Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) is found in ovarian follicular fluid, and its concentration is closely related to follicular health status. <em>N</em>evertheless, the molecular mechanisms underlying melatonin function in follicles are uncertain. In this study, melatonin concentration was measured in porcine follicular fluid at different stages of health. The melatonin concentration decreased as the follicles underwent atresia, suggesting that melatonin may participate in the maintenance of follicular health. The molecular pathway through which melatonin may regulate follicular development was further investigated. The pro-apoptotic protein BimEL (Bcl-2-interacting mediator of cell death-Extra Long), a key protein controlling granulosa cell apoptosis during follicular atresia, was selected as the target molecule. BimEL was downregulated when porcine granulosa cells were cultured in medium containing 10-9 M melatonin and isolated cumulus oocyte complexes (COCs) or follicle stimulating hormone (FSH). Interestingly, ERK-mediated phosphorylation was a prerequisite for the melatonin-induced decline in BimEL, and melatonin only promoted the ubiquitination of phosphorylated BimEL, and did not affect the activities of the lysosome or the proteasome. Moreover, the melatonin-induced downregulation of BimEL was independent of its receptor and its antioxidant properties. In conclusion, melatonin may maintain follicular health by inducing BimEL ubiquitination to inhibit the apoptosis of granulosa cells.

Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) plays important roles in plant developmental growth, especially in root architecture. The similarity in both chemical structure and biosynthetic pathway suggests a potential linkage between melatonin and auxin signaling. However the molecular mechanism regulating this melatonin-mediated root architecture changes is not yet elucidated. In the present study, we re-analyzed previously conducted transcriptome data and identified 16 auxin-related genes whose expression patterns were altered by treatment with melatonin. Several of these genes encoding important auxin transporters or strongly affecting auxin transport were significantly down regulated. In wild type Arabidopsis, Melatonin inhibited both primary root growth and hypocotyl elongation, but enhanced lateral root development in a dose dependent manner. However, the lateral-root-promoting role of melatonin was abolished when each individual null mutant affecting auxin transport including pin<em>5</em>, wag1, tt4 and tt<em>5</em>, was examined. Furthermore, melatonin acts synergistically with auxin to promote lateral root development in wild type Arabidopsis, but such synergistic effects were absent in knockout mutants of individual auxin transport related genes examined. These results strongly suggest that melatonin enhances lateral root development through regulation of auxin distribution via modulation of auxin transport. A working model is proposed to explain how melatonin and auxin act together to promote lateral root development. The present study deepens our understanding of the relationship between melatonin and auxin signaling in plant species.

Salt stress is one of the most serious limiting factors in worldwide agricultural production, resulting in huge annual yield loss. Since 199<em>5</em>, melatonin (<i><em>N</em></i>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>)-an ancient multi-functional molecule in eukaryotes and prokaryotes-has been extensively validated as a regulator of plant growth and development, as well as various stress responses, especially its crucial role in plant salt tolerance. Salt stress and exogenous melatonin lead to an increase in endogenous melatonin levels, partly via the phyto-melatonin receptor CA<em>N</em>D2/PMTR1. Melatonin plays important roles, as a free radical scavenger and antioxidant, in the improvement of antioxidant systems under salt stress. These functions improve photosynthesis, ion homeostasis, and activate a series of downstream signals, such as hormones, nitric oxide (<em>N</em>O) and polyamine metabolism. Melatonin also regulates gene expression responses to salt stress. In this study, we review recent literature and summarize the regulatory roles and signaling networks involving melatonin in response to salt stress in plants. We also discuss genes and gene families involved in the melatonin-mediated salt stress tolerance.

Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) plays important roles in plant defenses against a variety of biotic and abiotic stresses, including UV-B stress. Molecular mechanisms underlying functions of melatonin in plant UV-B responses are poorly understood. Here we show that melatonin effect on molecular signaling pathways, physiological changes, and UV-B stress resistance in Arabidopsis. Both exogenous and endogenous melatonin affected expression of UV-B signal transduction pathway genes. Experiments using UV-B signaling component mutants cop1-4 and hy<em>5</em>-21<em>5</em> revealed that melatonin not only acts as an antioxidant to promote UV-B stress resistance, but also regulates expression of several key components of UV-B signaling pathway, including ubiquitin-degrading enzyme (COP1), transcription factors (HY<em>5</em>, HYH), and RUP1/2. Our findings indicate that melatonin delays and subsequently enhances expression of COP1, HY<em>5</em>, HYH, and RUP1/2, which act as central effectors in UV-B signaling pathway, thus regulating their effects on antioxidant systems to protect the plant from UV-B stress. This article is protected by copyright. All rights reserved.

Keywords: Arabidopsis thaliana; UV radiation; UV-B signaling pathway; abiotic stress; hormones; melatonin.

Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>), which is synthesized from tryptophan, is formed during alcoholic fermentation, though its role in yeast is unknown. This study employed Saccharomyces cerevisiae as an eukaryote model to evaluate the possible effects of melatonin supplementation on endogenous cellular defense systems by measuring its effects on various cellular targets. Cell viability, intracellular reduced and oxidized glutathione levels (GSH and GSSG, respectively), reactive oxygen species (ROS) production, and expression of genes related to antioxidant defense in yeast, such as the glutathione system, catalase, superoxide dismutase, glutaredoxin, and thioredoxin, were assessed. Melatonin alone decreased GSH, increased GSSG, and activated antioxidant defense system genes, which reached maximum levels in the stationary phase. These results indicate that melatonin supplementation enables cells to resist better the stress generated in the stationary phase. However, when cells were subjected to oxidative stress induced by H2O2, melatonin was able to partially mitigate cell damage by decreasing ROS accumulation and GSH and increasing GSSG; this was followed by enhanced cell viability after stress exposure, mostly when occurring in the early stationary phase. Additionally, under such conditions, most genes related to endogenous antioxidant defense continued to be up-regulated with melatonin supplementation. The findings demonstrate that melatonin can act as antioxidant in S. cerevisiae.

Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) is synthesized from tryptophan by Saccharomyces cerevisiae and non-conventional yeast species. Antioxidant properties have been suggested as a possible role of melatonin in a S. cerevisiae wine strain. However, the possible antioxidant melatonin effect on non-Saccharomyces species and other strains of S. cerevisiae must be evaluated. The aim of this study was to determine the antioxidant capacity of melatonin in eight S. cerevisiae strains and four non-conventional yeasts (Torulaspora delbrueckii, Metschnikowia pulcherrima, Starmerella bacillaris, and Hanseniaspora uvarum). Therefore, the ROS formation, lipid peroxidation, catalase activity, fatty acid composition, and peroxisome proliferation were investigated. The results showed that the presence of melatonin increases peroxisome accumulation and slightly increases the catalase activity. When cells grown in the presence of melatonin were exposed to oxidative stress induced by H2O2, lower ROS accumulation and lipid peroxidation were observed in all tested strains. Therefore, the increased catalase activity that was a consequence of oxidative stress was lower in the presence of melatonin. Moreover, the presence of MEL modulates cell FA composition, increasing oleic and palmitoleic acids and leading to higher UFA/SFA ratios, which have been previously related to a higher tolerance to H2O2. These findings demonstrate that melatonin can act as an antioxidant compound in both S. cerevisiae and non-Saccharomyces yeasts.

Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>, MLT) is a neuroendocrine hormone, which is primarily synthesized by the pineal gland in vertebrates. Melatonin is a remarkable molecule with diverse biological and physiological actions and is involved in the regulation of various important functions such as circadian rhythm, energy metabolism, the reproductive system, the cardiovascular system, and the neuropsychiatric system. It also plays a role in disease by having anti-neoplastic and anti-osteoarthritic effects among others. Recently, research has focused on the roles of melatonin in oxidative stress, lipid metabolism, and hepatic steatosis and its potential therapeutic roles.

<AbstractText>Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) in plants, regulates shoot and root growth and alleviates environmental stresses. Melatonin and the phyto-hormone auxin are tryptophan-derived compounds. However, it largely remains controversial as to whether melatonin and auxin act through similar or overlapping signalling and regulatory pathways.</AbstractText><AbstractText>Here, we have used a promoter-activation study to demonstrate that, unlike auxin (1-naphthalene acetic acid, <em>N</em>AA), melatonin neither induces Direct repeat <em>5</em> DR<em>5</em> expression in Arabidopsis thaliana roots under normal growth conditions nor suppresses the induction of Alternative oxidase 1a AOX1a in leaves upon Antimycin A treatment, both of which are the hallmarks of auxin action. Additionally, comparative global transcriptome analysis conducted on Arabidopsis treated with melatonin or <em>N</em>AA revealed differences in the number and types of differentially expressed genes. Auxin (4.<em>5</em> μM) altered the expression of a diverse and large number of genes whereas melatonin at <em>5</em> μM had no significant effect but melatonin at 100 μM had a modest effect on transcriptome compared to solvent-treated control. Interestingly, the prominent category of genes differentially expressed upon exposure to melatonin trended towards biotic stress defence pathways while downregulation of key genes related to photosynthesis was observed.</AbstractText><AbstractText>Together these findings indicate that though they are both indolic compounds, melatonin and auxin act through different pathways to alter gene expression in Arabidopsis thaliana. Furthermore, it appears that effects of melatonin enable Arabidopsis thaliana to prioritize biotic stress defence signalling rather than growth. These findings clear the current confusion in the literature regarding the relationship of melatonin and auxin and also have greater implications of utilizing melatonin for improved plant protection.</AbstractText>

Melatonin is a multifunctional biomolecule found in both animals and plants. In this review, the biosynthesis, levels, signaling, and possible roles of melatonin and its metabolites in plants is summarized. Tryptamine <em>5</em>-hydroxylase (T<em>5</em>H), which catalyzes the conversion of tryptamine into serotonin, has been proposed as a target to create a melatonin knockout mutant showing a lesion mimic phenotype in rice. Due to lower anabolic capacity for melatonin biosynthesis and higher catabolic capacity for melatonin metabolism, plants generally maintain low melatonin levels. Some plants, including Arabidopsis and tobacco, do not possess tryptophan decarboxylase (TDC), the first committed step enzyme required for melatonin biosynthesis. Major melatonin metabolites include cyclic 3-hydroxymelatonin (3-OHM) and 2-hydroxymelatonin (2-OHM). Other melatonin metabolites such as <em>N</em>¹ -<em>acetyl</em>-<em>N</em>² -formyl-<em>5</em>-methoxykynuramine (AFMK), <em>N</em>-<em>acetyl</em>-<em>5</em>-methoxykynuramine (AMK), and <em>5</em>-<em>methoxytryptamine</em> (<em>5</em>-MT) are also produced when melatonin is applied to rice. The signaling pathways of melatonin and its metabolites act via the mitogen-activated protein kinase (MAPK) cascade, possibly by Cand2 as a melatonin receptor, although this hypothesis remains controversial. Melatonin mediates many important functions in growth stimulation and stress tolerance due to its potent antioxidant activity and function in activating the MAPK cascade. The concentration distribution of melatonin metabolites appears to be species-specific because corresponding enzymes such as M2H, M3H, catalases, indoleamine 2,3-dioxygenase (IDO), and <em>N</em>-<em>acetyl</em>serotonin de<em>acetyl</em>ase (ASDAC) are differentially expressed among plant species and even among different tissues within species. Differential levels of melatonin and its metabolites can lead to differential physiological effects among plants when melatonin is either applied exogenously or overproduced through ectopic overexpression.

Keywords: MAPK pathway; melatonin; melatonin metabolites; metabolism.

Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) is implicated in physiologic changes related to light-dark cycles and has been recently found to display antioxidant properties. It is known that the reaction of melatonin with certain reactive oxygen and nitrogen species, such as hydrogen peroxide and singlet oxygen, produces <em>N</em>1-<em>acetyl</em>-<em>N</em>2-formyl-<em>5</em>-methoxykynuramine (AFMK). We report herein on the development of a new liquid chromatography/tandem mass spectrometry (LC/ESI/MS-MS) assay to quantitatively determine melatonin and AFMK. The stable isotopic internal standard of melatonin-D3 was synthesized by the reaction of <em>5</em>-<em>methoxytryptamine</em> with deuterated <em>acetyl</em> chloride (CD3COCl). Labeled AFMK (AFMK-D3) was obtained after photooxidation of melatonin-D3. The predominant ion [M + H]+ in the full scan mass spectra of melatonin, melatonin-D3, AFMK and AFMK-D3 were located, respectively, at m/z = 233, 236, 26<em>5</em> and 268. The collision-induced dissociation of the molecules revealed a predominant fragment at m/z = 174 for melatonin and melatonin-D3 (loss of the <em>N</em>-<em>acetyl</em> group), and at m/z = 178 for AFMK and AFMK-D3 (loss of both the <em>N</em>-<em>acetyl</em> and the <em>N</em>-formyl groups). The m/z transitions from 233 to 174 (melatonin), from 236 to 174 (melatonin-D3), from 26<em>5</em> to 178 (AFMK), and from 268 to 178 (AFMK-D3) were therefore chosen for the multiple reaction monitoring detection experiments, ensuring a high specificity and an accurate quantification of melatonin and AFMK in human plasma.

Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>), a pleotropic molecule with a wide distribution, has received considerable attention in recent years, mostly because of its various major effects on tissues or cells since it has both receptor-dependent and receptor-independent actions over a wide range of concentrations. These biological and physiological functions of melatonin include regulation of circadian rhythms by modulating the expression of core oscillator genes, scavenging the reactive oxygen species and reactive nitrogen species, modulating the immune system and inflammatory response, and exerting cytoprotective and antiapoptotic effects. Given the multiple critical roles of melatonin, dysregulation of its production or any disruption in signaling through its receptors may have contributed in the development of a wide range of disorders including type 2 diabetes, aging, immune-mediated diseases, hypertension, and cancer. Herein, we focus on the modulatory effects of melatonin on angiogenesis and its implications as a therapeutic strategy in cancer and related diseases.

A reliable, sensitive and rapid assay has been developed for determining the activity of hydroxyindole-O-methyltransferase (HIOMT; S-adenosyl-L-methionine:<em>N</em>-<em>acetyl</em>serotonin-O-methyltransferase; EC 2.1.1.4), which catalyzes the final step in the melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) biosynthetic pathway. This method is based on the separation and detection of melatonin formed enzymatically from <em>N</em>-<em>acetyl</em>serotonin and S-adenosyl-L-methionine, by high-performance liquid chromatography with fluorometric detection. The detection limit for melatonin formed per sample was as low as 1<em>5</em>0 fmol, indicating that the sensitivity of this assay was comparable to that of a radioisotopic assay. The assay was applied to the determination of HIOMT activity in rat pineal gland. The HIOMT activity obtained in this study was comparable with, or slightly lower than those reported previously using radioisotopic assays.

The effects of pinealectomy and exogenous melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) on serum leptin levels were investigated in rats. Exogenous administration of melatonin to intact rats resulted in significant decreases in serum leptin levels (P < 0.0<em>5</em>) compared to those of the intact control group. Serum leptin levels were significantly elevated in the pinealectomised rats in comparison to the sham-pinealectomised animals (P < 0.001) and were significantly suppressed by exogenous administration of melatonin compared to those of non-treated pinealectomised rats (P < 0.001). Hormone concentrations in the melatonin-treated pinealectomised group were found to be similar to those seen in the sham-pinealectomised group. These results suggest that pineal gland has an effect on leptin release.

The melato<em>n</em>i<em>n</em>-bi<em>n</em>di<em>n</em>g protei<em>n</em> i<em>n</em> chicke<em>n</em> brai<em>n</em> membra<em>n</em>es was characterized usi<em>n</em>g both [12<em>5</em>I]-2-iodomelato<em>n</em>i<em>n</em> a<em>n</em>d [3H]-melato<em>n</em>i<em>n</em> as radioliga<em>n</em>ds. Saturatio<em>n</em> studies co<em>n</em>ducted at 2<em>5</em> degrees C revealed a si<em>n</em>gle class of bi<em>n</em>di<em>n</em>g site with dissociatio<em>n</em> co<em>n</em>sta<em>n</em>ts of 24 +/- 4.8 pM (<em>n</em> = 7) a<em>n</em>d 12<em>5</em> +/- 21 pM (<em>n</em> = 6) for the iodi<em>n</em>ated a<em>n</em>d tritiated liga<em>n</em>ds, respectively. Calculatio<em>n</em> of the affi<em>n</em>ity co<em>n</em>sta<em>n</em>t usi<em>n</em>g data from ki<em>n</em>etic experime<em>n</em>ts gave values of 2.2 +/- 0.4 pM a<em>n</em>d 13<em>5</em> +/- 1<em>5</em> pM for the iodi<em>n</em>ated a<em>n</em>d tritiated liga<em>n</em>ds, respectively. Competitio<em>n</em> studies showed that the ra<em>n</em>k order of i<em>n</em>hibitio<em>n</em> of bi<em>n</em>di<em>n</em>g by melato<em>n</em>i<em>n</em> a<em>n</em>alogues was similar for both radioliga<em>n</em>ds (2-iodomelato<em>n</em>i<em>n</em>>> melato<em>n</em>i<em>n</em>>> 2,3-dihydromelato<em>n</em>i<em>n</em>>> <em>N</em>-<em>acetyl</em>-<em>5</em>-methoxyky<em>n</em>ure<em>n</em>ami<em>n</em>e>> <em>N</em>-<em>acetyl</em>seroto<em>n</em>i<em>n</em>>> <em>5</em>-<em>methoxytryptamine</em>). The calculatio<em>n</em> of Ki, which depe<em>n</em>ds upo<em>n</em> the affi<em>n</em>ity co<em>n</em>sta<em>n</em>t, was 22 +/- 4.9 pM a<em>n</em>d 129 +/- 21 pM for 2-iodomelato<em>n</em>i<em>n</em> a<em>n</em>d melato<em>n</em>i<em>n</em>, respectively, whe<em>n</em> the affi<em>n</em>ity co<em>n</em>sta<em>n</em>t derived from the [12<em>5</em>I]-2-iodomelato<em>n</em>i<em>n</em> saturatio<em>n</em> experime<em>n</em>ts was used, but 4.9 +/- 1.<em>5</em> pM a<em>n</em>d 33 +/- <em>5</em>.<em>5</em> pM whe<em>n</em> the ki<em>n</em>etically derived co<em>n</em>sta<em>n</em>t was used. Whe<em>n</em> [3H]-melato<em>n</em>i<em>n</em> was used, the Ki for melato<em>n</em>i<em>n</em> was 72 +/- 8 pM a<em>n</em>d 20 +/- 4.6 pM for 2-iodomelato<em>n</em>i<em>n</em> a<em>n</em>d melato<em>n</em>i<em>n</em>. Bi<em>n</em>di<em>n</em>g of [12<em>5</em>I]-2-iodomelato<em>n</em>i<em>n</em> to the membra<em>n</em>es was partially reversible at 2<em>5</em> degrees C i<em>n</em> co<em>n</em>trast to the complete reversibility of [3H]-melato<em>n</em>i<em>n</em>. Exami<em>n</em>atio<em>n</em> of the effects of temperature o<em>n</em> bi<em>n</em>di<em>n</em>g i<em>n</em>dicated that at 37 degrees C both associatio<em>n</em> a<em>n</em>d dissociatio<em>n</em> of both liga<em>n</em>ds were accelerated. Closer exami<em>n</em>atio<em>n</em> showed that at 37 degrees C there was a loss of approximately 40% of the [12<em>5</em>I]-2-iodomelato<em>n</em>i<em>n</em> bi<em>n</em>di<em>n</em>g sites a<em>n</em>d little i<em>n</em>flue<em>n</em>ce upo<em>n</em> the affi<em>n</em>ity of bi<em>n</em>di<em>n</em>g with time. By co<em>n</em>trast, whe<em>n</em> [3H]-melato<em>n</em>i<em>n</em> was used, the affi<em>n</em>ity decreased fourfold, with o<em>n</em>ly a slight cha<em>n</em>ge i<em>n</em> the <em>n</em>umber of sites. If membra<em>n</em>es were i<em>n</em>cubated at 37 degrees C a<em>n</em>d the<em>n</em> switched 2<em>5</em> degrees C, bi<em>n</em>di<em>n</em>g i<em>n</em>creased, emphasizi<em>n</em>g the fact that the bi<em>n</em>di<em>n</em>g sites were <em>n</em>ot destroyed. Whereas there appears to be little doubt that 2-iodomelato<em>n</em>i<em>n</em> is a biologically active melato<em>n</em>i<em>n</em> ago<em>n</em>ist, the bi<em>n</em>di<em>n</em>g of the radioactive form of this ago<em>n</em>ist to the putative melato<em>n</em>i<em>n</em> receptor bi<em>n</em>di<em>n</em>g site is quite differe<em>n</em>t from that of the e<em>n</em>doge<em>n</em>ous liga<em>n</em>d. This may have serious co<em>n</em>seque<em>n</em>ces i<em>n</em> studies where receptor co<em>n</em>te<em>n</em>t is determi<em>n</em>ed followi<em>n</em>g physiological or pharmacological i<em>n</em>terve<em>n</em>tio<em>n</em>s.

Both in vitro and in vivo observations have suggested that melatonin modulates malignant cell growth. The present studies aimed to characterize the interactions of melatonin with cultured murine B16 melanoma cells. Time- and temperature-dependent specific melatonin accumulation by B16 murine melanoma cells was observed. B16 cells possessed a high affinity binding site (KD = 1.4 nM) which exhibited structural specificity in its affinity for analogues of melatonin (melatonin>> 6-hydroxymelatonin = <em>N</em>-<em>acetyl</em>-<em>5</em>-hydroxytryptamine>> <em>5</em>-<em>methoxytryptamine</em>>>) <em>5</em>-hydroxytryptamine). Evidence for a lower affinity uptake system without structural specificity was also observed. <em>N</em>inety-five per cent of the specific cell-associated melatonin in B16 cells was present in the soluble subcellular fraction of lysed cells; more than 97% of the cell-associated radioactivity was authentic melatonin. When the solubilized cell extracts from the binding assay were analysed by gel filtration immediately, all of the bound counts eluted at the void volume. Continuous exposure to melatonin for 48-120 h did not affect B16 cell proliferation as determined by cell counts, 3-[4,<em>5</em>-dimethylthiazol-2-yl]-2,<em>5</em>-diphenyltetrazolium bromide assay or [3H]thymidine incorporation. After 8-h pulse exposures to melatonin daily for 3 days, a 1<em>5</em>% stimulation of B16 cell proliferation (p < 0.02) was observed at melatonin concentrations of 0.1 and 1 nM. The anti-oestrogen, tamoxifen, inhibited B16 cell growth and increased specific melatonin accumulation by B16 cells at 1 x 10(-6) M (p < 0.02). Cultured B16 murine melanoma cells possessed a specific, high affinity uptake system for melatonin which appeared to be altered by anti-oestrogen exposure.

Matrix metalloproteinases (MMPs) maintain the crucial role in physiological turnover of extracellular matrix (ECM) proteins in gastric tissues. However, a little is known about the relationship of MMPs with ECM degradation during gastric ulceration and ECM remodeling during healing. Our objective was to investigate the effect of melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em> <em>methoxytryptamine</em>) on the regulation of MMP-9 and MMP-2 activity during prevention of gastric ulcer. In the present study, biochemical and zymographic methods were used to analyze the mechanism of melatonin in indomethacin-induced gastric ulcer in a rat model. Our studies reveal that melatonin dose-dependently downregulates the expression and secretion of pro-MMP-9 which is induced (approximately 10-fold) during indomethacin-induced gastric ulceration. Furthermore, melatonin prevents gastric ulceration in a dose-dependent manner through upregulation (approximately two- to threefold) of both pro-MMP-2 and active MMP-2 at the level of induction as well as secretion. It also prevents gastric ulcers by blocking glutathione depletion and lipid peroxidation in cytosolic and microsomal fractions. The novel findings of this study are attributed to the attenuation of the pro-MMP-9 and increase of MMP-2 activity by pretreatment with melatonin. The finding defines one of the MMP-mediated pathways for melatonin's action in gastric ulcer.

The i<em>n</em>flue<em>n</em>ces of seroto<em>n</em>i<em>n</em> (<em>5</em>-hydroxytryptami<em>n</em>e) o<em>n</em> the actio<em>n</em> of melato<em>n</em>i<em>n</em> (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) i<em>n</em> MIH (maturatio<em>n</em> i<em>n</em>duci<em>n</em>g hormo<em>n</em>e)-i<em>n</em>duced meiotic resumptio<em>n</em> were evaluated i<em>n</em> the oocytes of carp Catla catla usi<em>n</em>g a<em>n</em> i<em>n</em> vitro model. Oocytes from gravid female carp were isolated a<em>n</em>d i<em>n</em>cubated separately i<em>n</em> Medium 199 co<em>n</em>tai<em>n</em>i<em>n</em>g either (a) o<em>n</em>ly melato<em>n</em>i<em>n</em> (MEL; 100 pg/mL), or (b) o<em>n</em>ly seroto<em>n</em>i<em>n</em> (SER; 100 pg/mL), or (c) o<em>n</em>ly MIH (1 microg/mL), or (d) MEL a<em>n</em>d MIH (e) or MEL (4 h before) a<em>n</em>d MIH, or (f) MEL a<em>n</em>d SER, (g) or SER a<em>n</em>d MIH, or (h) SER (4 h before) a<em>n</em>d MIH, or (i) luzi<em>n</em>dole (L-a<em>n</em>tago<em>n</em>ist of MEL receptors; 10 microM) a<em>n</em>d MEL, or (j) MEL, L a<em>n</em>d MIH, or (k) MEL (4 h before), L a<em>n</em>d MIH, or (l) metoclopramide hydrochloride (M-a<em>n</em>tago<em>n</em>ist of SER receptors; 10 microM) a<em>n</em>d SER, or (m) M, MEL, SER, or (<em>n</em>) M, SER a<em>n</em>d MIH, or (o) M, SER (4 h before) a<em>n</em>d MIH, or (p) M, MEL SER a<em>n</em>d MIH, or (q) MEL, L, SER a<em>n</em>d M, or (r) MEL, L, SER, M, a<em>n</em>d MIH, or (s) MEL, SER, L a<em>n</em>d MIH. Co<em>n</em>trol oocytes were i<em>n</em>cubated i<em>n</em> the medium alo<em>n</em>e. Oocytes were i<em>n</em>cubated for 4, or 8, or 12, or 16 h a<em>n</em>d effects were evaluated by co<em>n</em>sideri<em>n</em>g the rate (%) of germi<em>n</em>al vesicle breakdow<em>n</em> (GVBD). At the e<em>n</em>d of 16 h i<em>n</em>cubatio<em>n</em>, 93.24+/-1.<em>5</em>7% oocytes u<em>n</em>derwe<em>n</em>t GVBD followi<em>n</em>g i<em>n</em>cubatio<em>n</em> with o<em>n</em>ly MIH, while i<em>n</em>cubatio<em>n</em> with o<em>n</em>ly MEL or o<em>n</em>ly SER resulted i<em>n</em> 77.1<em>5</em>+/-1.91% or 14.42+/-0.43% GVBD respectively. I<em>n</em>teresti<em>n</em>gly, i<em>n</em>cubatio<em>n</em> with MEL 4 h prior to additio<em>n</em> of MIH i<em>n</em> the medium, led to a<em>n</em> accelerated rate of GVBD (92.<em>5</em>8+/-1.10% at 12 h). I<em>n</em> co<em>n</em>trast, SER, irrespective of its time of applicatio<em>n</em> i<em>n</em> relatio<em>n</em> to MIH, resulted i<em>n</em> a maximum of 64.<em>5</em>7+/-0.86% GVBD. While L was fou<em>n</em>d to reduce the stimulatory actio<em>n</em>s of melato<em>n</em>i<em>n</em>, M suppressed the i<em>n</em>hibitory actio<em>n</em>s of seroto<em>n</em>i<em>n</em>. I<em>n</em> each case, both electrophoretic a<em>n</em>d immu<em>n</em>oblot studies revealed that the rate of GVBD was associated with the rate of formatio<em>n</em> of maturatio<em>n</em> promoti<em>n</em>g factor (a complex of two protei<em>n</em>s: a regulatory compo<em>n</em>e<em>n</em>t--cycli<em>n</em> B a<em>n</em>d the catalytic compo<em>n</em>e<em>n</em>t--Cdk1 or cdc2). Collectively, the prese<em>n</em>t study reports for the first time that SER <em>n</em>ot o<em>n</em>ly i<em>n</em>hibits the i<em>n</em>depe<em>n</em>de<em>n</em>t actio<em>n</em>s of MIH, but also the actio<em>n</em>s of MEL o<em>n</em> the MIH-i<em>n</em>duced oocytes maturatio<em>n</em> i<em>n</em> carp.

Oxygen consumption is a necessity for all aerobic organisms, but oxygen is also a toxic molecule that leads to the generation of free radicals. The brain consumes a high percentage of the oxygen inhaled (18.<em>5</em>%), and it contains large amounts of unsaturated fatty acids, which makes it highly susceptible to lipid peroxidation. Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>), the main secretory product of the pineal gland, is a free radical scavenger that was found to protect against lipid peroxidation in many experimental models. Another compound found in the pineal gland is pinoline (6-methoxy-1,2,3,4-tetrahydro-beta-carboline). Pinoline is structurally related to melatonin. Evidence suggests that pinoline may have an antioxidant capacity similar to that of melatonin. In this study, the ability of pinoline to protect against H2O2-induced lipid peroxidation of different rat brain homogenates (frontal cortex, striatum, cerebellum, hippocampus, and hypothalamus) was investigated. The degree of lipid peroxidation was assessed by estimating the levels of thiobarbituric acid reactive substances, malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA). Pinoline's antioxidant capacity was compared with that of melatonin. Both melatonin and pinoline reduced the level of MDA and 4-HDA in a dose-dependent manner in all brain regions tested. To compare the antioxidant capacities, percent-inhibition curves were created, and the IC<em>5</em>0 values were calculated. The IC<em>5</em>0 values for melatonin were higher in all brain regions than were those for pinoline. The IC<em>5</em>0 values for melatonin in the five different brain regions ranged from 0.16 mM-0.66 mM, and for pinoline, they ranged from 0.04 mM-0.13 mM. The possibility of synergistic interactions between melatonin and pinoline were also determined using the method of Berenbaum. Little evidence for either synergistic, additive, or antagonistic interactions between melatonin and pinoline was found.

One of the products of nitrogen-derived free radicals, peroxynitrite (O<em>N</em>OO(-)), is formed by the reaction of superoxide anion (O(2)(*-)) with nitric oxide (<em>N</em>O). O<em>N</em>OO(-) can cause damage to proteins and D<em>N</em>A through nitration. In particular, proteins and their constituent amino acids have been proven to be extremely sensitive to O<em>N</em>OO(-). However, the lack of specific endogenous defense enzymes to protect against O<em>N</em>OO(-) has prompted many researchers to search for endogenous scavengers. We previously found <em>5</em>-hydroxytryptamine (HT), which is an indole derivative (ID), to be an efficient O<em>N</em>OO(-) scavenger. In the present study, the interaction of several other indoles was further investigated: tryptophan (TRP), <em>5</em>-hydroxyL-tryptophan (HLT), HT, <em>N</em>-<em>acetyl</em>-<em>5</em>-hydroxytryptamine (AHT), <em>5</em>-methoxyindole-3-acetate (MIA), <em>5</em>-<em>methoxytryptamine</em> (MT), and melatonin. The O<em>N</em>OO(-) scavenging activity of ID was assayed by measuring the formation of oxidized dihydrorhodamine-123 (DHR-123). The scavenging efficacy was expressed as the IC(<em>5</em>0), denoting the concentration of each indole required to cause <em>5</em>0% inhibition of DHR-123 formation. In a separate in vitro study, the protective effect of IDs against O<em>N</em>OO(-)-induced nitration of bovine serum albumin was investigated. <em>N</em>itration was quantified using an immunoassay with a monoclonal anti-nitrotyrosine antibody, and a horseradish peroxidase-conjugated anti-mouse secondary antibody from sheep. The results revealed that the inhibitory activities of indoles were as follows: HLT, IC(<em>5</em>0) = 0.73 microM; HT, IC(<em>5</em>0) = 1.03 microM; and AHT, IC(<em>5</em>0) = 0.98 microM), showing relatively strong activities against O<em>N</em>OO(-). Interestingly, TRP, MIA, MT, and melatonin were less effective. Regarding the protection of albumin by IDs, the data showed that the formation of O<em>N</em>OO(-) was inhibited in a dose-dependent manner. Further probing of the mode of the interaction of indoles revealed that the hydroxyl groups in IDs are required for the enhanced scavenging action. It was concluded that several indole derivatives with hydroxyl groups are effective scavengers against O<em>N</em>OO(-), and that the scavenging efficacy depends on the presence of hydroxyl groups located within the indole ring structure.

Since melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) decreases locomotor activity and rearing and increases grooming behavior in a similar manner as somatostatin (SRIF), we examined if melatonin could induce these changes through somatostatinergic neurotransmission in the rat frontoparietal cortex. Male Wistar rats (200-2<em>5</em>0 g) received a single injection of melatonin (2<em>5</em> microg/kg per day) subcutaneously (s.c.) and were sacrificed <em>5</em> hr later. Melatonin treatment increased the number of 12<em>5</em>I-Tyr11-SRIF receptors in frontoparietal cortical membranes without any changes in the dissociation constant (Kd). The capacity of SRIF to inhibit basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activity was increased in melatonin-treated rats as compared to the control animals. Melatonin administration also induced a lower AC activity, both under basal conditions and after stimulation of the enzyme via stimulatory guanine nucleotide-binding proteins (Gs), or directly with FK. Functional inhibitory guanine nucleotide-binding protein (Gi) activity was increased in frontoparietal cortical membranes from melatonin-treated rats when compared to controls. Western blot analyzes showed that melatonin administration did not alter the amount of the Gialpha1, or Gialpha3 subunits, but reduced Gialpha2 levels in frontoparietal cortical membranes. <em>N</em>o significant changes in SRIF-like immunoreactivity content and SRIF mR<em>N</em>A levels were detected in this brain area after melatonin treatment. Administration of the melatonin receptor antagonist luzindole (10 mg/kg, s.c.) 30 min before melatonin injection did not change the melatonin-induced effects on the SRIF receptor effector system. In conclusion, the present results show that acute melatonin administration increases the activity of the SRIF receptor effector system and decreases Gialpha2 levels in the rat frontoparietal cortex. In addition, the coupling of Gs to AC is disturbed by melatonin.

Melatonin(<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) is synthesized from tryptophan and is intensively secreted into the blood only in darkness (nighttime) by the pineal gland. Melatonin is not only the most reliable marker of internal circadian phase but also a potent sleep-promoting and circadian phase regulatory agent in humans. There is evidence that daytime administered melatonin is able to exhibit short-acting hypnagogic effect and phase-shifting of the circadian rhythms such that sleep timing and associated various physiological functions realign at a new desired phase. Under favor of these properties, melatonin and melatonin receptor agonists have been shown to be potent therapeutic agents for the treatment of circadian rhythm sleep disorders and some type of insomnia.

Activity of vertebrate <em>N</em>a+,K+-ATPase was inhibited by <em>5</em>-hydroxytryptamine. Inhibition was reversible, and activity could be restored by dilution of <em>5</em>-hydroxytryptamine (<em>5</em>HT). The ability of indole derivatives to inhibit depended on the presence of a net charge on the third atom of the indole C-3 side chain. Indoles with a net positive charge, such as <em>5</em>HT, were stronger inhibitors than those with a net negative charge. Derivatives with a net charge of zero were not inhibitors. The ability of indole derivatives to inhibit <em>N</em>a+, K+-ATPase activity decreased in the order <em>5</em>HT>> tryptamine>> tryptophanamide>> tryptophol>> <em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>>> indole-3-acetic acid. Trp did not inhibit either ATPase or p<em>N</em>PPase activity. Charged indole derivatives also inhibited the p<em>N</em>PPase activity of <em>N</em>a+,K+-ATPase. <em>5</em>HT and tryptophanamide were stronger inhibitors of p<em>N</em>PPase activity than indole-3-acetic acid. Binding of [3H]ouabain was inhibited by <em>5</em>HT and tryptophanamide. Trp did not inhibit [3H]ouabain binding. The near equilibrium level of [3H]-ouabain binding in the presence of ATP, Mg2+, and <em>N</em>a+ was decreased by <em>5</em>HT in the manner characteristic of a competitive inhibitor. Enzyme-bound [3H]ouabain could be displaced by <em>5</em>HT. <em>5</em>HT was a complex mixed inhibitor of K+ with interactions at two sites, Tris was a competitive inhibitor with interactions at three sites, and ouabain was a simple noncompetitive inhibitor. The data are explained through a model indole binding site containing oppositely charged residues.

Many physiologic changes related to light-dark cycles and antioxidant effects have been related to melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) and its metabolites, <em>N</em>1-<em>acetyl</em>-<em>N</em>2-formyl-<em>5</em>-methoxykynuramine (AFMK) and <em>N</em>1-<em>acetyl</em>-<em>5</em>-methoxykynuramine (AMK). In this review, we discuss some methodologies, in particular, those employing high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) assays to quantitatively determine melatonin, AFMK, and AMK. These approaches offer a highly specific and an accurate quantification of melatonin and its metabolites. These characteristics are essential to point out correctly the biological effects of these compounds in physiological and pathological conditions.

Both the presence of receptors for gonadal steroids in the pineal gland and in vitro observations of direct action of melatonin upon Leydig cells, inhibiting testosterone secretion, indicate a direct connection between pineal gland and gonadal function. In the present study, we used a transmission electron microscope to analyze the morphologic parameters of Leydig cells from adult Swiss outbred white mice treated with daily subcutaneous injections of 100 micrograms of melatonin (<em>N</em>-<em>acetyl</em>, <em>5</em>-<em>methoxytryptamine</em>), during 22 consecutive days, compared with sham-control animals which had only received the melatonin vehicle. The melatonin group of mice showed a decrease in nuclear volume and fractional nuclear volume; smooth and rough endoplasmic reticulum; mitochondria; and Golgi complex. Our data also showed an increase in cytoplasmic volume, fractional cytoplasmic volume, and lysosomes in these same animals. The results suggest that melatonin, directly or indirectly, alters the ultrastructure of mouse Leydig cells and possibly influences their secretory activity by inhibiting their capacity to secrete steroids.