Effects of acute hypothyroidism on plasma melatonin and Aanat and Asmt expression in the pineal gland and gonads of rats.

Introduction: The reproductive system is tightly regulated by environmental and physiological signals. Melatonin, known as the hormone of darkness, plays a crucial role in regulating both the circadian and reproductive systems in mammals. Hypothyroidism is a key endocrine disorder that harms the reproductive system. Despite many studies on melatonin's effects on the reproductive system, there is conflicting information regarding melatonin synthesis modulation in hypothyroidism. The objective of this study was to investigate the modulation of plasma melatonin levels and gene expression of Aanat and Asmt in the pineal gland and gonads of rats with hypothyroidism at different times of the day. Methods: Female and male Wistar rats were divided into control and hypothyroid groups. Hypothyroidism was induced using propylthiouracil (PTU) for 15 days, rats were euthanized six hours after lights on (ZT6), before lights off (ZT11.5), and six hours after lights off (ZT18). Free thyroxine (FT4) and melatonin were quantified in plasma, and gene expressions of melatonin synthesizing enzymes (Aanat and Asmt) were measured in pineal and sexual organs (testis and ovary). Also, morphological analysis was performed in sexual organs. Results: The results reveal some disparities between the sexes. Hypothyroidism reduced antral and primary follicles in the ovary, and reduced the weight of testis, epididymis, and prostate. In relation to gene expression, we observed a reduction in Aanat expression in the pineal gland during the light phase (ZT6), and in males, this reduction occurred during the dark phase (ZT18). Regarding Asmt expression, there was a decrease in females also during the dark phase (ZT18). In the gonads, there was an increase in expression in both sexes at ZT11.5. Additionally, it was interesting to observe the association between FT4 levels and Asmt expression in the gonads. Conclusions: This study showed that acute hypothyroidism can affect components of the melatonergic system in gonads, particularly gene expression of melatonin synthesis enzymes (Aanat and Asmt) contributing to changes in reproduction organs during disease progression. These findings enhance our understanding of melatonin synthesis in the reproductive system during hypothyroidism, showing distinct responses in male and female rats, and suggest that hypothyroidism affects the circadian rhythmicity of melatonin synthesis in a sex-dependent manner.

AANAT1 regulates insect midgut detoxification through the ROS/CncC pathway.

Insecticide resistance has been a problem in both the agricultural pests and vectors. Revealing the detoxification mechanisms may help to better manage insect pests. Here, we showed that arylalkylamine N-acetyltransferase 1 (AANAT1) regulates intestinal detoxification process through modulation of reactive oxygen species (ROS)-activated transcription factors cap"n"collar isoform-C (CncC): muscle aponeurosis fibromatosis (Maf) pathway in both the oriental fruit fly, Bactrocera dorsalis, and the arbovirus vector, Aedes aegypti. Knockout/knockdown of AANAT1 led to accumulation of biogenic amines, which induced a decreased in the gut ROS level. The reduced midgut ROS levels resulted in decreased expression of CncC and Maf, leading to lower expression level of detoxification genes. AANAT1 knockout/knockdown insects were more susceptible to insecticide treatments. Our study reveals that normal functionality of AANAT1 is important for the regulation of gut detoxification pathways, providing insights into the mechanism underlying the gut defense against xenobiotics in metazoans.

Inhibition of hippocampal melatonin synthesis by siRNA induced learning and memory deficits in male rats.

Melatonin, the multi-functional neurohormone, is synthesized in the extra-pineal tissues such as the hippocampus. The key enzyme in hippocampal melatonin synthesis is arylalkylamine-N-acetyltransferase (AANAT). The importance of melatonin synthesis in the hippocampus has not yet been determined. We investigated hippocampal AANAT role in cognitive function using gene silencing small interference RNA (siRNA) technology. The hippocampal local melatonin synthesis was inhibited by AANAT-siRNA injection. The time-gene silencing profile of AANAT-siRNA was obtained by RT-PCR technique. The cytotoxicity of siRNA dose was determined by MTT assay on the B65 neural cells. Animals received the selected dosage of AANAT-siRNA. Then, the spatial working memory (Y maze), object recognition memory and spatial reference memory (Morris's water maze, MWM) were evaluated. The anxiety-like behaviors were evaluated by the elevated plus maze. After one week, following the probe test of MWM, the rats were sacrificed for histological analysis. The hippocampal melatonin levels were measured using the liquid chromatography-mass spectrometry technique. The hippocampal melatonin levels in the AANAT-siRNA group decreased. Animals receiving the AANAT-siRNA showed deficits in spatial learning and working memory which were verified by increased escape latency and reduced spontaneous alternations, respectively. There was an increase in anxiety-like behaviors as well as a deficit in recognition memory in the AANAT-siRNA group. The Nissl staining and immunohistochemistry of activated caspase-3 showed the neuronal loss and cell apoptosis in hippocampal tissue of the AANAT-siRNA group. The 18F-FDG-PET imaging displayed lower glucose metabolism following the reduction in AANAT mRNA. Data suggest that the AANAT mRNA and hippocampal melatonin synthesis might be an essential factor for learning, memory and some aspects of cognition, as well as homeostasis of hippocampal cells.

Two pathways regulate insulin-like growth factor genes in the brain and liver of the tropical damselfish Chrysiptera cyanea: A possible role for melatonin in the actions of growth and thyroid hormones.

External and internal factors are involved in controlling the growth of fishes. However, little is known about the mechanisms by which external factors trigger stimulus signals. This study explored the physiological roles of melatonin in the transcription of growth-related genes in the brain and liver of Chrysiptera cyanea, a tropical damselfish with long-day preference. In brain samples of this species collected at 4-h intervals, the transcript levels of arylalkylamine N-acetyltransferase2 (aanat2), the rate-limiting enzyme of melatonin synthesis, and growth hormone (gh) peaked at 20:00 and 00:00, respectively. Concomitantly, the transcript levels of insulin-like growth factors (igf1 and igf2) in the brain and liver were upregulated during the scotophase. Levels of iodothyronine deiodinases (dio2 and dio3), enzymes that convert thyroxine (T4) to triiodothyronine (T3) and reverse T3, respectively, increased in the brain (dio2 and dio3) and liver (dio2) during the photophase, whereas dio3 levels in the liver showed the opposite trend. Fish reared in melatonin-containing water exhibited significant increases in the transcription levels of gh and igf1 in the brain and igf1 in the liver, suggesting that growth in this fish is positively regulated by the GH/IGF pathway on a daily basis. Melatonin treatment also stimulated the transcript levels of dio2 and dio3 in the liver, but not in the brain. Fish consuming pellets containing T3, but not T4, showed significant increases in gh and igf1 in the brain and igf1 and igf2 in the liver, suggesting that the intercellular actions of the TH/IGF pathway have an impact on growth on a daily basis. In summary, IGF synthesis and action in the brain and liver undergo dual regulation by distinct hormone networks, which may also be affected by daily, seasonal, or nutritional factors.

Melatonin-Regulated Chaperone Binding Protein Plays a Key Role in Cadmium Stress Tolerance in Rice, Revealed by the Functional Characterization of a Novel Serotonin N-Acetyltransferase 3 (SNAT3) in Rice.

The study of the mechanisms by which melatonin protects against cadmium (Cd) toxicity in plants is still in its infancy, particularly at the molecular level. In this study, the gene encoding a novel serotonin N-acetyltransferase 3 (SNAT3) in rice, a pivotal enzyme in the melatonin biosynthetic pathway, was cloned. Rice (Oryza sativa) OsSNAT3 is the first identified plant ortholog of archaeon Thermoplasma volcanium SNAT. The purified recombinant OsSNAT3 catalyzed the conversion of serotonin and 5-methoxytryptamine to N-acetylserotonin and melatonin, respectively. The suppression of OsSNAT3 by RNAi led to a decline in endogenous melatonin levels followed by a reduction in Cd tolerance in transgenic RNAi rice lines. In addition, the expression levels of genes encoding the endoplasmic reticulum (ER) chaperones BiP3, BiP4, and BiP5 were much lower in RNAi lines than in the wild type. In transgenic rice plants overexpressing OsSNAT3 (SNAT3-OE), however, melatonin levels were higher than in wild-type plants. SNAT3-OE plants also tolerated Cd stress, as indicated by seedling growth, malondialdehyde, and chlorophyll levels. BiP4 expression was much higher in the SNAT3-OE lines than in the wild type. These results indicate that melatonin engineering could help crops withstand Cd stress, resulting in high yields in Cd-contaminated fields.

Variation in the AA-NAT gene G203A is associated with Awassi and Hamdani sheep fertility.

Arylalkylamine-N-acetyl-transferase (AA-NAT) is one of several genes that influence sheep reproduction. Thus, the objective of this study was to investigate whether genetic variability within the AA-NAT gene influenced the reproductive performance of Awassi and Hamdani ewes. A total of 99 twin and 101 single-progeny ewes were analyzed for genomic DNA. Polymerase chain reaction (PCR) was used to produce amplicons of 300, 313, and 287 bp from exons 1, 2, and 3 of the AA-NAT gene. A 300-bp amplicon was genotyped, resulting in two genotypes: GG and GA. Through sequence analysis, a mutation 203 G > A was identified in the GA genotype. The statistical analysis revealed a strong correlation between the single nucleotide polymorphism (SNP) 203 G > A and reproductive performance. Ewes carrying this mutation showed significantly increased litter sizes, twinning rates, lambing rates, and fewer days to lambing compared to those carrying GG. These findings demonstrate that the presence of the 203 G > A SNP variant has a significant positive impact on litter sizes and enhances the fertility of Awassi and Hamdani sheep.

Ovarian overexpression of ASMT gene increases follicle numbers in transgenic sheep: Association with lipid metabolism.

Melatonin plays an important role in mammalian reproductive activities, to further understand the effects of endogenous melatonin on functions of ovary, the transgenic sheep with overexpression of melatonin synthetic enzyme gene ASMT in ovary were generated. The results showed that total melatonin content in follicular fluid of transgenic sheep was significantly greater than that in the wild type. Accordingly, the follicle numbers of transgenic sheep were also significantly greater than those in the WT. The results of follicular fluid metabolites sequencing showed that compared with WT, the differential metabolites of the transgenic sheep were significantly enriched in several signaling pathways, the largest number of metabolites was lipid metabolism pathway and the main differential metabolites were lipids and lipoid molecules. SMART-seq2 were used to analyze the oocytes and granulosa cells of transgenic sheep and WT sheep. The main differential enrichment pathway was metabolic pathway, in which lipid metabolism genes accounted for the majority. In conclusion, this is the first report to show that ovary overexpression of ASMT increased local melatonin production and follicle numbers. These results may imply that ASMT plays an important role in follicle development and formation, and melatonin intervention may be a potential method to promote this process.

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.

The cutaneous stress response system in three-spined stickleback and European flounder exposed to oxidative stress: Different mode of action.

In fish, the skin is directly exposed to multiple environmental stressors and provides the first line of defense against harmful external factors. It turned out that cortisol and melatonin (Mel) are involved in fish cutaneous stress response system (CSRS) similar to mammalian. This study investigates the mode of action of CSRS in two teleost species of different biology and skin characteristics, the three-spined stickleback and the European flounder, after exposure to oxidative stress induced by a potassium dichromate solution. The cutaneous stress response system presents different ways of action in two studied species: Mel concentration increases in the skin of both species, but cortisol concentration increases in the skin only in sticklebacks. Data suggest that stickleback skin cells can produce cortisol. However, cortisol is not involved in the response to oxidative stress in flounders. In stickleback skin, two genes encoding AANAT and ASMT/HIOMT (enzymes involved in Mel synthesis), aanat1a and asmt2, are expressed, but in flounder skin, only one, asmtl. Because gene expression does not change in stickleback skin after exposure to stress, the source of increased Mel is probably outside the skin. A lack of expression of the gene encoding AANAT in flounder skin strongly suggests that Mel is transported to the skin by the bloodstream from other sites of synthesis. Pigment dispersion in the skin after exposure to oxidative stress is found only in sticklebacks.

Escherichia coli RimI Encodes Serotonin N-Acetyltransferase Activity and Its Overexpression Leads to Enhanced Growth and Melatonin Biosynthesis.

Serotonin N-acetyltransferase (SNAT) functions as the penultimate or final enzyme in melatonin biosynthesis, depending on the substrate. The Escherichia coli orthologue of archaeal SNAT from Thermoplasma volcanium was identified as RimI (EcRimI), with 42% amino acid similarity to archaeal SNAT. EcRimI has been reported to be an N-acetyltransferase enzyme. Here, we investigated whether EcRimI also exhibits SNAT enzyme activity. To achieve this goal, we purified recombinant EcRimI and examined its SNAT enzyme kinetics. As expected, EcRimI showed SNAT activity toward various amine substrates including serotonin and 5-methoxytryptamine, with Km and Vmax values of 531 µM and 528 pmol/min/mg protein toward serotonin and 201 µM and 587 pmol/min/mg protein toward 5-methoxytryptamine, respectively. In contrast to the rimI mutant E. coli strain that showed no growth defect, the EcRimI overexpression strain exhibited a 2-fold higher growth rate than the control strain after 24 h incubation in nutrient-rich medium. The EcRimI overexpression strain produced more melatonin than the control strain in the presence of 5-methoxytryptamine. The enhanced growth effect of EcRimI overexpression was also observed under cadmium stress. The higher growth rate associated with EcRimI expression was attributed to increased protein N-acetyltransferase activity, increased synthesis of melatonin, or the combined effects of both.

The melatonin system is expressed in the ovine uterus: effect of the day of the oestrous cycle and undernutrition.

CONTEXT: Melatonin influences female reproduction, but expression of the melatonin system has not been characterised in the ovine uterus. AIMS: We aimed to determine whether synthesising enzymes (arylalkylamine N-acetyltransferase (AANAT) and N-acetylserotonin-O-methyltransferase (ASMT)), melatonin receptors 1 and 2 (MT1 and MT2), and catabolising enzymes (myeloperoxidase (MPO) and indoleamine 2,3-dioxygenase 1 and 2 (IDO1 and 2)), are expressed in the ovine uterus, and if they are influenced by the oestrous cycle (Experiment 1) or by undernutrition (Experiment 2). METHODS: In Experiment 1, gene and protein expression was determined in sheep endometrium samples collected on days 0 (oestrus), 5, 10 and 14 of the oestrous cycle. In Experiment 2, we studied uterine samples from ewes fed either 1.5 or 0.5times their maintenance requirements. KEY RESULTS: We have demonstrated the expression of AANAT and ASMT in the endometrium of sheep. AANAT and ASMT transcripts, and AANAT protein were more elevated at day 10, then decreased to day 14. A similar pattern was observed for MT2 , IDO1 , and MPO mRNA, which suggests that the endometrial melatonin system might be influenced by ovarian steroid hormones. Undernutrition increased AANAT mRNA expression, but seemed to decrease its protein expression, and increased MT2 and IDO2 transcripts, whereas ASMT expression was unaffected. CONCLUSIONS: The melatonin system is expressed in the ovine uterus and is affected by oestrous cycle and undernutrition. IMPLICATIONS: The results help explain the adverse effects of undernutrition on reproduction in sheep, and the success of exogenous melatonin treatments in improving reproductive outcomes.

RP58 knockdown contributes to hypoxia-ischemia-induced pineal dysfunction and circadian rhythm disruption in neonatal rats.

Hypoxia-ischemia (HI) of the brain not only impairs neurodevelopment but also causes pineal gland dysfunction, which leads to circadian rhythm disruption. However, the underlying mechanism of circadian rhythm disruption associated with HI-induced pineal dysfunction remains unknown. The zinc finger protein repressor protein with a predicted molecular mass of 58 kDa (RP58) is involved in the development and differentiation of nerve cells. In this study, we established an HI model in neonatal rats to investigate the expression of RP58 and its role in pineal dysfunction and circadian rhythm disruption induced by HI. We demonstrated that RP58 was highly expressed in the pineal gland under normal conditions and significantly downregulated in the pineal gland and primary pinealocytes following HI. Knockdown of RP58 decreased the expression of enzymes in the melatonin (Mel) synthesis pathway (tryptophan hydroxylase 1 [TPH1], acetylserotonin O-methyltransferase [ASMT], and arylalkylamine N-acetyltransferase [AANAT]) and clock genes (circadian locomotor output cycles kaput [CLOCK] and brain and muscle ARNT-like 1 [BMAL1]), and it also reduced the production of Mel, caused pineal cell injury, and disrupted circadian rhythms in vivo and in vitro. Similarly, HI reduced the expression of Mel synthesis enzymes (TPH1, ASMT, and AANAT) and clock genes (CLOCK and BMAL1), and caused pineal injury and circadian rhythm disruption, which were exacerbated by RP58 knockdown. The detrimental effect of RP58 knockdown on pineal dysfunction and circadian rhythm disruption was reversed by the addition of exogenous Mel. Furthermore, exogenous Mel reversed HI-induced pineal dysfunction and circadian rhythm disruption, as reflected by improvements in Mel production, voluntary activity periods, and activity frequency, as well as a diminished decrease in the expression of Mel synthesis enzymes and clock genes. The present study suggests that RP58 is an endogenous source of protection against pineal dysfunction and circadian rhythm disruption after neonatal HI.

The Uterine Melatonergic Systems of AANAT and Melatonin Membrane Receptor 2 (MT2) Are Essential for Endometrial Receptivity and Early Implantation in Mice.

In the current study, using Aanat and Mt2 KO mice, we observed that the preservation of the melatonergic system is essential for successful early pregnancy in mice. We identified that aralkylamine N-acetyltransferase (AANAT), melatonin receptor 1A (MT1), and melatonin receptor 1B (MT2) were all expressed in the uterus. Due to the relatively weak expression of MT1 compared to AANAT and MT2, this study focused on AANAT and MT2. Aanat and Mt2 KO significantly reduced the early implantation sites and the abnormal morphology of the endometrium of the uterus. Mechanistical analysis indicated that the melatonergic system is the key player in the induction of the normal nidatory estrogen (E2) response for endometrial receptivity and functions by activating the STAT signaling pathway. Its deficiency impaired the interactions between the endometrium, the placenta, and the embryo. The reduction in melatonin production caused by Aanat KO and the impairment of signal transduction caused by Mt2 KO reduced the uterine MMP-2 and MMP-9 activity, resulting in a hyperproliferative endometrial epithelium. In addition, melatonergic system deficiency also increased the local immunoinflammatory reaction with elevated local proinflammatory cytokines leading to early abortion in the Mt2 KO mice compared to the WT mice. We believe that the novel data obtained from the mice might apply to other animals including humans. Further investigation into the interaction between the melatonergic system and reproductive effects in different species would be worthwhile.

Arylalkylamine N-acetyltransferase (AANAT): Blue light induction, nuclear translocation, and potential role in the survival of chicken retina neuronal cells.

In vertebrates, arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) is the time-keeping and key regulatory enzyme in melatonin (Mel) biosynthesis. AANAT is present in the pineal gland, retina, and other regions where it is controlled by light, cyclic adenosine monophosphate (cAMP) levels, and the molecular clock. AANAT converts serotonin to N-acetyl serotonin (NAS) and the last enzyme in the pathway, hydroxy-o-methyltransferase (HIOMT), forms Mel by NAS methylation. We have previously shown that AANAT is expressed in chicken retinal ganglion cells (RGCs) during daytime at the level of mRNA and enzyme activity. Here we investigated the presence of AANAT protein and mRNA throughout development in the chicken embryonic retina as well as AANAT expression, phosphorylation, and its sub-cellular localization in primary cultures of retinal neurons from E10 embryonic retinas exposed to blue light (BL) and controls kept in the dark (D). From embryonic days 7-10 (E7-10) AANAT mRNA and protein were visualized mainly concentrated in the forming ganglion cell layer (GCL), while from E17 through postnatal days, expression was detectable all through the different retinal cell layers. At postnatal day 10 (PN10) when animals were subjected to a 12:12 h LD cycle, AANAT was mainly expressed in the GCL and inner nuclear layer cells at noon (Zeitgeber Time (ZT 6)) and in the photoreceptor cell layer at night (ZT 21). Primary cultures of retinal neurons exhibited an induction of AANAT protein when cells were exposed to BL for 1 h as compared with D controls. After BL exposure, AANAT showed a significant change in intracellular localization from the cytoplasm to the nucleus in the BL condition, remaining in the nucleus 1-2 h in the D after BL stimulation. BL induction of nuclear AANAT was substantially inhibited when cultures were treated with the protein synthesis inhibitor cycloheximide (CHD). Furthermore, the phosphorylated form of the enzyme (pAANAT) increased after BL in nuclear fractions obtained from primary cultures as compared with D controls. Finally, the knockdown of AANAT by sh-RNA in primary cultures affected cell viability regardless of the light condition. AANAT knockdown also affected the redox balance, sh-AANAT treated cultures showing higher levels of reactive oxygen species (ROS) than in the sh-control. Our results support the idea that AANAT is a BL-sensing enzyme in the inner retina of diurnal vertebrates, undergoing phosphorylation and nuclear importation in response to BL stimulation. Moreover, it can be inferred that AANAT plays a novel role in nuclear function, cell viability, and, likely, through redox balance regulation.

Molecular Cloning and Characterization of a Serotonin N-Acetyltransferase Gene, xoSNAT3, from Xanthomonas oryzae pv. oryzae.

Rice bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the top ten bacterial plant diseases worldwide. Serotonin N-acetyltransferase (SNAT) is one of the key rate-limiting enzymes in melatonin (MT) biosynthesis. However, its function in pathogenic bacteria remains unclear. In this study, a Xoo SNAT protein (xoSNAT3) that showed 27.39% homology with sheep SNAT was identified from a collection of 24 members of GCN5-related N-acetyltransferase (GNAT) superfamily in Xoo. This xoSNAT3 could be induced by MT. In tobacco-based transient expression system, xoSNAT3 was found localized on mitochondria. In vitro studies indicated that xoSNAT3 showed the optima enzymatic activity at 50 °C. The recombinant enzyme showed Km and Vmax values of 709.98 µM and 2.21 nmol/min/mg protein, respectively. Mutant △xoSNAT3 showed greater impaired MT biosynthesis than the wild-type strain. Additionally, △xoSNAT3 showed 14.06% less virulence and 26.07% less biofilm formation. Collectively, our results indicated that xoSNAT3 services as a SNAT involved in MT biosynthesis and pathogenicity in Xoo.

Genome-wide identification, characterization of Serotonin N-acetyltransferase and deciphering its importance under development, biotic and abiotic stress in soybean.

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme involved in plant melatonin biosynthesis. Identifying its expression under development and stress will reveal the regulatory role in the soybean. To identify and characterize SNAT, we employed genome-wide analysis, gene structure, cis-acting elements, expression, and enzyme activity. We identified seven putative genes by genome-wide analysis and found chloroplast signal peptides in three GmSNATs. To elucidate GmSNATs role, expression datasets of more than a hundred samples related to circadian rhythm, developmental stages, and stress conditions were analysed. Notably, the expression of GmSNAT1 did not show significant expression during biotic and abiotic stress. The GmSNAT1 sequence showed 67.8 and 72.2 % similarities with OsSNAT and AtSNAT, respectively. The Km and Vmax of the purified recombinant GmSNAT1 were 657 µM and 3780 pmol/min/mg, respectively. To further understand the GmSNAT1 role, we supplemented different concentrations of serotonin and melatonin to in-vitro cultures and seed priming. These studies revealed that the GmSNAT1 expression was significantly up-regulated at higher concentrations of serotonin and down-regulated at higher melatonin concentrations. We speculate that a high concentration of melatonin during abiotic, biotic stress, and in-vitro cultures are responsible for regulating GmSNAT1 expression, which may regulate them at the enzyme level during stress in soybean.

ß-Estradiol inhibits melatonin synthesis and melatonin receptor expression in sheep granulosa cells.

Melatonin, an important regulator of mammalian reproduction, is mainly produced in the pineal gland, and granulosa cells (GCs), the main mammalian ovarian secretory cells, synthesize melatonin and express melatonin receptors (MRs) MT1 and MT2. However, studies on melatonin regulation in GCs are lacking in sheep. In this study, we explored the effects of ß-estradiol (E2) on melatonin production and MR expression in GCs. We cultured sheep GCs to analyze the expression of the melatonin rate-limiting enzymes AANAT and HIOMT and the effects of E2 on AANAT, HIOMT, and MR expression and melatonin synthesis. To determine whether estrogen receptors (ERs) mediated E2 action on melatonin secretion and MR expression, we assessed ERA and ERB expression in GCs and observed whether ER antagonists counterbalanced the effects of E2. GCs expressed AANAT and HIOMT mRNA, indicating that they transformed exogenous serotonin into melatonin. E2 inhibited melatonin production by downregulating AANAT, HIOMT, and MRs. GCs expressed ERA and ERB; ERA/ERB inhibitors abolished E2-mediated inhibition of melatonin secretion and MR expression. PHTPP upregulated melatonin secretion and MT1 expression in E2-treated GCs, but did not significantly affect AANAT and MT2 expression. In conclusion, melatonin secretion in GCs was inhibited by E2 through an ERA- and ERB-mediated process.

Downregulation of AANAT by c-Fos in tubular epithelial cells with membranous nephropathy.

Melatonin is a hormone majorly secreted by the pineal gland and contributes to a various type of physiological functions in mammals. The melatonin production is tightly limited to the AANAT level, yet the most known molecular mechanisms underlying AANAT gene transcription is limited in the pinealocyte. Here, we find that c-Fos and cAMP-response element-binding protein (CREB) decreases and increases the AANAT transcriptional activity in renal tubular epithelial cell, respectively. Notably, c-Fos knockdown significantly upregulates melatonin levels in renal tubular cells. Functional results indicate that AANAT expression is decreased by c-Fos and resulted in enhancement of cell damage in albumin-injury cell model. We further find an inverse correlation between c-Fos and AANAT levels in renal tubular cells from experimental membranous nephropathy (MN) samples and clinical MN specimens. Our finding provides the molecular basis of c-Fos in transcriptionally downregulating expression of AANAT and melatonin, and elucidate the protective role of AANAT in preventing renal tubular cells death in albumin-injury cell model and MN progression.

Serotonin and Melatonin Biosynthesis in Plants: Genome-Wide Identification of the Genes and Their Expression Reveal a Conserved Role in Stress and Development.

Serotonin (Ser) and melatonin (Mel) serve as master regulators of plant growth and development by influencing diverse cellular processes. The enzymes namely, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H) catalyse the formation of Ser from tryptophan. Subsequently, serotonin N-acetyl transferase (SNAT) and acetyl-serotonin methyltransferase (ASMT) form Mel from Ser. Plant genomes harbour multiple genes for each of these four enzymes, all of which have not been identified. Therefore, to delineate information regarding these four gene families, we carried out a genome-wide analysis of the genes involved in Ser and Mel biosynthesis in Arabidopsis, tomato, rice and sorghum. Phylogenetic analysis unravelled distinct evolutionary relationships among these genes from different plants. Interestingly, no gene family except ASMTs showed monocot- or dicot-specific clustering of respective proteins. Further, we observed tissue-specific, developmental and stress/hormone-mediated variations in the expression of the four gene families. The light/dark cycle also affected their expression in agreement with our quantitative reverse transcriptase-PCR (qRT-PCR) analysis. Importantly, we found that miRNAs (miR6249a and miR-1846e) regulated the expression of Ser and Mel biosynthesis under light and stress by influencing the expression of OsTDC5 and OsASMT18, respectively. Thus, this study may provide opportunities for functional characterization of suitable target genes of the Ser and Mel pathway to decipher their exact roles in plant physiology.

[Association of polymorphic variants of DDC (AADC), AANAT and ASMT genes encoding enzymes for melatonin synthesis with the higher risk of neuropsychiatric disorders]. / Assotsiatsiya polimorfizmov genov DDC (AADC), AANAT i ASMT, kodiruyushchikh fermenty sinteza melatonina, s riskom razvitiya psikhonevrologicheskikh rasstroistv.

Melatonin is the most well-known regulator of the circadian rhythms of all living organisms and the main substrate synthesized at night. There are 4 stages in the synthesis of melatonin. This review focuses on the 2nd, 3rd, and 4th stages. The review is aimed at analyzing publications on molecular genetic association studies on the role of single nucleotide polymorphisms (SNPs) of the DDC (AADC), AANAT and ASMT genes encoding melatonin synthesis enzymes in the pathogenesis of socially significant neuropsychiatric disorders in humans. The authors analyzed the available full-text articles from several databases, as well as materials from electronic resources. Search depth was 15 years. The analysis of these studies over the past decade show the association of some SNPs of the studied genes with the risk of neuropsychiatric disorders such as delayed sleep phase disorder, attention deficit hyperactivity disorder, autism spectrum disorder, migraine, Parkinson's disease, depression, anxiety, bipolar-affective disorder, schizophrenia.