Response to weaning and dietary L-glutamine supplementation: metabolomic analysis in piglets by gas chromatography/mass spectrometry.

A novel metabolomic method based on gas chromatography/mass spectrometry (GC-MS) was applied to determine the metabolites in the serum of piglets in response to weaning and dietary L-glutamine (Gln) supplementation. Thirty-six 21-d-old piglets were randomly assigned into three groups. One group continued to suckle from the sows (suckling group), whereas the other two groups were weaned and their diets were supplemented with 1% (w/w) Gln or isonitrogenous L-alanine, respectively, representing Gln group or control group. Serum samples were collected to characterize metabolites after a 7-d treatment. Results showed that twenty metabolites were down-regulated significantly (P<0.05) in control piglets compared with suckling ones. These data demonstrated that early weaning causes a wide range of metabolic changes across arginine and proline metabolism, aminosugar and nucleotide metabolism, galactose metabolism, glycerophospholipid metabolism, biosynthesis of unsaturated fatty acid, and fatty acid metabolism. Dietary Gln supplementation increased the levels of creatinine, D-xylose, 2-hydroxybutyric acid, palmitelaidic acid, and α-L-galactofuranose (P<0.05) in early weaned piglets, and were involved in the arginine and proline metabolism, carbohydrate metabolism, and fatty acid metabolism. A leave-one-out cross-validation of random forest analysis indicated that creatinine was the most important metabolite among the three groups. Notably, the concentration of creatinine in control piglets was decreased (P=0.00001) compared to the suckling piglets, and increased (P=0.0003) in Gln-supplemented piglets. A correlation network for weaned and suckling piglets revealed that early weaning changed the metabolic pathways, leading to the abnormality of carbohydrate metabolism, amino acid metabolism, and lipid metabolism, which could be partially improved by dietary Gln supplementation. These findings provide fresh insight into the complex metabolic changes in response to early weaning and dietary Gln supplementation in piglets.

Effect of double mutations K214/A-E215/Q of FRATide on GSK3ß: insights from molecular dynamics simulation and normal mode analysis.

Glycogen synthase kinase 3ß (GSK3ß) is a multifunctional serine/threonine protein kinase that is involved in several biological processes including insulin and Wnt signaling pathways. The Wnt signaling via FRAT-mediated displacement of axin inhibits GSK3ß activity toward non-primed substrates without affecting its activity toward primed substrates. Herein, molecular dynamics simulation, molecular mechanics generalized Born/surface area (MM_GBSA) calculation, and normal mode analysis are performed to explore the structural influence of the double mutations K214/A-E215/Q of FRATide on the GSK3ß-FRATide complex. The results reveal that the priming phosphate-binding site, the primed substrate-binding site, the alignment of the critical active site residues in the ATP-binding site, as well as the periodic open-closed conformational change of the ATP-binding site, which are critical for the catalytic activity of GSK3ß, are negligibly influenced in the mutated system compared with the wild-type (WT) system. This indicates that FRATide does not inhibit the GSK3ß activity toward primed substrates. Additionally, MM_GBSA calculation indicates that the less energy-favorable GSK3ß-FRATide complex is observed in the mutant than in the WT complex.

Molecular modeling and molecular dynamics simulation studies on pyrrolopyrimidine-based α-helix mimetic as dual inhibitors of MDM2 and MDMX.

Inhibition of the interactions between the tumor suppressor protein p53 and its negative regulators, the MDM2 and MDMX oncogenic proteins, is increasingly gaining interest in cancer therapy and drug design. In this study, we carry out molecular docking, molecular dynamics (MD) simulations, and molecular mechanics Poisson-Boltzmann and generalized Born/surface area (MM-PB/GBSA) binding free energy calculations on an active compound 3a and an inactive compound NC-1, which share a common pyrrolopyrimidine-based scaffold. MD simulations and MM-PB/GBSA calculations show that the compound NC-1 may not bind to MDM2 and MDMX, in agreement with the experimental results. Detailed MM-PB/GBSA calculations on the MDM2-3a and MDMX-3a complexes unravel that the binding free energies are similar for the two complexes. Furthermore, the van der Waals energy is the largest component of the binding free energy for both complexes, which indicates that the interactions between the compound 3a and MDM2 and MDMX are dominated by shape complementarity. In addition, the analysis of individual residue contribution and protein-ligand binding mode show that the three functional groups on R1, R2, and R3 of the compound 3a can mimic the spatial orientation of the side chains of Phe19, Trp23, and Leu26 of p53, respectively. The obtained computational results suggest that the compound 3a can act as a dual inhibitor of MDM2-p53 and MDMX-p53 interactions, consistent with the experimental results.

Molecular modeling and molecular dynamics simulation studies of the GSK3ß/ATP/substrate complex: understanding the unique P+4 primed phosphorylation specificity for GSK3ß substrates.

Substrate specificity of protein kinases is of fundamental importance for the integrity and fidelity of signaling pathways. Glycogen synthase kinase 3ß (GSK3ß) has a unique substrate specificity that prefers phosphorylation of its substrates at the P+4 serine before it can further phosphorylate the substrate at the P0 serine in the canonical motif SXXXS(p), where S(p) is the primed phosphorylation site. The detailed phosphorylation mechanism, however, is not clearly understood. In this study, a three-dimensional (3D) model of the ternary complex of GSK3ß, ATP, and the phosphorylated glycogen synthase (pGS), termed GSK3ß/ATP/pGS, is constructed using a hierarchical approach and by integrating molecular modeling and molecular dynamics (MD) simulations. Based on the 3D model, the substrate primed phosphorylation mechanism is investigated via two 12 ns comparative MD simulations of the GSK3ß/ATP/pGS and GSK3ß/ATP/GS systems, which differ in the phosphate group bound to the P+4 serine of GS. In agreement with structural analysis, computed binding free energies reveal that the binding of pGS to GSK3ß is favored in the prephosphorylated state compared with the GS native state. More importantly, comparison with the system simulated without primed phosphorylation in the GSK3ß/ATP/GS complex shows that for an optimal phosphorylation reaction to occur, the pGS priming phosphate in the GSK3ß/ATP/pGS system optimizes the proper orientation of the GSK3ß N- and C-terminal domains and clamps the P0 serine of pGS in the appropriate configuration for interaction with the ATP γ-phosphate within the catalytic groove.

Role of bridging water molecules in GSK3ß-inhibitor complexes: insights from QM/MM, MD, and molecular docking studies.

The role of water molecules is increasingly gaining interest in drug design, and several studies have highlighted their paramount contributions to the specificity and the affinity of ligand binding. In this study, we employ the two-layer ONIOM-based quantum mechanics/molecular mechanics (QM/MM) calculations, molecular dynamics (MD) simulations, and molecular docking studies to investigate the effect of bridging water molecules at the GSK3ß-inhibitors interfaces. The results obtained from the ONIOM geometry optimization and AIM analysis corroborated the presence of bridging water molecules that form hydrogen bonds with protein side chain of Thr138 and/or backbone of Gln185, and mediate interactions with inhibitors in the 10 selected GSK3ß-inhibitor complexes. Subsequently, MD simulations carried out on a representative system of 1R0E demonstrated that the bridging water molecule is stable at the GSK3ß-inhibitor interface and appears to contribute to the stability of the protein-inhibitor interactions. Furthermore, molecular docking studies of GSK3ß-inhibitor complexes indicated that the inhibitors can increase binding affinities and the better docked conformation of inhibitors can be obtained by inclusion of the bridging water molecules, especially for the flexible inhibitors, in docking experiments into individual protein conformations. Our results elucidate the importance of bridging water molecules at the GSK3ß-inhibitor interfaces and suggest that they might prove useful in rational drug design.

Dissection of the difference between the group I metal ions in inhibiting GSK3ß: a computational study.

Glycogen synthase kinase 3ß (GSK3ß) is a serine/threonine kinase that requires two cofactor Mg(2+) ions for catalysis in regulating many important cellular signals. Experimentally, Li(+) is a competitive inhibitor of GSK3ß relative to Mg(2+), while this mechanism is not experienced with other group I metal ions. Herein, we use native Mg(2)(2+)-Mg(1)(2+) GSK3ß and its Mg(2)(2+)-M(1)(+) (M = Li, Na, K, and Rb) derivatives to investigate the effect of metal ion substitution on the mechanism of inhibition through two-layer ONIOM-based quantum mechanics/molecular mechanics (QM/MM) calculations and molecular dynamics (MD) simulations. The results of ONIOM calculations elucidate that the interaction of Na(+), K(+), and Rb(+) with ATP is weaker compared to that of Mg(2+) and Li(+) with ATP, and the critical triphosphate moiety of ATP undergoes a large conformational change in the Na(+), K(+), and Rb(+) substituted systems. As a result, the three metal ions (Na(+), K(+), and Rb(+)) are not stable and depart from the active site, while Mg(2+) and Li(+) can stabilize in the active site, evident in MD simulations. Comparisons of Mg(2)(2+)-Mg(1)(2+) and Mg(2)(2+)-Li(1)(+) systems reveal that the inline phosphor-transfer of ATP and the two conserved hydrogen bonds between Lys85 and ATP, together with the electrostatic potential at the Li(1)(+) site, are disrupted in the Mg(2)(2+)-Li(1)(+) system. These computational results highlight the possible mechanism why Li(+) inhibits GSK3ß.

Insight into analysis of interactions of GW9508 to wild-type and H86F and H137F GPR40: a combined QM/MM study and pharmacophore modeling.

GPR40 is a novel potential target for the treatment of type 2 diabetes. In this work, a two-layered ONIOM based QM/MM approach was employed to study the interactions between GW9508 and GPR40: wild-type, H86F, and H137F mutated systems. The calculated results clearly indicated that His137 is directly involved in ligand recognition through the NH-π interaction with the GW9508. In contrast, His86 is not interacting with the GW9508 in the NH-π interaction. The interaction energies, calculated at the MP2/6-31(d, p) level, were performed to gain more insight into the energetic differences of the wild-type and two mutated systems at the atomistic level. In addition, the obtained pharmacophore model was well consistent with structure-functional requirements for the binding of GPR40 agonists and with per-residue energy decomposition of the ONIOM calculations.

Molecular docking and molecular dynamics simulation studies of GPR40 receptor-agonist interactions.

In order to explore the agonistic activity of small-molecule agonists to GPR40, AutoDock and GROMACS software were used for docking and molecular dynamics studies. A molecular docking of eight structurally diverse agonists (six carboxylic acids (CAs) agonist, and two non-carboxylic acids (non-CAs) agonist) was performed and the differences in their binding modes were investigated. Moreover, a good linear relationship based on the predicted binding affinities (pK(i)) determined by using AutoDock and experimental activity values (pEC50) was obtained. Then, the 10ns molecular dynamics (MD) simulations of three obtained ligand-receptor complexes embedded into the phospholipid bilayer were carried out. The position fluctuations of the ligands located inside the transmembrane domain were explored, and the stable binding modes of the three studied agonists were determined. Furthermore, the residue-based decomposition of interaction energies in three systems identified several critical residues for ligand binding.

Prebiotic oligosaccharides change the concentrations of short-chain fatty acids and the microbial population of mouse bowel.

The purpose of this study was to clarify effects of selected oligosaccharides on concentrations of cecal short-chain fatty acids (SCFAs), total large bowel wet weight and wall weight, and cecal microbiota levels in mice. Mice were respectively given gavage of selected fructooligosaccharides (FOS), galactooligosaccharides (GOS), mannanoligosaccharides (MOS), and chitooligosaccharides (COS) [1000 mg/(kg body weight.d)]. Control group was given physiological saline solution. After 14 d treatment, SCFAs and lactate in mice cecum were significantly increased (P<0.05) by intake of oligosaccharides, especially FOS and GOS. Thus, providing these oligosaccharides as ingredients in nutritional formulas may benefit the gastrointestinal tract.

Curcumin suppresses PPARdelta expression and related genes in HT-29 cells.

AIM: To investigate the effects of curcumin on the expression of peroxisome proliferator-activated receptordelta (PPARdelta) and related genes in HT-29 cells. METHODS: HT-29 cells were treated with curcumin (0-80 micromol/L) for 24 h. The effects of curcumin on the morphology of HT-29 cells were studied by Hoechst 33342 staining. The activity of caspase-3 was determined using DEVD-pNA as substrate. The levels of peroxisome PPARdelta, 14-3-3epsilon and vascular endothelial growth factor (VEGF) in HT-29 cells were determined by Western blotting analysis and their mRNA expression was determined by real-time quantitative RT-PCR. RESULTS: Treatment with 10-80 micromol/L curcumin induced typical features of apoptosis and activated the caspase-3 in HT-29 cells. The expression of PPARdelta, 14-3-3epsilon and VEGF was reduced and the activity of beta-catenin/Tcf-4 signaling was inhibited by curcumin treatment. CONCLUSION: Curcumin can induce apoptosis of HT-29 cells and down-regulate the expression of PPARdelta, 14-3-3epsilon and VEGF in HT-29.

Curcumin induces apoptosis through the mitochondria-mediated apoptotic pathway in HT-29 cells.

OBJECTIVE: To investigate the effects of curcumin on release of cytochrome c and expressions of Bcl-2, Bax, Bad, Bcl-xL, caspase-3, poly ADP-ribose polymerase (PARP), and survivin of HT-29 cells. METHODS: HT-29 cells were treated with curcumin (0 approximately 80 micromol/L) for 24 h. The release of cytochrome c from the mitochondria and the apoptosis-related proteins Bax, Bcl-2, Bcl-xL, Bad, caspase-3, PARP, and survivin were determined by Western blot analysis and their mRNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Curcumin significantly induced the growth inhibition and apoptosis of HT-29 cells. A decrease in expressions of Bcl-2, Bcl-xL and survivin was observed after exposure to 10 approximately 80 micromol/L curcumin, while the levels of Bax and Bad increased in the curcumin-treated cells. Curcumin also induced the release of cytochrome c, the activation of caspase-3, and the cleavage of PARP in a dose-dependent manner. CONCLUSION: These data suggest that curcumin induced the HT-29 cell apoptosis possibly via the mitochondria-mediated pathway.

Fatty acid profiles of muscle from large yellow croaker (Pseudosciaena crocea R.) of different age.

We investigated the fatty acid profiles of muscle from large yellow croaker (Pseudosciaena crocea R.) of different age. One- and two-year-old fish were cultured in floating net cages and sampled randomly for analysis. Moisture, protein, lipid and ash contents were determined by methods of Association of Analytical Chemist (AOAC) International. Fatty acid profile was determined by gas chromatography. Crude protein, fat, moisture and ash contents showed no significant differences between the two age groups. The contents of total polyunsaturated fatty acids and docosahexaenoic acid (DHA) were significantly higher and eicosapentaenoic acid (EPA) content was significantly lower in the two-year-old large yellow croaker than in the one-year-old (P<0.05). No significant differences were observed in the contents of total saturated fatty acids and monounsaturated fatty acids, or the ratio of n-3/n-6 fatty acids among the large yellow croakers of the two age groups. We conclude that large yellow croakers are good food sources of EPA and DHA.

Effects of fish protein hydrolysate on growth performance and humoral immune response in large yellow croaker (Pseudosciaena crocea R.).

We investigated the effects of fish protein hydrolysate (FPH) on growth performance and humoral immune response of the large yellow croaker (Pseudosciaena crocea R.). One thousand and two hundred large yellow croakers [initial average weight: (162.75+/-23.85) g] were divided into four groups and reared in floating sea cages (3 m x 3 m x 3 m). The animals were fed with 4 diets: basal diet only (control) or diets supplemented with 5%, 10% and 15% (w/w) FPH. The results show that dietary FPH levels significantly influenced the growth and immunity of the large yellow croaker. Compared with the control group, total weight gain (TWG) in all treatment groups, relative weight gain (RWG) and specific growth rate (SGR) in fish fed with diets supplemented with 10% and 15% FPH were significantly increased (P<0.05). Similar results were observed in immune parameters [lysozyme activity, serum complements, immunoglobulin M (IgM)]. Lysozyme activity, complement C4 and IgM were also significantly increased (P<0.05) in fish fed with diets supplemented with 10% and 15% FPH, while complement C3 level was significantly increased (P<0.05) in all treatment groups. In general, with the supplementation of FPH, particularly at dose of 10%, the growth performance and immunity of the large yellow croaker can be improved effectively.

Influence of dietary conjugated linoleic acid on growth, fatty acid composition and hepatic lipogenesis in large yellow croaker (Pseudosciaena crocea R.).

We examined the effects of conjugated linoleic acid (CLA) on growth, fatty acid composition and enzyme activity of fatty acid oxidation in the liver of large yellow croaker. We divided 1600 fish (average initial weight 150 g) into 4 groups and reared them in 8 cages. Four dietary treatments were formulated to contain 0%, 1%, 2% and 4% (w/w) CLA, respectively. The fish were fed for 10 weeks ad libitum twice daily. We found that the dietary CLA had no effect on growth, biometric parameters and whole body proximate (P>0.05), but showed some significant effects on the fatty acid composition in both muscle and the liver. The activities of lipogenic enzymes were slightly depressed in fish fed with increasing levels of CLA when compared with control (P>0.05). Dietary CLA supplementation had no effects on liver lipid content, but significantly increased the contents of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) (P<0.05) and decreased monounsaturated fatty acid (MUFA) content in both muscle and the liver. Dietary CLA inclusion resulted in significant increases of the biologically active cis-9, trans-11 and trans-10, cis-12 isomers in both tissues (P<0.05). The total accumulation of CLA was higher in the liver (3.83%, w/w) than in muscle (3.77%, w/w) when fed with 4% (w/w) CLA. This study demonstrates that large yellow croakers are capable of absorbing and depositing CLA and long-chain n-3 PUFA in the liver and muscle, showing that this species fed with CLA could be an important human food source for these healthful fatty acids.

Effects of alpha-tocopheryl acetate supplementation in preslaughter diet on antioxidant enzyme activities and fillet quality of commercial-size Sparus macrocephalus.

This study examined the effects of dietary alpha-tocopheryl acetate supplementation on antioxidant enzyme activities and fillet quality in commercial-size Sparus macrocephalus. Three hundred fish [main initial weight (350+/-12) g] were divided into three groups (E250, E500 and E1000) and reared in 9 cages. The fish were fed for 8 weeks with three diets containing different levels of dietary alpha-tocopheryl acetate (289, 553, 1 069 mg/kg). Over the experimental period, fish were fed to satiation and reached a final mean weight of (465+/-28) g without significant body weight difference and proximate composition difference. Fillet alpha-tocopherol was significantly (P<0.05) different between groups, reaching levels of 14.2, 22.1, 30.9 microg/mg fillet for groups E250, E500 and E1000, respectively. Total serum superoxide dismutase (SOD) activity increased significantly (P<0.05) in fish fed the diets high in alpha-tocopheryl acetate, but serum glutathione peroxidase (GPX) activity was unaffected. In storage on ice, fillets of fish fed the diets high in alpha-tocopheryl acetate exhibited significantly lower (P<0.05) levels of oxidation. These results suggested that increased dietary alpha-tocopheryl acetate could increase its flesh deposition, increase the activity of SOD and prevent lipid peroxidation of Sparus macrocephalus fillets in retail storage on ice.

Influence of fasting on muscle composition and antioxidant defenses of market-size Sparus macrocephalus.

The study was conducted to investigate fasting effects on flesh composition and antioxidant defenses of market-size Sparus macrocephalus. Two hundred fish (main initial weight 580 g) were divided into two groups (control and fasted) and reared in 6 cages. After two weeks of adaptation, group I fasted for 28 d; group II was fed normally as a control. In 3, 7, 14, 21 and 28 d, 6 fish per group were sampled for proximate flesh composition, liver antioxidant enzyme activities and malondialdehyde flesh content analyses. In fasted fish, the reduction of lipid content in muscle occurred after day 3, and, compared to controls, the content of protein decreased from day 14, the activities of liver antioxidative enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPX) increased from day 3, and flesh malondialdehyde levels increased from day 21. Flesh fat reduction shows that fasting may be used as a technique to reduce flesh lipid content in Sparus macrocephalus. However, considering flesh protein loss and the subsequent oxidative stress, the fasting technique should be used with precautions.

Effects of dietary supplementation with clostridium butyricum on the growth performance and humoral immune response in Miichthys miiuy.

The effects of dietary supplementation with Clostridium butyricum on growth performance and humoral immune response in Miichthys miiuy were evaluated. One hundred and fifty Miichthys miiuy weighing approximately 200-260 g were divided into five groups and reared in 15 tanks with closed circuiting culture system. The animals were fed 5 diets: basal diet only (control) or supplemented of the basal diet with C. butyricum at doses of 10(3) (CB1), 10(5) (CB2), 10(7) (CB3) or 10(9) (CB4) CFU/g. Compared with the control, the serum phenoloxidase activity was significantly increased by the supplementation (P<0.05), acid phosphatases activity was increased significantly (P<0.05) at the doses of 10(9) CFU/g. Serum lysozyme activity peaked at dose of 10(7) CFU/g and in the skin mucus at dose of 10(9) CFU/g. Immunoglobulin M level in the serum and skin mucus was increased except at dose of 10(3) CFU/g (P<0.05). The growth at the dose of 10(9) CFU/g was higher than that of the control (P<0.05). It is concluded that supplementation of C. butyricum can mediate the humoral immune responses and improve the growth performance in Miichthys miiuy.

Effects of the dietary supplementation with fructooligosaccharides on the excretion of nitrogen and phosphorus in Miichthys miiuy fries.

Effects of dietary supplementation with fructooligosaccharides on the excretion of nitrogen and phosphorus in Miichthys miiuy fries were investigated. Nine hundred Miichthys miiuy fries were divided into 3 groups, each with triplicates. The basal diet and the basal diet supplemented with carnitine groups were considered as the negative and positive controls respectively. Results showed that the nitrogen concentration in excreted feces decreased significantly in fries fed the diet supplementation with 1000 x 10(-6) fructooligosaccharides and 200 x 10(-6) carnitine (P<0.05). The ammonic-nitrogen concentration decreased significantly in the carnitine group only (P<0.05), indicating the decreasing tendency caused by the supplementation with fructooligosaccharides. Supplementation with both did not have significant effects on the concentration of phosphorus in feces of Miichthys miiuy fries.