Anti-melatonin monoclonal antibodies (MAbs) of high titer were prepared by coupling melatonin to bovine serum albumin with formaldehyde and by immunizing BALB/c mice with multifocal intradermal injections and by fusing high titer antibody producing spleen cells with myeloma cells of SP2/0 origin. Five MAbs were selected for further characterization as classes and subclasses. After four successive limiting dilutions, antibodies were produced by these five clones with high affinities ranging from 10(9) to 10(11)/m. These clones were found to be of the immunoglobulin Ig G1 and IgG(2b) subclass with kappa light chain. A systematic study of cross-reactions with seven compounds (indole, aromatic and imidazole derivatives) showed that the antibody had a high specificity for melatonin, low reactivity with 6-hydroxymelatonin and <em>N</em>-<em>acetyl</em>-<em>5</em>-hydroxytryptamine, and no detectable reactivity with tryptamine, l-tryptophan, <em>5</em>-<em>methoxytryptamine</em> and <em>N</em>-<em>acetyl</em>-L-tryptophan. The roles of the indole nucleus and the side chain in the determination of the antigenic properties of the molecule are discussed. One of the MAbs, 4C9D7, was used to establish a competitive enzyme-linked immunosorbent assay for the detection of melatonin in supernatant.

This study assessed the location of melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) and of a pinoline derivative (GWC22) [6-ethyl-1-(3-methoxyphenyl)-2-propyl-1,2,3,4-tetrahydro-beta-carboline], when present in lipid assemblies such as linoleate micelles, phosphatidylcholine liposomes or low density lipoproteins (LDL). The efficiency of radical scavenging by these compounds is highly dependent on their partitioning between the lipidic and aqueous phases. We determined the proportion of melatonin or GWC22 in the aqueous and lipid phases of each system (concentrations of the antioxidants ranging between 3 x 10(-<em>5</em>) and 10(-4) m) by assaying melatonin or GWC22 by HPLC/UV detection, or by fluorescence for melatonin in micelles. Our results show that melatonin and GWC22 were preferentially located in the aqueous phase of micelles (68.4% and <em>5</em>9.0%, respectively), whereas only 30.<em>5</em>% of melatonin and 39.0% of GWC22 were found in the lipid phase. By contrast, in phosphatidylcholine liposomes, both compounds were essentially present in the lipid phase (73.<em>5</em>% for melatonin and 79.1% for GWC22, versus 2<em>5</em>.9% and 19.<em>5</em>% in the aqueous phase, respectively). In the case of LDL, 99.9% of the melatonin added was found in the methanol/water extracting phase containing phospholipids, unesterified cholesterol and apolipoprotein B100. The partitioning of melatonin and GWC22 in linoleate micelles gave new insights on the marked protective effect of GWC22 towards radiation-induced lipid peroxidation and allowed us to determine more accurately the lower limit values of the reaction rate constants of the two molecules studied with lipid peroxyl radicals, i.e. k(LOO.+melatonin))>>or= 9.0 x 10(4)m(-1)s(-1) and k(LOO.+GWC22)>>or= 3.<em>5</em> x 10(<em>5</em>)m(-1)s(-1).

Determination of melatonin (MT) (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) and related indole compounds using standard capillary electrophoresis (CE) system with UV detection was investigated. Satisfactory separations of six analytes i.e. l-tryptophan (l-TRP), <em>5</em>-methoxyindoleacetic acid (<em>5</em>-MIAA), 6-hydroxymelatonin (6-HMT), MT, serotonin (SER) and <em>5</em>-<em>methoxytryptamine</em> (<em>5</em>-MTRA) were performed employing micellar electrokinetic chromatography (MEKC). The optimal background electrolytes (BGE) used for separations were 20mM tetraborate buffer (pH 9.2) and 20mM phosphate buffer (pH 3.3) when employing techniques with normal and reverse migration of micelles, respectively. Fifty millimolar sodium dodecyl sulfate (SDS) was employed as the pseudostationary phase and voltage of +/-20kV was used throughout the investigation. On-line preconcentration techniques, stacking and sweeping, were applied in order to overcome high detection limits that are a serious drawback of CE with UV detection. A comparison of used techniques, concerning enhancement factors and limits of detection (LOD), is presented. Obtained results show that the use of stacking with reverse migrating micelles (SRMM) as one of preconcentration techniques allows obtaining the lowest estimated LODs for MT at the level of 30ng/mL with injection time of 99s at 0.<em>5</em>psi. Estimated LODs for other analytes in these conditions were, 21, 26 and 100ng/mL for l-TRP, <em>5</em>-MIAA and 6-HMT, respectively. Signals of <em>5</em>-MTRA and SER obtainable only with 10s injection allowed reaching estimated LODs of 62.<em>5</em> and 130ng/mL, respectively. Analysis of spiked, diluted human serum was carried out as a preliminary application illustration of developed procedure.

Inactivation of a neurotransmitter, after its stimulated release, via high-affinity uptake mechanisms is an essential regulatory step of neurotransmission in both the central and peripheral nervous systems. To initiate explorations of the molecular mechanisms and the underlying biochemical architecture of high-affinity neurotransmitter uptake systems, we have used gene transfer technology to establish and identify novel cellular models that express these systems. Human genomic D<em>N</em>A was transfected into mouse L-M fibroblasts and two independently arising, clonal cell lines (L-S1 and L-S2) have been identified as expressing high-affinity serotonin (<em>5</em>-HT) uptake systems. The <em>5</em>-HT uptake characteristics of L-S1 and L-S2 are essentially comparable (in terms of <em>N</em>a+ dependence, temperature sensitivity, imipramine antagonizability, kinetic saturability and high affinities) and those of L-S1 have been reported previously. Furthermore, competition studies utilizing catecholamine neurotransmitters and their amino acid precursors demonstrated that these systems are highly specific for <em>5</em>-HT. Several known inhibitors of high-affinity <em>5</em>-HT uptake systems (including amitriptyline, desipramine, fluoxetine, imipramine, nortriptyline, tryptamine, <em>5</em>-<em>methoxytryptamine</em> and <em>N</em>-<em>acetyl</em> <em>5</em>-<em>methoxytryptamine</em>) were assessed in terms of their respective potencies to inhibit <em>5</em>-[3H]HT uptake by L-S1 and L-S2 cells. For L-S1 cells, the rank order of inhibitor potencies is imipramine greater than amitriptyline greater than fluoxetine greater than desipramine = nortriptyline greater than tryptamine greater than <em>5</em>-<em>methoxytryptamine</em> greater than <em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>. For L-S2, the rank order is similar to that of L-S1 except that fluoxetine is more potent than amitriptyline.(ABSTRACT TRU<em>N</em>CATED AT 2<em>5</em>0 WORDS)

Suprachiasmatic nucleus (SC<em>N</em>) controls various physiological, endocrine and behavioral functions by regulating conversion of serotonin (<em>5</em>-HT) to melatonin (MEL). Aging leads to alterations in the neural and temporal organization of the SC<em>N</em> leading to circadian dysfunction. Age-induced stoichiometric alterations in daily chronomics of various components of <em>5</em>-HT metabolism were studied by constructing interactomes between parameters. The levels of tryptophan (TRP), <em>5</em>-hydroxytryptophan (<em>5</em>-HTP), <em>5</em>-hydroxytryptamine (<em>5</em>-HT), <em>N</em>-<em>acetyl</em>serotonin (<em>N</em>AS), <em>N</em>-<em>acetyl</em> <em>5</em>-<em>methoxytryptamine</em> (MEL), <em>5</em>-hydroxyindoleacetic acid (<em>5</em>-HIAA), <em>5</em>-methoxyindole acetic acid (<em>5</em>-MIAA), <em>5</em>-hydroxytryptophol (<em>5</em>-HTOH), <em>5</em>-methoxytryptophol (<em>5</em>-MTOH) and <em>N</em>-<em>acetyl</em>tryptamine (<em>N</em>AT) were measured at (Zeitgeber time 0, 6, 12 and 18) in male rat SC<em>N</em> of 3, 12 and 24 months age groups. Age-induced decrease was observed in mean levels of <em>N</em>AS, MEL, <em>5</em>-HIAA, <em>5</em>-MIAA, <em>5</em>-MTOH, and <em>N</em>AT and increase was observed in TRP, <em>5</em>-HTP, <em>5</em>-HT and <em>5</em>-HTOH in rat SC<em>N</em>. Daily pulses decreased with aging significantly for TRP, <em>5</em>-HT, <em>N</em>AS, MEL, <em>5</em>-HIAA, <em>5</em>-MIAA and <em>N</em>AT. We report here, the age-induced change in interactions between various <em>5</em>-HT metabolism components by middle age (12 m) changing further by 24 m. The daily rhythms persisted with aging for <em>N</em>AS, MEL and <em>5</em>-HTOH. Though, rhythms were abolished for TRP, <em>5</em>-HTP, <em>5</em>-HIAA, <em>5</em>-MIAA, <em>5</em>-MTOH and <em>N</em>AT differentially at 12 and 24 m. The MEL administration showed restoration in <em>5</em>-HT ratio with <em>5</em>-HTP, MEL and <em>5</em>-HTOH in 24 m and <em>N</em>AS and <em>5</em>-HIAA in 12 m SC<em>N</em>. Thus, MEL administration effects alterations of age-induced stoichiometry in levels and chronomics of serotonin metabolic network interactomes.

Luteinizing hormone (LH) influences the secretion of melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) from the pineal gland. The present study examined the possible presence of LH/chorionic gonadotropin (CG) receptor in the pineal gland of adult female rats. Reverse transcriptase-polymerase chain reaction analyses demonstrated that LH/CG receptor mR<em>N</em>A is expressed in the pineal gland. Western blotting showed that the pineal gland, like the ovary, contains an 80 kDa receptor protein. Immunohistochemistry revealed that LH/CG receptor, arylalkylamine <em>N</em>-<em>acetyl</em>transferase (a regulatory enzyme in melatonin biosynthesis) and serotonin (a melatonin precursor) are localized primarily to the same cells of the pineal gland. We further found that the levels of pineal LH/CG receptor protein in normal cycling female rats change significantly during the estrous cycle, being lowest at early metestrus. These results demonstrate that LH/CG receptor is expressed in the pineal gland, primarily in melatonin-synthesizing cells, namely pinealocytes. Furthermore, it is suggested that LH influences pineal melatonin secretion through binding to this receptor. In addition, LH/CG receptor levels in the pineal gland are regulated during the estrous cycle under normal physiological conditions.

Hydroxyindole-O-methyltransferase (HIOMT; EC 2.1.1.4) catalyzes the final step in the melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) biosynthetic pathway. HIOMT-like activity was detected in the head of fifth last-instar larvae of the silkworm (Bombyx mori), and the optimum pH for this activity was 7.9. The apparent Michaelis constants (K(m)) for S-adenosyl-L-methionine and <em>N</em>-<em>acetyl</em>serotonin were 87.6 microM and 96.6 microM, respectively. When the silkworms were entrained to a 12 h light/12 h dark lighting schedule, the HIOMT-like activity showed a significant diurnal variation with high levels during the dark period. The diurnal variation in the activity persisted in constant darkness, but was suppressed by constant light. These findings demonstrate that HIOMT-like activity in the silkworm head occurs as a circadian rhythm and that the rhythm entrains to environmental light/dark cycles. The melatonin rhythm in the silkworm head may therefore be regulated not only by serotonin <em>N</em>-<em>acetyl</em>transferase (EC 2.3.1.87), but also by HIOMT.

Seroto<em>n</em>i<em>n</em> (<em>5</em>-HT) is a mediator (through <em>5</em>-HT1P receptors) of slow EPSPs i<em>n</em> mye<em>n</em>teric ga<em>n</em>glia of the small i<em>n</em>testi<em>n</em>e. The effect of <em>5</em>-HT ca<em>n</em> be mimicked by elevati<em>n</em>g cAMP; therefore, we tested the hypothesis that the slow EPSP-like respo<em>n</em>se to <em>5</em>-HT is cAMP-mediated. Gui<em>n</em>ea pig gut was e<em>n</em>zymatically dissociated; mye<em>n</em>teric ga<em>n</em>glia remai<em>n</em>ed i<em>n</em>tact a<em>n</em>d were collected by filtratio<em>n</em>. <em>N</em>euro<em>n</em>s i<em>n</em> the isolated ga<em>n</em>glia retai<em>n</em>ed their ability to ma<em>n</em>ifest the slow EPSP-like respo<em>n</em>se to <em>5</em>-HT. Exposure to <em>5</em>-HT raised the ga<em>n</em>glio<em>n</em>ic level of cAMP (ED<em>5</em>0 0.3 microM). This effect was <em>n</em>ot a<em>n</em>tago<em>n</em>ized by the <em>5</em>-HT1P a<em>n</em>tago<em>n</em>ist, <em>N</em>-<em>acetyl</em>-<em>5</em>-hydroxytryptophyl-<em>5</em>-hydroxytryptopha<em>n</em> amide (100.0 microM), or mimicked by the <em>5</em>-HT1P ago<em>n</em>ist, <em>5</em>-hydroxyi<em>n</em>dalpi<em>n</em>e (10.0 microM). I<em>n</em>creases i<em>n</em> cAMP were also evoked by the <em>5</em>-HT1 ago<em>n</em>ist, <em>5</em>-carboxyamidotryptami<em>n</em>e (10.0 microM), the <em>5</em>-HT2 ago<em>n</em>ist, (+-)-1-(2,<em>5</em>-dimethoxy-4-iodophe<em>n</em>yl)-2-ami<em>n</em>opropa<em>n</em>e (DOI; 1.0-10.0 microM), a<em>n</em>d by the <em>5</em>-HT4 ago<em>n</em>ists, re<em>n</em>zapride (1.0-10.0 microM) a<em>n</em>d <em>5</em>-<em>methoxytryptamine</em> (1.0-10.0 microM); however, <em>n</em>either the <em>5</em>-HT1/<em>5</em>-HT2 a<em>n</em>tago<em>n</em>ists, spipero<em>n</em>e, methysergide, a<em>n</em>d methiothepi<em>n</em>, <em>n</em>or the <em>5</em>-HT4 a<em>n</em>tago<em>n</em>ist, tropisetro<em>n</em> (ICS 20<em>5</em>-930; 10.0 microM), were able to i<em>n</em>hibit the rise i<em>n</em> cAMP evoked by these compou<em>n</em>ds or by <em>5</em>-HT (0.1-10.0 microM). The <em>5</em>-HT-evoked elevatio<em>n</em> of cAMP was a<em>n</em>tago<em>n</em>ized by keta<em>n</em>seri<em>n</em> (10.0 microM), which also blocked the effects of <em>5</em>-<em>methoxytryptamine</em> a<em>n</em>d DOI, but <em>n</em>ot those of re<em>n</em>zapride. The effective co<em>n</em>ce<em>n</em>tratio<em>n</em> of DOI, however, was higher tha<em>n</em> that <em>n</em>eeded for activatio<em>n</em> of <em>5</em>-HT2 receptors, a<em>n</em>d <em>N</em>orther<em>n</em> a<em>n</em>alysis usi<em>n</em>g a cD<em>N</em>A probe e<em>n</em>codi<em>n</em>g the rat <em>5</em>-HT2 receptor failed to reveal the prese<em>n</em>ce of <em>5</em>-HT2 mR<em>N</em>A i<em>n</em> mye<em>n</em>teric ga<em>n</em>glia, although it hybridizes with mR<em>N</em>A of the right size i<em>n</em> the gui<em>n</em>ea pig brai<em>n</em>. Compou<em>n</em>ds that failed to cha<em>n</em>ge levels of cAMP or to a<em>n</em>tago<em>n</em>ize the actio<em>n</em> of <em>5</em>-HT i<em>n</em>cluded 8-hydroxy-di-<em>n</em>-propylami<em>n</em>o tetrali<em>n</em>, R<em>5</em>8639, R88226, a<em>n</em>d sumatripta<em>n</em>. It is co<em>n</em>cluded that the receptor respo<em>n</em>sible for the <em>5</em>-HT-i<em>n</em>duced rise i<em>n</em> cAMP i<em>n</em> ga<em>n</em>glia isolated from the gui<em>n</em>ea pig mye<em>n</em>teric plexus is <em>n</em>ot a k<em>n</em>ow<em>n</em> subtype of <em>5</em>-HT receptor. Si<em>n</em>ce the pharmacology of this <em>n</em>ovel receptor is differe<em>n</em>t from that of the slow EPSP-like respo<em>n</em>se to <em>5</em>-HT, the receptor probably does <em>n</em>ot mediate the slow EPSP.

Methods were developed for the a<em>n</em>alysis of <em>5</em>-hydroxytryptami<em>n</em>e, related i<em>n</em>dolealkylami<em>n</em>es (tryptami<em>n</em>e, melato<em>n</em>i<em>n</em>, <em>5</em>-<em>methoxytryptamine</em>, <em>N</em>-<em>acetyl</em>-<em>5</em>-hydroxytryptami<em>n</em>e a<em>n</em>d 6-hydroxymelato<em>n</em>i<em>n</em>) a<em>n</em>d <em>5</em>-hydroxyi<em>n</em>dole-3-acetic acid (<em>5</em>HIAA) i<em>n</em> bovi<em>n</em>e reti<em>n</em>a, aqueous a<em>n</em>d vitreous humours. <em>5</em>-Hydroxytryptami<em>n</em>e a<em>n</em>d related i<em>n</em>dolealkylami<em>n</em>es were extracted a<em>n</em>d derivatized to form their correspo<em>n</em>di<em>n</em>g pe<em>n</em>tafluoropropio<em>n</em>yl spirocyclic derivatives. <em>5</em>HIAA was extracted a<em>n</em>d derivatized to the correspo<em>n</em>di<em>n</em>g pe<em>n</em>tafluoropropio<em>n</em>amide-trifluoroethyl derivative. Ide<em>n</em>tificatio<em>n</em> a<em>n</em>d qua<em>n</em>titatio<em>n</em> by gas chromatography-<em>n</em>egative io<em>n</em> chemical io<em>n</em>izatio<em>n</em> mass spectrometry was made with refere<em>n</em>ce to deuteriated i<em>n</em>ter<em>n</em>al sta<em>n</em>dards. <em>5</em>-Hydroxytryptami<em>n</em>e was prese<em>n</em>t i<em>n</em> all (<em>n</em> = 34) reti<em>n</em>al samples a<em>n</em>alysed (20.<em>5</em>3 +/- 1.64 <em>n</em>g) while <em>N</em>-<em>acetyl</em>-<em>5</em>-hydroxytryptami<em>n</em>e was detected i<em>n</em> half of the samples of reti<em>n</em>a (0.06 +/- 0.02 <em>n</em>g). Melato<em>n</em>i<em>n</em> (0.1<em>5</em> +/- 0.06 <em>n</em>g) a<em>n</em>d tryptami<em>n</em>e (0.78 +/- 0.34 <em>n</em>g) were detected i<em>n</em> o<em>n</em>ly a small <em>n</em>umber of reti<em>n</em>as. <em>5</em>-<em>Methoxytryptamine</em> was <em>n</em>ot prese<em>n</em>t i<em>n</em> reti<em>n</em>a. <em>5</em>-Hydroxytryptami<em>n</em>e was also prese<em>n</em>t i<em>n</em> aqueous (0.76 +/- 0.17 <em>n</em>g ml-1 a<em>n</em>d vitreous (0.3<em>5</em> +/- 0.0<em>5</em> <em>n</em>g ml-1' humours from bovi<em>n</em>e eye. Tryptami<em>n</em>e, melato<em>n</em>i<em>n</em>, <em>5</em>-<em>methoxytryptamine</em> a<em>n</em>d <em>N</em>-<em>acetyl</em>-<em>5</em>-hydroxytryptami<em>n</em>e were <em>n</em>ot detected i<em>n</em> bovi<em>n</em>e aqueous a<em>n</em>d vitreous humours. <em>5</em>HIAA was fou<em>n</em>d i<em>n</em> both bovi<em>n</em>e aqueous (2.03 +/- 0.38 <em>n</em>g ml-1) a<em>n</em>d vitreous (0.6<em>5</em> +/- 0.06 <em>n</em>g ml-1) humours, but its co<em>n</em>siste<em>n</em>t determi<em>n</em>atio<em>n</em> i<em>n</em> reti<em>n</em>a was obviated by i<em>n</em>terfere<em>n</em>ce from spurious peaks.

The hormone melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) is an indole derivative with a flexible peptide-like side chain attached at the C3 position. Using a combination of two-color resonant two-photon ionization (2C-R2PI), laser-induced fluorescence excitation (LIF), resonant ion-dip infrared spectroscopy (RIDIRS), fluorescence-dip infrared spectroscopy (FDIRS), and UV-UV hole-burning spectroscopy, the conformational preferences of melatonin in a molecular beam have been determined. Three major trans-amide conformers and two minor cis-amide conformers have been identified in the R2PI spectrum and characterized with RIDIRS and FDIRS. Structural assignments are made using the infrared spectra in concert with density functional theory and localized MP2 calculations. Observation of cis-amide melatonin conformers in the molecular beam, despite the large energy gap (approximately 3 kcal/mol) between trans- and cis-amides, is striking because there are at least nine lower-energy trans-amide minima that are not detected. The implications of this observation for cooling and trapping conformational population in a supersonic expansion are discussed.

It seems clear that the pineal hormone, melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>), is involved in the reproductive behavior of several animal species including humans. Moreover, several data also support a role for <em>5</em>-methoxytryptophol (ML), another pineal hormone, in the control of sexual processes. To test the role of ML in human reproductive axis, 128 healthy children, 68 boys and 60 girls, were studied. Each of these groups was divided in three age subgroups of 6, 11, and 14 years. A single blood sample (0900 hours) was obtained from each subject to determine melatonin, ML, FSH, LH, estradiol (girls), and testoterone (boys) by RIA. Statistical analysis of the data included A<em>N</em>OVA-II (factor I: age, factor II: sex) and an analysis of covariance with age as covariate. A similar plasma melatonin concentration, with a significant decrease between 6 and 11 years, was found in boys and girls. Melatonin concentrations correlate well with initiation of the pubertal development in these children, although no sex differences were found. Concentrations of ML are approximately <em>5</em>0% of those of melatonin. In contrast to melatonin, ML levels show significant age and sex differences. Plasma ML concentration significantly increased in boys (P < 0.001) and decreased in girls (P < 0.001) after 8 years of age. These results support the hypothesis that, besides melatonin, other pineal compounds such as ML may be involved in the maturation process in humans. The pineal indole ML may also be used as a marker of the different chronobiology in the pubertal development in boys and girls.

OBJECTIVE

Laryngoscopy and endotracheal intubation are considered as potent stimuli which lead to an increase in heart rate and blood pressure. Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) has been studied for pre-operative anxiolysis and sedation in Intensive Care Unit. We made a hypothesis that melatonin can provide haemodynamic stability during laryngoscopy and intubation when given 120 min before the procedure.

METHODS

Sixty American Society of Anesthesiologists physical status Grade I and II patients of either gender, 20-4<em>5</em> years old, 40-6<em>5</em> kg body weight, scheduled to undergo elective surgical procedures under general anaesthesia were assigned into two equal groups - Group C (control) and Group M (melatonin). They received oral placebo or melatonin tablets 6 mg, respectively, 120 min before surgery. The haemodynamic parameters were recorded preoperatively, during laryngoscopy and endotracheal intubation and thereafter at 1, 3, <em>5</em> and 10 min. Unpaired t-test was used for between-group comparison of ratio and interval scale data. For within-group comparison of ratio and interval scale data, repeated-measures A<em>N</em>OVA and post hoc Bonferroni t-tests were used.

RESULTS

It was observed that in the control group, there was a significant increase in heart rate and blood pressure at laryngoscopy and intubation and persisted till 10 min post-intubation. In melatonin group, there was an insignificant increase in heart rate at the time of laryngoscopy and intubation which however settled within 1 min post-intubation.

CONCLUSIONS

Melatonin is an effective drug for attenuation of cardiovascular responses to laryngoscopy and endotracheal intubation.

The aim of the present study was to investigate whether the ovulation-maintaining effect of melatonin in rats, exposed to continuous light (LL), was also exerted by other pineal indoles which have been reported to influence the reproductive processes of mammals. The effect of 10 micrograms melatonin was compared with that of similar amounts of either <em>N</em>-<em>acetyl</em>serotonin, <em>5</em>-methoxytryptophol, <em>5</em>-methoxyindole-3-acetic acid, <em>5</em>-hydroxytryptophol, <em>5</em>-<em>methoxytryptamine</em> or <em>5</em>-methoxytryptophan. All these compounds appeared to be significantly less effective than melatonin in preventing the effect of LL, ovulation being preserved in only 20--33% of the rats investigated, with melatonin this percentage being 60--7<em>5</em>%. Investigations were also carried out to assess the effect of these indole derivatives on HIOMT (hydroxyindole-O-methyl transferase) activity in synthesizing different <em>5</em>-methoxyindoles in the abnormally influenced pineal gland due to LL. Melatonin, the compound the effect of which on ovarian cyclicity is strongest, stimulates <em>5</em>-methoxytryptophol synthesis; while other less active compounds stimulate the synthesis of melatonin and inhibit that of O-<em>acetyl</em>-<em>5</em>-methoxytryptophol. The possibility that the effect of other indoles than melatonin on ovarian cyclicity might be due to stimulation of melatonin synthesis was considered. A possible functional relationship of the different indoles cannot be excluded.

The effects of native cyclodextrins (alpha, beta or gamma), hydroxypropyl-beta-cyclodextrin, beta-cyclodextrin solubilized in urea, soluble starch and glucose in water solution on the fluorescence behaviour of melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) (M) and <em>5</em>-<em>methoxytryptamine</em> [<em>5</em>-methoxy-3-(2-aminoethyl)indole] (<em>5</em>M) were determined. In addition, the effect of methanol and propanol with and without beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin was assessed. From the fluorescence changes with pH, the values of the pKa for the ground (9.9 +/- 0.2) and the excited state (7.7 +/- 0.2) for <em>5</em>M were determined. From the fluorescence changes with beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin, the association constants of M, <em>5</em>MH [<em>5</em>-methoxy-3-(2-ammoniumethyl)indole] and <em>5</em>M with the two hosts were determined. The values with beta-cyclodextrin were KAssoc<em>5</em>MH = (1.4 +/- 0.4) x 10(2) mol-1 dm3, KAssoc<em>5</em>M = (1.6 +/- 0.1) x 10(2) mol-1 dm3 and KAssocM = (1.1 +/- 0.2) x 10(2) mol-1 dm3, and with hydroxypropyl-beta-cyclodextrin KAssoc<em>5</em>MH = (1.1 +/- 0.3) x 10(2) mol-1 dm3, KAssoc<em>5</em>M = (2.<em>5</em> +/- 0.1) x 10(2) mol-1 dm3 and KAssocM = (1.<em>5</em>1 +/- 0.07) x 10(2) mol-1 dm3. The ratios of the fluorescence quantum yields for the bound and free substrate (phi b/phi f) were in the range 1.1<em>5</em>-1.48. The detection limits under the optimum conditions were 0.381 +/- 0.001 ng cm-3 for the complex <em>5</em>MH-hydroxypropyl-beta-cyclodextrin in water and 0.290 +/- 0.001 ng cm-3 for the complex M-hydroxypropyl-beta-cyclodextrin in water with <em>5</em>% of methanol. The recovery of melatonin from pharmaceutical preparations was 98-103% with an RSD of 2%. The recovery from rat pineals was also good. The method is direct, simple and accurate.

Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) is a hormon secreted mostly by the pineal gland in the brain which maintains the body's circadian rhythm. Interestingly, this indol derivative is produced by enterochromaffin-like cells (ECL) in the gastrointestinal tract (GIT) in amount about 400 fold greater than detected in the pinealocytes. Previous studies revealed that melatonin exerts beneficial action against acute gastric damage induced by stress ethanol, aspirin and ischemia-reperfusion. Hyperglycemia, which is the main symptom of diabetes mellitus, is known to induce mitochondrial dysfunction and endoplasmic reticulum stress, both promoting the generation of reactive oxygen species (ROS). ROS were shown to exhibit higher activity than molecular oxygen under basal conditions due to unpaired electron in its outermost shell of electrons. ROS lead to damage of cellular proteins, nucleic acids and membrane polyunsaturated fatty lipids. In this study, we induced diabetes mellitus by the application of strep. tozocin in presence of gastric ulcers. Male Wistar rats were used in this model. 9 days after gastric ulcers and diabetes mellitus induction, groups of rats were treated with saline or melatonin (20 mg/kg i.g.). At the termination of the experiment, rats were anesthetized, abdomen was opened and gastric blood flow (GBF) was measured. Stomachs were removed for determination of gastric ulcers area by planimetry. Tissue samples were collected for biochemical assays. We demonstrated that melatonin significantly accelerates gastric ulcers healing with and without coexistence of diabetes mellitus. This effect was accompanied by increase of GBF level. Moreover, we observed an increase in superoxide dismutase (SOD) activity and an decrease in lipid peroxidation products concentration within gastric tissue homogenates of animals treated with melatonin, as compared with control group. Melatonin application accelerates gastric ulcers healing with and without presence of diabetes mellitus. We conclude that melatonin can physiologically regulate anti-oxidative enzymes activity and increase GBF level.

The importance of fluoride as a natural and industrial toxicant is recognized worldwide. We evaluated the regulating role and biological effect of vesicular (liposomal and nanoencapsulated) melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) for drug delivery and controlled release on the depletion of inflammatory mediators, as well as oxidative damage in sodium fluoride (<em>N</em>aF)-treated lungs and liver. Hepatic and bronchial damage was induced in Swiss albino rats with a single acute ingestion of <em>N</em>aF (48 mg/kg body weight, oral gavage). <em>N</em>aF exposure caused the generation of reactive oxygen species (ROS); upregulation of T<em>N</em>F-α and TGF-β; decreased activities of antioxidant systems (glutathione, glutathione-S-transferase, superoxide dismutase, catalase), succinate dehydrogenase, membrane microviscosity, and membrane potential; increased activity of lipid peroxidation and nicotinamide adenine dinucleotide hydride oxidase; and increased hepatic and nephrite toxicities (P<0.001) compared to those in normal animals. Charge (-ve/+ve)-specific single liposomal (dicetyl phosphate/stearylamine) and nanoencapsulated melatonin (4.46 mg/kg body weight, intravenous) treatments (2 hours after <em>N</em>aF exposure) significantly (P<0.01/0.001) and maximally (P<0.001) inhibited all alterations developed in <em>N</em>aF-mediated oxidative injuries in rat liver (+ve) and lungs (-ve), demonstrating their strong free radical scavenging, antioxidant and antigenotoxic properties, and vesicular efficiencies of targeting. Overall, these results suggest that nanoencapsulated melatonin might be considered as a more powerful remedial therapy in comparison to liposomes, in terms of its efficacy in regulating <em>N</em>aF-intoxicated oxidative injury.

The signal pathway of target of rapamycin (TOR) plays an important role in regulating cell growth and proliferation, follicular development, and ovulation. Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) (MT) is involved in the regulation of many physiological functions in animals. Recent studies have shown that MT affects the number and the degree of maturation of follicles in the ovary, but there are few studies concerning its mechanism. Therefore, the aim of this study was to investigate the mechanism of TOR signal pathway in the regulation of ovarian function by MT in aging laying hens. In the present study, a total of 60 hens (70-week-old) were randomly divided into 2 groups: control group and melatonin group (M). Melatonin was administered intraperitoneally at a dose of 20 mg/kg/D for 28 D in the M group. The results showed that MT significantly increased the levels of the antioxidant enzymes superoxide dismutase and total antioxidant capacity (P < 0.01) as well as levels of immunoglobulin (IgA, IgG, and IgM) (P < 0.0<em>5</em>) and the reproductive hormones estradiol and luteinizing hormone (P < 0.01) in the plasma and also increased the numbers of middle white follicles and small white follicles (P < 0.0<em>5</em>) and decreased the level of reactive oxygen species in plasma (P < 0.01) in laying hens. There were higher expression levels in MT receptor A (P < 0.0<em>5</em>), melatonin receptor B (P < 0.01), and tuberous sclerosis complex 2 (P < 0.01). Activation of TOR, 4E binding protein-l (4E-BP1), and ribosomal protein 6 kinase (P < 0.01) was found in the M. The levels of mTOR and p-mTOR protein were increased in the M (P < 0.0<em>5</em>). The mTORC1-dependent 4E-BP1 and p-4E-BP1 were increased in the M (P < 0.0<em>5</em>). This study indicated that MT may enhance follicle growth by increasing levels of antioxidant enzymes and reproductive hormones and by activating the mTOR and downstream components in aging laying hens.

<strong class="sub-title"> Background: </strong> Sleep dysfunction is a common problem in people with schizophrenia, and side effects of treatment often exacerbate metabolic and cardiovascular risk and may induce extrapyramidal side effects. Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>) is an endogenously produced hormone which has demonstrated direct and indirect antioxidant and neuroprotective effects. Previous studies have explored the use of exogenous melatonin in improving sleep outcomes in the general population, yet indications for use in schizophrenia are unclear.

Aim: To synthesize the evidence from clinical trials investigating prescribed melatonin as an adjunctive therapy in patients with schizophrenia.

Methods: A systematic literature review of MEDLINE (Ovid), Embase, PsychINFO, and PubMed on the 27/08/20; and CINAHL and Cochrane Library databases, was conducted. Inclusion criteria were: a peer-reviewed clinical trial published in English; included a group of patients with schizophrenia; used melatonin as an adjunctive therapy; and reported any outcome of any duration. Exclusion criteria were: neurodegenerative diseases, primary sleep disorders, co-morbid substance use or animal studies.

Results: Fifteen studies were included in the current review with the following primary outcomes: sleep (n = 6), metabolic profile (n = 3), tardive dyskinesia (n = 3), cognitive function (n = 2) and benzodiazepine discontinuation (n = 1).

Conclusion: Adjunctive melatonin therapy has some positive outcomes for sleep, metabolic profile and tardive dyskinesia in patients with schizophrenia. No beneficial effect of melatonin was observed on outcomes of cognition or benzodiazepine discontinuation. Future studies utilizing larger samples and investigations specifically comparing the effect of melatonin as adjunctive therapy with different antipsychotics in patients with schizophrenia are required.

Keywords: Clinical trials; Melatonin; Metabolic syndrome; Schizophrenia; Sleep; Tardive dyskinesia.

<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em> (Melatonin), as a crucial messenger in plants, functions in adjusting biological rhythms, stress tolerance, plant growth and development. Several studies have shown the retardation effect of exogenous melatonin treatment on plant growth and development. However, the in vivo role of melatonin in regulating plant leaf growth and the underlying mechanism are still unclear. In this study, we found that high concentration of melatonin suppressed leaf growth in Arabidopsis by reducing both cell size and cell number. Further kinetic analysis of the fifth leaves showed that melatonin remarkably inhibited cell division rate. Additionally, flow cytometic analysis indicated that melatonin negatively regulated endoreduplication during leaf development. Consistently, the expression analysis revealed that melatonin regulated the transcriptional levels of key genes of cell cycle and ribosome. Taken together, this study suggests that high concentration of melatonin negatively regulated the leaf growth and development in Arabidopsis, through modulation of endoreduplication and the transcripts of cell cycle and ribosomal key genes.

Hypoxia-inducible factor-1 (HIF-1) plays an important role in cellular responses to hypoxia, including the transcriptional activation of several genes involved in tumor angiogenesis. Melatonin, also known as <em>N</em>-<em>acetyl</em>-<em>5</em>-methopxytryptamine, is produced naturally by the pineal gland and has anti-angiogenic effects in cancer through its ability to modulate HIF-1α activity. However, the use of melatonin as a therapeutic is limited by its low oral bioavailability and short half-life. Here, we synthesized melatonin-like molecules with enhanced HIF-1α targeting activity and less toxicity, and investigated their effects on tumor growth and angiogenesis, as well as the underlying molecular mechanisms. Among melatonin derivatives, <em>N</em>-butyryl-<em>5</em>-<em>methoxytryptamine</em> (<em>N</em>B-<em>5</em>-MT) showed the most potent HIF-1α targeting activity. This molecule was able to 1) reduce the expression of HIF-1α at the protein level, 2) reduce the transcription of HIF-1α target genes, 3) reduce reactive oxygen species (ROS) generation, 4) decrease angiogenesis in vitro and in vivo, and <em>5</em>) suppress tumor size and metastasis. In addition, <em>N</em>B-<em>5</em>-MT showed improved anti-angiogenic activity compared with melatonin due to its enhanced cellular uptake. <em>N</em>B-<em>5</em>-MT is thus a promising lead for the future development of anti-cancer compounds with HIF-1α targeting activity. Given that HIF-1α is overexpressed in the majority of human cancers, the melatonin derivative <em>N</em>B-<em>5</em>-MT could represent a novel potent therapeutic agent for cancer.

<strong class="sub-title"> Keywords: </strong> HIF-1α; <em>N</em>-butyryl-<em>5</em>-<em>methoxytryptamine</em>; VEGF; angiogenesis; melatonin; melatonin-like molecules.

In mammalian ovaries, the avascular environment within follicular cavity is supposed to cause hypoxic status in granulosa cells (GCs), leading to apoptotic cell death accompanied by cumulative reactive oxygen species (ROS) production. Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>, MT), a broad-spectrum antioxidant that exists in porcine follicle fluid, was suggested to maintain GCs survival under stress conditions. In this study, using the established hypoxic model (1% O₂) of cultured porcine GCs, we explored the effect of MT on GCs apoptosis. The results showed that MT restored cell viability and reduced the apoptosis of GCs during hypoxia exposure. In addition, GCs treated with MT exhibited decreased ROS levels and increased expression of antioxidant enzymes including heme oxygenase-1 (HO-1), glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and catalase (CAT) upon hypoxia incubation. Moreover, the hypoxia-induced expression of cleaved caspase 3, 8, and 9 was significantly inhibited after MT treatment. In contrast, blocking melatonin receptor 2 (MT<em>N</em>R1B) with a competitive antagonist 4-phenyl-2-propionamidotetralin (4P-PDOT) diminished the inhibitory effects of MT on caspase 3 activation. By detecting levels of protein kinase (PKA), a downstream kinase of MT<em>N</em>R1B, we further confirmed the involvement of MT-MT<em>N</em>R1B signaling in mediating GCs protection during hypoxia stress. Together, the present data provide mechanistic evidence suggesting the role of MT in defending GCs from hypoxia-induced apoptosis.

Keywords: apoptosis; granulosa cells; hypoxia; melatonin; reactive oxygen species.

Melatonin (<i><em>N</em></i>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em> MEL) is an indolamine that has antioxidant, anti-inflammatory and anti-tumor properties. Moreover, MEL is capable of exhibiting both anti-apoptotic and pro-apoptotic effects. In the normal cells, MEL possesses antioxidant property and has an anti-apoptotic effect, while in the cancer cells it has pro-apoptotic action. We investigated the combined effect of MEL and navitoclax (ABT-737), which promotes cell death, on the activation of proliferation in acute promyelocytic leukemia on a cell model HL-60. The combined effect of these compounds leads to a reduction of the index of mitotic activity. The alterations in the level of anti- and pro-apoptotic proteins such as BclxL, Bclw, Mcl-1, and BAX, membrane potential, Ca²⁺ retention capacity, and ROS production under the combined action of MEL and ABT-737 were performed. We obtained that MEL in combination with ABT-737 decreased Ca²⁺ capacity, dropped membrane potential, increased ROS production, suppressed the expression of anti-apoptotic proteins such as BclxL, Bclw, and Mcl-1, and enhanced the expression of pro-apoptotic BAX. Since, MEL modulates autophagy and endoplasmic reticulum (ER) stress in cancer cells, the combined effect of MEL and ABT-737 on the expression of ER stress and autophagy markers was checked. The combined effect of MEL and ABT-737 (0.2 μM) increased the expression of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), leading to a decrease in the level of binding immunoglobulin protein (BIP) followed by an increase in the level of C/EBP homologous protein (CHOP). In this condition, the expression of ERO1 decreased, which could lead to a decrease in the level of protein disulfide isomerase (PDI). The obtained data suggested that melatonin has potential usefulness in the treatment of cancer, where it is able to modulate ER stress, autophagy and apoptosis.

Keywords: HL-60 cells; acute promyelocytic leukemia; apoptosis; autophagy; endoplasmic reticulum stress; melatonin; navitoclax (ABT-737); permeability transition pore.

<em>N</em>-<em>Acetyl</em>-<em>5</em>-<em>methoxytryptamine</em> (melatonin, MT) at pharmacological concentrations promotes the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells; however, its role at physiological concentrations (1 pM-10 nM) remains unclear. We explored the effects of 1 pM-1 µM MT on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and its underlying mitochondrial dynamics-mediated mechanisms. T he PDLSC phenotype was detected by flow cytometry and evaluated for three-line differentiation. Alkaline phosphatase activity assay and Alizarin red staining were used to evaluate osteogenic differentiation. Osteogenesis-related gene and protein expression levels were measured by quantitative reverse transcription -polymerase chain reaction and western blotting. Mitochondrial function assays were performed using reactive oxygen species, ATP and <em>N</em>AD⁺/<em>N</em>ADH kits and molecular mechanisms of mitochondrial dynamics-related proteins were assessed by western blotting. Our results have shown that physiological MT concentrations induced differentiation of hPDLSCs and down-regulated osteopontin (OP<em>N</em>) and osteocalcin (OC<em>N</em>) expression levels, which were restored or even up-regulated by 1 µM MT (lowest pharmacological concentration). Compared to the osteogenic induction alone, this treatment decreased the intracellular ATP content, whereas the intracellular reactive oxygen species level and <em>N</em>AD+/<em>N</em>ADH ratio were increased. Mitochondrial function- and dynamics-related protein expression levels were consistent with those of osteogenic genes following osteogenic induction and MT treatment of hPDLSCs at various physiological concentrations. Physiological MT concentrations inhibited the osteogenic differentiation of hPDLSCs and simultaneously altered mitochondrial function. These findings provide insights into the stem cell tissue engineering and functions of MT.

To clarify the role of diluents in the preparation of molecularly imprinted polymers utilizing only hydrogen bonding, we investigated the effects of diluents by using different solvents. Melatonin (<em>N</em>-<em>acetyl</em>-<em>5</em>-<em>methoxytryptamine</em>), an amide bond and indole ring-containing hormone was chosen as the target molecule. <em>N</em>-Propionyl-<em>5</em>-<em>methoxytryptamine</em> was used as the pseudo template, methacrylic acid as the functional monomer, and solvents were used as diluents. Interactions between the template, the functional monomer, melatonin, and the solvents, were observed by 1H <em>N</em>MR spectroscopy. The polymers were evaluated by high-performance liquid chromatography. The results suggest the hydrogen bonding-acceptor capacity of the solvent is the most important factor in the preparation of molecularly imprinted polymers for hydrogen bonding-donating molecules. Hydrogen bonding between the template, the functional monomer, and solvent can be estimated from the chemical shifts in 1H <em>N</em>MR spectra of those molecules in the solvent.