Stress-induced changes in cognitive function and intestinal barrier integrity can be ameliorated by venlafaxine and synbiotic supplementations.

Stress profoundly impacts various aspects of both physical and psychological well-being. Our previous study demonstrated that venlafaxine (Vlx) and synbiotic (Syn) treatment attenuated learned fear-like behavior and recognition memory impairment in immobilized-stressed rats. In this study, we further investigated the physical, behavior, and cellular mechanisms underlying the effects of Syn and/or Vlx treatment on brain and intestinal functions in stressed rats. Adult male Wistar rats, aged 8 weeks old were subjected to 14 days of immobilization stress showed a decrease in body weight gain and food intake as well as an increase in water consumption, urinary corticosterone levels, and adrenal gland weight. Supplementation of Syn and/or Vlx in stressed rats resulted in mitigation of weight loss, restoration of normal food and fluid intake, and normalization of corticosterone levels. Behavioral analysis showed that treatment with Syn and/or Vlx enhanced depressive-like behaviors and improved spatial learning-memory impairment in stressed rats. Hippocampal dentate gyrus showed stress-induced neuronal cell death, which was attenuated by Syn and/or Vlx treatment. Stress-induced ileum inflammation and increased intestinal permeability were both effectively reduced by the supplementation of Syn. In addition, Syn and Vlx partly contributed to affecting the expression of the glial cell-derived neurotrophic factor in the hippocampus and intestines of stressed rats, suggesting particularly protective effects on both the gut barrier and the brain. This study highlights the intricate interplay between stress physiological responses in the brain and gut. Syn intervention alleviate stress-induced neuronal cell death and modulate depression- and memory impairment-like behaviors, and improve stress-induced gut barrier dysfunction which were similar to those of Vlx. These findings enhance our understanding of stress-related health conditions and suggest the synbiotic intervention may be a promising approach to ameliorate deleterious effects of stress on the gut-brain axis.

The role of the phosphate groups of trinitrophenyl adenosine 5'-triphosphate (TNP-ATP) in allosteric activation of pyruvate carboxylase and the inhibition of acetyl CoA-dependent activation.

A previous study showed that 2'-3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) was a weak allosteric activator of Rhizobium etli pyruvate carboxylase (RePC) in the absence of acetyl-CoA. On the other hand, TNP-ATP inhibited the allosteric activation of RePC by acetyl-CoA. Here, we aimed to study the role of triphosphate group of TNP-ATP on its allosteric activation of the enzyme and inhibition of acetyl-CoA-dependent activation of RePC using TNP-ATP and its derivatives, including TNP-ADP, TNP-AMP and TNP-adenosine. The pyruvate carboxylation activity was assayed to determine the effect of reducing the number of phosphate groups in TNP-ATP derivatives on allosteric activation and inhibition of acetyl-CoA activation of RePC and chicken liver pyruvate carboxylase (CLPC). Reducing the number of phosphate groups in TNP-ATP derivatives decreased the activation efficacy for both RePC and CLPC compared to TNP-ATP. The apparent binding affinity and inhibition of activation of the enzymes by acetyl-CoA were also diminished when the number of phosphate groups in the TNP-ATP derivatives was reduced. Whilst TNP-AMP activated RePC, it did not activate CLPC, but it did inhibit acetyl-CoA activation of both RePC and CLPC. Similarly, TNP-adenosine did not activate RePC; however, it did inhibit acetyl-CoA activation using a different mechanism compared to phosphorylated TNP-derivatives. These findings indicate that mechanisms of PC activation and inhibition of acetyl-CoA activation by TNP-ATP and its derivatives are different. This study provides the basis for possible drug development for treatment of metabolic diseases and cancers with aberrant expression of PC.

Biotin, a universal and essential cofactor: synthesis, ligation and regulation.

Biotin is a covalently attached enzyme cofactor required for intermediary metabolism in all three domains of life. Several important human pathogens (e.g. Mycobacterium tuberculosis) require biotin synthesis for pathogenesis. Humans lack a biotin synthetic pathway hence bacterial biotin synthesis is a prime target for new therapeutic agents. The biotin synthetic pathway is readily divided into early and late segments. Although pimelate, a 7-carbon α,ω-dicarboxylic acid that contributes 7 of the 10 biotin carbons atoms, was long known to be a biotin precursor, its biosynthetic pathway was a mystery until the Escherichia colipathway was discovered in 2010. Since then, diverse bacteria encode evolutionarily distinct enzymes that replace enzymes in the E. coli pathway. Two new bacterial pimelate synthesis pathways have been elucidated. In contrast to the early pathway, the late pathway, assembly of the fused rings of the cofactor, was long thought settled. However, a new enzyme that bypasses a canonical enzyme was recently discovered as well as homologs of another canonical enzyme that functions in synthesis of another protein-bound coenzyme, lipoic acid. Most bacteria tightly regulate transcription of the biotin synthetic genes in a biotin-responsive manner. The bifunctional biotin ligases which catalyze attachment of biotin to its cognate enzymes and repress biotin gene transcription are best understood regulatory system.

Biosynthetic sericin 1-like protein skews dendritic cells to tolerogenic-like phenotype.

Silkworm sericin has been widely exploited in biomaterials due to its favorable biological activities. However, the extraction processes of sericin from silkworm cocoons can alter the biological and biophysical properties, including a structural diversity of natural sericin. In addition, extracted natural sericin is often contaminated with fibroin that may be harmful to human cells. Induction of tolerogenic dendritic cell (DC) has become a strategy in biomaterial fields because this cell type plays a key role in immune modulation and wound healing. To overcome undesired effects of extracted natural sericin and to improve its biological properties, we biosynthesized sericin 1-like protein that contained only functional motifs and tested its biological activity and immunomodulatory properties in fibroblasts and DCs, respectively. In comparison to natural sericin, biosynthetic sericin 1 promoted collagen production in fibroblasts at a late time point. Furthermore, DCs treated with biosynthetic sericin 1 exhibited a tolerogenic-like phenotype with semimaturation and low production of proinflammatory cytokines, but high production of anti-inflammatory cytokine, IL-10. Biosynthetic sericin 1 might be developed as immunomodulator or immunosuppressant.

Investigation of the Roles of Allosteric Domain Arginine, Aspartate, and Glutamate Residues of Rhizobium etli Pyruvate Carboxylase in Relation to Its Activation by Acetyl CoA.

The mechanism of allosteric activation of pyruvate carboxylase by acetyl CoA is not fully understood. Here we have examined the roles of residues near the acetyl CoA binding site in the allosteric activation of Rhizobium etli pyruvate carboxylase using site-directed mutagenesis. Arg429 was found to be especially important for acetyl CoA binding as substitution with serine resulted in a 100-fold increase in the Ka of acetyl CoA activation and a large decrease in the cooperativity of this activation. Asp420 and Arg424, which do not make direct contact with bound acetyl CoA, were nonetheless found to affect acetyl CoA binding when mutated, probably through changed interactions with another acetyl CoA binding residue, Arg427. Thermodynamic activation parameters for the pyruvate carboxylation reaction were determined from modified Arrhenius plots and showed that acetyl CoA acts to decrease the activation free energy of the reaction by both increasing the activation entropy and decreasing the activation enthalpy. Most importantly, mutations of Asp420, Arg424, and Arg429 enhanced the activity of the enzyme in the absence of acetyl CoA. A main focus of this work was the detailed investigation of how this increase in activity occurred in the R424S mutant. This mutation decreased the activation enthalpy of the pyruvate carboxylation reaction by an amount consistent with removal of a single hydrogen bond. It is postulated that Arg424 forms a hydrogen bonding interaction with another residue that stabilizes the asymmetrical conformation of the R. etli pyruvate carboxylase tetramer, constraining its interconversion to the symmetrical conformer that is required for catalysis.

Residues in the acetyl CoA binding site of pyruvate carboxylase involved in allosteric regulation.

We have examined the roles of Asp1018, Glu1027, Arg469 and Asp471 in the allosteric domain of Rhizobium etli pyruvate carboxylase. Arg469 and Asp471 interact directly with the allosteric activator acetyl coenzyme A (acetyl CoA) and the R469S and R469K mutants showed increased enzymic activity in the presence and absence of acetyl CoA, whilst the D471A mutant exhibited no acetyl CoA-activation. E1027A, E1027R and D1018A mutants had increased activity in the absence of acetyl CoA, but not in its presence. These results suggest that most of these residues impose restrictions on the structure and/or dynamics of the enzyme to affect activity.

Mechanisms of inhibition of Rhizobium etli pyruvate carboxylase by L-aspartate.

L-aspartate is a regulatory feedback inhibitor of the biotin-dependent enzyme pyruvate carboxylase in response to increased levels of tricarboxylic acid cycle intermediates. Detailed studies of L-aspartate inhibition of pyruvate carboxylase have been mainly confined to eukaryotic microbial enzymes, and aspects of its mode of action remain unclear. Here we examine its inhibition of the bacterial enzyme Rhizobium etli pyruvate carboxylase. Kinetic studies demonstrated that L-aspartate binds to the enzyme cooperatively and inhibits the enzyme competitively with respect to acetyl-CoA. L-aspartate also inhibits activation of the enzyme by MgTNP-ATP. The action of L-aspartate was not confined to inhibition of acetyl-CoA binding, because the acetyl-CoA-independent activity of the enzyme was also inhibited by increasing concentrations of L-aspartate. This inhibition of acetyl-CoA-independent activity was demonstrated to be focused in the biotin carboxylation domain of the enzyme, and it had no effect on the oxamate-induced oxaloacetate decarboxylation reaction that occurs in the carboxyl transferase domain. L-aspartate was shown to competitively inhibit bicarbonate-dependent MgATP cleavage with respect to MgATP but also probably inhibits carboxybiotin formation and/or translocation of the carboxybiotin to the site of pyruvate carboxylation. Unlike acetyl-CoA, L-aspartate has no effect on the coupling between MgATP cleavage and oxaloacetate formation. The results suggest that the three allosteric effector sites (acetyl-CoA, MgTNP-ATP, and L-aspartate) are spatially distinct but connected by a network of allosteric interactions.