Hydroxylase Type Research Articles

Identification of New Non-BBB Permeable Tryptophan Hydroxylase Inhibitors for Treating Obesity and Fatty Liver Disease.

Serotonin (5-hydroxytryptophan) is a hormone that regulates emotions in the central nervous system. However, serotonin in the peripheral system is associated with obesity and fatty liver disease. Because serotonin cannot cross the blood-brain barrier (BBB), we focused on identifying new tryptophan hydroxylase type I (TPH1) inhibitors that act only in peripheral tissues for treating obesity and fatty liver disease without affecting the central nervous system. Structural optimization inspired by para-chlorophenylalanine (pCPA) resulted in the identification of a series of oxyphenylalanine and heterocyclic phenylalanine derivatives as TPH1 inhibitors. Among these compounds, compound 18i with an IC50 value of 37 nM was the most active in vitro. Additionally, compound 18i showed good liver microsomal stability and did not significantly inhibit CYP and Herg. Furthermore, this TPH1 inhibitor was able to actively interact with the peripheral system without penetrating the BBB. Compound 18i and its prodrug reduced body weight gain in mammals and decreased in vivo fat accumulation.

Increase of 5-HT levels is induced both in mouse brain and HEK-293 cells following their exposure to a non-viral tryptophan hydroxylase construct

Tryptophan hydroxylase type 2 (Tph2) is the rate-limiting enzyme for serotonin (5-HT) biosynthesis in the brain. Dysfunctional Tph2 alters 5-HT biosynthesis, leading to a deficiency of 5-HT, which could have repercussions on human behavior. In the last decade, several studies have associated polymorphisms of the TPH2 gene with suicidal behavior. Additionally, a 5-HT deficiency has been implicated in various psychiatric pathologies, including alcoholism, impulsive behavior, anxiety, and depression. Therefore, the TPH2 gene could be an ideal target for analyzing the effects of a 5-HT deficiency on brain function. The aim of this study was to use the construct pIRES-hrGFP-1a-Tph2-FLAG to treat CD1-male mice and to transfect HEK-293-cells and then to evaluate whether this treatment increases 5-HT production. 5-HT levels were enhanced 48 h post-transfection, in HEK-293 cells. Three days after the ocular administration of pIRES-hrGFP-1a-Tph2-FLAG to mice, putative 5-HT production was significantly higher than in the control in both hypothalamus and amygdala, but not in the brainstem. Further research will be needed on the possible application of this treatment for psychiatric diseases involving a Tph2 dysfunction or serotonin deficiency.

Differential localization of serotoninergic system elements in human amniotic epithelial cells†.

Serotonin or 5-hydroxytryptamine (5-HT) is a biogenic amine involved in regulating several functions, including development. However, its impact on human embryo development has been poorly studied. The present work investigated the expression and distribution of the main components of the serotoninergic system in human amniotic tissue and human amniotic epithelial cells (hAEC) in vitro, as an alternative model of early human embryo development. Amniotic membranes from full-term healthy pregnancies were used. Human amnion tissue or hAEC isolated from the amnion was processed for reverse transcription-polymerase chain reaction and immunofluorescence analyses of the main components of the serotoninergic system. We found the expression of tryptophan hydroxylase type 1 (TPH1), type 2 (TPH2), serotonin transporter (SERT), monoamine oxidase-A (MAOA), as well as HTR1D and HTR7 receptors at mRNA level in amnion tissue as well in hAEC. Interestingly, we found the presence of 5-HT in the nucleus of the cells in amnion tissue, whereas it was located in the cytoplasm of isolated hAEC. We detected TPH1, TPH2, and HTR1D receptor in both the nucleus and cytoplasm. SERT, MAOA, and HTR7 receptor were only observed in the cytoplasm. The results presented herein show, for the first time, the presence of the serotoninergic system in human amnion in vivo and in vitro.

Selective Serotonin Reuptake Inhibitors Increase Urinary Free Cortisol in Patients with Carney Complex and Primary Pigmented Nodular Adrenocortical Disease

Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent Cushing syndrome predominantly associated with Carney Complex (CNC), a multiple endocrine neoplasia syndrome primarily caused by inactivating defects in PRKAR1A. PPNAD, which has neuroendocrine features, demonstrates cortisol production in response to serotonin as well as increased expression of tryptophan hydroxylase type 2, a serotonin synthesizing enzyme, and the serotonin (5-HT) receptors types 4, 6, and 7. This creates an autocrine/paracrine serotonergic regulatory loop that activates cortisol production. PPNAD can be diagnosed through a 6-day Liddle test (LT) showing a paradoxical increase of >50% from baseline in 24-h urinary free cortisol (UFC) on the 2nd day of high-dose dexamethasone administration (Day 6). Selective serotonin reuptake inhibitors (SSRIs) inhibit the reuptake of serotonin and are widely used for the treatment of depression. We performed a retrospective cohort study of patients with CNC and PPNAD that underwent a LT to evaluate the effect of SSRIs on UFCs, with the hypothesis that SSRI use leads to an exaggerated increase in UFC through presumed activation of the described serotonergic regulatory loop. Of the 34 patients (4–65 y) with CNC and PPNAD that underwent a LT at our institution between 2004 and 2018, 4 took an SSRI during testing. No differences were observed between the SSRI (S) group and the non-SSRI (NS) group in baseline UFCs and the percent increase in UFC on D6. Specifically, the median (IQR) baseline UFC in the S group was 36 (13–252) mcg/24h (nl 4–56) vs 35 (13–98) mcg/24h in the NS group (P=0.95). The percent change in UFC was 208 (93–683)% in the S group and 185 (28–364)% in the NS group (P=0.89). However, there was a difference for overall UFC measurement (across days 1–6 of the LT) in the S group vs the NS group (P=0.03). Age <18 vs 18+ and sex did not have an effect on the outcomes (P=0.17 and P=0.74, respectively). Thus, we conclude that though the percent change in UFC during the LT was similar in both groups, there was a significant difference in overall UFC in the S group when compared to the NS group. These data support an effect of SSRIs and serotonin on UFC and consequently cortisol production in PPNAD. This interesting observation has to be confirmed in more patients with CNC and PPNAD to further elucidate the effects of SSRIs on cortisol production in patents with PPNAD-caused Cushing syndrome, as this may have significant diagnostic and therapeutic implications.

Functional Identification of Two Types of Carotene Hydroxylases from the Green Alga Dunaliella bardawil Rich in Lutein.

The salt-tolerant unicellular alga Dunaliella bardawil FACHB-847 can accumulate large amounts of lutein, but the underlying cause of massive accumulation of lutein is still unknown. In this study, genes encoding two types of carotene hydroxylases, i.e., β-carotene hydroxylase (DbBCH) and cytochrome P450 carotenoid hydroxylase (DbCYP97s; DbCYP97A, DbCYP97B, and DbCYP97C), were cloned from D. bardawil. Their substrate specificities and enzyme activities were tested through functional complementation assays in Escherichia coli. It was showed that DbBCH could catalyze the hydroxylation of the β-rings of both β- and α-carotene, and displayed a low level of ε-hydroxylase. Unlike CYP97A from higher plants, DbCYP97A could not hydroxylate β-carotene. DbCYP97A and DbCYP97C showed high hydroxylase activity toward the β-ring and ε-ring of α-carotene, respectively. DbCYP97B displayed minor activity toward the β-ring of α-carotene. The high accumulation of lutein in D. bardawil may be due to the multiple pathways for lutein biosynthesis generated from α-carotene with zeinoxanthin or α-cryptoxanthin as intermediates by DbBCH and DbCYP97s. Taken together, this study provides insights for understanding the underlying reason for high production of lutein in the halophilic green alga D. bardawil FACHB-847.

Tuning of pKa values activates substrates in flavin-dependent aromatic hydroxylases

Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation. Herein, we employed 19F NMR in conjunction with fluorinated substrate analogs to directly measure pKa values and to monitor protein events in hydroxylase active sites. We found that the single-component monooxygenase 3-hydroxybenzoate 6-hydroxylase (3HB6H) depresses the pKa of the bound substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) by 1.6 pH units, consistent with previously proposed mechanisms. 19F NMR was applied anaerobically to the two-component monooxygenase 4-hydroxyphenylacetate 3-hydroxylase (HPAH), revealing depression of the pKa of 3-fluoro-4-hydroxyphenylacetate by 2.5 pH units upon binding to the C2 component of HPAH. 19F NMR also revealed a pKa of 8.7 ± 0.05 that we attributed to an active-site residue involved in deprotonating bound substrate, and assigned to His-120 based on studies of protein variants. Thus, in both types of hydroxylases, we confirmed that binding favors the phenolate form of substrate. The 9 and 14 kJ/mol magnitudes of the effects for 3HB6H and HPAH-C2, respectively, are consistent with pKa tuning by one or more H-bonding interactions. Our implementation of 19F NMR in anaerobic samples is applicable to other two-component flavin-dependent hydroxylases and promises to expand our understanding of their catalytic mechanisms.

Gut-brain communication in hyperfunction of 5-hydroxytryptamine induced by oral zinc oxide nanoparticles exposure in young mice

Zinc oxide nanoparticles (ZnONPs) have been widely used in food storage containers and food additives in daily life. However, the impact of oral intake of ZnONPs on nervous system is extremely limited, especially on children and adolescents. In this study, four weeks old mice were treated with either vehicle or ZnONPs suspension solution at 26 mg/kg by intragastric administration for 30 days. Our results demonstrated that oral ZnONPs exposure could induce pathological changes in gut and abnormal excitement of enteric neurons. Interestingly, we found that ZnONPs caused enhancement of 5-hydroxytryptamine (5-HT) in gut by activation of its biosynthesis, transport and receptors, and subsequently resulting in increased level of 5-HT in brain via gut-brain communication by blood. Our data also showed that there were no apparent changes on the expressions of interleukin (Il)-6, Il-1β, C–C motif chemokine ligand 2 (Ccl2), tumor necrosis factor (Tnf) in gut and zinc chelator Mt2 in gut and cortex. Meanwhile, no significant changes were observed on the expressions of tryptophan hydroxylase type 1, 5-HT receptor 3A (Htr3a) and Htr4 in hippocampus and cortex. Our study indicate that oral ZnONPs exposure causes hyperfunction of 5-HT in gut in young mice which may further spread to brain via gut-brain communication.

SUN-356 The Effects of Selective Serotonin Reuptake Inhibitors on Urinary Free Cortisol in Patients with Carney Complex and Primary Pigmented Nodular Adrenocortical Disease

PPNAD is a rare cause of ACTH-independent Cushing syndrome mainly associated with Carney complex (CNC). It is diagnosed by histologic examination or a 6-day Liddle test (LT) showing a paradoxical increase of >50% in 24-h urinary free cortisol (UFC) on the 2nd day of high-dose dexamethasone administration (Day 6 [D6]). PPNAD tissue has neuroendocrine features and can overexpress the serotonin (5-HT) synthesizing enzyme tryptophan hydroxylase type 2 and the 5-HT receptors types 4, 6, and 7, with formation of an illicit stimulatory serotonergic loop whose pharmacological inhibition in vitro decreases cortisol production [1]. SSRIs inhibit the reuptake of 5-HT and are used as 1st-line antidepressants. This is a retrospective cohort study of patients with CNC and PPNAD that underwent a LT with the objective to evaluate the effect of SSRIs on UFCs in these patients. Of the 34 patients (4-65 y) with CNC and PPNAD that had a LT at our institution between 2004 and 2018, 4 patients took an SSRI during testing. No statistically significant differences were demonstrated between the SSRI (S) group and the non-SSRI (NS) group in baseline UFCs and urine 17-hydroxycorticosteroids (17OHS) and the percent increase in UFC and 17OHS on D6. Specifically, the median (IQR) baseline UFC in the S group was 36 (13-252) mcg/24h (nl 4-56) vs 35 (13-98) mcg/24h in the NS group (P=0.95). Baseline 17OHS were 11 (8-18) mg/mg creatinine (Cr) (nl ≤6) in the S group vs 8 (5-11) mg/mgCr in the NS group (P=0.27). The percent change in UFC was 208 (93-683)% in the S group and 185 (28-364)% in the NS group (P=0.89), while the percent change in 17OHS was 56 (40-87)% in the S group and 67 (16-92)% in the NS group (P=0.91). On D6, the S group appeared to have higher UFC [118 (63-635) mcg/24h vs 112 (58-201) mcg/24h; P=0.64] and 17OHS [16 (10-39) mg/mgCr vs 13 (7-17) mg/mgCr; P=0.25] than the NS group, but these were not statistically significant. Though these data do not confirm an effect of SSRIs on UFC and ultimately cortisol production in PPNAD, and the percent change in UFC during the LT was similar between both groups, the higher D6 UFC in the S group, when the baseline UFCs between the 2 groups were similar may suggest a degree of increased cortisol production in the S group. The data are limited by the small number of patients in the S group (n=4). In addition, 50% of patients in the S group vs 77% in the NS group underwent adrenalectomy, which is indicative of more severe disease in the NS group. More data are needed from this cohort to determine if SSRI use affects cortisol production in vivo, as this may have significant diagnostic and therapeutic implications for patients with CNC and PPNAD. Reference: 1. Bram, Z., et al., PKA regulatory subunit 1A inactivating mutation induces serotonin signaling in primary pigmented nodular adrenal disease. JCI Insight, 2016. 1(15): p. e87958.

The CH25H-CYP7B1-RORα axis of cholesterol metabolism regulates osteoarthritis.

Osteoarthritis-the most common form of age-related degenerative whole-joint disease1-is primarily characterized by cartilage destruction, as well as by synovial inflammation, osteophyte formation and subchondral bone remodelling2,3. However, the molecular mechanisms that underlie the pathogenesis of osteoarthritis are largely unknown. Although osteoarthritis is currently considered to be associated with metabolic disorders, direct evidence for this is lacking, and the role of cholesterol metabolism in the pathogenesis of osteoarthritis has not been fully investigated4-6. Various types of cholesterol hydroxylases contribute to cholesterol metabolism in extrahepatic tissues by converting cellular cholesterol to circulating oxysterols, which regulate diverse biological processes7,8. Here we show that the CH25H-CYP7B1-RORα axis of cholesterol metabolism in chondrocytes is a crucial catabolic regulator of the pathogenesis of osteoarthritis. Osteoarthritic chondrocytes had increased levels of cholesterol because of enhanced uptake, upregulation of cholesterol hydroxylases (CH25H and CYP7B1) and increased production of oxysterol metabolites. Adenoviral overexpression of CH25H or CYP7B1 in mouse joint tissues caused experimental osteoarthritis, whereas knockout or knockdown of these hydroxylases abrogated the pathogenesis of osteoarthritis. Moreover, retinoic acid-related orphan receptor alpha (RORα) was found to mediate the induction of osteoarthritis by alterations in cholesterol metabolism. These results indicate that osteoarthritis is a disease associated with metabolic disorders and suggest that targeting the CH25H-CYP7B1-RORα axis of cholesterol metabolism may provide a therapeutic avenue for treating osteoarthritis.

Two Subjects with Congenital Adrenal Hyperplasia due to 21-OH Hydroxylase Deficiency and Type 1 Diabetes Mellitus: Is there a Genetic Connection?

Two Subjects with Congenital Adrenal Hyperplasia due to 21-OH Hydroxylase Deficiency and Type 1 Diabetes Mellitus: Is there a Genetic Connection?

Polymorphisms of serotonin transporter gene and psychological status in patients with multiple sclerosis

Background: Multiple sclerosis (MS) is the most common neuroinflammatory disease in young adults. Anxiety and depression may predispose individuals to MS and flare-ups. Serotonin transmission is modified in some brain regions of patients with MS, and these changes may contribute to their psychiatric abnormalities. We studied the frequencies of common polymorphisms of the serotonin reuptake transporter (SERT) gene in patients with MS according to their psychological status. Methods: The 5-HTTLPR, rs25531, and STin2VNTR polymorphisms of the SERT gene were genotyped by polymerase chain reaction (PCR)-based methods in 100 patients with MS and 100 healthy controls. Results: There were no remarkable differences in SERT gene polymorphisms between patients with MS and healthy controls. Unlike the control group, 41% of the patients showed some degree of depression based on Beck Depression Inventory (BDI), but no association was observed between SERT gene polymorphisms after the patients were stratified by depression status. Conclusion: In addition to SERT gene polymorphisms, modulation of serotonin at the synapses may also be regulated by genetic variations in tryptophan hydroxylase type 2 and serotonin receptors. Further studies with functional brain imaging of the serotonergic system in patients with MS can provide information on the role of serotonin in this disease.

Selective Inhibitors of a Human Prolyl Hydroxylase (OGFOD1) Involved in Ribosomal Decoding.

Human prolyl hydroxylases are involved in the modification of transcription factors, procollagen, and ribosomal proteins, and are current medicinal chemistry targets. To date, there are few reports on inhibitors selective for the different types of prolyl hydroxylases. We report a structurally informed template‐based strategy for the development of inhibitors selective for the human ribosomal prolyl hydroxylase OGFOD1. These inhibitors did not target the other human oxygenases tested, including the structurally similar hypoxia‐inducible transcription factor prolyl hydroxylase, PHD2.

A novel splicing site IRP1 somatic mutation in a patient with pheochromocytoma and JAK2V617F positive polycythemia vera: a case report

BackgroundThe role of the hypoxia signaling pathway in the pathogenesis of pheochromocytoma/paraganglioma (PPGL)-polycythemia syndrome has been elucidated. Novel somatic mutations in hypoxia-inducible factor type 2A (HIF2A) and germline mutations in prolyl hydroxylase type 1 and type 2 (PHD1 and PHD2) have been identified to cause upregulation of the hypoxia signaling pathway and its target genes including erythropoietin (EPO) and its receptor (EPOR). However, in a minority of patients presenting with this syndrome, the genetics and molecular pathogenesis remain unexplained. The aim of the present study was to uncover novel genetic causes of PPGL-polycythemia syndrome.Case presentationA female presented with a history of JAK2V617F positive PV, diagnosed in 2007, and right adrenal pheochromocytoma diagnosed and resected in 2011. Her polycythemia symptoms and hematocrit levels continued to worsen from 2007 to 2011, with an increased frequency of phlebotomies. Postoperatively, until early 2013, her hematocrit levels remained normalized. Following this, the hematocrit levels ranged between 46.4 and 48.9% [35–45%]. Tumor tissue from the patient was further tested for mutations in genes related to upregulation of the hypoxia signaling pathway including iron regulatory protein 1 (IRP1), which is a known regulator of HIF-2α mRNA translation. Functional studies were performed to investigate the consequences of these mutations, especially their effect on the HIF signaling pathway and EPO. Indel mutations (c.267-1_267delGGinsTA) were discovered at the exon 3 splicing site of IRP1. Minigene construct and splicing site analysis showed that the mutation led to a new splicing site and a frameshift mutation of IRP1, which caused a truncated protein. Fluorescence in situ hybridization analysis demonstrated heterozygous IRP1 deletions in tumor cells. Immunohistochemistry results confirmed the truncated IRP1 and overexpressed HIF-2α, EPO and EPOR in tumor cells.ConclusionsThis is the first report which provides direct molecular genetic evidence of association between a somatic IRP1 loss-of-function mutation and PHEO and secondary polycythemia. In patients diagnosed with PHEO/PGL and polycythemia with negative genetic testing for mutations in HIF2A, PHD1/2, and VHL, IRP1 should be considered as a candidate gene.

Tetrahydrobiopterin treatment reduces brain L-Phe but only partially improves serotonin in hyperphenylalaninemic ENU1/2 mice.

Hyperphenylalaninemia (HPA) caused by hepatic phenylalanine hydroxylase (PAH) deficiency has severe consequences on brain monoamine neurotransmitter metabolism. We have studied monoamine neurotransmitter status and the effect of tetrahydrobiopterin (BH4) treatment in Pahenu1/enu2 (ENU1/2) mice, a model of partial PAH deficiency. These mice exhibit elevated blood L-phenylalanine (L-Phe) concentrations similar to that of mild hyperphenylalaninemia (HPA), but brain levels of L-Phe are still ~5-fold elevated compared to wild-type. We found that brain L-tyrosine, L-tryptophan, BH4 cofactor and catecholamine concentrations, and brain tyrosine hydroxylase (TH) activity were normal in these mice but that brain serotonin, 5-hydroxyindolacetic acid (5HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) content, and brain TH protein, as well as tryptophan hydroxylase type 2 (TPH2) protein levels and activity were reduced in comparison to wild-type mice. Parenteral L-Phe loading conditions did not lead to significant changes in brain neurometabolite concentrations. Remarkably, enteral BH4 treatment, which normalized brain L-Phe levels in ENU1/2 mice, lead to only partial recovery of brain serotonin and 5HIAA concentrations. Furthermore, indirect evidence indicated that the GTP cyclohydrolase I (GTPCH) feedback regulatory protein (GFRP) complex may be a sensor for brain L-Phe elevation to ameliorate the toxic effects of HPA. We conclude that BH4 treatment of HPA toward systemic L-Phe lowering reverses elevated brain L-Phe content but the recovery of TPH2 protein and activity as well as serotonin levels is suboptimal, indicating that patients with mild HPA and mood problems (depression or anxiety) treated with the current diet may benefit from supplementation with BH4 and 5-OH-tryptophan.

PKA regulatory subunit 1A inactivating mutation induces serotonin signaling in primary pigmented nodular adrenal disease

Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent hypercortisolism. The disease is primarily caused by germline mutations of the protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene, which induces constitutive activation of PKA in adrenocortical cells. Hypercortisolism is thought to result from PKA hyperactivity, but PPNAD tissues exhibit features of neuroendocrine differentiation, which may lead to stimulation of steroidogenesis by abnormally expressed neurotransmitters. We hypothesized that serotonin (5-HT) may participate in the pathophysiology of PPNAD-associated hypercortisolism. We show that PPNAD tissues overexpress the 5-HT synthesizing enzyme tryptophan hydroxylase type 2 (Tph2) and the serotonin receptors types 4, 6, and 7, leading to formation of an illicit stimulatory serotonergic loop whose pharmacological inhibition in vitro decreases cortisol production. In the human PPNAD cell line CAR47, the PKA inhibitor H-89 decreases 5-HT4 and 5-HT7 receptor expression. Moreover, in the human adrenocortical cell line H295R, inhibition of PRKAR1A expression increases the expression of Tph2 and 5-HT4/6/7 receptors, an effect that is blocked by H-89. These findings show that the serotonergic process observed in PPNAD tissues results from PKA activation by PRKAR1A mutations. They also suggest that Tph inhibitors may represent efficient treatments of hypercortisolism in patients with PPNAD.

Expression and functional analysis of citrus carotene hydroxylases: unravelling the xanthophyll biosynthesis in citrus fruits.

BackgroundXanthophylls are oxygenated carotenoids and fulfill critical roles in plant growth and development. In plants, two different types of carotene hydroxylases, non-heme di-iron and heme-containing cytochrome P450, were reported to be involved in the biosynthesis of xanthophyll. Citrus fruits accumulate a high amount of xanthophylls, especially β,β-xanthophylls. To date, however, the roles of carotene hydroxylases in regulating xanthophyll content and composition have not been elucidated.ResultsIn the present study, the roles of four carotene hydroxylase genes (CitHYb, CitCYP97A, CitCYP97B, and CitCYP97C) in the biosynthesis of xanthophyll in citrus fruits were investigated. Phylogenetic analysis showed that the four citrus carotene hydroxylases presented in four distinct clusters which have been identified in higher plants. CitHYb was a non-heme di-iron carotene hydroxylase, while CitCYP97A, CitCYP97B, and CitCYP97C were heme-containing cytochrome P450-type carotene hydroxylases. Gene expression results showed that the expression of CitHYb increased in the flavedo and juice sacs during the ripening process, which was well consistent with the accumulation of β,β-xanthophyll in citrus fruits. The expression of CitCYP97A and CitCYP97C increased with a peak in November, which might lead to an increase of lutein in the juice sacs during the ripening process. The expression level of CitCYP97B was much lower than that of CitHYb, CitCYP97A, and CitCYP97C in the juice sacs during the ripening process. Functional analysis showed that the CitHYb was able to catalyze the hydroxylation of the β-rings of β-carotene and α-carotene in Escherichia coli BL21 (DE3) cells. Meanwhile, when CitHYb was co-expressed with CitCYP97C, α-carotene was hydroxylated on the β-ring and ε-ring sequentially to produce lutein.ConclusionsCitHYb was a key gene for β,β-xanthophyll biosynthesis in citrus fruits. CitCYP97C functioned as an ε-ring hydroxylase to produce lutein using zeinoxanthin as a substrate. The results will contribute to elucidating xanthophyll biosynthesis in citrus fruits, and provide new strategies to improve the nutritional and commercial qualities of citrus fruits.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0840-2) contains supplementary material, which is available to authorized users.

Tryptophan hydroxylase type 2 variants modulate severity and outcome of addictive behaviors in Parkinson's disease

BackgroundImpulse control disorders and compulsive medication intake may occur in a minority of patients with Parkinson's disease (PD). We hypothesize that genetic polymorphisms associated with addiction in the general population may increase the risk for addictive behaviors also in PD. MethodsSixteen polymorphisms in candidate genes belonging to five neurotransmitter systems (dopaminergic, catecholaminergic, serotonergic, glutamatergic, opioidergic) and the BDNF were screened in 154 PD patients with addictive behaviors and 288 PD control subjects. Multivariate analysis investigated clinical and genetic predictors of outcome (remission vs. persistence/relapse) after 1 year and at the last follow-up (5.1 ± 2.5 years). ResultsAddictive behaviors were associated with tryptophan hydroxylase type 2 (TPH2) and dopamine transporter gene variants. A subsequent analysis within the group of cases showed a robust association between TPH2 genotype and the severity of addictive behaviors, which survived Bonferroni correction for multiple testing. At multivariate analysis, TPH2 genotype resulted the strongest predictor of no remission at the last follow-up (OR[95%CI], 7.4[3.27–16.78] and 13.2[3.89–44.98] in heterozygous and homozygous carriers, respectively, p < 0.001). The extent of medication dose reduction was not a predictor. TPH2 haplotype analysis confirmed the association with more severe symptoms and lower remission rates in the short- and the long-term (p < 0.005 for all analyses). ConclusionThe serotonergic system is likely to be involved in the pathophysiology of addictive behaviors in PD, modulating the severity of symptoms and the rate of remission at follow-up. If confirmed in larger independent cohorts, TPH2 genotype may become a useful biomarker for the identification of at-risk individuals.

Immunohistochemical detection and gene expression of tyrosine hydroxylase and vesicular monoamine transporter type 2 in intrinsic cardiac ganglia of socially isolated rats

Social isolation induced a significant increase in resting heart rate and reduction in heart rate variability. Dysfunction of the intrinsic cardiac nervous system is implicated in the genesis of cardiovascular diseases. Previous evidence suggests that cardiac ganglia contain noradrenergic neurons. Thus, immunohistochemical expression of catecholaminesynthesizing enzyme tyrosine hydroxylase (TH) and vesicular monoamine transporter 2 (VMAT2) were analyzed, as well as the effects of social isolation stress on mRNA and protein levels of this enzyme and transporter in the intrinsic cardiac nervous system of adult rats. Our results indicate that cardiac ganglion neurons express TH and VMAT2 immunoreactivity. Chronic isolated stress of rats caused a decrease in TH mRNA and VMAT2 mRNA in the neurons of intrinsic cardiac ganglia. No significant alterations in the protein levels of TH and VMAT2 were observed in these neurons. These data indicate that the neurons of intrinsic cardiac ganglia express TH as well as VMAT2 but that social isolation stress does not change their protein levels.

Intake of melatonin increases tryptophan hydroxylase type 1 activity in aged rats: Preliminary study

Pineal melatonin is important not only for synchronization of biological rhythms, but also in the ageing process as a potential drug to relieve oxidative damage. During ageing, the nocturnal melatonin production decreases resulting in an increased incidence of disorders. Present in vivo experiments were performed to study the effects of exogenous melatonin chronically administered to old rats on the pineal biosynthesis of melatonin and the precursor serotonin (5-HT) mediated by tryptophan hydroxylase type 1 (TPH-1). Accumulation of 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition was used as a measure of the TPH-1 activity. 5-HT and its metabolite 5-HIAA were also quantified by HPLC-ED. As expected, ageing resulted in worsening of different neurochemical parameters. However, chronic intake of melatonin (1mg/kg/day, diluted in drinking water, 4weeks) increased TPH-1 activity and significantly improved the age-induced deficits in nocturnal melatonin content in the pineal gland. Results suggest that melatonin intake (or melatonin rich foods) may contribute to recover the pineal function preventing the nocturnal descent of 5-HT and melatonin biosynthesis that normally occur in pineal gland as a consequence of ageing.

The genome of the moderate halophile Amycolicicoccus subflavus DQS3-9A1(T) reveals four alkane hydroxylation systems and provides some clues on the genetic basis for its adaptation to a petroleum environment.

The moderate halophile Amycolicicoccus subflavus DQS3-9A1T is the type strain of a novel species in the recently described novel genus Amycolicicoccus, which was isolated from oil mud precipitated from oil produced water. The complete genome of A. subflavus DQS3-9A1T has been sequenced and is characteristic of harboring the genes for adaption to the harsh petroleum environment with salinity, high osmotic pressure, and poor nutrient levels. Firstly, it characteristically contains four types of alkane hydroxylases, including the integral-membrane non-heme iron monooxygenase (AlkB) and cytochrome P450 CYP153, a long-chain alkane monooxygenase (LadA) and propane monooxygenase. It also accommodates complete pathways for the response to osmotic pressure. Physiological tests proved that the strain could grow on n-alkanes ranging from C10 to C36 and propane as the sole carbon sources, with the differential induction of four kinds of alkane hydroxylase coding genes. In addition, the strain could grow in 1–12% NaCl with the putative genes responsible for osmotic stresses induced as expected. These results reveal the effective adaptation of the strain DQS3-9A1T to harsh oil environment and provide a genome platform to investigate the global regulation of different alkane metabolisms in bacteria that are crucially important for petroleum degradation. To our knowledge, this is the first report to describe the co-existence of such four types of alkane hydroxylases in a bacterial strain.