Chitin deacetylases are necessary for insect femur muscle attachment and mobility.

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.

Glycogen phosphorylase revisited: extending the resolution of the R- and T-state structures of the free enzyme and in complex with allosteric activators.

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.

Impact of metal nanoparticles on the structure and function of metabolic enzymes.

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.

Role of Endogenous Cathepsin L in Muscle Protein Degradation in Olive Flounder (Paralichthys olivaceus) Surimi Gel.

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.

The linear ubiquitin E3 ligase-Relish pathway is involved in the regulation of proteostasis in Drosophila muscle during aging.

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.

Application of proteomics to understand the molecular mechanisms determining meat quality of beef muscles during postmortem aging.

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.

Structural studies of human muscle FBPase.

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).

Loss of metabolic flexibility as a result of overexpression of pyruvate dehydrogenase kinases in muscle, liver and the immune system: Therapeutic targets in metabolic diseases.

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 Ca2+ into mitochondria through mitochondria-associated endoplasmic reticulum membranes are discussed. Recent efforts to find small molecule PDK inhibitors with therapeutic potential are also reviewed.

Why Are Branched-Chain Amino Acids Increased in Starvation and Diabetes?

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.

Purification and Regulation of Pyruvate Kinase from the Foot Muscle of the Anoxia and Freeze Tolerant Marine Snail, Littorina littorea.

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.

Enzymatic, non enzymatic antioxidants and glucose metabolism enzymes response differently against metal stress in muscles of three fish species depending on different feeding niche.

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.

Dichloroacetate reveals the presence of metabolic inertia at the start of exercise in rainbow trout (Oncorhynchus mykiss, Walbaum 1792).

A lag in the increase in oxygen consumption (MO2 ) 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 MO2 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 MO2 thereafter or at rest. This indicates that a delay in PDH activation ("metabolic inertia") contributes to the lag phenomenon.

Comparative assessment of biomarker response to tissue metal concentrations in urban populations of the land snail Helix pomatia (Pulmonata: Helicidae).

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.

Marker enzyme activities in hindleg from creatine-deficient AGAT and GAMT KO mice - differences between models, muscles, and sexes.

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.

Regulation of the α-ketoglutarate dehydrogenasecomplex during hibernation in a small mammal, the Richardson's ground squirrel (Urocitellus richardsonii).

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 CO2 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 Ca2+ 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.

Correct Fish Muscle AChE Extraction for Use in Environmental Risk Assessment and Ecotoxicological Studies Has Been Neglected.

Several articles published in the field of ecotoxicology and environmental risk assessment have failed to adequately use fish muscle cholinesterases as biomarkers. This letter seeks to underline the importance of developing and applying correct protocols for the extraction of these enzymes from the tissues of these animals.

Pressure tolerance of deep-sea enzymes can be evolved through increasing volume changes in protein transitions: a study with lactate dehydrogenases from abyssal and hadal fishes.

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 ( ΔVPP∘ ). 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 ΔVPP∘ 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.

Banded tetra (Astyanax aeneus) as bioindicator of trace metals in aquatic ecosystems of the Yucatan Peninsula, Mexico: Experimental biomarkers validation and wild populations biomonitoring.

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 (CuSO4) 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.

Hydrolysis of oxidized phosphatidylcholines by crude enzymes from chicken, pork and beef muscles.

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.

Comprehensive in silico Study of GLUT10: Prediction of Possible Substrate Binding Sites and Interacting Molecules.

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.