Protein dynamics governs most of the fundamental processes in the human body. Particularly, the dynamics of loops located near an active site can be involved in the positioning of the substrate and the reaction mechanism. The understanding of the functioning of dynamic loops is therefore a challenge, and often requires the use of a multi-disciplinary approach mixing, for example, crystallographic experiments and molecular dynamics simulations. In the present work, the dynamic behavior of the JK-loop of the human indoleamine 2,3-dioxygenase 1 hemoprotein, a target for immunotherapy, is investigated. To overcome the lack of knowledge on this dynamism, the study reported here is based on 3 crystal structures presenting different conformations of the loop, completed with molecular dynamics trajectories and MM-GBSA analyses, in order to trace the reaction pathway of the enzyme. In addition, the crystal structures identify an exo site in the small unit of the enzyme, that is populated redundantly by the substrate or the product of the reaction. The role of this newer reported exo site still needs to be investigated.
Background: To establish a method to prevent and manage fatigue caused by psychological and physical stress in young females, early detection factors, such as understanding of fatigue and causes of psychological and physical stress, as well as a review of early management of psychiatric disease, are important. With increasing knowledge regarding the diverse causes of stress, it is important to select biomarkers with consideration of the types of stress burden and mechanisms underlying the development of physical symptoms. The methods used to search for stress characteristics is an issue that needs to be addressed. However, consensus regarding objective assessment methods for impaired mental health is lacking.
Methods: We examined the effects of an objective structured clinical examination (OSCE), considered to be a uniform source of psychological and physical stress, on biomarkers of oxidative stress and fatigue in 16 third-year female medical university students (21.3 ± 2.1 years old) in Japan with a normal menstrual cycle. A self-administered questionnaire consisting of Zung's Self-rating Depression Scale (SDS) and State-Trait Anxiety Inventory (STAI) was used to assess subjective stress. Furthermore, stress-related biomarkers (urinary 8-hydroxy-2'-deoxyguanosine [u-8-OHdG], urinary 5-hydroxytryptamine [u-5-HT], and salivary human herpesvirus-6 [s-HHV-6]) were examined at 1 month, 1 week, and 1 day before, and 1 week after the OSCE.
Results: The results indicated that the OSCE did not have effects on u-8-OHdG, a biomarker of oxidative stress. However, u-5-HT and s-HHV-6 were found to be elevated in examinations performed prior to the OSCE.
Conclusions: The present findings suggest that u-5-HT and s-HHV-6 levels can be used for objective assessment of mental and physical fatigue in young females, including that produced not only by knowledge regarding an upcoming OSCE, but also by skill and attitude aspects related to that examination.
Tryptophan and its bioactive metabolites are associated with health conditions such as systemic inflammation, cardiometabolic diseases, and neurodegenerative disorders. There are dynamic interactions among metabolites of tryptophan. The interactions between metabolites, particularly those that are strong and temporally reproducible could be of pathophysiological relevance. Using a targeted metabolomics approach, the concentration levels of tryptophan and 18 of its metabolites across multiple pathways was quantified in 24-hours urine samples at 2 time-points, age 17 years (baseline) and 18 years (follow-up) from 132 (52% female) apparently healthy adolescent participants of the DOrtmund Nutritional and Anthropometric Longitudinally Designed (DONALD) Study. In sex-specific analyses, we applied 2 network approaches, the Gaussian graphical model and Bayesian network to (1) explore the network structure for both time-points, (2) retrieve strongly related metabolites, and (3) determine whether the strongly related metabolites were temporally reproducible. Independent of selected covariates, the 2 network approaches revealed 5 associations that were strong and temporally reproducible. These were novel relationships, between kynurenic acid and indole-3-acetic acid in females and between kynurenic acid and xanthurenic acid in males, as well as known relationships between kynurenine and 3-hydroxykynurenine, and between 3-hydroxykynurenine and 3-hydroxyanthranilic acid in females and between tryptophan and kynurenine in males. Overall, this epidemiological study using network-based approaches shed new light into tryptophan metabolism, particularly the interaction of host and microbial metabolites. The 5 observed relationships suggested the existence of a temporally stable pattern of tryptophan and 6 metabolites in healthy adolescent, which could be further investigated in search of fingerprints of specific physiological states. The metabolites in these relationships may represent a multi-biomarker panel that could be informative for health outcomes.
The immunomodulatory capacity of mental stress is one of the basic concepts of psychoneuroimmunology. The current prospective longitudinal study was designed to evaluate the effect of acute mental stress on neurotransmitter precursor amino acid levels in individuals with depression at 2 time points. Ten physically healthy patients with a diagnosis of major depressive episode and Montgomery-Åsberg Depression Rating Scale scores (MADRAS) ⩾20 points at inclusion were assessed on 2 study days (once with higher MADRAS scores, once with lower MADRAS scores; median 34.5 days apart) and subjected to a standardized acute mental stress test on each study day. Blood was collected at 4 time points: once prior to and at 3 time points (0, 30 minutes, 60 minutes) following mental stress. Neurotransmitter precursor amino acid levels, that is kynurenine/tryptophan (KYN/TRP) and phenylalanine/tyrosine (PHE/TYR), as well as neopterin and nitrite were analyzed in a total of 80 individual blood samples. Regression and correlation analyses were performed. Regression analyses of PHE/TYR (R 2 = .547) and KYN/TRP (R 2 = .440) in relation to MADRAS depression severity showed a quadratic curve fit. This was reflected by a negative linear correlation between MADRAS scores and PHE/TYR as well as KYN/TRP in the lower score range (r = -.805, P < .001 and r = -.586, P < .001 respectively) and a positive correlation in the higher MADRAS score range (r = .713, P < .001 and r = .379, P = .016 respectively). No effect of acute mental stress was found. This analysis exemplifies the implications of sampling as well as data distributions on results. The crosstalk of biological mechanisms that orchestrate metabolic and immunological signaling may vary depending on depression severity resulting in non-linear associations that may explain the heterogeneity of results found in the literature.
Excess of brain kynurenic acid (KYNA), a neuroactive metabolite of the kynurenine pathway, is known to elicit cognitive dysfunction. In the present study, we investigated spatial working memory in mice with elevated levels of KYNA, induced by targeted deletion of kynurenine 3-monooxygenase (KMO), as well as long-term potentiation (LTP) of field excitatory postsynaptic potentials (fEPSPs) in hippocampal brain slices from these mice. The KMO knock-out (KMO-/-) mice performed more poorly in the spatial working memory task as compared to their wild-type (WT) counterparts, as reflected by fewer correct choices in a T-maze. Both fEPSPs, or LTP, did not significantly differ between the 2 mouse strains. However, administration of PF-04859989, a kynurenine aminotransferase (KAT) II inhibitor, limiting the production of KYNA, facilitated fEPSP and enhanced LTP to a greater extent in hippocampal slices from KMO-/- mice compared to WT mice. The results of the present study point to an essential role for KYNA in modulating LTP in the hippocampus of KMO-/- mice which may account for their dysfunctional spatial working memory.
Nowadays, development of analytical methods responding to a need for rapid and accurate determination of human metabolites is highly desirable. Herein, an electrochemical method employing a Nafion-coated glassy carbon electrode (Nafion/GCE) has been developed for reliable determination of kynurenine (a key tryptophan metabolite) using a differential pulse adsorptive stripping voltammetry. To our knowledge, this is the first analytical method to allow for kynurenine determination at the Nafion-coated electrode. The methodology involves kynurenine pre-concentration in 0.1 M H2SO4 in the Nafion film at the potential of +0.5 V and subsequent stripping from the electrode by differential pulse voltammetry. Under optimal conditions, the sensor can detect 5 nM kynurenine (for the accumulation time of 60 seconds), but the limit of detection can be easily lowered to 0.6 nM by prolonging the accumulation time to 600 seconds. The sensor shows sensitivity of 36.25 μAμM-1cm-2 and 185.50 μAμM-1cm-2 for the accumulation time of 60 and 600 seconds, respectively. The great advantage of the proposed method is easy sensor preparation, employing drop coating method, high sensitivity, short total analysis time, and no need for sample preparation. The method was validated for linearity, precision, accuracy (using a high-performance liquid chromatography), selectivity (towards tryptophan metabolites and different amino acids), and recovery. The comprehensive microscopic and electrochemical characterization of the Nafion/GCE was also conducted with different methods including atomic force microscopy (AFM), optical profilometry, time-of-flight secondary ion mass spectrometry (TOF-SIMS), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). The method has been applied with satisfactory results for determination of kynurenine concentration in a culture medium collected from the human ovarian carcinoma cells SK-OV-3 and to measure IDO enzyme activity in the cancer cell extracts.
Viral infection and chronic maternal inflammation during pregnancy are correlated with a higher prevalence of autism spectrum disorder (ASD). However, the pathoetiology of ASD is not fully understood; moreover, the key molecules that can cross the placenta following maternal inflammation and contribute to the development of ASD have not been identified. Recently, the pro-inflammatory cytokine, interleukin-17A (IL-17A) was identified as a potential mediator of these effects. To investigate the impact of maternal IL-17A on offspring, C57BL/6J dams were injected with IL-17A-expressing plasmids via the tail vein on embryonic day 12.5 (E12.5), and maternal IL-17A was expressed continuously throughout pregnancy. By adulthood, IL-17A-injected offspring exhibited behavioral abnormalities, including social and cognitive defects. Additionally, maternal IL-17A promoted metabolism of the essential amino acid tryptophan, which produces several neuroactive compounds and may affect fetal neurodevelopment. We observed significantly increased levels of kynurenine in maternal serum and fetal plasma. Thus, we investigated the effects of high maternal concentration of kynurenine on offspring by continuously administering mouse dams with kynurenine from E12.5 during gestation. Obviously, maternal kynurenine administration rapidly increased kynurenine levels in the fetal plasma and brain, pointing to the ability of kynurenine to cross the placenta and change the KP metabolites which are affected as neuroactive compounds in the fetal brain. Notably, the offspring of kynurenine-injected mice exhibited behavioral abnormalities similar to those observed in offspring of IL-17A-conditioned mice. Several tryptophan metabolites were significantly altered in the prefrontal cortex of the IL-17A-conditioned and kynurenine-injected adult mice, but not in the hippocampus. Even though we cannot exclude the possibility or other molecules being related to ASD pathogenesis and the presence of a much lower degree of pathway activation, our results suggest that increased kynurenine following maternal inflammation may be a key factor in changing the balance of KP metabolites in fetal brain during neuronal development and represents a therapeutic target for inflammation-induced ASD-like phenotypes.
Objectives: Major depressive disorder (MDD) is associated with dysregulations of leptin and tryptophan-kynurenine (Trp-Kyn) (TKP) pathways. Leptin, a pro-inflammatory cytokine, activates Trp conversion into Kyn. However, leptin association with down-stream Kyn metabolites in MDD is unknown.
Methods: Fasting plasma samples from 29 acutely ill drug-naïve (n = 16) or currently non-medicated (⩾6 weeks; n = 13) MDD patients were analyzed for leptin, Trp, Kyn, its down-stream metabolites (anthranilic [AA], kynurenic [KYNA], xanthurenic [XA] acids and 3-hydroxykynurenine [3HK]), C-reactive protein (CRP), neopterin, body mass index (BMI), and insulin resistance (HOMA-IR). Depression severity was assessed by HAM-D-21.
Results: In female (n = 14) (but not in male) patients HAM-D-21 scores correlated with plasma levels of AA (but not other Kyn metabolites) (rho = -0.644, P = .009) and leptin (Spearman's rho = -0.775, P = .001). Inclusion of AA into regression analysis improved leptin prediction of HAM-D from 48.5% to 65.9%. Actual HAM-D scores highly correlated with that calculated by formula: HAM-D = 34.8518-(0.5660 × leptin [ng/ml] + 0.4159 × AA [nmol/l]) (Rho = 0.84, P = .00015). In male (n = 15) (but not in female) patients leptin correlated with BMI, waist circumference/hip ratio, CRP, and HOMA-IR.
Conclusions: Present findings of gender specific AA/Leptin correlations with HAM-D are important considering that AA and leptin are transported from plasma into brain, and that AA formation is catalyzed by kynureninase-the only TKP gene associated with depression according to genome-wide analysis. High correlation between predicted and actual HAM-D warrants further evaluation of plasma AA and leptin as an objective laboratory test for the assessment of depression severity in female MDD patients.
Low levels of the neurotransmitter serotonin have been associated with the onset of depression. While traditional treatments include antidepressants, physical exercise has emerged as an alternative for patients with depressive disorders. Yet there remains the fundamental question of how exercise is sensed by the brain. The existence of a muscle-brain endocrine loop has been proposed: according to this scenario, exercise modulates metabolization of tryptophan into kynurenine within skeletal muscle, which in turn affects the brain, enhancing resistance to depression. But the breakdown of tryptophan into kynurenine during exercise may also alter serotonin synthesis and help limit depression. In this study, we investigated whether peripheral serotonin might play a role in muscle-brain communication permitting adaptation for endurance training. We first quantified tryptophan metabolites in the blood of 4 trained athletes before and after a long-distance trail race and correlated changes in tryptophan metabolism with physical performance. In parallel, to assess exercise capacity and endurance in trained control and peripheral serotonin-deficient mice, we used a treadmill incremental test. Peripheral serotonin-deficient mice exhibited a significant drop in physical performance despite endurance training. Brain levels of tryptophan metabolites were similar in wild-type and peripheral serotonin-deficient animals, and no products of muscle-induced tryptophan metabolism were found in the plasma or brains of peripheral serotonin-deficient mice. But mass spectrometric analyses revealed a significant decrease in levels of 5-hydroxyindoleacetic acid (5-HIAA), the main serotonin metabolite, in both the soleus and plantaris muscles, demonstrating that metabolization of tryptophan into serotonin in muscles is essential for adaptation to endurance training. In light of these findings, the breakdown of tryptophan into peripheral but not brain serotonin appears to be the rate-limiting step for muscle adaptation to endurance training. The data suggest that there is a peripheral mechanism responsible for the positive effects of exercise, and that muscles are secretory organs with autocrine-paracrine roles in which serotonin has a local effect.
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