D-amino Acid Oxidase Activity Research Articles

Development of growth selection system and pocket engineering of d-amino acid oxidase to enhance selective deamination activity toward d-phosphinothricin.

D-amino acid oxidase (DAAO)-catalyzed selective oxidative deamination is a very promising process for synthesizing l-amino acids including l-phosphinothricin ( l-PPT, a high-efficiency and broad-spectrum herbicide). However, the wild-type DAAO's low activity toward unnatural substrates like d-phosphinothricin ( d-PPT) hampers its application. Herein, a DAAO from Caenorhabditis elegans (CeDAAO) was screened and engineered to improve the catalytic potential on d-PPT. First, we designed a novel growth selection system, taking into account the intricate relationship between the growth of Escherichia coli (E. coli) and the catalytic mechanism of DAAO. The developed system was used for high-throughput screening of gene libraries, resulting in the discovery of a variant (M6) with significantly increased catalytic activity against d-PPT. The variant displays different catalytic properties on substrates with varying hydrophobicity and hydrophilicity. Analysis using Alphafold2 modeling and molecular dynamic simulations showed that the reason for the enhanced activity was the substrate-binding pocket with enlarged size and suitable charge distribution. Further QM/MM calculations revealed that the crucial factor for enhancing activity lies in reducing the initial energy barrier of the reductive half reaction. Finally, a comprehensive binding-model index to predict the enhanced activity of DAAO toward d-PPT, and an enzymatic deracemization approach was developed, enabling the efficient synthesis of l-PPT with remarkable efficiency.

D-serine and D-amino acid oxidase levels in patients with schizophrenia spectrum disorders in the first episode and 6-month follow-up

BackgroundD-serine and the D-amino acid oxidase (DAO) enzyme, which breaks down d-amino acids, may be involved in the pathophysiology of schizophrenia by affecting the N-methyl-D-aspartate (NMDA) receptor. The exact role of D-serine and DAO, as well as the consequences of increased DAO activity in patients with schizophrenia, remain unclear. We aimed to investigate D-serine and DAO levels in patients with first-episode schizophrenia spectrum disorders before treatment and after six months of treatment. MethodComparisons for the serum levels of D-serine and DAO were made between 81 healthy controls and 89 patients with first-episode schizophrenia spectrum disorders without a history of treatment. Further comparisons were made after 6 months for changes in these levels in the 41 patients in follow-up. The Positive and Negative Syndrome Scale (PANNS), Calgary Scale for Depression in Schizophrenia (CDSS), Montreal Cognitive Assessment Scale (MoCA), Global Assessment Scale (GAS), and Clinical Global Impression Scale (CGI) were used to evaluate the symptom severity and functionality. Secondary results included comparisons related to antipsychotic equivalent doses. ResultsBefore treatment, patients had significantly lower levels of D-serine, DAO, and D-serine/DAO ratio compared to healthy individuals (p < 0.001; p < 0.001; p = 0.004). DAO and D-serine levels of the patients were higher after six months of treatment (p = 0.025; p = 0.001). There was correlation of DAO levels with antipsychotic dosage and with PANSS negative and total subscale scores (rho = 0.421, p = 0.01; rho = 0.280, p = 0.008; rho = 0.371, p = 0.000). No correlation was found between serum D-serine level, DAO level, and the D-serine/DAO ratio with cognitive function. ConclusionsThe results suggest that D-serine and DAO may play a role that is sensitive to treatment effects in schizophrenia spectrum disorders. To gain a more comprehensive understanding of the impact antipsychotic drugs have on NMDA receptor dysfunction, there is a requirement for studies that directly evaluates the activity of the DAO enzyme.

Evaluation of D-amino acid oxidase activity in rat kidney using a D-kynurenine derivative, 6-methylthio-D-kynurenine: An in vivo microdialysis study.

D-Amino acid oxidase (DAO), a D-amino acid metabolizing enzyme, is reportedly associated with the psychiatric disease schizophrenia, suggesting a role for DAO inhibitors in its treatment. We have previously reported that DAO catalyzes the conversion of nonfluorescent 6-methylthio-D-kynurenine (MeS-D-KYN) to fluorescent 5-methylthiokynurenic acid (MeS-KYNA) in vitro. The present study aimed to determine the potential of MeS-D-KYN in evaluating DAO activity in vivo using renal microdialysis technique in rats. Male Sprague-Dawley rats were subjected to linear microdialysis probe implantation in the left kidney. Continuous perfusion of MeS-D-KYN was maintained, and DAO activity in the kidney cortex was evaluated by measuring the MeS-KYNA content in the microdialysate. The microdialysate was collected every 30 min and analyzed by high-performance liquid chromatography with fluorescence detection, monitored at 450 nm with an excitation wavelength of 364 nm. A significant production of MeS-KYNA was observed during, but not before, infusion of MeS-D-KYN, indicating that this compound is not endogenous. MeS-KYNA production was suppressed by the co-infusion of DAO inhibitor, 5-chlorobenzo[d]isoxazol-3-ol (CBIO), suggesting that MeS-D-KYN was converted to MeS-KYNA by renal DAO. Moreover, oral administration of CBIO effectively suppressed DAO activity in a dose-dependent manner. DAO converted MeS-D-KYN to MeS-KYNA in vivo, suggesting the potential of this compound in evaluating DAO activity. The use of the renal microdialysis technique developed in this study facilitates the monitoring of DAO activity in live experimental animals.

Oxygen-consumption based quantification of chemogenetic H2O2 production in live human cells.

Reactive Oxygen Species (ROS) in the form of H2O2 can act both as physiological signaling molecules as well as damaging agents, depending on their concentration and localization. The downstream biological effects of H2O2 were often studied making use of exogenously added H2O2, generally as a bolus and at supraphysiological levels. But this does not mimic the continuous, low levels of intracellular H2O2 production by for instance mitochondrial respiration. The enzyme d-Amino Acid Oxidase (DAAO) catalyzes H2O2 formation using d-amino acids, which are absent from culture media, as a substrate. Ectopic expression of DAAO has recently been used in several studies to produce inducible and titratable intracellular H2O2. However, a method to directly quantify the amount of H2O2 produced by DAAO has been lacking, making it difficult to assess whether observed phenotypes are the result of physiological or artificially high levels of H2O2. Here we describe a simple assay to directly quantify DAAO activity by measuring the oxygen consumed during H2O2 production. The oxygen consumption rate (OCR) of DAAO can directly be compared to the basal mitochondrial respiration in the same assay, allowing to estimate whether the ensuing level of H2O2 production is within the range of physiological mitochondrial ROS production. In the tested monoclonal RPE1-hTert cells, addition of 5 mM D-Ala to the culture media amounts to a DAAO-dependent OCR that surpasses ∼5% of the OCR that stems from basal mitochondrial respiration and hence produces supra-physiological levels of H2O2. We show that the assay can also be used to select clones that express differentially localized DAAO with the same absolute level of H2O2 production to be able to discriminate the effects of H2O2 production at different subcellular locations from differences in total oxidative burden. This method therefore greatly improves the interpretation and applicability of DAAO-based models, thereby moving the redox biology field forward.

Enhancement of the substrate specificity of D-amino acid oxidase based on tunnel-pocket engineering.

D-Amino acid oxidase (DAAO) selectively catalyzes the oxidative deamination of D-amino acids, making it one of the most promising routes for synthesizing optically pure L-amino acids, including L-phosphinothricin( L-PPT), a chiral herbicide with significant market potential. However, the native DAAOs that have been reported have low activity against unnatural acid substrate D-PPT. Herein, we designed and screened a DAAO from Rhodotorula taiwanensis (RtwDAAO), and improved its catalytic potential toward D-PPT through protein engineering. A semirational design approach was employed to create a mutation library based on the tunnel-pocket engineering. After three rounds of iterative saturation mutagenesis, the optimal variant M3rd -SHVG was obtained, exhibiting a >2000-fold increase in relative activity. The kinetic parameters showed that M3rd -SHVG improved the substrate binding affinity and turnover number. This is the optimal parameter reported so far. Further, molecular dynamics simulation revealed that the M3rd -SHVG reshapes the tunnel-pocket and corrects the direction of enzyme-substrate binding, allowing efficiently catalyze unnatural substrates. Our strategy demonstrates that the redesign of tunnel-pockets is effective in improving the activity and kinetic efficiency of DAAO, which provides a valuable reference for enzymatic catalysis. With the M3rd -SHVG as biocatalyst, 500 mM D, L-PPT was completely converted and the yield reached 98%. The results laid the foundation for further industrial production.

Association Between Risperidone Use and Kidney Function Decline in Patients with Schizophrenia: A Retrospective Cohort Study

PurposeSeveral D-amino acids have been shown to be protective against kidney injury in mice. Risperidone, a currently used atypical antipsychotic agent for schizophrenia, is also known to inhibit the activity of D-amino acid oxidase, which degrades certain D-amino acids. Based on the hypothesis that risperidone would prevent kidney disease progression, this study investigated the association between risperidone use and kidney function decline in patients with schizophrenia. MethodsThis retrospective cohort study included patients who were diagnosed with schizophrenia and had data available from two or more serum creatinine measurements between April 1, 2010, and March 31, 2020. Patients who used risperidone for at least 30 days were included in the risperidone group, whereas those who had no record of risperidone use were included in the control group. Cox regression models were used to evaluate the risk for 40% decline in estimated glomerular filtration rate (eGFR) in patients treated with risperidone compared to that in the control group. FindingsOverall, 212 patients used risperidone and 1468 patients had no record of risperidone use. The mean age was 55 years, 759 (45%) of the patients were male, and the mean eGFR at baseline was 88 mL/min/1.73 m2. The mean age in the risperidone group was less than that in the control group (52 vs 56 years); other baseline characteristics were comparable between the two groups. During a mean follow-up of 1.6 years, 267 patients (16%) had a 40% eGFR decline. The incidence rate of 40% eGFR decline was lower in the risperidone group than in the control group (60 vs 104 per 1000 person-years). After adjustment for baseline age, sex, and eGFR, risperidone use was associated with a decreased risk for 40% eGFR decline (hazard ratio = 0.54; 95% CI, 0.33–0.87; P = 0.01). ImplicationsRisperidone use may be associated with decreased risk for kidney function decline in patients with schizophrenia. Further studies are warranted to validate these findings.

P-159 Impact of the symbiotic metabolites D-amino acids on preimplantation development

Abstract Study question Are the metabolites derived from the symbiotic microbiota of reproductive tract involved in preimplantation development? Summary answer The metabolites derived from the symbiotic microbiota of reproductive tract, D-amino acid（D-AA）, contributed to preimplantation development. What is known already It has been known that Lactobacillus exist in the vagina as symbiotic bacteria. Though it was thought that Lactobacillus keep the pH environment acidic and prevent infection by pathogenic bacteria, thereby keeping the uterus and fallopian tubes sterile in mammal, we have identified Staphylococcus aureus in oviductal lavage fluid from female mice and confirmed that the antibiotics disrupt the implantation pattern. In human, the vaginal microbiota is reported to be dominated by Lactobacillus, which produces D-amino acids (D-AA). These findings suggest the involvement of bacterial metabolites D-AA in the establishment of human pregnancy. Study design, size, duration 23 women undergoing in vitro fertilization (IVF) treatment at Keio University Hospital from 2019 to 2021 were assessed for eligibility. 3 were excluded due to spontaneous pregnancy or infectious disease. Twenty subjects were allocated to the successful pregnancy group (S-group (8); mean ± standard deviation 35.5 ± 2.8 years) defined as gestational sac detected on ultrasound at 5 weeks' gestation; and to the unsuccessful pregnancy group (U- group (12); 35.5 ± 2.7 years)). Participants/materials, setting, methods Genomic DNA (gDNA) extracted from vaginal and intrauterine samples at each menstrual cycle was used for metagenome analysis at the genus and the species levels by next-generation sequencing, and by quantitative real-time PCR. Amino acids were analyzed using a two-dimensional HPLC. Immunocytochemical staining by a polyclonal antibody to D-AA oxidase (DAO), which catabolizes D-AA, was performed using B6D2F1 mice and mice with a natural point-mutation G181R in DAO gene, which abolishes DAO activity. Main results and the role of chance Both vaginal/uterine bacterial counts were higher in the luteal phase than in the follicular phase. Vaginal bacterial counts in the S-group were lower than those in the U- group. The composition of microbiota of the vaginal samples (N = 23) shifted from the follicular to the luteal phase, with Lactobacillus spp. predominating. However, there was no significant difference in the ratio of Lactobacillus spp. between the S- and U-groups. The intrauterine samples of the S-group contained a high abundance of L. crispatus, L. gasseri, and L. ultunensis, while the U-group contained a high abundance of L. jensenii. Next, we analyzed the Lactobacillus metabolite, D-AA, and detected D-Ser, D-Ala, D-Asn, and D-Pro in the vaginal, uterine, and follicular fluid of the S-group. Follicular fluid that developed into blastocysts contained more D-AA than that of arrested embryos (N = 2). To investigate the significance of D-AA in the preimplantation development, mouse embryos were cultured in amino acid-depleted medium. All of them arrested (0.0%: 0/78), whereas 41% (25/61) of the embryos with D-AA added to the amino acid-depleted medium developed into blastocysts. We confirmed the expression of DAO in oocytes and blastocysts. DAO-null mice had fewer births than wild type. Limitations, reasons for caution The main limitation is the differences of symbiotic microbiota in reproductive tract and embryonic development between mice and humans; it may be difficult to apply the results of this analysis to humans. Wider implications of the findings Our results display a correlation between species-level microbiome profiling and IVF outcomes. In addition, microbiota-derived metabolites D-AA are suggested to be crucial mediators of embryo development. These findings could give rise to the development of new diagnostic techniques and drug. Trial registration number Not applicable

PHYSIOLOGICAL ROLE OF D-AMINO ACIDS AND BIOANALYTICAL POTENTIAL OF D-AMINO ACID OXIDASES

D-amino acid oxidase (DAAO) plays an important role in the functioning of both prokaryotes and eukaryotes. DAAO is increasingly being used in practice, including for the determination of D-amino acids in complex samples, including human tissues and fl uids. There are generally two types of DAAO in all organisms. The fi rst type is an enzyme highly specifi c for D-aspartate and has its own name D-aspartate oxidase (DASPO). DAAO of the second type is characterized by a wide spectrum of substrate specificity, with preference for one or another D-amino acid varying from source to source. The activity of DAAO with a large number of substrates greatly complicates the selective determination of a particular D-amino acid. The problem is often solved by choosing an enzyme that, under the conditions of analysis, has low or no activity with other D-amino acids present in the sample. For the convenience of selecting a particular enzyme, we have collected and analyzed literature data on the catalytic parameters of known DAAOs with the most important D-amino acids. In addition, similar data are presented for novel recombinant DAAOs from the methylotrophic yeast Ogataea parapolymorpha DL-1. Analysis of the data shows that, with the D-amino acid series, the new OpaDASPO and OpaDAAO have the highest catalytic parameters.

Chiral Amino Acid Analysis in the Plasma of B6DAO&lt;sup&gt;-/-&lt;/sup&gt; Mice Lacking D-Amino Acid Oxidase Activity

A B6DAO-/- mouse strain lacking D-amino acid oxidase (DAO) activity with the genetic background of C57BL strain, frequently used to produce disease model mice in the current medical/life science areas, was newly developed. In the plasma of the control C57BL/6J and mutant B6DAO-/- mice, five D-amino acids widely found in mammals were determined using a highly-selective two-dimensional LC-MS/MS system. As the targets, D-alanine (Ala), D-aspartic acid (Asp), D-leucine (Leu), D-proline (Pro) and D-serine (Ser) were selected. All five chiral amino acids were clearly observed, and the D-enantiomers of the neutral amino acids (Ala, Leu, Pro and Ser) significantly increased in the mutant B6DAO-/- mice (3.69-36.91 nmol/mL) compared to those in the control C57BL/6J mice (0.24-2.03 nmol/mL), whereas the amounts of D-Asp were almost equal between the two strains. The obtained values are consistent with those in previous reports using ddY/DAO-/- mice having spontaneous DAO deficiency and the control ddY/DAO+/+ mice. These results indicated that the newly-developed B6DAO-/- strain is practically useful to clarify the controlling mechanism of intrinsic D-amino acids that are expected as new bio-functional molecules and/or biomarkers in mammals.

An integrated strategy to study the combination mechanisms of Bupleurum chinense DC and Paeonia lactiflora Pall for treating depression based on correlation analysis between serum chemical components profiles and endogenous metabolites profiles

Ethnopharmacological relevanceBupleurum chinense DC-Paeonia lactiflora Pall (BCD-PLP) is a common clinical herb pair in traditional Chinese medicine (TCM) prescriptions commonly used to treat depression. However, its combination mechanisms with its anti-depressive effects remain highly unclear. Aim of the studyHere, an effective strategy has been developed to study the combination mechanisms of Bupleurum chinense DC (BCD) and Paeonia lactiflora Pall (PLP) by integrating serum pharmacochemistry analysis, metabolomics technology, and molecular docking technology. Materials and methodsFirst, the depression model rats were replicated by the chronic unpredictable mild stress (CUMS) procedure, and the difference in the chemical composition in vivo before and after the combination of BCD and PLP was analyzed by integrating background subtraction and multivariate statistical analysis techniques. Then, UPLC/HRMS-based serum metabolomics was performed to analyze the synergistic effect on metabolite regulation before and after the combination of BCD and PLP. Further, the correlation analysis between the differential exogenous chemical components and the differential endogenous metabolites before and after the combination was employed to dissect the combination mechanisms from a global perspective of combining metabolomics and serum pharmacochemistry. Finally, the molecular docking between the differential chemical components and the key metabolic enzymes was applied to verify the regulatory effect of the differential exogenous chemical components on the differential endogenous metabolites. ResultsThe serum pharmacochemistry analysis results demonstrated that the combination of BCD and PLP could significantly affect the content of 10 components in BCD (including 5 prototype components were significantly decreased and 5 metabolites were significantly increased) and 8 components in PLP (including 4 prototype components and 3 metabolites were significantly increased, 1 metabolite was significantly decreased), which indicated that the combination could enhance BCD prototype components' metabolism and the absorption of the PLP prototype components. Besides, metabolomics results indicated that the BCD-PLP herb pair group significantly reversed more metabolites (8) than BCD and PLP single herb group (5 & 4) and has a stronger regulatory effect on metabolite disorders caused by CUMS. Furthermore, the correlation analysis results suggested that saikogenin F and saikogenin G were significantly positively correlated with the endogenous metabolite itaconate, an endogenous anti-inflammatory metabolite; and benzoic acid was significantly positively correlated with D-serine, an endogenous metabolite with an antidepressant effect. Finally, the molecular docking results further confirmed that the combination of BCD and PLP could affect the activities of cis-aconitic acid decarboxylase and D-amino acid oxidase by increasing the in vivo concentration of saikogenin F and benzoic acid, which further enhances its anti-inflammatory activity and anti-depressive effect. ConclusionsIn this study, an effective strategy has been developed to study the combination mechanisms of BCD and PLP by integrating serum pharmacochemistry analysis, multivariate statistical analysis, metabolomics technology, and molecular docking technology. Based on this strategy, the present study indicated that the combination of BCD and PLP could affect the activities of cis-aconitic acid decarboxylase and D-amino acid oxidase by increasing the concentration of saikogenin F and benzoic acid in vivo, which further enhances its anti-depressive effect. In short, this strategy will provide a reliable method for elucidating the herb-herb compatibility mechanism of TCM.

Direct Fluorescence Evaluation of d-Amino Acid Oxidase Activity Using a Synthetic d-Kynurenine Derivative.

d-Amino acid oxidase (DAO) has been suggested to be associated with the central nervous system diseases, such as schizophrenia. We newly synthesized a nonfluorescent 5-methylthio-d-kynurenine (MeS-d-KYN), which was converted to blue-fluorescent 6-MeS-kynurenic acid (MeS-KYNA, λex = 364 nm, λem = 450 nm) through a one-step reaction by incubation with DAO. It was revealed that fluorescence intensity increased accompanied by commercial porcine kidney DAO activity (unit) with a good correlation (R2 = 0.9972), suggesting that the fluorometric evaluation of DAO activity using MeS-d-KYN is feasible. MeS-d-KYN was applied to fluorescent DAO imaging in cultured LLC-PK1 cells, and the blue fluorescence of MeS-KYNA overlapped considerably with the location of peroxisomes, which was suggested to be the location of DAO in the cells. Because fluorescence was diminished in the presence of 6-chloro-1,2-benzisoxazol-3(2H)-one (CBIO), a DAO inhibitor, it was considered that DAO activity in cells could be directly evaluated using MeS-d-KYN as the substrate.

Targeting D-Amino Acid Oxidase (DAAO) for the Treatment of Schizophrenia: Rationale and Current Status of Research.

In the brain, D-amino acid oxidase (DAAO) is a peroxisomal flavoenzyme. Through oxidative deamination by DAAO, D-serine, the main coagonist of synaptic N-methyl-D-aspartate receptors (NMDARs), is degraded into α-keto acids and ammonia; flavin adenine dinucleotide (FAD) is simultaneously reduced to dihydroflavine-adenine dinucleotide (FADH2), which is subsequently reoxidized to FAD, with hydrogen peroxide produced as a byproduct. NMDAR hypofunction is implicated in the pathogenesis of schizophrenia. In previous studies, compared with control subjects, patients with schizophrenia had lower D-serine levels in peripheral blood and cerebrospinal fluid but higher DAAO expression and activity in the brain. Inhibiting DAAO activity and slowing D-serine degradation by using DAAO inhibitors to enhance NMDAR function may be a new strategy for use in the treatment of schizophrenia. The aim of this leading article is to review the current research in DAAO inhibitors.

Precolumn derivatization LC/MS method for observation of efficient hydrogen sulfide supply to the kidney via d-cysteine degradation pathway

d-Cysteine (d-Cys) is metabolized to hydrogen sulfide (H2S) by d-amino acid oxidase (DAO)/3-mercaptopyruvate sulfurtransferase pathway. The pathway is required for H2S supplementation that ameliorates acute kidney injury after the oral administration of d-Cys in mice. However, whether the rate-limiting activity of DAO regulates the tissue-selectivity or the extent of d-Cys degradation and H2S supplementation remains unclear. Here, to analyze the levels of d-Cys and H2S, we use two derivatization methods, a new method with no detectable isomerization of Cys and an established method for H2S. The derivatives were determined by LC/MS using a C18 column. With the methods, we show that inhibition of DAO significantly suppresses the H2S supplementation and d-Cys degradation in the mouse kidney. Additionally, we found that d-Cys is more efficiently metabolized into H2S than l-Cys in the kidney. Our results reveal the utility of the method and support the advantage of d-Cys administration in improving the supply of H2S to the kidneys.

Off-line two-dimensional LC-MS/MS determination of tryptophan enantiomers in mammalian urine and alteration of their amounts in d-amino acid oxidase deficient mice

D-Tryptophan (D-Trp) is one of the minor D-enantiomers of amino acids discovered in microbes and mollusca. In the present study, a highly-selective 2D chiral LC-MS/MS method has been designed and developed focusing on the determination of Trp enantiomers to investigate the presence and regulation of free D-Trp in mammals. The developed system consisted of a reversed-phase separation for the first dimension, an enantioselective separation for the second dimension and also the detection using a triple quadrupole mass spectrometer for the third/fourth dimensions. Using the present method, urinary D-Trp in mammals, including healthy human volunteers and mice, were successfully determined. Although only l-Trp was observed in a mixed urine sample of healthy volunteers, small amounts of D-Trp were detected in the C57BL/6J mice (n = 5, %D=6.18 ± 0.47). In B6DAO- mice lacking the activity of d-amino acid oxidase (DAO), relatively high levels of D-Trp were observed (n = 6, %d=27.43 ± 3.26). The obtained %d values of Trp in the urine of the C57BL/6J mice and B6DAO- mice were confirmed using various enantioselective columns having different separation properties. These results indicate that the urinary D-Trp level is regulated by DAO in mammals, and further investigations, such as tissue distribution and physiological significance of the intrinsic D-Trp, are expected.

P 13 Application of antipsychotic drugs in the treatment of schizophrenia according to single nucleotide polymorphisms of risk genes

Introduction: Schizophrenia is a chronic mental disease which has a genetic etiology in 80% of the cases. In the last years, about 260 risk genes with a predisposition to schizophrenia have been described, and correlations between single nucleotide polymorphisms (SNPs) of risk genes and the therapeutic efficacy of an antipsychotic treatment/pharmacotherapy resistance have been reported. Methods: In schizophrenia, important risk genes, such as catechol-O-methyl transferase (COMT), monoamine oxidase (MAO A/B), glutamic acid decarboxylase 67 (GAD 67), dysbindin-1 and neuregulin-1 are mentioned. To describe the function of these risk genes, neural networks in the ventral tegmental area, hippocampus and prefrontal cortex are developed. Results: An association between the SNPs of some risk genes and the efficacy of an antipsychotic treatment is reported: SNPs such as rs165599 ( COMT gene), rs1801028 ( D2 receptor gene) and rsSer9 Gly ( D3 receptor gene) are associated with a better antipsychotic treatment efficacy. In this case, a treatment of positive and negative schizophrenic symptoms with risperidone is secured. The rs4680 SNP ( COMT and D2 receptor genes) is associated with antipsychotic-induced dopamine hypersensitivity and pharmacotherapy resistance. The function of the COMT and MAO A/B risk genes is described. In schizophrenia, through a reduced activity in the corresponding enzymes, a decrease in dopamine degradation occurs and hence, via D2 receptors, and dopamine hyperactivity has been observed. The GAD 67 risk gene is linked with GABAergic dysfunction and consequently GABAergic neurons weakly presynaptically inhibit D2 dopaminergic neurons. The D-amino acid oxidase activator (DAOA) risk gene is connected with glutamatergic dysfunction via NMDA receptors. Glutamatergic neurons might exert a weak presynaptic inhibition upon 5-HT2A serotonergic neurons located in the ventral tegmental area and hippocampus. Conclusion: It is important to examine the SNPs of the risk genes involved in schizophrenia to establish a correlation between these SNPs and the efficacy of a determined antipsychotic drug. Thus, schizophrenic patients with a good response to a determined antipsychotic treatment and patients with resistance to this treatment could be well differentiated. The latter patients with pharmacoresistance might be treated with the antipsychotic drug clozapine and an additional treatment with cariprazine, a partial agonist at D2 and D3 receptors.

D-Amino Acids and pLG72 in Alzheimer's Disease and Schizophrenia.

Numerous studies over the last several years have shown that d-amino acids, especially d-serine, have been related to brain and neurological disorders. Acknowledged neurological functions of d-amino acids include neurotransmission and learning and memory functions through modulating N-methyl-d-aspartate type glutamate receptors (NMDARs). Aberrant d-amino acids level and polymorphisms of genes related to d-amino acids metabolism are associated with neurodegenerative brain conditions. This review summarizes the roles of d-amino acids and pLG72, also known as d-amino acid oxidase activator, on two neurodegenerative disorders, schizophrenia and Alzheimer’s disease (AD). The scope includes the changes in d-amino acids levels, gene polymorphisms of G72 genomics, and the role of pLG72 on NMDARs and mitochondria in schizophrenia and AD. The clinical diagnostic value of d-amino acids and pLG72 and the therapeutic importance are also reviewed.

Utilization of different lysine isomers: A case study on the growth, metabolic enzymes, antioxidant capacity and muscle amino acid composition in Macrobrachium rosenbergii

An 8-week feeding trial was conducted to compare the effects of different lysine isomers on growth performance, amino acid metabolism-related enzyme activities, antioxidant capacity and muscle amino acid composition of giant river prawn (Macrobrachium rosenbergii). The basal diet was formulated to be low in lysine and contained only 10.9 g/kg lysine. The other three experimental diets were supplemented with 12 g/kg D-lysine, L-lysine, and DL-lysine, respectively. Dietary lysine supplementation improved the growth rate and feed utilization of M. rosenbergii, but significant differences were only found between the prawn fed the control diet and the L-lysine diet. L-lysine supplementation significantly improved the intestinal and hepatic trypsin activities, but unaffected the lipase and amylase activities. The highest hepatic aspartate aminotransferase and alanine aminotransferase activities were observed in prawn fed the L-lysine diet, whereas the highest γ-glutamyl transferase activity was found in prawn fed the D-lysine diet. D-lysine supplementation significantly increased the intestinal and hepatic D-amino acid oxidase activities. However, L-lysine supplementation improved the hepatic lysozyme activity and total antioxidant capacity, depressed the hepatic lactate dehydrogenase and alkaline phosphatase activities. Conversely, D-lysine supplementation generally depressed the hepatic superoxide dismutase and peroxidase activities, and nitric oxide content. Furthermore, L-lysine supplementation increased the muscle aspartic acid, serine, glutamic acid, alanine, lysine, arginine, delicious amino (DAA), non-essential amino acids, total amino acids (TAA) contents and DAA/TAA ratio. These results indicate that M. rosenbergii can effectively utilize L-lysine for growth; D-lysine may partly be used but reveal specific toxic effects on antioxidant response.

Mouse strain specificity of DAAO inhibitors-mediated antinociception.

D‐Amino acid oxidase (DAAO) specifically catalyzes the oxidative deamination of neutral and polar D‐amino acids and finally yields byproducts of hydrogen peroxide. Our previous work demonstrated that the spinal astroglial DAAO/hydrogen peroxide (H2O2) pathway was involved in the process of pain and morphine antinociceptive tolerance. This study aimed to report mouse strain specificity of DAAO inhibitors on antinociception and explore its possible mechanism. DAAO inhibitors benzoic acid, CBIO, and SUN significantly inhibited formalin‐induced tonic pain in Balb/c and Swiss mice, but had no antinociceptive effect in C57 mice. In contrast, morphine and gabapentin inhibited formalin‐induced tonic pain by the same degrees among Swiss, Balb/c and C57 mice. Therefore, mouse strain difference in antinociceptive effects was DAAO inhibitors specific. In addition, intrathecal injection of D‐serine greatly increased spinal H2O2 levels by 80.0% and 56.9% in Swiss and Balb/c mice respectively, but reduced spinal H2O2 levels by 29.0% in C57 mice. However, there was no remarkable difference in spinal DAAO activities among Swiss, Balb/c and C57 mice. The spinal expression of glutathione (GSH) and glutathione peroxidase (GPx) activity in C57 mice were significantly higher than Swiss and Balb/c mice. Furthermore, the specific GPx inhibitor D‐penicillamine distinctly restored SUN antinociception in C57 mice. Our results reported that DAAO inhibitors produced antinociception in a strain‐dependent manner in mice and the strain specificity might be associated with the difference in spinal GSH and GPx activity.

Risk Genes in Schizophrenia and Their Importance in Choosing the Appropriate Antipsychotic Treatment.

Schizophrenia is a chronic mental illness that in 80% of cases has a genetic etiology. In the last years, 260 risk genes with a predisposition to schizophrenia have been discovered and correlations between risk genes and the therapeutic efficacy of an antipsychotic treatment/pharmacotherapy resistance have been reported. The objective of this review is to update the main risk genes involved in schizophrenia and to establish an association between the single nucleotide polymorphisms (SNPs) of these genes and pharmacotherapy resistance/efficacy of a determined antipsychotic treatment. Besides, neural networks in the brain centers involved in schizophrenia will be updated to point out the altered functions of classical neurotransmitters and neuropeptides due to risk genes. In schizophrenia, important risk genes, such as catechol-O-methyl transferase (COMT), monoamine oxidase (MAO A/B), glutamic acid decarboxylase 67 (GAD 67), dysbindin-1 and neuregulin-1 will be mentioned. To describe the function of these risk genes, neural networks in the ventral tegmental area, hippocampus and prefrontal cortex will also be developed. An association between the SNPs of some risk genes and the efficacy of an antipsychotic treatment is reported: SNPs such as rs165599 (COMT gene), rs1801028 (D2 receptor gene) and rsSer9Gly (D3 receptor gene) are associated with a better antipsychotic treatment efficacy (e.g., treatment of negative schizophrenic symptoms with risperidone). The rs4680 SNP (COMT and D2 receptor genes) is associated with antipsychoticinduced dopamine hypersensitivity and pharmacotherapy resistance. The function of risk genes is described: COMT and MAO A/B genes, with reduced activity in the corresponding enzymes, are associated with a decrease in dopamine degradation and hence dopamine hyperactivity occurred via D2 receptors. The GAD 67 risk gene is linked with GABAergic dysfunction and consequently GABAergic neurons weakly presynaptically inhibit D2 dopaminergic neurons. The D-amino acid oxidase activator (DAOA) risk gene is connected with glutamatergic dysfunction via NMDA receptors. Glutamatergic neurons might exert a weak presynaptic inhibition upon 5- HT2A serotonergic neurons located in the ventral tegmental area and hippocampus. Neural networks in the latter two regions and in the prefrontal cortex are updated. It is important to examine the SNPs of the risk genes involved in schizophrenia to establish a correlation between these SNPs and the efficacy of a determined antipsychotic drug. In the future, after examining these SNPs, it might be possible to choose the most appropriate antipsychotic drug. Thus, schizophrenic patients with a good response to a determined antipsychotic treatment and patients with resistance to this treatment could be well differentiated.

Development of a Combination Fermentation Strategy to Simultaneously Increase Biomass and Enzyme Activity of D-amino Acid Oxidase Expressed in Escherichia coli.

D-amino acid oxidase (DAAO) is widely used in the industrial preparation of L-amino acids, and cultivating Escherichia coli (E. coli) expressing DAAO for the biosynthesis of L-phosphinothricin (L-PPT) is very attractive. At present, the biomass production of DAAO by fermentation is still limited in large-scale industrial applications because the expression of DAAO during the fermentation process inhibits the growth of host cells, which limits higher cell density. In this study, the factors that inhibit the growth of bacterial cells during a 5-L fed-batch fermentation process were explored, and the fermentation process was optimized by co-expressing catalase (CAT), by balancing the biomass and the enzyme activity, and by adding exogenous D-alanine (D-Ala) to relieve the limitation of DAAO on the cells and optimize fermentation. Under optimal conditions, the DO-STAT feeding mode with DO controlled at 30% ± 5% and the addition of 27.5 g/L lactose mixed with 2 g/L D-Ala during induction at 28 °C resulted in the production of 26.03 g dry cell weight (DCW)/L biomass and 390.0 U/g DCW specific activity of DAAO; an increase of 78% and 84%, respectively, compared with the initial fermentation conditions. The fermentation strategy was successfully scale-up to a 5000-L fermenter.