Microbial liberation of N-methylserotonin from orange fiber in gnotobiotic mice and humans.

Plant fibers in byproduct streams produced by non-harsh food processing methods represent biorepositories of diverse, naturally occurring, and physiologically active biomolecules. To demonstrate one approach for their characterization, mass spectrometry of intestinal contents from gnotobiotic mice, plus in vitro studies, revealed liberation of N-methylserotonin from orange fibers by human gut microbiota members including Bacteroides ovatus. Functional genomic analyses of B. ovatus strains grown under permissive and non-permissive N-methylserotonin "mining" conditions revealed polysaccharide utilization loci that target pectins whose expression correlate with strain-specific liberation of this compound. N-methylserotonin, orally administered to germ-free mice, reduced adiposity, altered liver glycogenesis, shortened gut transit time, and changed expression of genes that regulate circadian rhythm in the liver and colon. In human studies, dose-dependent, orange-fiber-specific fecal accumulation of N-methylserotonin positively correlated with levels of microbiome genes encoding enzymes that digest pectic glycans. Identifying this type of microbial mining activity has potential therapeutic implications.

pH-Controlled Chemoselective Rapid Azo-Coupling Reaction (CRACR) Enables Global Profiling of Serotonylation Proteome in Cancer Cells.

Serotonylation has been identified as a novel protein posttranslational modification for decades, where an isopeptide bond is formed between the glutamine residue and serotonin through transamination. Transglutaminase 2 (also known as TGM2 or TGase2) was proven to act as the main "writer" enzyme for this PTM, and a number of key regulatory proteins (including small GTPases, fibronectin, fibrinogen, serotonin transporter, and histone H3) have been characterized as the substrates of serotonylation. However, due to the lack of pan-specific antibodies for serotonylated glutamine, the precise enrichment and proteomic profiling of serotonylation still remain challenging. In our previous research, we developed an aryldiazonium probe to specifically label protein serotonylation in a bioorthogonal manner, which depended on a pH-controlled chemoselective rapid azo-coupling reaction. Here, we report the application of a photoactive aryldiazonium-biotin probe for the global profiling of serotonylation proteome in cancer cells. Thus, over 1,000 serotonylated proteins were identified from HCT 116 cells, many of which are highly related to carcinogenesis. Moreover, a number of modification sites of these serotonylated proteins were determined, attributed to the successful application of our chemical proteomic approach. Overall, these findings provided new insights into the significant association between cellular protein serotonylation and cancer development, further suggesting that to target TGM2-mediated monoaminylation may serve as a promising strategy for cancer therapeutics.

Neuroprotective and Neurite Outgrowth Stimulating Effects of New Low-Basicity 5-HT7 Receptor Agonists: In Vitro Study in Human Neuroblastoma SH-SY5Y Cells.

There is some evidence that the serotonin receptor subtype 7 (5-HT7) could be new therapeutic target for neuroprotection. The aim of this study was to compare the neuroprotective and neurite outgrowth potential of new 5-HT7 receptor agonists (AH-494, AGH-238, AGH-194) with 5-CT (5-carboxyamidotryptamine) in human neuroblastoma SH-SY5Y cells. The results revealed that 5-HT7 mRNA expression was significantly higher in retinoic acid (RA)-differentiated cells when compared to undifferentiated ones and it was higher in cell cultured in neuroblastoma experimental medium (DMEM) compared to those placed in neuronal (NB) medium. Furthermore, the safety profile of compounds was favorable for all tested compounds at concentration used for neuroprotection evaluation (up to 1 µM), whereas at higher concentrations (above 10 µM) the one of the tested compounds, AGH-194 appeared to be cytotoxic. While we observed relatively modest protective effects of 5-CT and AH-494 in UN-SH-SY5Y cells cultured in DMEM, in UN-SH-SY5Y cells cultured in NB medium we found a significant reduction of H2O2-evoked cell damage by all tested 5-HT7 agonists. However, 5-HT7-mediated neuroprotection was not associated with inhibition of caspase-3 activity and was not observed in RA-SH-SY5Y cells exposed to H2O2. Furthermore, none of the tested 5-HT7 agonists altered the damage induced by 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenylpyridinium ion (MPP +) and doxorubicin (Dox) in UN- and RA-SH-SY5Y cells cultured in NB. Finally we showed a stimulating effect of AH-494 and AGH-194 on neurite outgrowth. The obtained results provide insight into neuroprotective and neurite outgrowth potential of new 5-HT7 agonists.

N-Acetylserotonin Alleviates Retinal Autophagy via TrkB/AKT/Nrf2 Signaling Pathway in Retinal Ischemia-Reperfusion Injury Rats.

INTRODUCTION: The objective of this study was to investigate the impact of N-acetylserotonin (NAS) on the autophagy of retinal cells in rats with retinal ischemia-reperfusion injury (RIRI) and to explore the mechanisms by which NAS administration can alleviate RIRI through the tropomyosin-related kinase receptor B (TrkB)/protein kinase B (Akt)/nuclear factor erythroid-derived factor 2-related factor (Nrf2) signaling pathway. METHODS: Healthy adult male rats were randomly assigned to four groups: sham, RIRI, RIRI+NAS, and RIRI+NAS+ANA-12. The RIRI group was induced by elevating intraocular pressure, and changes in retinal structure and edema were assessed using H&E staining. The RIRI+NAS and RIRI+NAS+ANA-12 groups received intraperitoneal injections of NAS before and after modeling. The RIRI+NAS+ANA-12 group was also administered ANA-12, a TrkB antagonist. Immunohistochemical staining and Western blot analysis were used to evaluate phosphorylated TrkB (p-TrkB), phosphorylated Akt (p-Akt), Nrf2, sequestosome 1 (P62), and microtubule-associated protein 1 light chain 3 (LC3-II) levels in the retinas of each group. Electroretinogram was recorded to detect retinal function in each group of rats 24 h after modeling. RESULTS: The RIRI+NAS group had a thinner retina and more retinal ganglion cells (RGCs) than RIRI and RIRI+NAS+ANA-12 groups (p < 0.05). Immunohistochemical staining and Western blot results showed that p-TrkB, p-Akt, n-Nrf2, and P62 levels in the RIRI+NAS group were higher compared with those in RIRI and RIRI+NAS+ANA-12 groups (p < 0.05). Also, lower LC3-II levels were observed in the RIRI+NAS group compared with that in RIRI and RIRI+NAS+ANA-12 groups (p < 0.05). Electroretinogram recording results showed that 24 h after retinal ischemia-reperfusion, the magnitude of b-wave changes was attenuated in the RIRI+NAS group compared with the RIRI group (p < 0.05). CONCLUSION: The administration of NAS activates the TrkB/Akt/Nrf2 signaling pathway, reduces autophagy, alleviates retinal edema, promotes the survival of retinal ganglion cells (RGCs), and provides neuroprotection against retinal injury.

Short communication: An alternative pathway for melatonin synthesis in the skin of European flounder (Platichthys flesus).

The classic melatonin biosynthesis pathway (Mel; N-acetyl-5-methoxytryptamine) involves two consecutive enzymatic steps that are decisive in hormone production: conversion of serotonin (5-hydroxytryptamine; 5-HT) to N-acetylserotonin (NAS) and the methylation of the last compound to Mel. This pathway requires the activity of the enzymes: the first is of the category of N-acetyltransferases (AANAT, SNAT, or NAT) and the second is N-acetylserotonin O-methyltransferase (ASMT; also known as HIOMT). However, quite recently, new information has been provided on the possibility of an alternative Mel synthesis pathway; it would include a two-step action by these enzymes, but in reverse order, where ASMT (or ASMTL, the enzyme related to ASMT) methylates 5-HT to 5-methoxytryptamine (5-MT), and then the last compound is acetylated by an enzyme of the category of N-acetyltransferases to Mel. In our study on the activity of enzymes in the Mel biosynthesis pathway in flounder skin, we have found an increase in 5-MT level, as a result of the increase in 5-HT concentration, which is followed by a growing concentration of Mel. However, we have not found any increase in Mel concentration, despite an increase in NAS in the samples. Our data strongly suggest an alternative way of Mel production in flounder skin in which 5-HT is first methylated to 5-MT, which is then acetylated to Mel.

Positive allosteric modulation of indoleamine 2,3-dioxygenase 1 restrains neuroinflammation.

l-tryptophan (Trp), an essential amino acid for mammals, is the precursor of a wide array of immunomodulatory metabolites produced by the kynurenine and serotonin pathways. The kynurenine pathway is a paramount source of several immunoregulatory metabolites, including l-kynurenine (Kyn), the main product of indoleamine 2,3-dioxygenase 1 (IDO1) that catalyzes the rate-limiting step of the pathway. In the serotonin pathway, the metabolite N-acetylserotonin (NAS) has been shown to possess antioxidant, antiinflammatory, and neuroprotective properties in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, little is known about the exact mode of action of the serotonin metabolite and the possible interplay between the 2 Trp metabolic pathways. Prompted by the discovery that NAS neuroprotective effects in EAE are abrogated in mice lacking IDO1 expression, we investigated the NAS mode of action in neuroinflammation. We found that NAS directly binds IDO1 and acts as a positive allosteric modulator (PAM) of the IDO1 enzyme in vitro and in vivo. As a result, increased Kyn will activate the ligand-activated transcription factor aryl hydrocarbon receptor and, consequently, antiinflammatory and immunoregulatory effects. Because NAS also increased IDO1 activity in peripheral blood mononuclear cells of a significant proportion of MS patients, our data may set the basis for the development of IDO1 PAMs as first-in-class drugs in autoimmune/neuroinflammatory diseases.

Changes in the Metabolic Profile of Melatonin Synthesis-Related Indoles during Post-Embryonic Development of the Turkey Pineal Organ.

Research on age-dependent changes in pineal activity has been limited almost exclusively to melatonin (MLT). This study determined, for the first time, the alterations occurring in the metabolic profile of MLT synthesis-related indoles during the post-embryonic development period in birds. Turkeys reared under a 12 h light/dark cycle were euthanized at 2 h intervals for 24 h at the ages of 2, 7, 14, and 28 days and 10, 20, 30, and 45 weeks. The results showed prominent changes in the metabolic profile of indoles during development and could be distinguished in four stages. The first stage, from hatching to the age of 2 weeks, was characterized by a decrease in the 5-hydroxytryptophan concentration and an increase in the concentrations of serotonin (5-HT), MLT, 5-methoxyindoleacetic acid, and 5-methoxytryptamine (5-MTAM). During the second stage, around the age of 1 month, the concentrations of N-acetylserotonin (NAS) and MLT reached a maximum. The synthesis and degradation of 5-HT were also the highest. The third stage, around the age of 10 weeks, was characterized by decreased levels of 5-HT (approximately 50%) and 5-hydroxyindoleacetic acid and a high level of 5-MTAM. The last stage, covering the age of 20 to 45 weeks, was characterized by a large decrease in the synthesis, content, and degradation of 5-HT. Despite these changes, there were no prominent differences in the nocturnal levels of NAS and MLT between the third and fourth stages. The concentrations of all tryptophan derivatives showed daily fluctuations until the age of 45 weeks.

Serotonergic Drugs Inhibit Chikungunya Virus Infection at Different Stages of the Cell Entry Pathway.

Chikungunya virus (CHIKV) is an important reemerging human pathogen transmitted by mosquitoes. The virus causes an acute febrile illness, chikungunya fever, which is characterized by headache, rash, and debilitating (poly)arthralgia that can reside for months to years after infection. Currently, effective antiviral therapies and vaccines are lacking. Due to the high morbidity and economic burden in the countries affected by CHIKV, there is a strong need for new strategies to inhibit CHIKV replication. The serotonergic drug 5-nonyloxytryptamine (5-NT) was previously identified as a potential host-directed inhibitor for CHIKV infection. In this study, we determined the mechanism of action by which the serotonin receptor agonist 5-NT controls CHIKV infection. Using time-of-addition and entry bypass assays, we found that 5-NT predominantly inhibits CHIKV in the early phases of the replication cycle, at a step prior to RNA translation and genome replication. Intriguingly, however, no effect was seen during virus-cell binding, internalization, membrane fusion and genomic RNA (gRNA) release into the cell cytosol. In addition, we show that the serotonin receptor antagonist methiothepin mesylate (MM) also has antiviral properties toward CHIKV and specifically interferes with the cell entry process and/or membrane fusion. Taken together, pharmacological targeting of 5-HT receptors may represent a potent way to limit viral spread and disease severity.IMPORTANCE The rapid spread of mosquito-borne viral diseases in humans puts a huge economic burden on developing countries. For many of these infections, including those caused by chikungunya virus (CHIKV), there are no specific treatment possibilities to alleviate disease symptoms. Understanding the virus-host interactions that are involved in the viral replication cycle is imperative for the rational design of therapeutic strategies. In this study, we discovered an antiviral compound, elucidated its mechanism of action, and propose serotonergic drugs as potential host-directed antivirals for CHIKV.

Sex and race differences of cerebrospinal fluid metabolites in healthy individuals.

INTRODUCTION: Analyses of cerebrospinal fluid (CSF) metabolites in large, healthy samples have been limited and potential demographic moderators of brain metabolism are largely unknown. OBJECTIVE: Our objective in this study was to examine sex and race differences in 33 CSF metabolites within a sample of 129 healthy individuals (37 African American women, 29 white women, 38 African American men, and 25 white men). METHODS: CSF metabolites were measured with a targeted electrochemistry-based metabolomics platform. Sex and race differences were quantified with both univariate and multivariate analyses. Type I error was controlled for by using a Bonferroni adjustment (0.05/33 = .0015). RESULTS: Multivariate Canonical Variate Analysis (CVA) of the 33 metabolites showed correct classification of sex at an average rate of 80.6% and correct classification of race at an average rate of 88.4%. Univariate analyses revealed that men had significantly higher concentrations of cysteine (p < 0.0001), uric acid (p < 0.0001), and N-acetylserotonin (p = 0.049), while women had significantly higher concentrations of 5-hydroxyindoleacetic acid (5-HIAA) (p = 0.001). African American participants had significantly higher concentrations of 3-hydroxykynurenine (p = 0.018), while white participants had significantly higher concentrations of kynurenine (p < 0.0001), indoleacetic acid (p < 0.0001), xanthine (p = 0.001), alpha-tocopherol (p = 0.007), cysteine (p = 0.029), melatonin (p = 0.036), and 7-methylxanthine (p = 0.037). After the Bonferroni adjustment, the effects for cysteine, uric acid, and 5-HIAA were still significant from the analysis of sex differences and kynurenine and indoleacetic acid were still significant from the analysis of race differences. CONCLUSION: Several of the metabolites assayed in this study have been associated with mental health disorders and neurological diseases. Our data provide some novel information regarding normal variations by sex and race in CSF metabolite levels within the tryptophan, tyrosine and purine pathways, which may help to enhance our understanding of mechanisms underlying sex and race differences and potentially prove useful in the future treatment of disease.

N-acetylserotonin alleviated the expression of interleukin-1ß in retinal ischemia-reperfusion rats via the TLR4/NF-κB/NLRP3 pathway.

This study aimed to explore the effects of N-acetylserotonin (NAS) on the expression of interleukin-1ß (IL-1ß) in the retina of retinal ischemia-reperfusion injury (RIRI) rats via the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB)/nod-like receptor pyrin domain containing 3 (NLRP3) signaling pathway. In this study, adult male Sprague Dawley rats were randomly divided into the sham, RIRI, RIRI + NAS and RIRI + TAK-242 + NAS groups. The rats in the RIRI + NAS and RIRI + TAK-242 + NAS groups were intraperitoneally injected with NAS 30 min before and after modeling. TAK-242, a selective TLR4 inhibitor, was administered by intraperitoneal injection in RIRI + TAK-242 + NAS group. The RIRI rat model was established by elevating the intraocular pressure to 110 mmHg for 60 min. The retinal structure and edema were assessed by H&E staining. The expression levels of TLR4, phosphorylated NF-κB (p-NF-κB), NLRP3, cleaved Caspase-1, and IL-1ß in the retina of each group were detected using immunohistochemistry and Western blot. The correlations of the differences of TLR4+ and cleaved Caspase-1+ with IL-1ß+ cells (between the NAS and the RIRI groups) were analyzed, using linear regression in the RIRI + NAS group. Results showed that thinner retina, more RGCs, and less TLR4+, p-NF-κB+, NLRP3+, cleaved Caspase-1+, and IL-1ß+ cells in the retina were observed in the RIRI + NAS and RIRI + TAK-242 + NAS groups compared with the RIRI group 12 h after RIRI (all P < 0.01). Western blot analysis results showed that the expression of IL-1ß in the RIRI + NAS group began to increase 6 h after RIRI, and it reached a high level 12 h after RIRI, and then decreased. And it was lower at each time point in the RIRI + NAS group than in the RIRI group, and there existed significant difference (all P < 0.01). Besides, the expression levels of TLR4, p-NF-κB, NLRP3, and cleaved Caspase-1 proteins in the RIRI + NAS and RIRI + TAK-242 + NAS groups decreased 12 h after RIRI compared with those in the RIRI group (all P < 0.01). The difference in IL-1ß+ cells was significantly correlated with those of TLR4+ and cleaved Caspase-1+ cells in the RIRI + NAS group (r2 = 0.9054 or 0.7431, P < 0.01). In conclusion, NAS could attenuate the expression of IL-1ß by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway, reduce the retina edema, and promote the survival of RGCs, thereby alleviating the retinal injury and exert its neuroprotective effect.

N-acetylserotonin Derivative Exerts a Neuroprotective Effect by Inhibiting the NLRP3 Inflammasome and Activating the PI3K/Akt/Nrf2 Pathway in the Model of Hypoxic-Ischemic Brain Damage.

Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the main causes of neonatal disability and death. As a derivative of N-acetylserotonin, N-[2-(5-hydroxy-1H-indol-3-yl) ethyl]-2-oxopiperidine-3-carboxamide (HIOC) can easily cross the blood-brain barrier and have a long half-life in the brain. In this study, the hypothesis was verified that HIOC plays a neuroprotective role in the HIE model and its potential mechanism was evaluated. Firstly, an HIE rat model was established to deliver HIOC, revealing that it can reduce cerebral infarction volume, cerebral edema, and neuronal apoptosis. The results of immunofluorescence staining, Western blots and RT-PCR further showed that HIOC could inhibit the activation of the NLRP3 inflammasome and the expression of related proteins. Finally, the activation of the phosphatidylinositol-3-kinase (PI3K)/Akt/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway by HIOC was verified in vitro and in vivo. It was discovered that HIOC could increase the nuclear translocation of Nrf2, and that this induction can be reversed by the PI3K/Akt pathway inhibitor LY294002. In general terms, the neuroprotective effect of HIOC was confirmed in the HIE model, which is related to the activation of the Pi3k/Akt/Nrf2 signal pathway and the inhibition of the NLRP3 inflammasome.

Melatonin synthesis genes N-acetylserotonin methyltransferases evolved into caffeic acid O-methyltransferases and both assisted in plant terrestrialization.

Terrestrialization is one of the most momentous events in the history of plant life, which leads to the subsequent evolution of plant diversity. The transition species, in this process, had to acquire a range of adaptive mechanisms to cope with the harsh features of terrestrial environments compared to that of aquatic habitat. As an ancient antioxidant, a leading regulator of ROS signaling or homeostasis, and a presumed plant master regulator, melatonin likely assisted plants transition to land and their adaption to terrestrial ecosystems. N-acetylserotonin methyltransferases (ASMT) and caffeic acid O-methyltransferases (COMT), both in the O-methyltransferase (OMT) family, catalyze the core O-methylation reaction in melatonin biosynthesis. How these two enzymes with close relevance evolved in plant evolutionary history and whether they participated in plant terrestrialization remains unknown. Using combined phylogenetic evidence and protein structure analysis, it is revealed that COMT likely evolved from ASMT by gene duplication and subsequent divergence. Newly emergent COMT gained a significantly higher ASMT activity to produce greater amounts of melatonin for immobile plants to acclimate to the stressful land environments after evolving from the more environmentally-stable aquatic conditions. The COMT genes possess more conserved substrate-binding sites at the amino acid level and more open protein conformation compared to ASMT, and getting a new function to catalyze the lignin biosynthesis. This development directly contributed to the dominance of vascular plants among the Earth's flora and prompted plant colonization of land. Thus, ASMT, together with its descendant COMT, might play key roles in plant transition to land. The current study provides new insights into plant terrestrialization with gene duplication contributing to this process along with well-known horizontal gene transfer.

A Cornflower Extract Containing N-Feruloylserotonin Reduces Inflammation in Human Skin by Neutralizing CCL17 and CCL22 and Inhibiting COX-2 and 5-LOX.

Thymus and Activation-Regulated Chemokine (TARC/CCL17) and Macrophage-Derived Chemokine (MDC/CCL22) are two key chemokines exerting their biological effect via binding and activating a common receptor CCR4, expressed at the surface of type 2 helper T (Th2) cells. By recruiting Th2 cells in the dermis, CCL17 and CCL22 promote the development of inflammation in atopic skin. The aim of this research was to develop a plant extract whose biological properties, when applied topically, could be beneficial for people with atopic-prone skin. The strategy which was followed consisted in identifying ligands able to neutralize the biological activity of CCL17 and CCL22. Thus, an in silico molecular modeling and a generic screening assay were developed to screen natural molecules binding and blocking these two chemokines. N-Feruloylserotonin was identified as a neutraligand of CCL22 in these experiments. A cornflower extract containing N-feruloylserotonin was selected for further in vitro tests: the gene expression modulation of inflammation biomarkers induced by CCL17 or CCL22 in the presence or absence of this extract was assessed in the HaCaT keratinocyte cell line. Additionally, the same cornflower extract in another vehicle was evaluated in parallel with N-feruloylserotonin for cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymatic cellular inhibition. The cornflower extract was shown to neutralize the two chemokines in vitro, inhibited COX-2 and 5-LOX, and demonstrated anti-inflammatory activities due mainly to the presence of N-feruloylserotonin. Although these findings would need to be confirmed in an in vivo study, the in vitro studies lay the foundation to explain the benefits of the cornflower extract when applied topically to individuals with atopic-prone skin.

Synergistic neuroprotection by coffee components eicosanoyl-5-hydroxytryptamide and caffeine in models of Parkinson's disease and DLB.

Hyperphosphorylated α-synuclein in Lewy bodies and Lewy neurites is a characteristic neuropathological feature of Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). The catalytic subunit of the specific phosphatase, protein phosphatase 2A (PP2A) that dephosphorylates α-synuclein, is hypomethylated in these brains, thereby impeding the assembly of the active trimeric holoenzyme and reducing phosphatase activity. This phosphatase deficiency contributes to the accumulation of hyperphosphorylated α-synuclein, which tends to fibrillize more than unmodified α-synuclein. Eicosanoyl-5-hydroxytryptamide (EHT), a fatty acid derivative of serotonin found in coffee, inhibits the PP2A methylesterase so as to maintain PP2A in a highly active methylated state and mitigates the phenotype of α-synuclein transgenic (SynTg) mice. Considering epidemiologic and experimental evidence suggesting protective effects of caffeine in PD, we sought, in the present study, to test whether there is synergy between EHT and caffeine in models of α-synucleinopathy. Coadministration of these two compounds orally for 6 mo at doses that were individually ineffective in SynTg mice and in a striatal α-synuclein preformed fibril inoculation model resulted in reduced accumulation of phosphorylated α-synuclein, preserved neuronal integrity and function, diminished neuroinflammation, and improved behavioral performance. These indices were associated with increased levels of methylated PP2A in brain tissue. A similar profile of greater PP2A methylation and cytoprotection was found in SH-SY5Y cells cotreated with EHT and caffeine, but not with each compound alone. These findings suggest that these two components of coffee have synergistic effects in protecting the brain against α-synuclein-mediated toxicity through maintenance of PP2A in an active state.

Embryonic Development of Avian Pineal Secretory Activity-A Lesson from the Goose Pineal Organs in Superfusion Culture.

The embryonic ontogeny of pineal secretory activity in birds has been investigated almost exclusively in chickens. This study aimed to characterize this process in domestic geese. The pineal organs of embryos aged 18-28 days were incubated in superfusion culture under different light conditions for 4-5 days and treated with norepinephrine (NE). Melatonin (MLT) was measured by radioimmunoassay and other indoles by HPLC with fluorescence detection. Additionally, pineal organs were collected from embryos at 14-28 days of age and used to measure catecholamines by HPLC with electrochemical detection. MLT secretion increased with embryo age, most intensively between the 22nd and 24th days of life. The daily changes in MLT secretion under the 12 L:12D cycle occurred on the first day of culture, starting from an embryonic age of 24 days. MLT secretion was controlled by the light-dark cycle in all age groups studied. However, exposure to light during the scotophase did not alter the secretion of MLT. The endogenous oscillator expressed its activity in regulating MLT secretion in the pineal organs of embryos aged 24 days and older but could not generate a rhythm after one cycle. The rhythm of 5-hydroxytryptophan release during the first day of culture was found in the pineal organs of all embryos, while the rhythmic release of N-acetylserotonin and 5-methoxyindole acetic acid started at the age of 24 days. The proportion of released indoles changed with embryo age. NE caused a decrease in MLT secretion and provoked an increase in serotonin release. Incubation of the pineal organs induced the development of MLT secretory machinery and its diurnal rhythmicity. The pineal content of catecholamines increased prominently at the end of embryonic development.

Comparative analysis of metabolome of rice seeds at three developmental stages using a recombinant inbred line population.

Plants are considered an important food and nutrition source for humans. Despite advances in plant seed metabolomics, knowledge about the genetic and molecular bases of rice seed metabolomes at different developmental stages is still limited. Here, using Zhenshan 97 (ZS97) and Minghui 63 (MH63), we performed a widely targeted metabolic profiling in seeds during grain filling, mature seeds and germinating seeds. The diversity between MH63 and ZS97 was characterized in terms of the content of metabolites and the metabolic shifting across developmental stages. Taking advantage of the ultra-high-density genetic map of a population of 210 recombinant inbred lines (RILs) derived from a cross between ZS97 and MH63, we identified 4681 putative metabolic quantitative trait loci (mQTLs) in seeds across the three stages. Further analysis of the mQTLs for the codetected metabolites across the three stages revealed that the genetic regulation of metabolite accumulation was closely related to developmental stage. Using in silico analyses, we characterized 35 candidate genes responsible for 30 structurally identified or annotated compounds, among which LOC_Os07g04970 and LOC_Os06g03990 were identified to be responsible for feruloylserotonin and l-asparagine content variation across populations, respectively. Metabolite-agronomic trait association and colocation between mQTLs and phenotypic quantitative trait loci (pQTLs) revealed the complexity of the metabolite-agronomic trait relationship and the corresponding genetic basis.

Classical and non-classical melatonin receptor agonist-directed micellization of bipyridinium-based supramolecular amphiphiles in water.

The addition of molecular recognition units into structures of amphiphiles is a means by which soft matter capable of undergoing template-directed micellization can be obtained. These supramolecular amphiphiles can bind with molecular templates using non-covalent bonding interactions, forming host-guest complexes that hold the amphiphiles together as they undergo micellization. In most cases, such templates are synthesized and designed for a specific molecular recognition motif. It is not clear, however, to what extent these types of amphiphile systems are responsive to members of a biologically derived class of molecular targets, for example, melatonin receptor agonists and their numerous isosteres. Herein, we describe the template-directed micellization and arrangement at the air-water interface of a bipyridinium-based gemini surfactant, driven by the influence of donor-acceptor CT interactions with a series of bioactive classical and non-classical melatonin isosteres. Under the conditions of templation by either 5-methoxytryptophol, N-acetylserotonin, N-acetyltryptamine, or the pharmaceutical agent agomelatine, favorable Gibbs free energies of micellization were observed with decreases in CMC by up to 70%, and concomitant increases of 28% in surface pressure, and decreases of 20% in contact angle versus untemplated solutions. Solid state thermochromic transition temperatures for inkjet-printed patterns of the templated amphiphile solutions were inversely correlated with trends observed for their respective CMCs, and exhibited no correlation to their binding constants. These findings contend for the generalizable use of melatonin receptor agonists as targets and/or templates for chemical systems, which rely on π-stacking donor-acceptor CT interactions in water to facilitate the actions of binding, sequestration, or template-directed self-assembly.

Characterization of serotonin and N-acetylserotonin systems in the human epidermis and skin cells.

Tryptophan hydroxylase (TPH) activity was detected in cultured epidermal melanocytes and dermal fibroblasts with respective Km of 5.08 and 2.83 mM and Vmax of 80.5 and 108.0 µmol/min. Low but detectable TPH activity was also seen in cultured epidermal keratinocytes. Serotonin and/or its metabolite and precursor to melatonin, N-acetylserotonin (NAS), were identified by LC/MS in human epidermis and serum. Endogenous epidermal levels were 113.18 ± 13.34 and 43.41 ± 12.45 ng/mg protein for serotonin (n = 8/8) and NAS (n = 10/13), respectively. Their production was independent of race, gender, and age. NAS was also detected in human serum (n = 13/13) at a concentration 2.44 ± 0.45 ng/mL, while corresponding serotonin levels were 295.33 ± 17.17 ng/mL (n = 13/13). While there were no differences in serum serotonin levels, serum NAS levels were slightly higher in females. Immunocytochemistry studies showed localization of serotonin to epidermal and follicular keratinocytes, eccrine glands, mast cells, and dermal fibrocytes. Endogenous production of serotonin in cultured melanocytes, keratinocytes, and dermal fibroblasts was modulated by UVB. In conclusion, serotonin and NAS are produced endogenously in the epidermal, dermal, and adnexal compartments of human skin and in cultured skin cells. NAS is also detectable in human serum. Both serotonin and NAS inhibited melanogenesis in human melanotic melanoma at concentrations of 10-4 -10-3  M. They also inhibited growth of melanocytes. Melanoma cells were resistant to NAS inhibition, while serotonin inhibited cell growth only at 10-3  M. In summary, we characterized a serotonin-NAS system in human skin that is a part of local neuroendocrine system regulating skin homeostasis.

Two indoleamines are secreted from rat pineal gland at night and act on melatonin receptors but are not night hormones.

INTRODUCTION: At night, the pineal gland produces the indoleamines, melatonin, N-acetylserotonin (NAS), and N-acetyltryptamine (NAT). Melatonin is accepted as a hormone of night. Could NAS and NAT serve that role too? METHODS: Concentration-response measurements with overexpressed human melatonin receptors MT1 and MT2 ; mass spectrometry analysis of norepinephrine-stimulated secretions from isolated rat pineal glands; analysis of 24-hour periodic samples of rat blood. RESULTS: We show that NAT and NAS do activate melatonin receptors MT1 and MT2 , although with lower potency than melatonin, and that in vitro, melatonin and NAS are secreted from stimulated, isolated pineal glands in roughly equimolar amounts, but secretion of NAT was much less. All three were found at roughly equal concentrations in blood during the night. However, during the day, serum melatonin fell to very low values creating a high-amplitude circadian rhythm that was absent after pinealectomy, whereas NAS and NAT showed only small or no circadian variation. CONCLUSION: Blood levels of NAS and NAT were insufficient to activate peripheral melatonin receptors, and they were invariant, so they could not serve as circulating hormones of night. However, they could instead act in paracrine circadian fashion near the pineal gland or via other higher-affinity receptors.

AmOctα2R: Functional Characterization of a Honeybee Octopamine Receptor Inhibiting Adenylyl Cyclase Activity.

The catecholamines norepinephrine and epinephrine are important regulators of vertebrate physiology. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they use the phenolamines tyramine and octopamine for similar physiological functions. These biogenic amines activate specific members of the large protein family of G protein-coupled receptors (GPCRs). Based on molecular and pharmacological data, insect octopamine receptors were classified as either α- or ß-adrenergic-like octopamine receptors. Currently, one α- and four ß-receptors have been molecularly and pharmacologically characterized in the honeybee. Recently, an α2-adrenergic-like octopamine receptor was identified in Drosophila melanogaster (DmOctα2R). This receptor is activated by octopamine and other biogenic amines and causes a decrease in intracellular cAMP ([cAMP]i). Here, we show that the orthologous receptor of the honeybee (AmOctα2R), phylogenetically groups in a clade closely related to human α2-adrenergic receptors. When heterologously expressed in an eukaryotic cell line, AmOctα2R causes a decrease in [cAMP]i. The receptor displays a pronounced preference for octopamine over tyramine. In contrast to DmOctα2R, the honeybee receptor is not activated by serotonin. Its activity can be blocked efficiently by 5-carboxamidotryptamine and phentolamine. The functional characterization of AmOctα2R now adds a sixth member to this subfamily of monoaminergic receptors in the honeybee and is an important step towards understanding the actions of octopamine in honeybee behavior and physiology.