaerobic metabolites in L. littorea. Furthermore, PK purified from anoxic and frozen animals exhibited greater stability compared to the non-stressed control animals, determined through high-temperature incubation studies. Phosphorylation of threonine and tyrosine residues was also assessed and demonstrated that levels of threonine phosphorylation of PK from anoxic animals were significantly higher than those of PK from control and frozen animals, suggesting a potential mechanism for regulating PK activity. Taken together, these results suggest that PK plays a role in suppressing metabolic rate in these animals during environmental anoxia exposure.

The citric acid cycle (CAC) is a central metabolic pathway that links carbohydrate, lipid, and amino acid metabolism in the mitochondria and, hence, is a crucial target for metabolic regulation. The α-ketoglutarate dehydrogenase complex (KGDC) is the rate-limiting step of the CAC, the three enzymes of the complex catalyzing the transformation of α-ketoglutarate to succinyl-CoA with the release of CO 2 and reduction of NAD to NADH. During hibernation, the metabolic rate of small mammals is suppressed, in part due to reduced body temperature but also active controls that suppress aerobic metabolism. The present study examined KGDC regulation during hibernation in skeletal muscle of the Richardson's ground squirrel (Urocitellus richardsonii). The KGDC was partially purified from skeletal muscle of euthermic and hibernating ground squirrels and kinetic properties were evaluated at 5°, 22°, and 37 °C. KGDC from hibernator muscle at all temperatures compared with euthermic controls exhibited a decreased affinity for CoA as well as reduced activation by Ca 2+ ions at 5 °C from both euthermic and hibernating conditions. Co-immunoprecipitation was employed to isolate the E1, E2 and E3 enzymes of the complex (OGDH, DLST, DLD) to allow immunoblot analysis of post-translational modifications (PTMs) of each enzyme. The results showed elevated phospho-tyrosine content on all three enzymes during hibernation as well as increased ADP-ribosylation and succinylation of hibernator OGDH. Taken together these results show that the KGDC is regulated by posttranslational modifications and temperature effects to reorganize enzyme activity and mitochondrial function to aid suppression of mitochondrial activity during hibernation., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The traffic pressure is increasing, resulting in the emission of atmospheric pollution. Soil organisms will need to respond to pollution stressors. Among them, land snails are valuable indicators of ecosystem disturbance. In this study, land snails Helix pomatia were sampled from three city localities with different traffic intensity. Oxidative stress biomarkers catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione-S-transferase (GST) in the foot muscle (FM) and hepatopancreas (HP) tissue were determined. Also, five heavy metal (Cd, Cu, Ni, Pb, and Zn) concentrations were quantified in soil and tissue samples. According to the results, the highway induces the strongest contamination on the surrounding environment, with the highest metal concentrations measured in soil and snails. At the most polluted locality, only Cd exceeded some soil guidelines authorities that we referred to in this study. In addition, tissue Cd concentrations exceeded the United States Environmental Protection Agency (USEPA) value (1 mg kg -1 ) for soil invertebrate toxicity at all localities making it likely responsible for generating adverse effects in snails. Regarding HP, the CAT and GST are the most sensitive parameters that could be useful as oxidative stress biomarkers in snails exposed to the actual metals in the environment. On the other hand, in FM tissue, the most pronounced changes were recorded for GPX and GR. Based on tissue-specific enzyme responses, three urban populations were clearly separated. Therefore land snails are the promising candidates for quick field-based biomarker studies after showing a tissue-specific concentration-dependent induction of certain enzymes to heavy metals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bioindicator organisms are important tools in environmental monitoring studies. Understanding this, the overall goal of the present study was to evaluate the sensitivity and viability of the native fish species Banded tetra, (Astyanax aeneus; Günther, 1860), widely spread in the aquatic ecosystems of the Yucatan Peninsula in Mexico, as a bioindicator organism. In order to do this, we performed a bioassay at sublethal concentrations using copper (CuSO 4 ) to experimentally evaluate and validate the relationship between the trace metals and oxidative stress biomarkers response [(catalase (CAT), lipoperoxidation content (LPO)], detoxification [(glutathione S-transferase (GST), metallothionein content (MT)] and neurotoxicity (AChE) in muscle of A. aeneus. Results showed changes in biomarkers after 96 h: Catalase activity (CAT) was significantly higher above 1.5 and 2 mg/L (154.35 and 172.50% increase, respectively); lipid peroxidation contents (TBARS), GST activity, and MT content were very similar to CAT activity at 1.5 and 2 mg/L of Cu. In terms of neurotoxicity, AChE activity was significantly inhibited at 0.1 mg/L (64%; p < 0.001) and 0.5 mg/L (44%; p < 0.001) of Cu. Based on the bioassay results, we performed a trace metal monitoring campaign in muscle of A. aeneus caught in 15 sites with different anthropogenic activities, during the summer of 2017, to establish a baseline of trace metals pollution in the state of Campeche. A. aeneus showed the highest trace metal accumulation in the following order: Al > Fe > Mn > Zn > Cu > Hg > Cr > Pb > Cd > V > As, while sites were arrange as follows: Xnoha lagoon > Palizada River > Candelaria River > Ululmal > Maravillas > López Mateos. PCA showed a cluster between biomarkers (GST, CAT, TBARS, and MT) and concentration of metals (Cd, Cu, Fe, Zn, Hg and Cr). Conversely, AChE inhibition was not related to a specific metal, but highest inhibitions (>50%) were present in those sites with intensive agricultural practices. These results determined that, based on its physiological response and trace metal bioaccumulation, Astyanax aeneus can be considered a good bioindicator for evaluating the presence of trace metals in tropical aquatic systems of the Yucatan Peninsula., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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 A 2 (PLA 2 ) 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Creatine kinase (CK) functions as an energy buffer in muscles. Its substrate, creatine, is generated by L-arginine:glycine amidinotransferase (AGAT) and guanidinoacetate N-methyltransferase (GAMT). Creatine deficiency has more severe consequences for AGAT than GAMT KO mice. In the present study, to characterize their muscle phenotype further, we recorded the weight of tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius (GAS), plantaris (PLA) and soleus (SOL) from creatine-deficient AGAT and GAMT, KO and WT mice. In GAS, PLA and SOL representing glycolytic, intermediate and oxidative muscle, respectively, we recorded the activities of pyruvate kinase (PK), lactate dehydrogenase (LDH), citrate synthase (CS) and cytochrome oxidase (CO). In AGAT KO compared to WT mice, muscle atrophy and differences in marker enzyme activities were more pronounced in glycolytic than oxidative muscle. In GAMT KO compared to WT, the atrophy was modest, differences in PK and LDH activities were minor, and CS and CO activities were slightly higher in all muscles. SOL from males had higher CS and CO activities compared to females. Our results add detail to the characterization of AGAT and GAMT KO skeletal muscle phenotypes and illustrate the importance of taking into account differences between muscles, and differences between sexes.

Objectives: The Arterial Tortuosity Syndrome (ATS) is an autosomal recessive connective tissue disorder, mainly characterized by tortuosity and stenosis of the arteries with a propensity towards aneurysm formation and dissection. It is caused by mutations in the SLC2A10 gene that encodes the facilitative glucose transporter GLUT10. The molecules transported by and interacting with GLUT10 have still not been unambiguously identified. Hence, the study attempts to identify both the substrate binding site of GLUT10 and the molecules interacting with this site., Methods: As High-resolution X-ray crystallographic structure of GLUT10 was not available, 3D homology model of GLUT10 in open conformation was constructed. Further, molecular docking and bioinformatics investigation were employed., Results and Discussion: Blind docking of nine reported potential in vitro substrates with this 3D homology model revealed that substrate binding site is possibly made with PRO531, GLU507, GLU437, TRP432, ALA506, LEU519, LEU505, LEU433, GLN525, GLN510, LYS372, LYS373, SER520, SER124, SER533, SER504, SER436 amino acid residues. Virtual screening of all metabolites from the Human Serum Metabolome Database and muscle metabolites from Human Metabolite Database (HMDB) against the GLUT10 revealed possible substrates and interacting molecules for GLUT10, which were found to be involved directly or partially in ATS progression or different arterial disorders. Reported mutation screening revealed that a highly emergent point mutation (c. 1309G>A, p. Glu437Lys) is located in the predicted substrate binding site region., Conclusion: Virtual screening expands the possibility to explore more compounds that can interact with GLUT10 and may aid in understanding the mechanisms leading to ATS., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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 ± 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., (Copyright © 2019. Published by Elsevier B.V.)

Background: Polymorphonuclear (PMN) elastase plays an important role in a variety of inflammatory disorders. Our aim was to analyse PMN elastase in idiopathic inflammatory myopathies (IIMs) and its association with disease activity., Methods: PMN elastase levels were measured using enzyme-linked immunosorbent assay in serum samples obtained from 74 patients with myositis (58 with dermatomyositis [DM] and 16 with polymyositis [PM]) and 22 healthy controls. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the discriminant capacity of PMN elastase level and PMN elastase-to-neutrophil ratio (ENR) in patients with active and remission myositis. The association of serum PMN elastase level and ENR with disease variables was evaluated in patients with IIMs. The disease specificity of PMN elastase level and ENR was further examined in 60 patients with other systemic autoimmune diseases., Results: PMN elastase level and ENR were significantly higher in patients with active IIMs, DM, and PM than in patients with remission. ROC curve analysis revealed that PMN elastase level and ENR both outperformed creatine kinase (CK), the currently used laboratory marker, and strongly discriminated patients with active disease and those with remission of IIMs, DM, and PM (area under the ROC curve [AUC] 0.9, 0.9, and 0.88 for PMN elastase; AUC 0.96, 0.96, and 1.0 for ENR; AUC 0.72, 0.70, and 0.80 for CK, respectively). PMN elastase level and ENR were positively correlated with myositis disease activity assessment, CK, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, C-reactive protein, and erythrocyte sedimentation rate. PMN elastase level and ENR were higher in the anti-PM-Scl positive myositis group than those in the anti-PM-Scl negative myositis group. Nevertheless, PMN elastase was not a specific disease marker for IIMs when compared with other autoimmune diseases., Conclusions: PMN elastase, particularly ENR, were significantly correlated with disease activity and could serve as useful biochemical markers for evaluating the disease activity of patients with IIMs. Thus, they are potentially helpful in monitoring disease progression and guiding treatment.

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 (Fe 2+ , Cu 2+ , Co 2+ , Pb 2+ Hg 2+ and As 3+ ) 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 IC 50 values in the range of 0.21-13.84 mM and 3.78-70.58 mM, respectively., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

AMP-activated protein kinase (AMPK) is considered as the master cellular metabolism regulator that activates various proteins, including O-GlcNAc transferase (OGT). Physiological roles of AMPK and OGT, including the relationship between their mRNA expression and food intake, are poorly understood in channel catfish. This study examined the tissue distribution of AMPK and OGT mRNA and changes in their expression in response to changes in food intake in channel catfish. Expression of all AMPK subunit and OGT mRNA was detectable in the whole brain, liver, heart, spleen, white muscle, and kidney of channel catfish. The OGT mRNA was highly localized in the brain compared to other tissues. 28-day fasting increased hepatic expression of AMPK α1, β1, and OGT mRNA while refeeding fish for 14 days after the 14-day fast decreased their expression to the level similar to that of fish that were fed daily. No changes were noted in the expression of muscle and brain AMPK mRNA or OGT mRNA by fasting and refeeding. Hepatic AMPK α1, α2 and β1 mRNA decreased in response to increased feeding frequency, whereas no changes in the expression of AMPK or OGT mRNA were noted in the brain or the muscle. Results of the current study indicated that the hepatic expression of AMPK and OGT mRNA appeared to be more sensitive to changes in food intake in channel catfish. However, further studies are needed to clearly demonstrate if food intake influences the expression of AMPK and OGT mRNA in various tissues, including the hypothalamus., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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 LC 50 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.

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 ± 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., (Copyright © 2019. Published by Elsevier Inc.)

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.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

Glycogen phosphorylase enzymes (GP) catalyse reversible reactions; the glucose transfer from glycogen to inorganic phosphate (P i , 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/P i , respectively. Substrate specificity was studied using different maltooligosaccharides and glycogen. Based on the K M 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 K i of 23 ± 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. K i values obtained in the direction of synthesis (1.92 ± 0.14 mM for caffeine and 11.5 ± 2.0 μM for GTH) have been shown to be in good agreement with the K i values obtained in the direction of phosphorolysis (4.05 ± 0.26 mM for caffeine and 13.8 ± 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The toxicity of titanium dioxide nanoparticles (TiO 2 -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 TiO 2 -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. TiO 2 -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 TiO 2 -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 TiO 2 -NP are metabolized and how its bioaccumulation may affect fish homeostasis and survival in the environment., (© 2019 Wiley Periodicals, Inc.)

Ischemia-reperfusion injury is caused by a period of ischemia followed by massive blood flow into a tissue that had experienced restricted blood flow. The severity of the injury is dependent on the time the tissue was restricted from blood flow, becoming more severe after longer ischemia times. This can lead to many complications such as tissue necrosis, cellular apoptosis, inflammation, metabolic and mitochondrial dysfunction, and even organ failure. One of the emerging therapies to combat ischemic reperfusion injury complications is hydrogen sulfide, which is a gasotransmitter that diffuses across cell membranes to exert effects on various signaling pathways regulating cell survival such as Akt, mitochondrial activity, and apoptosis. Although commonly thought of as a toxic gas, low concentrations of hydrogen sulfide have been shown to be beneficial in promoting tissue survival post-ischemia, and modulate a wide variety of cellular responses. This review will detail the mechanisms of hydrogen sulfide in affecting the Akt signaling pathway, mitochondrial function, and apoptosis, particularly in regards to ischemic reperfusion injury in muscle tissue. It will conclude with potential clinical applications of hydrogen sulfide, combinations with other therapies, and perspectives for future studies.

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.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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 (pCMVβ) expressible in eukaryotic cells before being elaborated. The plasmid is characterized by the insertion of the reporter gene lacZ, encoding for the bacterial enzyme β-galactosidase (β-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 β-gal was detected and quantified in muscle, liver and intestine; on the other, specific IgM against β-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 (β-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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2018. Published by Elsevier B.V.)

Esterase activity found in muscle extracts is useful to evaluate harmful effects of anticholinesterase pollutants. Yet, most procedures applied in the extraction of fish muscle esterases in order to investigate their activity as a biomarker of environmental exposure comprise the homogenization of muscle tissue in low-salt solutions, followed by centrifugation to separate the supernatant as the enzyme source. However, acetylcholinesterase (AChE), the main target in these monitoring efforts, is a membrane-bound protein and is only present in muscle extracts if homogenization is carried out using chaotropic high-salt solutions. In this context, four extraction procedures using muscle tissue from six fish species were evaluated in order to establish a reproducible and reliable AChE assay for the determination of this biomarker. Results indicate that over 80% of AChE activity might be lacking in low-salt supernatants, and that the highest activities are obtained after extraction with solutions containing either 1molL -1 NaCl or 1molL -1 NaCl plus 3% Triton X-100, preserving almost 100% esterase activity over acetylthiocholine as substrate after centrifugation. Thus, many studies in the literature suffer from theoretical flaws and report erroneous AChE activity, since typical muscle AChE activity, the end-point biomarker for anticholinesterase pollutants, may have not been consistently assayed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

Spinosad and temephos are two of the most used pesticides in Mexico for the control of vector causing disease such as dengue, chikungunya and Zika. The aim of this study was to compare the neurotoxic effects of these two pesticides using guppy fish (Poecilia reticulata) as a model organism. Guppies were exposed for 7 and 21 days to technical grade temephos and spinosad at 1.0 and 0.07 g/L, respectively, (10 and 0.5 mg/L of active substance; concentrations recommended by the Ministery of Health of the State (Secretaría de Salud de Nayarit (SSN) Mexico)). Subsequently, acetylcholinesterase activity (AChE) and acetylcholine concentrations (ACh) in muscle tissue were determined. Temephos exposure decreased AChE activity and increased ACh concentration, whereas exposure to spinosad only increased ACh concentration. Though cholinergic alterations were more severe in fish exposed to temephos, both pesticides were equally lethal during the first seven days after exposure. Nonetheless, temephos was more lethal after 21 days.

Over the last years, cholinesterase (ChE) and carboxylesterase (CbE) activities have been increasingly used in environmental biomonitoring to detect the exposure to anticholinesterase insecticides such as organophosphorates (OPs) and carbamates (CBs). The aim of this study was to determine ChE and CbE enzymatic activities present in liver and muscle of yellow-legged gulls (Larus michahellis), a seabird species considered suitable to monitor environmental pollution. In order to provide reference data for further biomonitoring studies, the influence of different factors, such as gender, age, sampling mode, and tissue, was considered in the present study. Our data report a statistically significant difference in CbE enzymatic activity comparing liver and muscle samples (P < 0.05) along with an age-related CbE activity in liver samples (P < 0.05). Moreover, according to our results, capture method might influence CbE and ChE activity in both liver and muscle samples (P < 0.05). These findings underline the importance to assess basal levels of ChE and CbE activity considering, among other factors, gender-, age- and organ-related differences and confirm the suitability of Larus michahellis as a sentinel species especially within an urban environment.

In this work, a new strategy for electrochemical analysis of enzyme has been proposed based on a self-assembled lipid bilayer on an electrode surface mediated by hydrazone chemistry. Taking aldolase as an example, the enzyme can catalyze the formation of products containing carbonyl groups. These groups can react with hydrazine groups of the functional lipid derivative, resulting in the self-assembly of a lipid bilayer on a guanidinium modified electrode surface. The lipid bilayer will then prevent the movement of hydrophilic electrochemical probes. Consequently, the catalytic reaction of the enzyme may result in the change of the obtained electrochemical peak current. Experimental results reveal that aldolase activity can be analyzed over a widely linear detection range from 5 mU/L to 100 U/L with a low detection limit of 1 mU/L. Meanwhile, the method can exhibit good precision and reproducibility and it can be applied for real sample analysis. What is more, because the lipid bilayer is the universal basis for cell-membrane structure, while hydrazone chemistry is popular in nature, this work may also provide a new insight for the development of electrochemical analysis and electrochemical biosensors.

Tributyltin (TBT) is reported to induce adipogenesis in fish, which might affect nutritional qualities and health status. Muscle tissues account for the majority of body mass, and have been described as a major site of fat deposition and an immunologically active organ. Therefore, the present study aims to evaluate whether chronic exposures of TBT, at environmental concentrations of 1, 10 and 100 ng/L, affects lipid accumulation, oxidative stress and immune status in muscle tissues of rare minnow (Gobiocypris rarus). After 60 d of exposure, TBT increased contents of total lipid, total cholesterol, triglyceride and fatty acids in muscle tissues. Interestingly, TBT exposure disrupted fatty acid composition and increased contents of unsaturated fatty acids (such as eicosapentaenoic acid and docosahexaenoic acid) in muscle tissues, which might be a response to preserve membrane functions from TBT exposure. Meanwhile, the concentrations of hepatic fatty acid desaturase 2 (Δ6-desaturase) and stearoyl-CoA desaturase (Δ9-desaturase) were increased after TBT exposure, which might contribute the increase of unsaturated fatty acids. Furthermore, TBT increased muscle lipid peroxidation products, antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase), and the expression of immune-related molecules (tumor necrosis factor alpha, interleukin 1 beta and nuclear factor kappa B) in muscle tissues. The disruption of TBT on the lipid accumulation, oxidative stress and immune-toxic effects in muscle tissues of fish might reduce nutritional qualities, and affect growth and health status, which might pose a constant and serious threat to fish and result in economic loss in aquaculture., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The current NRC selenium (Se) requirement for turkeys is 0.2 μg Se/g diet. We previously fed turkey poults a Se-deficient diet (0.005 μg Se/g) supplemented with 10 graded levels of Se (0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1.0 μg Se/g as Na2SeO3, 5/treatment) for 4 wk, and found that the minimum dietary Se requirement was 0.3 μg Se/g based on selenoprotein enzyme activity in blood, liver, gizzard and pancreas. Because the turkey is primarily a production animal, we expanded this analysis to kidney, heart, breast and thigh. Se concentrations in Se-deficient poults were 5.0, 9.8, 33, and 15% of levels in poults fed 0.4 μg Se/g in liver, kidney, thigh and breast, respectively. Increasing Se supplementation resulted in hyperbolic response curves for all tissues; breakpoint analysis indicated minimum Se requirements of 0.34-0.36 μg Se/g based on tissue Se levels in liver, kidney and thigh. Similarly, GPX1 activity in muscle tissues and kidney responded hyperbolically to increasing dietary Se, reaching well-defined plateaus with breakpoints at 0.30-0.36 μg Se/g. Minimum Se requirements based on GPX4 activity were 0.30-0.32 μg Se/g for breast and thigh. Selenoprotein transcript expression decreased significantly in Se deficiency for only 2, 3, 5, and 6 mRNA in breast, thigh, heart, and kidney, respectively, out of 24 known avian selenoproteins. Se response curves for regulated selenoprotein transcripts were hyperbolic, and reached well-defined plateaus with breakpoints in a narrow range of 0.08-0.19 μg Se/g. No selenoprotein transcript was altered by supernutritional Se. In summary, these results clearly indicate that the NRC dietary Se requirement should be raised to 0.4 μg Se/g, at least for poults, to meet the nutritional needs of the young turkey. The Se response curve plateaus further show that limits for turkey supplementation with selenite could safely be raised to 0.5 μg Se/g diet.