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Tryptophan plays a key role in several neurological and psychiatric disorders. In this study, we investigated the transport mechanisms of tryptophan in brain capillary endothelial (TR-BBB) cell lines and motor neuron-like (NSC-34) cell lines. The uptake of [3H]l-tryptophan was stereospecific, and concentration- and sodium-dependent in TR-BBB cell lines. Transporter inhibitors and several neuroprotective drugs inhibited [3H]l-tryptophan uptake by TR-BBB cell lines. Gabapentin and baclofen exerted a competitive inhibitory effect on [3H]l-tryptophan uptake. Additionally, l-tryptophan uptake was time- and concentration-dependent in both NSC-34 wild type (WT) and mutant type (MT) cell lines, with a lower transporter affinity and higher capacity in MT than in WT cell lines. Gene knockdown of LAT1 (l-type amino acid transporter 1) and CAT1 (cationic amino acid transporter 1) demonstrated that LAT1 is primarily involved in the transport of [3H]l-tryptophan in both TR-BBB and NSC-34 cell lines. In addition, tryptophan uptake was increased by TR-BBB cell lines but decreased by NSC-34 cell lines after pro-inflammatory cytokine pre-treatment. However, treatment with neuroprotective drugs ameliorated tryptophan uptake by NSC-34 cell lines after inflammatory cytokines pretreatment. The tryptophan transport system may provide a therapeutic target for treating or preventing neurodegenerative diseases.

It has been demonstrated, that long-term chronic tryptophan deficiency, results in decreased serotonin synthesis, which may lead to low bone mass and low bone formation. Findings from studies in male patients with idiopathic osteoporosis suggested a decreased transport of tryptophan in erythrocytes of osteoporotic patients, indicating that serotonin system defects may be involved in the etiology of low bone mass. Tryptophan is the precursor of serotonin, and a disturbed transport of tryptophan is implicated in altered serotonin synthesis. However, no study has investigated the tryptophan transport kinetics in MIO patients. The aim of this study is to investigate the kinetic parameters of tryptophan transport in fibroblasts derived from MIO patients compared to age and sex matched controls. Fibroblast cells were cultured from skin biopsies obtained from 14 patients diagnosed with Male Idiopathic Osteoporosis and from 13 healthy age-sex matched controls, without a diagnosis of osteoporosis. Transport of the amino acid tryptophan across the cell membrane was measured by the cluster tray method. The kinetic parameters, maximal transport capacity (Vmax) and affinity constant (Km) were determined by using the Lineweaver-Burke plot equation. The results of this study have shown a significantly lower mean value for Vmax (p = 0.0138) and lower Km mean value (p = 0.0009) of tryptophan transport in fibroblasts of MIO patients compared to the control group. A lower Vmax implied a decreased tryptophan transport availability in MIO patients. In conclusion, reduced cellular tryptophan availability in MIO patients might result in reduced brain serotonin synthesis and its endogenous levels in peripheral tissues, and this may contribute to low bone mass/formation. The findings of the present study could contribute to the etiology of idiopathic osteoporosis and for the development of novel approaches for diagnosis, treatment and management strategies of MIO., Highlights • MIO patients have a decreased in transport of tryptophan, a precursor of serotonin. • Decreased tryptophan could reduce brain serotonin synthesis and its endogenous levels in peripheral tissues. • Decreased availability of cellular tryptophan can have a direct effect on bone cells and contribute to low bone mass. • The findings of the present study provide further information's of the etiology of idiopathic osteoporosis.

Tryptophan is an essential amino acid in humans and an important serotonin and melatonin precursor. Monocarboxylate transporter MCT10 is a member of the SLC16A family proteins that mediates low-affinity tryptophan transport across basolateral membranes of kidney, small intestine, and liver epithelial cells, although the precise transport mechanism remains unclear. Here we developed a simple functional assay to analyze tryptophan transport by human MCT10 using a deletion mutant for the high-affinity tryptophan permease Tat2 in Saccharomyces cerevisiae. tat2Δtrp1 cells are defective in growth in YPD medium because tyrosine present in the medium competes for the low-affinity tryptophan permease Tat1 with tryptophan. MCT10 appeared to allow growth of tat2Δtrp1 cells in YPD medium, and accumulate in cells deficient for Rsp5 ubiquitin ligase. These results suggest that MCT10 is functional in yeast, and is subject to ubiquitin-dependent quality control. Whereas growth of Tat2-expressing cells was significantly impaired by neutral pH, that of MCT10-expressing cells was nearly unaffected. This property is consistent with the transport mechanism of MCT10 via facilitated diffusion without a need for pH gradient across the plasma membrane. Single-nucleotide polymorphisms (SNPs) are known to occur in the human MCT10 coding region. Among eight SNP amino acid changes in MCT10, the N81K mutation completely abrogated tryptophan import without any abnormalities in the expression or localization. In the MCT10 modeled structure, N81 appeared to protrude into the putative trajectory of tryptophan. Plasma membrane localization of MCT10 and the variant proteins was also verified in human embryonic kidney 293T cells.

Aim Serotonin is crucial for proper foetal development, and the placenta has been described as a 'donor' of serotonin for the embryo/foetus. However, in later stages of gestation the foetus produces its own serotonin from maternally-derived tryptophan and placental supply is no longer needed. We propose a novel model of serotonin homeostasis in the term placenta with special focus on the protective role of organic cation transporter 3 (OCT3/SLC22A3). Methods Dually perfused rat term placenta was employed to quantify serotonin/tryptophan transport and metabolism. Placental membrane vesicles isolated from human term placenta were used to characterize serotonin transporters on both sides of the syncytiotrophoblast. Results We obtained the first evidence that serotonin is massively taken up from the foetal circulation by OCT3. This uptake is concentration-dependent and inhibitable by OCT3 blockers of endogenous (glucocorticoids) or exogenous (pharmaceuticals) origin. Population analyses in rat placenta revealed that foetal sex influences placental extraction of serotonin from foetal circulation. Negligible foetal serotonin levels were detected in maternal-to-foetal serotonin/tryptophan transport and metabolic studies. Conclusion We demonstrate that OCT3, localized on the foetus-facing membrane of syncytiotrophoblast, is an essential component of foeto-placental homeostasis of serotonin. Together with serotonin degrading enzyme, monoamine oxidase-A, this offers a protective mechanism against local vasoconstriction effects of serotonin in the placenta. However, this system may be compromised by OCT3 inhibitory molecules, such as glucocorticoids or antidepressants. Our findings open new avenues to explore previously unsuspected/unexplained complications during pregnancy including prenatal glucocorticoid excess and pharmacotherapeutic risks of treating pregnant women with OCT3 inhibitors.

Introduction: Butyrate is a short-chain fatty acid metabolite produced by microbiota in the colon. With its antioxidant properties, butyrate has also been shown to alter the neurological functions in affective disorder models, suggesting it as a key mediator in gut-brain interactions. Objective: Here, we evaluated the negative effect of oxidative stress on the transport of the serotonin precursor tryptophan as present in affective disorders. Butyrate was hypothesized to be able to rescue these deficits due to its antioxidative capacities and its effect on transmembrane transport of tryptophan. Human skin-derived fibroblasts were used as cellular models to address these objectives. Methods: Human fibroblasts were treated with hydrogen peroxide to induce oxidative stress. Stressed as well as control cells were treated with different concentrations of butyrate. Tryptophan (3H) was used as a tracer to measure the transport of tryptophan across the cell membranes (n = 6). Furthermore, gene expression profiles of different amino acid transporters were analyzed (n = 2). Results: As hypothesized,oxidative stress significantly decreased the uptake of tryptophan in fibroblast cells, while butyrate counteracted this effect. Oxidative stress did not alter the gene expression profile of amino acid transporters. However, treatment of stressed and control cells with different concentrations of butyrate differentially regulated the gene expression of large amino acid transporters 1 and 2, which are the major transporters of tryptophan. Conclusions:Gut microbiota-derived butyrate may have therapeutic potential in affective disorders characterized by either aberrant serotonergic activity or neuroinflammation due to its role in rescuing the oxidative stress-induced perturbations of tryptophan transport.

Moxibustion has been widely accepted as an alternative therapy for major depressive disease (MDD). However, the efficacy of moxibustion treatment on MDD is highly variable because of its irregular operation. This study was designed to investigate how therapeutic duration and extent influence the anti-depression effect of moxibustion and the underlying mechanism involved. Rats with lipopolysaccharide-induced depression-like behavior were treated by moxibustion treatment. The anti-depression effect was determined by forced swimming test and open field test. Tryptophan (Trp) transport and its metabolism to serotonin (5-HT) and kynurenine (Kyn) were evaluated to explore the anti-depression mechanism. The results showed that moxibustion treatment could alleviate the depression-like behavior in rats. Trp transport and 5-HT generation were significantly increased, and the Trp-Kyn pathway was moderately inhibited by moxibustion. Prolonged therapy could be beneficial to the anti-depression effect by promoting the brain uptake of Trp and shifting the Trp metabolism to 5-HT. An enhanced therapeutic extent could increase 5-HT generation. In conclusion, this study determined that the anti-depression effect of moxibustion involves improved Trp transport and metabolism. The therapeutic duration benefits antidepressant effects, but the complex influence of the therapeutic extent on moxibustion efficacy requires further studies.

Mechanisms by which beneficial rhizobacteria promote plant growth include tryptophan-dependent indole-3-acetic acid (IAA) synthesis. The abundance of tryptophan in the rhizosphere, however, may influence the level of benefit provided by IAA-producing rhizobacteria. This study examined the cucumber-Bacillus amyloliquefaciens SQR9 system and found that SQR9, a bacterium previously shown to enhance the growth of cucumber, increased root secretion of tryptophan by three- to fourfold. Using a split-root system, SQR9 colonization of roots in one chamber not only increased tryptophan secretion from the noninoculated roots but also increased the expression of the cucumber tryptophan transport gene but not the anthranilate synthesis gene in those roots. The increased tryptophan in isolated rhizosphere exudates was sufficient to support increased IAA production by SQR9. Moreover, SQR9 colonization of roots in one chamber in the split-root system resulted in sufficient tryptophan production by the other roots to upregulate SQR9 IAA biosynthesis genes, including a 27-fold increase in the indole-3-acetonitrilase gene yhcX during subsequent colonization of those roots. Deletion of yhcX eliminated SQR9-mediated increases in root surface area, likely by reducing IAA-stimulated lateral root growth. This study demonstrates a chemical dialogue between B. amyloliquefaciens and cucumber in which this communication contributes to bacteria-mediated plant-growth enhancement.

Summary Background The incidence of basal cell carcinoma (BCC) is increasing and the costs for care rising. Therefore, the need for simplified and cost‐effective treatment choices is substantial. Aberrant signalling in several pathways, induced by ultraviolet radiation, is of importance in the development of BCC. Alterations in tumour metabolic activity are part of general carcinogenesis; however, these alterations are only partially recognized in skin cancer. Objectives To study expression profiles in BCCs compared with individually matched nontumour skin, with a focus on finding differences associated with tumour metabolism. Materials and methods Gene expression in biopsies from BCCs (n = 14) compared with biopsies from nontumour gluteal skin was analysed with microarrays (n = 4 + 4) and/or quantitative real‐time polymerase chain reaction (qPCR, n = 14 + 14). Protein expression and localization was assessed using immunohistochemistry (IHC) in formalin‐fixed and paraffin‐embedded BCC samples. Results Microarray analysis revealed increased expression of the amino acid transporters SLC7A5, SLC7A7 and SLC7A8 as well as the cytosolic enzyme tryptophan 2,3‐dioxygenase (TDO) 2 in BCC. Higher expression of SLC7A5 (P < 0·001), SLC7A8 (P < 0·001) and TDO2 (P = 0·002), but not SLC7A7 (P = 0·50), was confirmed by qPCR, and IHC demonstrated correlating tumour cell protein expression of SLC7A5 and SLC7A8. Protein expression of SLC7A7 was observed in the stratum granulosum, and TDO2 in immune cells. Conclusions This study highlights the upregulation of SLC7A5, SLC7A8 and TDO2 in BCC compared with nontumour skin. Our findings imply that amino acid transporters may be further explored as potential targets for future medical treatment., What's already known about this topic? The incidence of basal cell carcinoma (BCC) is increasing and consequently also the costs of care.The transport and metabolism of amino acids are often altered in tumours although the knowledge of whether this applies to BCCs is limited. What does this study add? Alterations of amino acid transporters SLC7A5 and SLC7A8 and the cytosolic enzyme TDO2 is suggested in BCC and are possible potential targets for treatment.SLC7A7 (transporter of e.g. lysine) is expressed in the stratum granulosum of normal epidermis and may be involved in the cornification process. What is the translational message? We have found tumour‐specific changes in proteins involved in nutrient transport and metabolism.These changes may be of importance for carcinogenesis and should be explored further for future drug development. Linked Comment: O’Shaughnessy. Br J Dermatol 2019; 180:16–17. Plain language summary available online Respond to this article

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-[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 (GTVEleven patients completed presurgical AMT-PET scan, seven of whom had progressive disease after initial therapy. GTVWe 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.

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.

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.

[EN] Cold is one of the most important abiotic stresses, severely affects the growth and development of plants, and limits their geographical distribution. Given that cold, salinity and drought stresses overlap some of their molecular mechanisms of response, a cDNA library of sugar beet (Beta vulgaris), generated from leaves of plants subjected to saline stress, was used to try to identify genes for tolerance to cold stress by its overexpression in yeast. In this way it was identified, among others, the CRIO4 gene whose characterization has been the work of this thesis. CRIO4 encodes a SEC14-like protein, and it is ortholog to PATL family of Arabidopsis thaliana and it shares a conserved domain with SEC14 and SFH family of yeast. CRIO4 overexpression confers to the yeast cells the ability to grow at low temperatures and tolerance to tunicamycin (an inhibitor of protein glycosylation). The in silico analysis of the CRIO4 sequence confirms several motifs and domains conserved in the protein that seem to direct its function toward the vesicular trafficking, and it reveals multiple potential phosphorylation sites in CRIO4 suggesting a possible role in signaling. Sec14 domain is a phospholipid binding and transfer domain, and the obtained results indicate that CRIO4 binds to phosphatidylinositol and phosphatidylethanolamine. CRIO4 does not complement the function of Sec14p of yeast, and this could be due to the lack of phosphatidylcholine affinity by CRIO4. Tryptophan transport is the greatest limiting factor for growth at low temperatures in yeast. The results of the yeast coexpression assay of CRIO4 and TAT2 (a high-affinity tryptophan permease) displayed in this thesis, seem to indicate stimulation of Tat2p transport to the plasma membrane by CRIO4, which could develop into an enhanced tryptophan absorption, thereby explaining the greater cold tolerance conferred by overexpression of CRIO4 in yeast.The subcellular localization study has shown that CRIO4 is associated with the plasma membrane and intracellular membranes, being part of vesicles, which seems to confirm the role of CRIO4 in intracellular trafficking, function that it would share with its orthologous genes. The expression pattern of CRIO4 in sugar beet has shown that CRIO4 is expressed in all analyzed tissues, focusing its highest expression in leaf and root. It has also been shown that CRIO4 expression increases after shorter exposure to cold stress and heat stress.Phenotypic studies of several mutant lines of PATLs genes from Arabidopsis thaliana have been carried out and high variability in results was obtained, this could be due to functional redundancy among the members of this multigenic family. Phenotypic studies of transgenic lines of Arabidopsis thaliana overexpressing the CRIO4 gene have also been performed. These lines show an improvement in their growth and development at low temperatures, thereby increasing their tolerance to cold stress. Furthermore, expression of CRIO4, in A. thaliana transgenic lines, increases considerably after cold stress and heat shock. Moreover, accumulation points of CRIO4 were observed along the whole cell, after heat shock.Intracellular recycling of PIN2, a gravitropic response indicator, is affected by decreasing temperature. The results obtained in this thesis suggest that overexpression of CRIO4 reactivates intracellular transport of PIN2 under cold stress, which would imply that its phenotype is related to auxin traffic., [ES] El frío es uno de los estreses abióticos más importantes, afecta severamente al crecimiento y desarrollo de las plantas, y limita su distribución geográfica. Puesto que el estrés por frío, por salinidad y por sequía solapan algunos de sus mecanismos moleculares de respuesta, se usó una biblioteca de ADNc de remolacha (Beta vulgaris) generada con hojas de plantas sometidas a estrés salino, para tratar de identificar genes de tolerancia a estrés por frío mediante su sobreexpresión en levadura. De este modo se identificó, entre otros, el gen CRIO4 cuya caracterización ha sido el trabajo de esta tesis. CRIO4 codifica una proteína tipo Sec14, y es ortólogo a la familia PATL de Arabidopsis thaliana y tiene un dominio conservado con SEC14 y la familia SFH de levadura. La sobreexpresión de CRIO4 confiere a las células de levadura la capacidad de crecer a temperaturas bajas y tolerancia a tunicamicina (inhibidor de la glicosilación de proteínas). El análisis in silico de la secuencia de CRIO4 confirma diversos motivos y dominios conservados en la proteína que parecen dirigir su función hacia el tráfico vesicular, y revela múltiples potenciales sitios de fosforilación en CRIO4 sugiriendo una posible función en señalización. El dominio Sec14 es un dominio de unión y transferencia de fosfolípidos, y los resultados obtenidos indican que CRIO4 establece unión con fosfatidiolinositol y fosfatidiletanolamina. CRIO4 no complementa la función de Sec14p de levadura, y esto podría ser debido a la falta de afinidad de CRIO4 por fosfatidilcolina. En levadura, el transporte de triptófano es el mayor factor limitante para el crecimiento a temperaturas bajas. Los resultados del ensayo de coexpresión de CRIO4 con TAT2 (una permeasa de alta afinidad para triptófano) en levadura mostrados en esta tesis, parecen indicar una estimulación del transporte de Tat2p hacia la membrana plasmática por parte de CRIO4, que podría devenir en una absorción de triptófano mejorada, explicando así la mayor tolerancia a frío conferida por la sobreexpresión de CRIO4 en levadura.El estudio de localización subcelular ha mostrado que CRIO4 está asociada a la membrana plasmática y en membranas intracelulares, formando parte de vesículas, lo que parece confirmar el papel de CRIO4 en el tráfico intracelular, función que compartiría con sus ortólogos. El patrón de expresión de CRIO4 en remolacha ha mostrado que CRIO4 se expresa en todos los tejidos analizados, concentrándose su mayor expresión en hoja y raíz. También se ha mostrado que la expresión de CRIO4 aumenta tras tiempos cortos de exposición a estrés por frío y estrés por calor.Se han realizado estudios fenotípicos de diferentes líneas mutantes en genes PATLs de Arabidopsis thaliana, obteniéndose una gran variabilidad en los resultados que podría ser debida a la redundancia funcional entre los miembros de esta familia multigénica. También se han realizado estudios fenotípicos de líneas transgénicas de Arabidopsis thaliana que sobreexpresan el gen CRIO4. Estas líneas muestran una mejoría en su crecimiento y desarrollo a temperaturas bajas, aumentando por tanto su tolerancia al estrés por frío. Además, la expresión de CRIO4, en las líneas transgénicas de A. thaliana, aumenta considerablemente tras el estrés por frío y tras un choque térmico, observándose en este último caso, acúmulos de CRIO4 a lo largo de toda la célula.El reciclaje intracelular de PIN2, indicador de la respuesta gravitrópica, se ve afectado por la disminución de la temperatura. Los resultados obtenidos en esta tesis parecen indicar que la sobreexpresión de CRIO4 reactiva el transporte intracelular de PIN2 bajo estrés por frío, lo que implicaría que su fenotipo está relacionado con el tráfico de auxinas., [CAT] El fred és un dels estressos abiòtics més importants, afecta severament al creixement i desenvolupament de les plantes, i limita la seua distribució geogràfica. Ja que l'estrès per fred, per salinitat i per sequera solapen alguns dels seus mecanismes moleculars de resposta, es va usar una biblioteca d'ADNc de remolatxa (Beta vulgaris) generada amb fulles de plantes sotmeses a estrès salí, per tractar d'identificar gens de tolerància a estrès per fred mitjançant la seua sobreexpressió en llevat. D'aquesta manera es va identificar, entre d'altres, el gen CRIO4 la caracterització del qual ha estat el treball d'aquesta tesi. CRIO4 codifica una proteïna tipus Sec14, i és ortòleg a la família PATL d'Arabidopsis thaliana i té un domini conservat amb SEC14 i la família SFH de llevat. La sobreexpressió de CRIO4 confereix a les cèl·lules de llevat la capacitat de créixer a temperatures baixes i tolerància a tunicamicina (inhibidor de la glicosilació de proteïnes). L'anàlisi in silico de la seqüència de CRIO4 confirma diversos motius i dominis conservats en la proteïna que semblen dirigir la seua funció cap al tràfic vesicular, i revelen múltiples potencials llocs de fosforilació en CRIO4 suggerint una possible funció en senyalització. El domini Sec14 és un domini d'unió i transferència de fosfolípids i els resultats obtinguts indiquen que CRIO4 estableix unió amb fosfatidiolinositol i fosfatidiletanolamina. CRIO4 no complementa la funció de Sec14p de llevat, i això podria ser a causa de la falta d'afinitat de CRIO4 per fosfatidilcolina. En llevat, el transport de triptòfan és el major factor limitant per al creixement a temperatures baixes. Els resultats de l'assaig de coexpressió de CRIO4 amb TAT2 (una permeasa d'alta afinitat per triptòfan) en llevat mostrats en aquesta tesi, semblen indicar una estimulació del transport de Tat2p cap a la membrana plasmàtica per part de CRIO4, que podria esdevindre en una absorció de triptòfan millorada, explicant així la major tolerància a fred conferida per la sobreexpressió de CRIO4 en llevat.L'estudi de localització subcel·lular ha mostrat que CRIO4 està associada a la membrana plasmàtica i en membranes intracel·lulars, formant part de vesícules, la qual cosa sembla confirmar el paper de CRIO4 en el tràfic intracel·lular, funció que compartiria amb els seus homòlegs. El patró d'expressió de CRIO4 en remolatxa ha mostrat que CRIO4 s'expressa en tots els teixits analitzats, concentrant-se la seua major expressió en fulla i arrel. També s'ha mostrat que l'expressió de CRIO4 augmenta després de temps curts d'exposició a estrès per fred i estrès per calor.S'han realitzat estudis fenotípics de diferents línies mutants en gens PATLs d'Arabidopsis thaliana, obtenint-se una gran variabilitat en els resultats que podria ser deguda a la redundància funcional entre els membres d'aquesta família multigènica. També s'han realitzat estudis fenotípics de línies transgèniques d'Arabidopsis thaliana que sobreexpressen el gen CRIO4. Aquestes línies mostren una millora en el seu creixement i desenvolupament a temperatures baixes, augmentant per tant la seua tolerància a l'estrès per fred. A més, l'expressió de CRIO4, en les línies transgèniques d'A. thaliana, augmenta considerablement després de l'estrès per fred i després d'un xoc tèrmic, observant-se en aquest últim cas, cúmuls de CRIO4 al llarg de tota la cèl·lula.El reciclatge intracel·lular de PIN2, indicador de la resposta gravitrópica, es veu afectat per la disminució de la temperatura. Els resultats obtinguts en aquesta tesi semblen indicar que la sobreexpressió de CRIO4 reactiva el transport intracel·lular de PIN2 sota estrès per fred, la qual cosa implicaria que el seu fenotip està relacionat amb el tràfic d'auxines., Izquierdo García, AC. (2016). Caracterización del gen CRIO4 y su implicación en tolerancia a estrés por frío [Tesis doctoral no publicada]. Universitat Politècnica de València. doi:10.4995/Thesis/10251/62826., TESIS

Tryptophan oxidation via the kynurenine pathway is an important mechanism of tumoral immunoresistance. Increased tryptophan metabolism via the serotonin pathway has been linked to malignant progression in breast cancer. In this study, we combined quantitative positron emission tomography (PET) with tumor immunohistochemistry to analyze tryptophan transport and metabolism in breast cancer.Dynamic α-[(11)C]methyl-l-tryptophan (AMT) PET was performed in nine women with stage II-IV breast cancer. PET tracer kinetic modeling was performed in all tumors. Expression of L-type amino acid transporter 1 (LAT1), indoleamine 2,3-dioxygenase (IDO; the initial and rate-limiting enzyme of the kynurenine pathway) and tryptophan hydroxylase 1 (TPH1; the initial enzyme of the serotonin pathway) was assessed by immunostaining of resected tumor specimens.Tumor AMT uptake peaked at 5-20 min postinjection in seven tumors; the other two cases showed protracted tracer accumulation. Tumor standardized uptake values (SUVs) varied widely (2.6-9.8) and showed a strong positive correlation with volume of distribution values derived from kinetic analysis (P.01). Invasive ductal carcinomas (n=6) showed particularly high AMT SUVs (range, 4.7-9.8). Moderate to strong immunostaining for LAT1, IDO and TPH1 was detected in most tumor cells.Breast cancers show differential tryptophan kinetics on dynamic PET. SUVs measured 5-20 min postinjection reflect reasonably the tracer's volume of distribution. Further studies are warranted to determine if in vivo AMT accumulation in these tumors is related to tryptophan metabolism via the kynurenine and serotonin pathways.

Purpose:Serotonin, a neurotransmitter synthesized from tryptophan, has been proposed to play a key role in central fatigue. In this study, we examined whether tryptophan itself and/or its two metabolites, kynurenic acid (KYNA) and quinolinic acid (QUIN), are involved in central fatigue.Materials and Methods:Experiments were conducted using Sprague-Dawley rats (SDR) and Nagase analbuminemic rats (NAR). Central fatigue was assessed by treadmill running and a Morris water maze test. Microdialysis was used to collect samples for measurement of extracellular concentration of tryptophan, serotonin and 5-hydroxyindoleacetic acid (5-HIAA) and to infuse test agents. To examine the kinetics of release, synaptosomes in the striatum were prepared in vitro to measure intra- and extrasynaptosomal concentration of tryptophan, serotonin and 5-HIAA.Results:The concentration of tryptophan secreted into the extracellular space of the striatum was higher during fatigue only, and quickly returned to basal levels with recovery from fatigue. Running time to exhaustion was reduced by activation of tryptophan receptors. Time to exhaustion was shorter in NAR, which maintain a higher extracellular level of striatum tryptophan than SDR. Impaired memory performance in a water maze task after tryptophan treatment was attributable to high levels of KYNA and QUIN in the hippocampus acting synergistically on N-methyl-D-aspartic acid receptors. When branched-chain amino acids were administered, tryptophan transport to the extracellular space of the striatum was drastically inhibited.Conclusion:Our findings demonstrate that the increase in fatigue which occurs because of excessively elevated brain tryptophan can be further amplified by the use of synthetic KYNA and QUIN.

Dysembryoplastic neuroepithelial tumors (DNTs) are typically hypometabolic but can show increased amino acid uptake on positron emission tomography (PET). To better understand mechanisms of amino acid accumulation in epileptogenic DNTs, we combined quantitative α-[(11)C]methyl-L: -tryptophan (AMT) PET with tumor immunohistochemistry. Standardized uptake values (SUVs) of AMT and glucose were measured in 11 children with temporal lobe DNT. Additional quantification for AMT transport and metabolism was performed in 9 DNTs. Tumor specimens were immunostained for the L: -type amino acid transporter 1 (LAT1) and indoleamine 2,3-dioxygenase (IDO), a key enzyme of the immunomodulatory kynurenine pathway. All 11 tumors showed glucose hypometabolism, while mean AMT SUVs were higher than normal cortex in eight DNTs. Further quantification showed increased AMT transport in seven and high AMT metabolic rates in three DNTs. Two patients showing extratumoral cortical increases of AMT SUV had persistent seizures despite complete tumor resection. Resected DNTs showed moderate to strong LAT1 and mild to moderate IDO immunoreactivity, with the strongest expression in tumor vessels. These results indicate that accumulation of tryptophan in DNTs is driven by high amino acid transport, mediated by LAT1, which can provide the substrate for tumoral tryptophan metabolism through the kynurenine pathway, that can produce epileptogenic metabolites. Increased AMT uptake can extend to extratumoral cortex, and presence of such cortical regions may increase the likelihood of recurrent seizures following surgical excision of DNTs.

Quinine is a major drug of choice for the treatment of malaria. However, the primary mode of quinine action is unclear, and its efficacy is marred by adverse reactions among patients. To help address these issues, a genome-wide screen for quinine sensitivity was carried out using the yeast deletion strain collection. Quinine-sensitive mutants identified in the screen included several that were defective for tryptophan biosynthesis (trp strains). This sensitivity was confirmed in independent assays and was suppressible with exogenous Trp, suggesting that quinine caused Trp starvation. Accordingly, quinine was found to inhibit [(3)H]Trp uptake by cells, and the quinine sensitivity of a trp1Delta mutant could be rescued by overexpression of Trp permeases, encoded by TAT1 and TAT2. The site of quinine action was identified specifically as the high affinity Trp/Tyr permease, Tat2p, with which quinine associated in a Trp-suppressible manner. A resultant action also on Tyr levels was reflected by the Tyr-suppressible quinine hypersensitivity of an aro7Delta deletion strain, which is auxotrophic for Tyr (and Phe). The present genome-wide dataset provides an important resource for discovering modes of quinine toxicity. That potential was validated with our demonstration that Trp and Tyr uptake via Tat2p is a major target of cellular quinine toxicity. The results also suggest that dietary tryptophan supplements could help to avert the toxic effects of quinine.

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

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 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. 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

Molecular imaging of the kynurenine pathway can be achieved by positron emission tomography (PET) of radiolabeled tryptophan derivatives. While tryptophan is the substrate of protein synthesis, the PET tracer 11C-alpha-methyl-L-tryptophan (AMT) is not incorporated into proteins; rather it is a substrate of indoleamine 2,3-dioxygenase (IDO), the initial and rate-limiting enzyme of the kynurenine pathway. Recent studies of AMT-PET have demonstrated high AMT uptake and accumulation in a variety of WHO grade II–IV gliomas, glioneuronal tumors, and meningiomas. Increased AMT uptake can also readily detect lung cancers and breast cancers. Tracer kinetic analysis of dynamic PET images can differentiate between tryptophan transport and metabolic rates, thus enhancing the clinical utility of AMT-PET. Studies of resected tumor specimens have shown high expression of LAT1, a key transporter of tryptophan, and also IDO and tryptophan 2,3-dioxygenase, in AMT-accumulating tumors. This chapter discusses the mechanisms of high AMT uptake on PET and provides a summary of potential clinical applications, including pretreatment tumor characterization, treatment planning, prognostication, as well as posttreatment tumor detection in various intra- and extracranial tumors.

Expression of the Bacillus subtilis tryptophan biosynthetic genes ( trpEDCFBA and pabA [ trpG ]) is regulated in response to tryptophan by TRAP, the trp RNA-binding attenuation protein. TRAP-mediated regulation of the tryptophan biosynthetic genes includes a transcription attenuation and two distinct translation control mechanisms. TRAP also regulates translation of trpP ( yhaG ), a single-gene operon that encodes a putative tryptophan transporter. Its translation initiation region contains triplet repeats typical of TRAP-regulated mRNAs. We found that regulation of trpP and pabA is unaltered in a rho mutant strain. Results from filter binding and gel mobility shift assays demonstrated that TRAP binds specifically to a segment of the trpP transcript that includes the untranslated leader and translation initiation region. While the affinities of TRAP for the trpP and pabA transcripts are similar, TRAP-mediated translation control of trpP is much more extensive than for pabA . RNA footprinting revealed that the trpP TRAP binding site consists of nine triplet repeats (five GAG, three UAG, and one AAG) that surround and overlap the trpP Shine-Dalgarno (S-D) sequence and translation start codon. Results from toeprint and RNA-directed cell-free translation experiments indicated that tryptophan-activated TRAP inhibits TrpP synthesis by preventing binding of a 30S ribosomal subunit. Taken together, our results establish that TRAP regulates translation of trpP by blocking ribosome binding. Thus, TRAP coordinately regulates tryptophan synthesis and transport by three distinct mechanisms: attenuation transcription of the trpEDCFBA operon, promoting formation of the trpE S-D blocking hairpin, and blocking ribosome binding to the pabA and trpP transcripts.

Eating and appetite disorders are frequent complications of the uremic syndrome which contribute to malnutrition in dialysis patients. The data suggest that uremic anorexia may occur with or without abdominal and visceral fat accumulation despite a lower food intake. This form of obesity (i.e., with low food intake and malnutrition) is more common in dialysis patients than obesity with high food intake. This article reviews the current knowledge regarding mechanisms responsible for appetite regulation in normal conditions and in uremic patients. Anorexia in dialysis patients has been historically considered as a sign of uremic toxicity due to "inadequate" dialysis as judged by uncertain means ("middle molecule" accumulation, Kt/V, "peak-concentration hypothesis," and others). We propose the tryptophan-serotonin hypothesis, based on a uremia-induced disorder in patients' amino acid profile--low concentrations of large neutral and branched-chain amino acids with high tryptophan levels. A high rate of tryptophan transport across the blood-brain barrier increases the synthesis of serotonin, a major appetite inhibitor. Inflammation may also play a role in the genesis of anorexia and malnutrition. For example, silent infection with Helicobacter pylori may be a source of cytokines with cachectic action; its eradication improves appetite and nutrition. The evaluation of appetite should take into account cultural and social aspects. Uremic patients showed a universal trend to carbohydrate preference and red meat refusal compared to healthy people. In contrast, white meat was less problematic. Uremic patients also have a remarkable attraction for citrics and strong flavors in general. Eating preferences or refusals have been related to the predominance of some appetite peptide modulators. High levels of cholecystokinin (CCK) (a powerful anorexigen) are associated with early satiety for carbohydrates and neuropeptide Y (NPY) (an orexigen) with repeated food intake. Obesity and elevated body mass index often falsely suggest a good nutritional status. In uremic patients (a hyperinsulinemia state), disorders in the regulation of fat distribution (insulin, leptin, insulin-like growth factor [IGF]-1, fatty acids, and disorders in receptors for insulin, lipoprotein lipase, mitochondrial uncoupling protein-2, and beta 3 adrenoreceptors) may cause abdominal fat accumulation without an increase in appetite. Finally, appetite regulation in uremia is highly complex. Disorders in adipose tissue, gastrointestinal and neuropeptides, retained or hyperproduced inflammatory end products, and central nervous system changes may all play a role. Uremic anorexia may be explained by a hypothalamic hyperserotoninergic state derived from a high concentration of tryptophan and low branched-chain amino acids.

Amino acid determinations in plasma have been used for diagnostic purposes and as markers of malnutrition since the 1960s. Biochemical research has given an insight into biochemical pathways and their organ specificity, allowing better understanding of the role of amino acids in the organism. In nutrition, amino acid concentrations are used as markers of excess, deficiency or imbalance for optimising the amount and composition of proteins in prepared nutrients. For useful results to be obtained in such investigations correct timing of sampling relative to the end of food intake is essential. Using data from a study in which a reduction of protein to 1.9 g/100 kcal in starter formulas was tested, it is shown that not only does this allow amino acid concentrations quite close to those of breast-fed babies to be achieved; in addition, the levels reached with the optimised composition lead to a balance with less competition from large neutral amino acids with tryptophan transport at the blood-brain barrier (LAT1 transporter), which probably enhances the supply to the brain even when less protein is fed than in conventional formula and when there is no increase of tryptophan in the blood.

The Antarctic bacterial isolate Sphingomonas sp. strain Ant 17 utilized a wide range of L-isomer amino acids as the sole carbon and energy source for growth. The pH and temperature optima for growth on amino acids were pH 7.0 and 15°C, respectively. Growth on serine and tryptophan was inhibited by uncouplers and inhibitors of oxidative phosphorylation, but not by monensin, a Na+/H+ antiporter, suggesting that sodium gradients were not specifically required for growth on these amino acids. Serine transport was via a high-affinity (apparent Km of 8 μM) permease specific for both the L- and D-isomer. Tryptophan transport exhibited biphasic kinetics with both high-affinity (apparent Km of 2.5 μM) and low-affinity (non-saturable) uptake systems detected. The high-affinity system was specific for L-tryptophan, L-tyrosine, and L-phenylalanine whereas the low-affinity permease was specific for L-tryptophan and L-phenylalanine, but not L-tyrosine. Neither orthovanadate nor sodium arsenate, inhibitors of ATP-dependent permeases, had any significant inhibitory effect on the rate of serine and tryptophan transport. The protonophore carbonyl cyanide m-chlorophenylhydrazone completely abolished serine and tryptophan transport; maximum rates of solute uptake were observed at acidic pH values (pH 4.0–5.0) for both amino acids. These results suggest that an electrochemical potential of protons is the driving force for serine and tryptophan transport by Ant 17. These high-affinity proton-driven permeases function over environmental extremes (e.g. broad temperature and pH range) that are likely to prevail in the natural habitat of this bacterium.

The availability of amino acids in the brain is regulated by the blood-brain barrier (BBB) large neutral amino acid transporter type 1 (LAT1) isoform, which is characterized by a high affinity (low Km) for substrate large neutral amino acids. The hypothesis that brain amino acid transport activity can be altered with single nucleotide polymorphisms was tested in the present studies with site-directed mutagenesis of the BBB LAT1. The rabbit has a high Km LAT1 large neutral amino acid transporter, as compared to the low Km neutral amino acid transporter at the human or rat BBB. The rabbit LAT1 was cloned from a rabbit brain capillary cDNA library. Alignment of the amino acid sequences of rabbit, human, and rat LAT1 revealed two radical amino acid residues that differ in the rabbit relative to the rat or human LAT1. The G219D mutation had a modest effect on the Km and Vmax of tryptophan transport via cloned rabbit LAT1 in frog oocytes, but the W234L variant reduced the Km by 64% and the Vmax by 96%. Conversely, LAT1 transport of either tryptophan or phenylalanine was nearly normalized when the double mutation W234L/G219D variant was produced. These studies show that marked changes in the affinity and capacity of the LAT1 are caused by single nucleotide polymorphisms and that phenotype can be restored with a double mutation.

The isoprenoid pathway produces an endogenous membrane sodium-potassium ATPase inhibitor, digoxin. Digoxin can regulate neurotransmitter transport in the brain with upregulation of tryptophan transport over tyrosine. The pathway was assessed in individuals with addiction, as well as in those with differing hemispheric dominance. The isoprenoid pathway was upregulated with increased digoxin synthesis in addiction. There was an increase in tryptophan catabolites--quinolinic acid, serotonin, nicotine, and strychnine--in patients with addiction, and a reduction in tyrosine catabolites--dopamine, noradrenatine, and morphine. The pattern seen in addiction individuals was similar to that in right hemispheric chemical dominance. Addiction represents a state of right hemispheric chemical dominance, hyperdigoxinemia, and endogenous morphine deficiency state.

Alteration in the isoprenoid metabolites--digoxin, ubiquinone, and dolichol--have been reported in neuronal degeneration (Parkinson's disease), oncogenesis (central nervous system glioma), functional neuropsychiatric disorders (schizophrenia and epilepsy), and immune-mediated disorders (multiple sclerosis). The coexistence of these disorders has been documented in literature and a central dysfunction related to digoxin and the isoprenoid pathway may underlie all these disorders. A family with a high prevalence of Parkinson's disease, schizophrenia, neoplasms, syndrome X, rheumatoid arthritis, and epilepsy has been described. The psychological behavioral patterns of the family were: creativity and high IQ, hypersexual behavior, reduced appetite and eating behavior, insomnia and reduced sleep patterns, increased tendency for spirituality, increased tendency for addiction, less bonding and affectionate behavior, and left handedness/right hemispheric dominance. Digoxin, an endogenous Na(+)-K+ ATPase inhibitor secreted by the hypothalamus, was found to be elevated and red blood cell (RBC) membrane Na(+)-K+ ATPase activity was found to be reduced in all the disorders and in the indexed family studied. Hypothalamic digoxin can modulate conscious perception and its dysfunction may lead to schizophrenia. Digoxin can also preferentially upregulate tryptophan transport over tyrosine, resulting in increased levels of depolarizng tryptophan catabolites, serotonin, quinolinic acid, strychnine, and nicotine, and decreased levels of hyperpolarizing tyrosine catabolites, dopamine, noradrenaline, and morphine, contributing to membrane Na(+)-K+ ATPase inhibition in all the above disorders and the indexed family. Digoxin-induced membrane Na(+)-K+ ATPase inhibition can result in increased intracellular Ca2+ and reduced Mg2+ levels, leading on to glutamate excitotoxicity, oncogene activation, and immune activation. Digoxin-induced altered Ca2+/Mg2+ ratios, reduced ubiquinone, and increased dolichol can affect glycoconjugate metabolism, membrane formation and structure, and mitochondrial function, leading to the diverse disorders described above, including those in the indexed family. The isoprenoid pathway and neurotransmitter patterns were compared in right-handed/LH dominant and left-handed/RH dominant individuals. The left-handed/RH dominant individuals compared to right-handed/LH dominant individuals had elevated hydroxymethylglutarylcoenzyme A reductase activity, with increased serum digoxin and dolichol levels. The serum ubiquinone, serum Mg2+ and RBC Na(+)-K+ ATPase activity were reduced in left-handed/RH dominant individuals. The left-handed/RH dominant individuals compared to right-handed/LH dominant individuals had elevated levels of serum tryptophan, quinolinic acid, serotonin, nicotine, and strychnine. The levels of tyrosine, dopamine, noradrenaline, and morphine were low in left-handed/RH dominant compared to right-handed/LH dominant individuals. The hyperdigoxinemic state indicates right hemispheric dominance. Hypothalamic digoxin can thus function as the master conductor of the neuroimmunoendocrine orchestra and coordinate the functions of various cellular organelles.

Most proteins of Bacillus subtilis contain the three aromatic amino acids--phenylalanine, tyrosine, and tryptophan--all of which are synthesized from a common precursor, chorismate. The known and putative B. subtilis genes of aromatic amino acid metabolism, their location and presumed organization in operons, and their likely enzyme products or function are summarized in this chapter. Important distinctions between B. subtilis and Escherichia coli with regard to aromatic amino acid metabolism are the existence of a multifunctional aromatic supraoperon in B. subtilis and this organism's use of cross-pathway regulation of gene expression. Trp RNA-binding attenuation protein (TRAP) activation is reduced when the hydrogen bond between Tht52 and the catboxyl group of tryptophan is prevented. The affinity of TRAP for tryptophan increases in the presence of bound RNA. In B. subtilis, the biosynthetic pathways for folate and tryptophan are highly interconnected. First, both pathways use chorismate and glutamine to synthesize the p-aminobenzoic acid and o-aminobenzoic acid precursors of folate and tryptophan, respectively. Next, the TrpG polypeptide functions as the glutamine amidotransferase component of two enzyme complexes. Further, the mtrAB operon encodes TRAP (mtrB) and GTP cyclohydrolase I (mrrA). Finally, TRAP negatively regulates both biosynthetic pathways in response to tryptophan. The chapter talks about tRNA trp regulation of trp gene expression, a gene coding for a putative tryptophan transport protein, and homologous polypeptides in some other bacterial species.

In vivo recombination has been used to make a series of AroP-PheP chimeric proteins. Analysis of their respective substrate profiles and activities has identified a small region within span III of AroP which can confer on a predominantly PheP protein the ability to transport tryptophan. Site-directed mutagenesis of the AroP-PheP chimera, PheP, and AroP has established that a key residue involved in tryptophan transport is tyrosine at position 103 in AroP. Phenylalanine is the residue at the corresponding position in PheP. The use of PheP-specific antisera has shown that the inability of certain chimeras to transport any of the aromatic amino acids is not a result of instability or a failure to be inserted into the membrane. Site-directed mutagenesis has identified two significant AroP-specific residues, alanine 107 and valine 114, which are the direct cause of loss of transport activity in chimeras such as A152P. These residues replace a glycine and an alanine in PheP and flank a highly conserved glutamate at position 110. Some suggestions are made as to the possible functions of these residues in the tertiary structure of the proteins.

Objective. To analyze the genotypes of the promoter region of the serotonin transporter gene (5-HTT) in patients with fibromyalgia (FM). Methods. Genomic DNA from 62 patients meeting the American College of Rheumatology 1990 criteria for FM and 110 healthy controls was analyzed by polymerase chain reaction. Additionally, the psychopathologic state of 52 of the FM patients was evaluated using the Beck Depression Inventory (BDI) and the Symptom Checklist-90-Revised (SCL-90-R). Results. The 5-HTTLPR genotypes in FM patients versus controls were distributed as follows: L/L 27% versus 34%, L/S 42% versus 50%, and S/S 31% versus 16%. FM patients with the S/S genotype had higher mean scores on the BDI and the SCL-90-R compared with those in the L/L and L/S groups. Conclusion. A higher frequency of the S/S genotype of 5-HTT was found in FM patients compared with healthy controls. The S/S subgroup exhibited higher mean levels of depression and psychological distress. These results support the notion of altered serotonin metabolism in at least a subgroup of patients with FM. Fibromyalgia (FM) is a syndrome of unknown etiology characterized by chronic widespread pain, increased tenderness on palpation, and additional symptoms such as disturbed sleep, stiffness, fatigue, and psychological distress (1). The pathophysiologic mechanisms underlying FM have been hypothetically linked to disturbances in serotonin (5-HT) metabolism and transmission (2). This hypothesis is based on several recent findings. Studies have shown that levels of tryptophan (the precursor of serotonin) and other amino acids are decreased in sera of FM patients (3), that FM patients have an abnormal serum tryptophan transport ratio (4), and that 5-HT serum levels in FM patients are lower compared with controls (2,5). Similar abnormalities regarding 5-HT metabolism have been found in the cerebrospinal fluid of FM patients (6,7).

1. The sodium-dependent amino acid transport systems responsible for proline, glycine and glutamine transport, together with the sodium-independent systems for leucine and tryptophan, have been investigated in isolated bovine chondrocytes by inhibition studies and ion replacement. Each system was characterized kinetically. 2. Transport via system A was identified using the system-specific analogue alpha-methylaminoisobutyric acid (MeAIB) as an inhibitor of proline, glycine and glutamine transport. 3. Uptake of proline, glycine and glutamine via system ASC was identified by inhibition with alanine or serine. 4. System Gly was identified by the inhibition of glycine transport with excess sarcosine (a substrate for system Gly) whilst systems A and ASC were inhibited. This system, having a very limited substrate specificity and tissue distribution, was also shown to be Na+ and Cl- dependent. Evidence for expression of the system Gly component GLYT-1 was obtained using the reverse transcriptase-polymerase chain reaction (RT-PCR). 5. System N, also of narrow substrate specificity and tissue distribution, was shown to be present in chondrocytes. Na+-dependent glutamine uptake was inhibited by high concentrations of histidine (a substrate of system N) in the presence of excess MeAIB and serine. 6. System L was identified using the system specific analogue 2-aminobicyclo(2,2, 1)heptane-2-carboxylic acid (BCH) and D-leucine as inhibitors of leucine and tryptophan transport. 7. The presence of system T was tested by using leucine, tryptophan and tyrosine inhibition. It was concluded that this system was absent in the chondrocyte. 8. Kinetic analysis showed the Na+-independent chondrocyte L system to have apparent affinities for leucine and tryptophan of 125 +/- 27 and 36 +/- 11 microM, respectively. 9. Transport of the essential amino acids leucine and tryptophan into bovine chondrocytes occurs only by the Na+-independent system L, but with a higher affinity than the conventional L system.

The human glucose transporter GLUT1 is abundant in red blood cells, the blood-brain barrier and epithelial cells, where it mediates the transport of the energy metabolite, glucose. In the present work some properties of GLUT1, including affinity binding of both substrates and inhibitors, transport rates as well as permeabilities of aromatic amino acids and drug-membrane interactions were analyzed by chromatographic methods.Reconstitution by size-exclusion chromatography on Superdex 75 from a detergent with a low CMC that provides monomeric GLUT1 was examined regarding D-glucose- and CB binding as well as D-glucose transport. Upon steric immobilization in Superdex 200 gel beads, residual detergent could be washed away and dissociation constants in the same range as reported for binding to GLUT1 reconstituted from other detergents were obtained. The transport rate into the GLUT1 proteoliposomes was low, probably due to residual detergent. Binding to GLUT1 at different pH was analyzed and the affinity of glucose and GLUT1 inhibitors was found to decrease with increasing pH (5–8.7). The average number of cytochalasin B-binding sites per GLUT1 monomers was, in most cases, approximately 0.4. GLUT1 may work as a functional monomer, dimer or oligomer. To determine whether GLUT1 was responsible for the transport of the aromatic amino acids tyrosine and tryptophan, uptake values and permeabilities of these amino acids into liposomes and GLUT1 proteoliposomes were compared to the permeabilities of D- and L- glucose in the same systems. Dihydrocytochalasin B was identified to be a new inhibitor of tyrosine and tryptophan transport into red blood cells. Ethanol turned out to inhibit the specific binding between CB and GLUT1 and also to decrease the partitioning of CB and drugs into lipid bilayers. A capacity factor for drug partitioning into membranes that allows comparison between columns with different amount of immobilized lipids was validated, and turned out to be independent of flow rate, amount of lipids and drug concentration in the ranges tested.

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.

PURPOSE PET studies with α[C-11]methyl-L-tryptophan (AMT) have shown decreased serotonin synthesis based on a decrease of the unidirectional uptake rate (K-complex) in neuropsychiatric conditions such as autism and depression. Increased AMT K-complex in tumors can indicate increased tryptophan metabolism via the immunosuppressive kynurenine pathway. Moreover, apparent AMT volume of distribution (VD') reflects net tryptophan transport from blood to tissue. We evaluated if kinetic parameters (K-complex, VD') of AMT, measured by PET, can predict the proliferative activity of glioma, and if these AMT parameters are altered in the remote cortex. METHODS We evaluated dynamic AMT PET images of 30 adult patients with grade 2 to 4 gliomas according to the World Health Organization's classification to determine tumoral AMT VD' and K-complex values, which were correlated with tumor proliferative activity as assessed by the Ki-67 labeling index in resected tumor specimens. We also compared cortical VD' and K-complex values between patients with glioma and healthy controls. RESULTS Both VD' and K-complex values were significantly higher in gliomas than in the contralateral cortex (VD', P < 0.001; K-complex, P < 0.001). Tumoral VD' values and tumor/cortex VD' ratios, but not the K-complex, showed strong positive correlations with the proliferative activity of glioma (P ≤ 0.001). The contralateral frontal cortex showed decreased AMT VD' and K-complex in patients with glioma compared with those in controls (P ≤ 0.01). CONCLUSIONS Increased net amino acid transport into tumor tissue, quantified by PET, can serve as an imaging marker of the proliferative activity of glioma. The data also suggest a glioma-induced down-regulation of cortical serotonin synthesis, likely mediated by shunting of tryptophan from serotonin synthesis to kynurenine metabolism.

The white, brown, and scarlet genes of Drosophila melanogaster encode three proteins that belong to the Traffic ATPase superfamily of transmembrane permeases and are involved in the transport of guanine and tryptophan (precursors of the red and brown eye pigments). We have determined the nucleotide sequences of two mutant white alleles (wco2 and wBwx) that cause reduced red pigmentation but have no effect on brown pigmentation. In wco2 the effect is only observed when interacting with the bw6 allele or a newly isolated allele (bwT50). These alleles of the brown gene were cloned and sequenced. In wco2 the codon for glycine 588 is changed to encode serine; in wBwx the triplet ATC encoding isoleucine 581 is deleted; asparagine 638 is changed to threonine in bw6, and glycine 578 is changed to aspartate in bwT50. No other relevant changes to the gene structures were detected. P-element-mediated germline transduction was used to construct a fly strain containing a white gene with a mutation of the nucleotide binding domain. Such flies had white eyes, indicating that the mutated white gene was unable to support either guanine or tryptophan transport. The implications of these mutations are discussed in terms of a model of the Drosophila pigment precursor transport system.

Transport of zwitterionic tryptophan was enhanced by formation of a pseudocrown ether containing a Cu(I)·bipyridine complex, and cooperative binding of the macroring and the complex moiety to the guest was strongly suggested.

The production of L-tryptophan through chemical synthesis, direct fermentation, bioconversion and enzymatic conversion has been reported. However, the role of transport system for aromatic amino acids in L-tryptophan producing strains has not been fully explored. In this study, the fact was revealed that L-tryptophan production and cell growth were affected by the modification of transport systems based on YddG functioning as aromatic amino acid excretion and AroP functioning as general aromatic amino acid permease. Through comparing glucose conversion rates of recombinant strains such as Escherichia coli TRTH ΔaroP, E. coli TRTH-Y, and E. coli TRTH ΔaroP-Y, the moderate modification of transport system resulted in the metabolic flux redistribution of L-tryptophan biosynthesis pathway. In the fed-batch fermentation by E. coli TRTH and E. coli TRTH-Y in 30-liter fermentor, the final production of L-tryptophan fermented by E. coli TRTH-Y was 36.3 g/L, which was 12.6% higher than fermentation by E. coli TRTH.

The purpose of this paper is to discuss the mechanisms of α-[11C]methyl-L-tryptophan (AMT) PET as an in vivo biomarker for detection of epileptogenic cortex. AMT was originally designed as a tracer to measure the serotonin synthesis rate. This tracer was first applied in patients with medically refractory epilepsy in an attempt to detect changes in serotonin synthesis based upon reports of increased serotonergic innervation in cortical specimens obtained following epilepsy surgery. The first group of epilepsy patients undergoing AMT PET scans were patients with tuberous sclerosis complex. Studies of brain tissue subsequent to epilepsy surgery in these patients with tuberous sclerosis complex implicated the kynurenine pathway of tryptophan metabolism as a primary mechanism of increased brain tissue retention of AMT in epileptogenic brain regions, rather than alterations in serotonin synthesis. Kinetic analyses of AMT in brain tumors indicate changes in tryptophan transport and tissue retention in other pools as well. These studies indicate that AMT PET may be a biomarker of immune activation in the epileptogenic process.

There are indications that serotonergic neurotransmission is disturbed in several psychiatric disorders. One explanation may be disturbed transport of tryptophan (precursor for serotonin synthesis) across cell membranes. Human fibroblast cells offer an advantageous model to study the transport of amino acids across cell membranes, since they are easy to propagate and the environmental factors can be controlled. The aim of this study was to functionally characterize tryptophan transport and to identify the main transporters of tryptophan in fibroblast cell lines from healthy controls.Tryptophan kinetic parameters ( Vmaxand Km) at low and high concentrations were measured in fibroblasts using the cluster tray method. Uptake of3H (5)-L-tryptophan at different concentrations in the presence and absence of excess concentrations of inhibitors or combinations of inhibitors of amino acid transporters were also measured. Tryptophan transport at high concentration (0.5 mM) had low affinity and high Vmaxand the LAT1 isoform of system-L was responsible for approximately 40% of the total uptake of tryptophan. In comparison, tryptophan transport at low concentration (50 nM) had higher affinity, lower Vmaxand approximately 80% of tryptophan uptake was transported by system-L with LAT1 as the major isoform. The uptake of tryptophan at the low concentration was mainly sodium (Na+) dependent, while uptake at high substrate concentration was mainly Na+independent. A series of different transporter inhibitors had varying inhibitory effects on tryptophan uptake.This study indicates that tryptophan is transported by multiple transporters that are active at different substrate concentrations in human fibroblast cells. The tryptophan transport trough system-L was mainly facilitated by the LAT1 isoform, at both low and high substrate concentrations of tryptophan.

Expression of the tryptophanase (tna) operon in Escherichia coli is regulated by catabolite repression and transcription attenuation. Expression is induced by the presence of elevated levels of tryptophan in a growth medium devoid of a catabolite-repressing carbon source. Induction requires the translation of a 24-residue coding region, tnaC, located in the 319-nucleotide transcribed leader region preceding tnaA, the structural gene for tryptophanase. Multicopy plasmids carrying the tnaC leader region were found to inhibit induction of the chromosomal tna operon. Mutational studies established that this inhibition was not due to inhibited transcription initiation, translation initiation, tryptophan transport, or enzyme activity. Rather, multicopy tnaC plasmids inhibited induction by preventing tryptophan-induced transcription antitermination in the leader region of the tna operon. Translation of the single Trp codon in tnaC of the multicopy plasmids was shown to be essential for this inhibition. We hypothesize that translation of the Trp codon of the leader peptide titrates out a trans-acting factor that is essential for tryptophan-induced antitermination in the chromosomal tna operon. We postulate that this factor is an altered form of tRNATrp.

Sterols are major lipids in eukaryotes and differ in their specific structure between species. Both cholesterol and ergosterol can form liquid ordered domains in artificial membranes. We reasoned that substituting the main sterol ergosterol by cholesterol in yeast should permit domain formation and discriminate between physical and sterol structure-dependent functions. Using a cholesterol-producing yeast strain, we show that solute transporters for tryptophan and arginine are functional, whereas the export of weak organic acids via Pdr12p, a multi-drug resistance family member, is not. The latter reveals a sterol function that is probably dependent upon a precise sterol structure. We present a series of novel yeast strains with different sterol compositions as valuable tools to characterize sterol function and use them to refine the sterol requirements for Pdr12p. These strains will also be improved hosts for heterologous expression of sterol-dependent proteins and safe sources to obtain pure cholesterol and other sterols.

Ceramide is produced by the condensation of a long chain base with a very long chain fatty acid. In Saccharomyces cerevisiae, one of the two major long chain bases is called phytosphingosine (PHS). PHS has been shown to cause toxicity in tryptophan auxotrophic strains of yeast because this bioactive ceramide precursor causes diversion of the high affinity tryptophan permease Tat2 to the vacuole rather than the plasma membrane. Loss of the integral membrane protein Rsb1 increased PHS sensitivity, which was suggested to be due to this protein acting as an ATP-dependent long chain base efflux protein. More recent experiments demonstrated that loss of the genes encoding the ATP-binding cassette transporter proteins Pdr5 and Yor1 elevated PHS tolerance. This increased resistance was suggested to be due to increased expression of RSB1. Here, we provide an alternative view of PHS resistance influenced by Rsb1 and Pdr5/Yor1. Rsb1 has a seven-transmembrane domain topology more consistent with that of a regulatory protein like a G-protein-coupled receptor rather than a transporter. Importantly, an rsb1Δ cell does not exhibit higher internal levels of PHS compared with isogenic wild-type cells. However, tryptophan transport is increased in pdr5Δ yor1 strains and reduced in rsb1Δ cells. Localization and vacuolar degradation of Tat2 are affected in these genetic backgrounds. Finally, internalization of FM4-64 dye suggests that loss of Pdr5 and Yor1 slows normal endocytic rates. Together, these data argue that Rsb1, Pdr5, and Yor1 regulate the endocytosis of Tat2 and likely other membrane transporter proteins.

The tryptophan operon (trp operon), paradigm for repressible operons, is regulated by three different negative-feedback mechanisms of repression, transcription attenuation and enzyme inhibition. Initiation of transcription is controlled by the interaction of the tryptophan repressor with its target site on the operator. Moreover, the interaction among the regulator genes, the operator genes and the structure genes and excretion of tryptophan play an important factor according to previous works. In this study, an expended mathematical model for the tryptophan operon regulation on the effects of repression, feedback enzyme inhibition, attenuation, interaction among genes and excretion of tryptophan is presented. The new model is first translated into the corresponding S-system version. The robustness of this model is then discussed by using the S-system model and the sensitivity analysis shows that the model is robust enough. The influences of cell growth rate on the biosynthesis of tryptophan, stability and dynamic behavior of the trp operon are also well investigated. The transportation of tryptophan through cytoplasmic membranes, especially the inhibition of tryptophan transport can influence the level of intracellular tryptophan significantly. The theoretical analysis indicates that an increase of inhibition constant of tryptophan transport is favorable for the biosynthesis of tryptophan. Furthermore, a steady-state optimization model is established based on trp operon models. The optimization results indicate that it is possible to attain a stable and robust steady state with a rate of tryptophan production increased more than 4.8 times in which the growth rate is kept as 0.00624h-1 and some key parameters is modulated.

The binding of L-triiodothyronine (T3) to rat erythrocyte membranes (ghosts and peripheral protein-depleted vesicles) was studied under equilibrium conditions. Ghosts contained high-affinity T3 binding sites whose dissociation constant (21 nM) was similar to the equilibrium-exchange Michaelis constant of T3 transport measured in ghosts. Each ghost contained about 8.10(3) high-affinity binding sites. The high-affinity T3 binding was stereospecific and was inhibited by L-tryptophan (Trp) but not by L-leucine. The iodothyronine and amino acid specificity of binding is therefore similar to that of System T, the erythrocyte T3/Trp transporter. These Trp-inhibitable high-affinity T3-binding sites were also present in peripheral protein-depleted membrane vesicles, indicating that they are integral part of the membrane. Ghosts prepared from human erythrocytes, which have very low System T transport activities, contained no detectable Trp-inhibitable high-affinity T3-binding sites. In rat erythrocyte ghosts, N-ethylmaleimide inactivated both the binding and the transport of T3. This inactivation was blocked by T3 and Trp with similar efficiencies. Phenylglyoxal, an arginine residue modifier, also inhibited both high-affinity T3 binding and System T transport activity. It is concluded that the Trp-inhibitable high-affinity T3-binding sites in the rat erythrocyte membrane are likely to be associated with System T.