Tryptophan Transport Research Articles

NIMG-37IMAGING CEREBRAL TRYPTOPHAN METABOLISM IN BRAIN TUMOR-ASSOCIATED DEPRESSION

Background: Depression in patients with brain tumors is associated with impaired quality of life and shorter survival. Altered metabolism of tryptophan to serotonin and kynurenine metabolites may play a role in tumorassociated depression. Our recent studies with alpha[ 11 C]methyl-L-tryptophan (AMT)-PET in brain tumor patients indicated abnormal tryptophan metabolism not only in the tumor mass but also in normal-appearing contralateral brain. In the present study, we explored if tryptophan metabolism in such brain regions is associated with depression. Methods: Twenty-one patients (mean age: 57 years) with a brain tumor (10 meningiomas, 8 gliomas, and 3 brain metastases) underwent AMT-PET scanning. MRI and AMT-PET images were co-registered, and AMT kinetic parameters, including volume of distribution (VD’, an estimate of net tryptophan transport) and K (unidirectional uptake, related to tryptophan metabolism), were measured in the tumor mass and in unaffected cortical and subcortical regions contralateral to the tumor. Depression scores (based on the Beck Depression Inventory-II [BDI-II]) were correlated with tumor size, grade, type, and AMT-PET variables. Results: The mean BDI-II score was 12 ± 10 (range: 2–33); clinical levels of depression were identified in seven patients (33 %). High BDI-II scores were most strongly associated with high thalamic AMT K values both in the whole group (Spearman’s rho = 0.63, p= 0.004) and in the subgroup of 18 primary brain tumors (r= 0.68, p = 0.004). Frontal and striatal VD’ values were higher in the depressed subgroup than in non-depressed patients (p< 0.05); the group difference was even more robust when moderately/severely depressed patients were compared to patients with no/mild depression (frontal: p= 0.005; striatal: p< 0.001). Tumor size, grade, and tumor type were not related to depression scores. Conclusions: Abnormalities of tryptophan transport and metabolism in the thalamus, striatum, and frontal cortex, measured by PET, are associated with depression in patients with brain tumor. These changes may indicate an imbalance between the serotonin and kynurenine pathways and serve as a molecular imaging marker of brain tumor-associated depression. Trial registration: ClinicalTrials.gov NCT02367469

Imaging cerebral tryptophan metabolism in brain tumor-associated depression.

BackgroundDepression in patients with brain tumors is associated with impaired quality of life and shorter survival. Altered metabolism of tryptophan to serotonin and kynurenine metabolites may play a role in tumor-associated depression. Our recent studies with alpha[11C]methyl-L-tryptophan (AMT)-PET in brain tumor patients indicated abnormal tryptophan metabolism not only in the tumor mass but also in normal-appearing contralateral brain. In the present study, we explored if tryptophan metabolism in such brain regions is associated with depression.MethodsTwenty-one patients (mean age: 57 years) with a brain tumor (10 meningiomas, 8 gliomas, and 3 brain metastases) underwent AMT-PET scanning. MRI and AMT-PET images were co-registered, and AMT kinetic parameters, including volume of distribution (VD’, an estimate of net tryptophan transport) and K (unidirectional uptake, related to tryptophan metabolism), were measured in the tumor mass and in unaffected cortical and subcortical regions contralateral to the tumor. Depression scores (based on the Beck Depression Inventory-II [BDI-II]) were correlated with tumor size, grade, type, and AMT-PET variables.ResultsThe mean BDI-II score was 12 ± 10 (range: 2–33); clinical levels of depression were identified in seven patients (33 %). High BDI-II scores were most strongly associated with high thalamic AMT K values both in the whole group (Spearman’s rho = 0.63, p = 0.004) and in the subgroup of 18 primary brain tumors (r = 0.68, p = 0.004). Frontal and striatal VD’ values were higher in the depressed subgroup than in non-depressed patients (p < 0.05); the group difference was even more robust when moderately/severely depressed patients were compared to patients with no/mild depression (frontal: p = 0.005; striatal: p < 0.001). Tumor size, grade, and tumor type were not related to depression scores.ConclusionsAbnormalities of tryptophan transport and metabolism in the thalamus, striatum, and frontal cortex, measured by PET, are associated with depression in patients with brain tumor. These changes may indicate an imbalance between the serotonin and kynurenine pathways and serve as a molecular imaging marker of brain tumor-associated depression.Trial registrationClinicalTrials.gov NCT02367469Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-015-0136-9) contains supplementary material, which is available to authorized users.

Leucine blocks lipopolysaccharide-induced depression-like behavior by interfering with kynurenine influx into the brain.

Inflammation activates the tryptophan-kynurenine metabolism pathway. Circulating kynurenine is transported into the brain at the vascular blood–brain barrier (BBB) by the large amino-acid transporter LAT-1, where it is further metabolized into neurotoxic metabolites responsible for depressive symptoms. LAT-1 transports tryptophan, kynurenine and the amino acid leucine. We sought to competitively inhibit kynurenine blood-to-brain transport with leucine in order to attenuate lipopolysaccharide-induced depression-like behavior in CD-1 mice. Given that the concentration of circulating kynurenine is 100 fold less than kynurenine, we hypothesized that leucine should inhibit kynurenine more than tryptophan transport. We had already demonstrated that inflammation increases luminal-to-abluminal transport of tryptophan using an in vitro model of BBB transport. Here we show that lipopolysaccharide-treated mice (0.83 mg/kg, ip) develop depression-like behavior at 24 h (reduced sucrose preference and increased forced swim test immobility; p p p p

The ArfGAP2/3 Glo3 and ergosterol collaborate in transport of a subset of cargoes.

ABSTRACTProteins reach the plasma membrane through the secretory pathway in which the trans Golgi network (TGN) acts as a sorting station. Transport from the TGN to the plasma membrane is maintained by a number of different pathways that act either directly or via the endosomal system. Here we show that a subset of cargoes depends on the ArfGAP2/3 Glo3 and ergosterol to maintain their proper localization at the plasma membrane. While interfering with neither ArfGAP2/3 activity nor ergosterol biosynthesis individually significantly altered plasma membrane localization of the tryptophan transporter Tat2, the general amino acid permease Gap1 and the v-SNARE Snc1, in a Δglo3 Δerg3 strain those proteins accumulated in internal endosomal structures. Export from the TGN to the plasma membrane and recycling from early endosomes appeared unaffected as the chitin synthase Chs3 that travels along these routes was localized normally. Our data indicate that a subset of proteins can reach the plasma membrane efficiently but after endocytosis becomes trapped in endosomal structures. Our study supports a role for ArfGAP2/3 in recycling from endosomes and in transport to the vacuole/lysosome.

Study of Three Single Nucleotide Polymorphisms in the SLC6A14 Gene in Association with Male Infertility.

Although several genetic causes of male infertility are known, the condition in around 60.0–75.0% of infertile male patients appears to be idiopathic. In some, genetic causes may be polygenic and require several low-penetrance genes to produce a phenotype outcome. In others, pleiotropy, when a gene can produce several phenotypic traits, may be involved. We have investigated whether single nucleotide polymorphisms (SNPs) in the SLC6A14 [solute carrier family 6 (amino acid transporter), member 14] gene are associated with male infertility. This gene has previously been linked with obesity and cystic fibrosis, which are associated with male infertility. It has a role in the transport of tryptophan and synthesis of serotonin that are important for normal spermatogenesis and testicular function. We have analyzed three SNPs (rs2312054, rs2071877 and rs2011162) in 370 infertile men and 241 fertile controls from two different populations (Macedonian and Slovenian). We found that the rs2011162(G) allele and rs2312054(A)-rs2071877(C)-rs2011162(G) haplotype are present at lower frequencies in the infertile rather than the fertile men (p = 0.044 and p = 0.0144, respectively).We concluded that the SLC6A14 gene may be a population-specific, low-penetrance locus which confers susceptibility to male infertility/subfertility. Additional follow-up studies of a large number of infertile men of different ethnic backgrounds are needed to confirm such a susceptibility.

Interferon-γ induces a tryptophan-selective amino acid transporter in human colonic epithelial cells and mouse dendritic cells

IDO1, which encodes the immunosuppressive and tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase-1 (IDO1), is a target for interferon-γ (IFN-γ). IDO1-mediated tryptophan catabolism in dendritic cells and macrophages arrests T cell proliferation, thereby providing a molecular basis for the immunosuppressive function of IDO1. Whether the entry of tryptophan into IDO1-expressing cells is also regulated by IFN-γ is not known. Here we used a human colonic epithelial cell line (CCD841) and a mouse dendritic cell line (DC2.4) to test the hypothesis that IFN-γ, which induces IDO1, also induces a tryptophan transporter to promote substrate availability to IDO1. Upon treatment with IFN-γ, there was a marked increase in IDO1 mRNA and a concomitant increase in tryptophan uptake in both cell lines. The induced uptake system was selective for tryptophan and saturable with a Michaelis constant of 36±3μM in CCD841 cells and 0.5±0.1μM in DC2.4 cells. The induction by IFN-γ and the tryptophan-selectivity of the induced transport system were demonstrable even in the presence of physiologic concentrations of all other amino acids. Since kynurenine, the catabolic end product of IDO1, is a signaling molecule as an agonist for the aryl hydrocarbon receptor (AhR), we examined if AhR signaling induces the tryptophan-selective transporter. Treatment of the cells with kynurenine and other AhR agonists increased tryptophan uptake. The present studies demonstrate that IFN-γ coordinately induces IDO1 and a tryptophan-selective transporter to maximize tryptophan depletion in IDO1-expressing cells and that the process involves a positive feedback mechanism via kynurenine-AhR signaling.

84. Targeting the kynurenine blood-to-brain transport system to treat inflammation-induced fatigue and depression

Inflammation is associated with activation of the tryptophan-kynurenine metabolism pathway. Circulating kynurenine is transported into the brain at the vascular blood–brain barrier (BBB) by the large neutral amino-acid transporter LAT-1. LAT-1 transports tryptophan and kynurenine. To determine how LAT-1 responds to inflammation, we used mouse microvascular endothelial cells that form tight junctions in vitro and constitutively express the tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO) and LAT-1. We first showed that addition of lipopolysaccharide and interferon-γ to the culture significantly increased endothelial cell IDO mRNA and the transport capacity of LAT-1 as measured with tryptophan from the luminal to the abluminal side of an in vitro model of the BBB. Before using leucine as a competitive inhibitor of LAT-1-mediated transport of kynurenine, we determined whether leucine can block the transport of tryptophan across BBB endothelial cells. Whereas leucine had no effect on tryptophan in the untreated BBB, it inhibited a portion of the enhanced transport of tryptophan conferred by pretreatment with lipopolysaccharide and interferon-γ. Consistent with this ability to block LPS-mediated brain uptake of tryptophan, leucine (administered 500 mg/kg IP before and 6 h after lipopolysaccharide) was also effective in abrogating lipopolysaccharide-induced decreases in locomotor activity and sucrose preference. Experiments are under way to assess the effect of leucine on kynurenine transport into the brain both in vivo and in vitro .

Loss of hydroxyl groups from the ceramide moiety can modify the lateral diffusion of membrane proteins in S. cerevisiae

In the yeast Saccharomyces cerevisiae, structural diversities of complex sphingolipids [inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide] are often observed in the presence or absence of hydroxyl groups on the C-4 position of long-chain base (C4-OH) and the C-2 position of very long-chain fatty acids (C2-OH), but the biological significance of these groups remains unclear. Here, we evaluated cellular membrane fluidity in hydroxyl group-defective yeast mutants by fluorescence recovery after photobleaching. The lateral diffusion of enhanced green fluorescent protein-tagged hexose transporter 1 (Hxt1-EGFP) was influenced by the absence of C4-OH and/or C2-OH. Notably, the fluorescence recovery of Hxt1-EGFP was dramatically decreased in the sur2Δ mutant (absence of C4-OH) under the csg1Δcsh1Δ background, in which mannosylation of IPC is blocked leading to IPC accumulation, while the recovery in the scs7Δ mutant (absence of C2-OH) under the same background was modestly decreased. In addition, the amount of low affinity tryptophan transporter 1 (Tat1)-EGFP was markedly decreased in the sur2Δcsg1Δcsh1Δ mutant and accumulated in intracellular membranes in the scs7Δcsg1Δcsh1Δ mutant without altering its protein expression. These results suggest that C4-OH and C2-OH are most probably critical factors for maintaining membrane fluidity and proper turnover of membrane molecules in yeast containing complex sphingolipids with only one hydrophilic head group.

Automated synthesis of 18F-fluoropropoxytryptophan for amino acid transporter system imaging.

Objective. This study was to develop a cGMP grade of [18F]fluoropropoxytryptophan (18F-FTP) to assess tryptophan transporters using an automated synthesizer. Methods. Tosylpropoxytryptophan (Ts-TP) was reacted with K18F/kryptofix complex. After column purification, solvent evaporation, and hydrolysis, the identity and purity of the product were validated by radio-TLC (1M-ammonium acetate : methanol = 4 : 1) and HPLC (C-18 column, methanol : water = 7 : 3) analyses. In vitro cellular uptake of 18F-FTP and 18F-FDG was performed in human prostate cancer cells. PET imaging studies were performed with 18F-FTP and 18F-FDG in prostate and small cell lung tumor-bearing mice (3.7 MBq/mouse, iv). Results. Radio-TLC and HPLC analyses of 18F-FTP showed that the Rf and Rt values were 0.9 and 9 min, respectively. Radiochemical purity was >99%. The radiochemical yield was 37.7% (EOS 90 min, decay corrected). Cellular uptake of 18F-FTP and 18F-FDG showed enhanced uptake as a function of incubation time. PET imaging studies showed that 18F-FTP had less tumor uptake than 18F-FDG in prostate cancer model. However, 18F-FTP had more uptake than 18F-FDG in small cell lung cancer model. Conclusion. 18F-FTP could be synthesized with high radiochemical yield. Assessment of upregulated transporters activity by 18F-FTP may provide potential applications in differential diagnosis and prediction of early treatment response.

Tryptophan PET-defined gross tumor volume offers better coverage of initial progression than standard MRI-based planning in glioblastoma patients.

Glioblastoma is an infiltrative malignancy that tends to extend beyond the MRI-defined tumor volume. We utilized positron emission tomography (PET) imaging with the radiotracer alpha-[11C]methyl-L -tryptophan (AMT) to develop a reliable high-risk gross tumor volume (HR-GTV) method for delineation of glioblastoma. AMT can detect solid tumor mass and tumoral brain infiltration by increased tumoral tryptophan transport and metabolism via the immunosuppressive kynurenine pathway. We reviewed all patients in our database with histologically proven glioblastoma who underwent preoperative AMT-PET scan prior to surgery and chemoradiation. Treated radiotherapy volumes were derived from the simulation CT with MRI fusion. High-GTV with contrast enhanced T1-weighted MRI alone (GTVMRI) was defined as the postoperative cavity plus any residual area of enhancement on postcontrast T1-weighted images. AMT-PET images were retrospectively fused to the simulation CT, and a high-risk GTVs generated by both AMT-PET alone (GTVAMT) was defined using a threshold previously established to distinguish tumor tissue from peritumoral edema. A composite volume of MRI and AMT tumor volume was also created (combination of MRI fused with AMT-PET data; GTVMRI+AMT). In patients with definitive radiographic progression, follow-up MRI demonstrating initial tumor progression was fused with the pretreatment images and a progression volume was contoured. The coverage of the progression volume by GTVMRI, GTVAMT, and GTVMRI+AMT was determined and compared using the Wilcoxon's signed-rank test. Eleven patients completed presurgical AMT-PET scan, seven of whom had progressive disease after initial therapy. GTVMRI (mean, 50.2 cm3) and GTVAMT (mean, 48.9 cm3) were not significantly different. Mean concordance index of the volumes was 39±15 %. Coverage of the initial recurrence volume by HR-GTVMRI (mean, 52 %) was inferior to both GTVAMT (mean, 68 %; p =0.028) and GTVMRI+AMT (mean 73 %; p =0.018). The AMT-PET-exclusive coverage was up to 41 % of the recurrent volume. There was a tendency towards better recurrence coverage with GTVMRI+AMT than with GTVAMT alone (p =0.068). Addition of 5 mm concentric margin around GTVMRI, GTVAMT, and GTVMRI+AMT would have completely covered the initial progression volume in 14, 57, and 71 % of the patients, respectively. We found that a GTV defined by AMT-PET produced similar volume, but superior recurrence coverage than the treated standard MRI-determined volume. A prospective study is necessary to fully determine the usefulness of AMT-PET for volume definition in glioblastoma radiotherapy planning.

Optimisation of engineered Escherichia coli biofilms for enzymatic biosynthesis of l-halotryptophans

Engineered biofilms comprising a single recombinant species have demonstrated remarkable activity as novel biocatalysts for a range of applications. In this work, we focused on the biotransformation of 5-haloindole into 5-halotryptophan, a pharmaceutical intermediate, using Escherichia coli expressing a recombinant tryptophan synthase enzyme encoded by plasmid pSTB7. To optimise the reaction we compared two E. coli K-12 strains (MC4100 and MG1655) and their ompR234 mutants, which overproduce the adhesin curli (PHL644 and PHL628). The ompR234 mutation increased the quantity of biofilm in both MG1655 and MC4100 backgrounds. In all cases, no conversion of 5-haloindoles was observed using cells without the pSTB7 plasmid. Engineered biofilms of strains PHL628 pSTB7 and PHL644 pSTB7 generated more 5-halotryptophan than their corresponding planktonic cells. Flow cytometry revealed that the vast majority of cells were alive after 24 hour biotransformation reactions, both in planktonic and biofilm forms, suggesting that cell viability was not a major factor in the greater performance of biofilm reactions. Monitoring 5-haloindole depletion, 5-halotryptophan synthesis and the percentage conversion of the biotransformation reaction suggested that there were inherent differences between strains MG1655 and MC4100, and between planktonic and biofilm cells, in terms of tryptophan and indole metabolism and transport. The study has reinforced the need to thoroughly investigate bacterial physiology and make informed strain selections when developing biotransformation reactions.

Low tryptophan uptake in contrast-enhancing lesions predicts long-term survival in patients with a previously treated glioblastoma: A pet study

Background: MRI has limited prognostic value in patients with a previously treated glioblastoma. Positron emission tomography (PET) imaging with alpha[11C]methyl-L-tryptophan (AMT), a radiotracer tracking tryptophan transport and metabolism via the immunosuppressive kynurenine pathway, can effectively differentiate glioma recurrence from radiation injury.

A Mathematical Model of Tryptophan Metabolism via the Kynurenine Pathway Provides Insights into the Effects of Vitamin B-6 Deficiency, Tryptophan Loading, and Induction of Tryptophan 2,3-Dioxygenase on Tryptophan Metabolites

Vitamin B-6 deficiency is associated with impaired tryptophan metabolism because of the coenzyme role of pyridoxal 5′-phosphate (PLP) for kynureninase and kynurenine aminotransferase. To investigate the underlying mechanism, we developed a mathematical model of tryptophan metabolism via the kynurenine pathway. The model includes mammalian data on enzyme kinetics and tryptophan transport from the intestinal lumen to liver, muscle, and brain. Regulatory mechanisms and inhibition of relevant enzymes were included. We simulated the effects of graded reduction in cellular PLP concentration, tryptophan loads and induction of tryptophan 2,3-dioxygenase (TDO) on metabolite profiles and urinary excretion. The model predictions matched experimental data and provided clarification of the response of metabolites in various extents of vitamin B-6 deficiency. We found that moderate deficiency yielded increased 3-hydroxykynurenine and a decrease in kynurenic acid and anthranilic acid. More severe deficiency also yielded an increase in kynurenine and xanthurenic acid and more pronounced effects on the other metabolites. Tryptophan load simulations with and without vitamin B-6 deficiency showed altered metabolite concentrations consistent with published data. Induction of TDO caused an increase in all metabolites, and TDO induction together with a simulated vitamin B-6 deficiency, as has been reported in oral contraceptive users, yielded increases in kynurenine, 3-hydroxykynurenine, and xanthurenic acid and decreases in kynurenic acid and anthranilic acid. These results show that the model successfully simulated tryptophan metabolism via the kynurenine pathway and can be used to complement experimental investigations.

Toxicological effect of dexamethasone treatment on the transport of both amino-acids, tyrosine and tryptophan, across the blood brain barrier (BBB)

Toxicological effect of dexamethasone treatment on the transport of both amino-acids, tyrosine and tryptophan, across the blood brain barrier (BBB)

Oral branched-chain amino acid supplements that reduce brain serotonin during exercise in rats also lower brain catecholamines

Exercise raises brain serotonin release and is postulated to cause fatigue in athletes; ingestion of branched-chain amino acids (BCAA), by competitively inhibiting tryptophan transport into brain, lowers brain tryptophan uptake and serotonin synthesis and release in rats, and reputedly in humans prevents exercise-induced increases in serotonin and fatigue. This latter effect in humans is disputed. But BCAA also competitively inhibit tyrosine uptake into brain, and thus catecholamine synthesis and release. Since increasing brain catecholamines enhances physical performance, BCAA ingestion could lower catecholamines, reduce performance and thus negate any serotonin-linked benefit. We therefore examined in rats whether BCAA would reduce both brain tryptophan and tyrosine concentrations and serotonin and catecholamine synthesis. Sedentary and exercising rats received BCAA or vehicle orally; tryptophan and tyrosine concentrations and serotonin and catecholamine synthesis rates were measured 1 h later in brain. BCAA reduced brain tryptophan and tyrosine concentrations, and serotonin and catecholamine synthesis. These reductions in tyrosine concentrations and catecholamine synthesis, but not tryptophan or serotonin synthesis, could be prevented by co-administering tyrosine with BCAA. Complete essential amino acid mixtures, used to maintain or build muscle mass, were also studied, and produced different effects on brain tryptophan and tyrosine concentrations and serotonin and catecholamine synthesis. Since pharmacologically increasing brain catecholamine function improves physical performance, the finding that BCAA reduce catecholamine synthesis may explain why this treatment does not enhance physical performance in humans, despite reducing serotonin synthesis. If so, adding tyrosine to BCAA supplements might allow a positive action on performance to emerge.

Differentiation of Glioblastomas from Metastatic Brain Tumors by Tryptophan Uptake and Kinetic Analysis: A Positron Emission Tomographic Study with Magnetic Resonance Imaging Comparison

Differentiating high-grade gliomas from solitary brain metastases is often difficult by conventional magnetic resonance imaging (MRI); molecular imaging may facilitate such discrimination. We tested the accuracy of α[11C]methyl-l-tryptophan (AMT)-positron emission tomography (PET) to differentiate newly diagnosed glioblastomas from brain metastases. AMT-PET was performed in 36 adults with suspected brain malignancy. Tumoral AMT accumulation was measured by standardized uptake values (SUVs). Tracer kinetic analysis was also performed to separate tumoral net tryptophan transport (by AMT volume of distribution [VD]) from unidirectional uptake rates using dynamic PET and blood input function. Differentiating the accuracy of these PET variables was evaluated and compared to conventional MRI. For glioblastoma/metastasis differentiation, tumoral AMT SUV showed the highest accuracy (74%) and the tumor/cortex VD ratio had the highest positive predictive value (82%). The combined accuracy of MRI (size of contrast-enhancing lesion) and AMT-PET reached up to 93%. For ring-enhancing lesions, tumor/cortex SUV ratios were higher in glioblastomas than in metastatic tumors and could differentiate these two tumor types with > 90% accuracy. These results demonstrate that evaluation of tryptophan accumulation by PET can enhance pretreatment differentiation of glioblastomas and metastatic brain tumors. This approach may be particularly useful in patients with a newly diagnosed solitary ring-enhancing mass.

Genetic engineering of Escherichia coli to enhance production of l-tryptophan

Reducing the accumulation of acetate in Escherichia coli cultures can decrease carbon efflux as by-products and reduce acetate toxicity, and therefore enable high cell density cultivation. The concentration of intracellular amino acids can be decreased by genetic modifications of the corresponding amino acid transport systems. This can increase the levels of amino acids in the fermentation broth by decreasing the feedback inhibition on the corresponding biosynthetic pathways. Here, the effects of genetic manipulation of phosphate acetyltransferase (pta), high affinity tryptophan transporter (mtr) and aromatic amino acid exporter (yddG) on L-tryptophan production were investigated. The pta mutants accumulated less acetate and showed higher capacity for producing L-tryptophan as compared with the parental strain. The strains lacking mtr, or overexpressed yddG, or with the both mtr knockout and yddG overexpression, accumulated lower concentrations of intracellular tryptophan but higher production of extracellular L-tryptophan. In the L-tryptohan fed-batch fermentation of an E. coli derived from TRTH0709/pMEL03 having deletion of pta-mtr and overexpression of yddG in a 30-L fermentor, the maximum concentration of L-tryptophan (48.68g/L) was obtained, which represented a 15.96% increase as compared with the parental strain. Acetate accumulated to a concentration of 0.95g/L. The intracellular concentration of L-tryptophan was low, and the glucose conversion rate reached a high level of 21.87%, which was increased by 15.53% as compared with the parent strain.

Effects of Parabolic Flight on Serotonin‐related Gene Expression in the Mouse Brain

Exposure to uncontrollable stress triggers a wide range of adaptive changes in the CNS including the elevation of serotonin metabolism and an increased susceptibility to affective disorders. The purpose of the present experiment is to elucidate whether gene expression levels, especially serotonin‐related genes, are altered in the mouse brain exposed to gravity‐changing stress using a competitive reverse transcription‐polymerase chain reaction. Mice were exposed to gravity‐changing stress during 8 times repeated by parabolic flight. Serotonin and tryptophan transporter, and tryptophan hydroxylase‐2 mRNA levels in the midbrain 6 h after the flight were significantly increased compared with pre‐parabolic flight control. In contrast, those of 5‐HT1A receptor, GAD65/67, and tyrosine hydroxylase were not altered by the flight. These results suggest that the serotonergic system, particularly presynaptically synthetic pathway, might be activated in the CNS by gravity‐changing stress.

In vivo metabolism of tryptophan in meningiomas is mediated by indoleamine 2,3-dioxygenase 1

Expression and activity of indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting step of the kynurenine pathway of tryptophan catabolism, can enable tumor cells to effectively evade the host’s immune response. The potential role of this system was investigated in meningiomas. Surgical specimens from 22 patients with meningiomas were used for cellular, immunological and molecular techniques (immunofluorescence, western blotting, RT-PCR and biochemical assay of enzyme activity) to investigate the expression and activity of IDO. In addition, PET imaging was obtained preoperatively in 10 patients using the tracer α-[11C]methyl-L-tryptophan (AMT) which interrogates the uptake and metabolism of tryptophan. Strong AMT accumulation was noted in all meningiomas by PET imaging indicating in vivo tryptophan uptake. Freshly-resected meningiomas expressed both LAT1, the tryptophan transporter system and IDO, demonstrating an active kynurenine pathway. Dissociated meningioma cells lost IDO expression. Following exposure to interferon-γ (IFNγ), IDO expression was reinduced and could be blocked by a selective IDO1 inhibitor. IDO activity may represent an element of local self-protection by meningiomas and could be targeted by emerging IDO1 inhibitors.

Indole production by the tryptophanase TnaA in Escherichia coli is determined by the amount of exogenous tryptophan

The signalling molecule indole occurs in significant amounts in the mammalian intestinal tract and regulates diverse microbial processes, including bacterial motility, biofilm formation, antibiotic resistance and host cell invasion. In Escherichia coli, the enzyme tryptophanase (TnaA) produces indole from tryptophan, but it is not clear what determines how much indole E. coli can produce and excrete, making it difficult to interpret experiments that investigate the biological effects of indole at high concentrations. Here, we report that the final yield of indole depends directly, and perhaps solely, on the amount of exogenous tryptophan. When supplied with a range of tryptophan concentrations, E. coli converted this amino acid into an equal amount of indole, up to almost 5 mM, an amount well within the range of the highest concentrations so far examined for their physiological effects. Indole production relied heavily on the tryptophan-specific transporter TnaB, even though the alternative transporters AroP and Mtr could import sufficient tryptophan to induce tnaA expression. This TnaB requirement proceeded via tryptophan transport and was not caused by activation of TnaA itself. Bacterial growth was unaffected by the presence of TnaA in the absence of exogenous tryptophan, suggesting that the enzyme does not hydrolyse significant quantities of the internal anabolic amino acid pool. The results imply that E. coli synthesizes TnaA and TnaB mainly, or solely, for the purpose of converting exogenous tryptophan into indole, under conditions and for signalling purposes that remain to be fully elucidated.