Engineering the Activity of a Newly Identified Arylalkylamine N-Acetyltransferase in the Acetylation of 5-Hydroxytryptamine.

Arylalkylamine N-acetyltransferase (AANAT) serves as a key enzyme in the biosynthesis of melatonin by transforming 5-hydroxytryptamine (5-HT) to N-acetyl-5-hydroxytryptamine (NAS), while its low activity may hinder melatonin yield. In this study, a novel AANAT derived from Sus scrofa (SsAANAT) was identified through data mining using 5-HT as a model substrate, and a rational design of SsAANAT was conducted in the quest to improving its activity. After four rounds of mutagenesis procedures, a triple combinatorial dominant mutant M3 was successfully obtained. Compared to the parent enzyme, the conversion of the whole-cell reaction bearing the best variant M3 exhibted an increase from 50 % to 99 % in the transformation of 5-HT into NAS. Additionally, its catalytic efficiency (kcat/Km) was enhanced by 2-fold while retaining the thermostability (Tm>45 °C). In the up-scaled reaction with a substrate loading of 50 mM, the whole-cell system incorporating variant M3 achieved a 99 % conversion of 5-HT in 30 h with an 80 % yield. Molecular dynamics simulations were ultilized to shed light on the origin of improved activity. This study broadens the repertoire of AANAT for the efficient biosynthesis of melatonin.

Biosynthetic Pathways of Tryptophan Metabolites in Saccharomyces cerevisiae Strain: Insights and Implications.

Tryptophan metabolites, such as 5-hydroxytryptophan (5-HTP), serotonin, and melatonin, hold significant promise as supplements for managing various mood-related disorders, including depression and insomnia. However, their chemical production via chemical synthesis and phytochemical extraction presents drawbacks, such as the generation of toxic byproducts and low yields. In this study, we explore an alternative approach utilizing S. cerevisiae STG S101 for biosynthesis. Through a series of eleven experiments employing different combinations of tryptophan supplementation, Tween 20, and HEPES buffer, we investigated the production of these indolamines. The tryptophan metabolites were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Notably, setups replacing peptone in the YPD media with tryptophan (Run 3) and incorporating tryptophan along with 25 mM HEPES buffer (Run 4) demonstrated successful biosynthesis of 5-HTP and serotonin. The highest 5-HTP and serotonin concentrations were 58.9 ± 16.0 mg L-1 and 0.0650 ± 0.00211 mg L-1, respectively. Melatonin concentrations were undetected in all the setups. These findings underscore the potential of using probiotic yeast strains as a safer and conceivably more cost-effective alternative for indolamine synthesis. The utilization of probiotic strains presents a promising avenue, potentially offering scalability, sustainability, reduced environmental impact, and feasibility for large-scale production.

Role of the circadian nuclear receptor REV-ERBα in dorsal raphe serotonin synthesis in mood regulation.

Affective disorders are frequently associated with disrupted circadian rhythms. The existence of rhythmic secretion of central serotonin (5-hydroxytryptamine, 5-HT) pattern has been reported; however, the functional mechanism underlying the circadian control of 5-HTergic mood regulation remains largely unknown. Here, we investigate the role of the circadian nuclear receptor REV-ERBα in regulating tryptophan hydroxylase 2 (Tph2), the rate-limiting enzyme of 5-HT synthesis. We demonstrate that the REV-ERBα expressed in dorsal raphe (DR) 5-HTergic neurons functionally competes with PET-1-a nuclear activator crucial for 5-HTergic neuron development. In mice, genetic ablation of DR 5-HTergic REV-ERBα increases Tph2 expression, leading to elevated DR 5-HT levels and reduced depression-like behaviors at dusk. Further, pharmacological manipulation of the mice DR REV-ERBα activity increases DR 5-HT levels and affects despair-related behaviors. Our findings provide valuable insights into the molecular and cellular link between the circadian rhythm and the mood-controlling DR 5-HTergic systems.

Association study of the TPH2 Gene with major depressive disorder in the Han chinese population

BACKGROUND AND OBJECTIVES: Tryptophan hydroxylase 2 (TPH2) catalyzes the rate-limiting step in serotonin biosynthesis in the nervous system. Several variants of human TPH2 have been reported to be associated with a spectrum of neuropsychiatric disorders such as unipolar major depression, bipolar disorder and suicidality etc. Recent studies suggested that two variants (T212 and A375) in the exon 7 and exon 9 were associated with major depressive disorder (MDD). METHODS: To replicate these findings, two polymorphisms located in exons 7 and 9 of TPH2 (rs7305115 and rs4290270, respectively) were analysed by DNA sequence in the case-control sample study in 191 MDD and 191 healthy volunteers. Statistical analyses were carried out using the program SPSS. The comparison of allele and genotype frequencies of each polymorphism between case and control groups was carried out on the online software SHEsis. All subjects were unrelated southern Han Chinese. RESULTS: No difference was observed on the allelic or genotypic distribution of TPH2 gene polymorphisms between the groups. However, the two-marker haplotype covering components T212 (rs7305115) A and A375 (rs4290270) T were observed to have a significantly protective effect MDD in female (corrected p = 0.0032; OR = 0.241[95% CI = 0.099-0.587]). CONCLUSIONS: The results suggest that TPH2 might be associated with a lower risk of female MDD. However, confirmatory studies in independent samples are needed

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Age related interaction of dopamine and serotonin synthesis in striatal synaptosomes

Tyrosine hydroxylase and tryptophan hydroxylase are key rate limiting enzymes in the biosynthesis of dopamine and serotonin, respectively. Since both enzymes are active in striatum, and affected by age, this study was undertaken to investigate interaction between dopamine and serotonin synthesis in brain striatal synaptosomes of aging rat. Male Wistar rats (3 and 30 month old) were killed by decapitation and brain striatal synaptosomes were prepared by discontinuous Ficoll/sucrose gradient technique. Synaptosomes were incubated in the presence of added pargiline (monoamineoxidase inhibitor), dopamine or serotonin synthesized during 25 min was measured by HPLC, employing electrochemical detection. Dopamine synthesis in synaptosomes prepared from young animals was markedly inhibited by addition of 5 microM serotonin concentrations (30%) and increasing serotonin concentrations up to 50 microM caused only a smaller additional inhibition. Dopamine synthesis in synaptosomes obtained from old rats was significantly lower than that of youg animals and addition of serotonin concentrations up to 50 microM had little effect on these preparations. In case of serotonin synthesis, exogenously added 5 microM dopamine inhibited serotonin synthesis in the synaptosomes of both ages by about 40%, whereas with higher concentration of dopamine (10-50 microM) the rate of inhibition was highly pronounced in old rats as compared to that of young animals. It is concluded that dopamine and serotonin interaction may be significant, and that these should be considered in long-term treatments of Parkinson's disease with L-DOPA.

Age related interaction of dopamine and serotonin synthesis in striatal synaptosomes

Tyrosine hydroxylase and tryptophan hydroxylase are key rate limiting enzymes in the biosynthesis of dopamine and serotonin, respectively. Since both enzymes are active in striatum, and affected by age, this study was undertaken to investigate interaction between dopamine and serotonin synthesis in brain striatal synaptosomes of aging rat. Male Wistar rats (3 and 30 month old) were killed by decapitation and brain striatal synaptosomes were prepared by discontinuous Ficoll/sucrose gradient technique. Synaptosomes were incubated in the presence of added pargiline (monoamineoxidase inhibitor), dopamine or serotonin synthesized during 25 min was measured by HPLC, employing electrochemical detection. Dopamine synthesis in synaptosomes prepared from young animals was markedly inhibited by addition of 5 microM serotonin concentrations (30%) and increasing serotonin concentrations up to 50 microM caused only a smaller additional inhibition. Dopamine synthesis in synaptosomes obtained from old rats was significantly lower than that of youg animals and addition of serotonin concentrations up to 50 microM had little effect on these preparations. In case of serotonin synthesis, exogenously added 5 microM dopamine inhibited serotonin synthesis in the synaptosomes of both ages by about 40%, whereas with higher concentration of dopamine (10-50 microM) the rate of inhibition was highly pronounced in old rats as compared to that of young animals. It is concluded that dopamine and serotonin interaction may be significant, and that these should be considered in long-term treatments of Parkinsons disease with L-DOPA.(AU)

Caracterización de células serotoninérgicas en el tejido cardiaco fetal / Characterization of serotonergic cells in fetal heart tissue

Objetivo: Caracterizar morfológica y bioquímicamente células productoras de serotonina durante el desarrollo del tejido cardiaco. Material y métodos: Se utilizaron ratas gestantes de la cepa Wistar. A los días 10, 12, 16 y 20 de gestación se obtuvieron los fetos por cesárea, a los cuales se les disecaron los corazones, que se fijaron para los ensayos de inmunohistoquímica para triptófano- 5-hidroxilasa (Tph), además se efectuó Western Blot para la enzima; se determinó la concentración de serotonina y la actividad de Tph por cromatografía de líquidos de alta resolución. Los resultados fueron analizados mediante U de Mann-Whitney, aceptando un nivel de significación de p < 0.05. Resultados: En los cortes de corazón fetal, desde el día 10 de gestación se observaron células inmunorreactivas para Tph, con metacromasia en su interior. Fibras nerviosas inmunorreactivas para la misma enzima que hacen contacto probablemente con las células serotoninérgicas. La actividad enzimática y la concentración de la serotonina aumentaron con la edad gestacional, además, se encontró la proteína enzimática por Western- Blot en el corazón fetal de 16 días de gestación. Conclusiones: Se demostró la presencia de células productoras de serotonina en el miocardio fetal, cuyo fenotipo corresponde a mastocitos cardiacos, lo cual sugiere que la serotonina puede ser importante en el desarrollo del tejido cardiaco y que también participa en los mecanismos fisiopatológicos de los defectos cardiacos estructurales o en la predisposición a enfermedades cardiovasculares en el adulto.

BACKGROUND: We undertook this study to present biochemical and morphological characterization of serotonergic cells during fetal heart development. METHODS: Wistar rats (10, 12, 16 and 20 days of gestation) were used. After obtaining the fetuses by Cesarean section, the hearts were removed and fixed for immunohistochemical assay of tryptophan-5-hydroxylase (Tph), in addition to Western blot for enzyme. Serotonin concentration and Tph were also evaluated with high-performance liquid chromatography. Results were analyzed using Mann-Whitney U test with a significance level of p <0.05. RESULTS: Metachromatic cells immunoreactive for Tph were observed from day 10 of gestation. Nerve fibers were also labeled, apparently making contact with serotonergic cells. Tph activity was measurable and serotonin levels increased with gestational age. The presence of Tph protein was confirmed by Western blot on day 16. CONCLUSIONS: The present results support the existence of cells located in the fetal myocardium, capable of producing serotonin whose phenotype belongs to cardiac mast cells. Their presence in this tissue strongly suggests that serotonin may play a key role in normal and abnormal development of cardiac tissue.

Vardenafil Increases Cell Proliferation in the Dentate Gyrus through Enhancement of Serotonin Expression in the Rat Dorsal Raphe

This study was conducted to evaluate the effects of vardenafil (Levitra), a phosphodiesterase-5 (PDE-5) inhibitor, on cell proliferation in the hippocampal dentate gyrus and on 5-hyroxytryptamine (5-HT, serotonin) synthesis and tryptophan hydroxylase (TPH) expression in the rat dorsal raphe nucleus. Male Sprague-Dawley rats were divided into 6 groups (n=5 in each group): a control group, a 0.5 mg/kg-1 day vardenafil-treated group, a 1 mg/kg-1 day vardenafil-treated group, a 2 mg/kg-1 day vardenafil-treated group, a 1 mg/kg-3 day vardenafil-treated group, and a 1 mg/kg-7 day vardenafil-treated group. 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry was then performed to evaluate cell proliferation in the dentate gyrus. In addition, 5-HT and TPH immunohistochemistry was conducted to evaluate serotonin expression in the dorsal raphe. The results revealed that treatment with vardenafil increased cell proliferation in the dentate gyrus and enhanced 5-HT synthesis and TPH expression in the dorsal raphe in a dose- and duration-dependent manner. The findings demonstrate that the increasing effect of vardenafil on cell proliferation is closely associated with the enhancing effect of vardenafil on serotonin expression under normal conditions.

Cambio en la biosíntesis de serotonina cerebral en ratas con diabetes mellitus inducida por estreptozotocina: efecto del tratamiento con insulina / Changes of brain serotonin synthesis in rats with diabetes mellitus insulin-dependent submitted to treatment with insulin

Objetivo. Investigar si los cambios en la actividad de la triptófano-5-hidroxilasa y la síntesis de serotonina cerebral provocados por diabetes mellitus, persisten o regresan a lo normal en el animal diabético tratado con insulina. Metodología. Se usaron ratas diabéticas por la administración de estreptozotocina y ratas diabéticas tratadas con insulina. A los días 7, 14 y 21 de evolución, se pesaron y se les determinó en el cerebro: la actividad de la triptófano-5-hidroxilasa y las concentraciones de L-triptófano y serotonina. En el plasma se les midió glucosa, albúmina y L-triptófano. Resultados. Los diabéticos mostraron un déficit significativo de peso corporal. El mismo patrón se observó en la albúmina y en el L-triptófano plasmático. En la corteza cerebral se observó que el L-triptófano, la 5-hidroxitriptamina y la actividad de la enzima fueron significativamente menores. Además, la actividad de la enzima en el tallo cerebral fue mayor, acompañada de una elevación de la 5-hidroxitriptamina. Los animales diabéticos tratados con insulina tuvieron una recuperación física y un retorno a lo normal del L-triptófano en el cerebro y en la sangre. Sin embargo, la actividad de la triptófano-5-hidroxilasa permaneció elevada a pesar del tratamiento con insulina y persistió un aumento de la síntesis del neurotransmisor tanto en el tallo como en la corteza cerebral. Conclusión. Los resultados confirman que la diabetes mellitus produce desnutrición crónica y una disminución en la síntesis de serotonina en el cerebro. Además, apoyan que el tratamiento con insulina en los diabéticos produce una recuperación física y un regreso a lo normal del precursor de la serotonina el L-triptófano plasmático y cerebral, pero interesantemente, a pesar de este hecho, la actividad de la triptófano-5-hidroxilasa permaneció elevada acompañada de un aumento del neurotransmisor. Estos hallazgos en conjunto, sugieren que el mecanismo de activación de la biosíntesis de la serotonina cerebral puede ser debido a un cambio relacionado a la cinética de la triptófano-5-hidroxilasa, más que a los cambios de la concentración plasmática y cerebral del L-triptófano inducidos por la diabetes mellitus. Esta alteración en el metabolismo de un neurotransmisor puede participar en los mecanismos fisiopatológicos de las alteraciones neuropsicológicas en los enfermos diabéticos.

Age related interaction of dopamine and serotonin synthesis in striatal synaptosomes. / Age related interaction of dopamine and serotonin synthesis in striatal synaptosomes.

Tyrosine hydroxylase and tryptophan hydroxylase are key rate limiting enzymes in the biosynthesis of dopamine and serotonin, respectively. Since both enzymes are active in striatum, and affected by age, this study was undertaken to investigate interaction between dopamine and serotonin synthesis in brain striatal synaptosomes of aging rat. Male Wistar rats (3 and 30 month old) were killed by decapitation and brain striatal synaptosomes were prepared by discontinuous Ficoll/sucrose gradient technique. Synaptosomes were incubated in the presence of added pargiline (monoamineoxidase inhibitor), dopamine or serotonin synthesized during 25 min was measured by HPLC, employing electrochemical detection. Dopamine synthesis in synaptosomes prepared from young animals was markedly inhibited by addition of 5 microM serotonin concentrations (30

) and increasing serotonin concentrations up to 50 microM caused only a smaller additional inhibition. Dopamine synthesis in synaptosomes obtained from old rats was significantly lower than that of youg animals and addition of serotonin concentrations up to 50 microM had little effect on these preparations. In case of serotonin synthesis, exogenously added 5 microM dopamine inhibited serotonin synthesis in the synaptosomes of both ages by about 40

, whereas with higher concentration of dopamine (10-50 microM) the rate of inhibition was highly pronounced in old rats as compared to that of young animals. It is concluded that dopamine and serotonin interaction may be significant, and that these should be considered in long-term treatments of Parkinsons disease with L-DOPA.

Elevación crónica de la síntesis de serotonina cerebral en rata adulta desnutrida in utero y resuperada nutricionalmente durante el amamantamiento / Chronic elevated brain serotonin synthesis in adult rats undernourished in utero and nutritionally recovered with breast-feeding

Introducción. Las ratas desnutridas in utero tienen la síntesis de 5-hidroxitriptamina (5-HT) cerebral acelerada y su concentración elevada, secundaria a un aumento del L-triptófano (L-Trp) y de la actividad de la triptófano-5-hidroxilasa (TrpOH). Además de un cambio cinético de la TrpOH, que consiste en un aumento de la afinidad por el L-Trp y mayor actividad por mecanismos de fosforilación. Por otra parte, cuando estos desnutridos fueron sometidos a un esquema de recuperación nutricia neonatal, mostraron completa recuperación física y retorno a lo normal del L-Trp durante el amamantamiento; pero a pesar de este hecho, la actividad de la TrpOH permaneció elevada y persistió un aumento de la 5-HT cerebral. El objetivo del presente estudio fue investigar si los cambios en la actividad de la TrpOH y en la síntesis de 5-HT cerebral, persisten o regresan a lo normal en el animal adulto nutricionalmente recuperado. Material y métodos. Se utilizaron ratas Wistar; adaptadas durante 2 semanas a condiciones ambientales estándar. Al término del período se formaron 2 grupos: uno con desnutrición (D) y el otro control (C). Después de 2 semanas, las hembras fueron pareadas con machos normales. Al nacimiento las crías fueron redistribuidas a madres del mismo grupo D y C. Además, se realizó un cruzamiento de las crías desnutridas a madres controles, lo que formó el grupo desnutrido recuperado (DR). Al día 21 de edad todas las crías fueron destetadas y se les continuó con el mismo esquema de alimentación. En los días 21, 60, 90 y 120, se obtuvo el tallo y la corteza cerebral en donde se determinó la TrpOH y las concentraciones de L-Trp y 5-HT. Además se les tomó sangre en donde se cuantificó el L-Trp y albúmina. También se les midió la ingesta de comida cada 24 horas y se les determinó el peso corpora, cerebral y de la longitud céfalo-sacra. Resultados...

Effects of acute and chronic toluene inhalation on behavior, monoamine metabolism and specific binding (3H-serotonin and 3H-norepinephrine) of rat brain

The present study deals with the effect of chronic toluene inhalation (30,000 - 40,000 ppm in air, 15 min/day for 30 days) that induced abnormal behavior states resembling the serotonin syndrome in rats: resting tremor, hindlimb abduction, Straub tail, head weaving and rigidity. The head weaving latencies were significantly decreased when assessed at 15 and 30 days of exposure totoluene vapors. The sequence pattern sign of serotonin syndrome were changed after 15 and 30 days of exposure, indicating possible comulative effects and/or tolerance development. There were no changes in concentrations of indolamine and catecholamine compounds in different parts of the rat brain (cerebral cortex, modbrain, brainstem and cerebellum) as influence of chronic toluene exposure. Examination of specific serotonin ((3H)-5HT) to crude synaptic membranes prepared from rat brains and subjected to chronic toluene inhalation revealed a very high increased value in apparent Kd (30.7 ñ 15) with respect to its air control (9.7 ñ 2.3) and baseline control (5.8 ñ 3.2). This difference was highly significant (p <0.02). There were no changes in apparent Bmax of specific (3H).5HT binding sites. On the other hand (3H)-NE binding of rat brain studies did not show any difference either in apparent Kd or apparent Bmasx. These results indicate that serotonin syndrome may be a consequence of changes of serotonergic mechanism, specifically a reduced affinity in specific (3H)-5HT binding sites

Alteraciones en la síntesis de serotonina cerebral inducidas por diabetes mellitus insulino dependiente / Changes of brain serotonin synthesis in rats with diabetes mellitus insulin depend

Objetivo. Determinar si las ratas con diabetes mellitus insulino-dependiente tienen una menor actividad de la vía serotoninérgica a través de la medición de la fracción libre del L-triptófano plasmático. Metodología. Se utilizó el modelo de la administración de estreptozotocina en ratas para el desarrollo de diabetes mellitus insulino-dependiente. A los 7, 14 y 21 días se les determinó la actividad de la vía serotoninérgica. Resultados. El grupo diabético mostró disminución del peso corporal y del nivel de L-triptófano (libre, unido y total) en el plasma. En el tejido nervioso también se presentó disminución del nivel de L-triptófano, de la serotonina y de la actividad de la triptófano-5-hidroxilasa. Por el contrario, la actividad de la hidroxilasa estuvo alta en el tallo cerebral desde el día 14, acompañada de una elevación de la síntesis de serotonina. Conclusiones. Se confirma que la diabetes mellitus insulino-dependiente produce una desnutrición crónica. La baja de L-triptófano en la sangre sugiere que su ingreso al cerebro está disminuido y que hay una menor síntesis del neurotransmisor, tal como se demostró en el cerebro de estos animales diabéticos. Sin embargo, el hecho de que la actividad de la triptófano-5-hidroxilasa se eleva en el tallo cerebral, con un consecuente aumento de la síntesis de serotonina, nos permite plantear que existe cambio en la síntesis del neurotransmisor dependiendo de la región, con las consecuencias funcionales respectivas