Melatonin: Inhibition of Microtubule-Based Oral Morphogenesis in Stentor coeruleus

SYNOPSIS. The temporal separation of macromolecular syntheses from protein assembly into microtubular structures has permitted the use of the Stentor ciliated oral membranellar band regenerating system as an assay for mitotic spindle inhibitors (Banerjee & Margulis, 1971). Melatonin, the pineal gland hormone, is additive to Colcemid in this assay; like Colcemid it specifically inhibits band migration as a sensitive function of developmental stage and concentration. Altho the entire process of band formation and cilia regeneration (stages 0–8) is inhibited by melatonin, stage 3 is especially sensitive.
Delay in morphogenesis at stage 3 can be measured as a precise function of inhibitors (Colcemid, podophyllotoxin, melatonin) of the form y=kxⁿ, where y is delay in hours and k is the concentration of inhibitor in moles. Riboflavin (0.2 μ M ) and nicotinamide (0.2 μM) in combination reversed Colcemid-induced delay in band regeneration, but (unlike melatonin) the vitamins alone were totally without effect on the regenerating system. Therefore melatonin probably interacts with microtubule protein whereas the B vitamins interact in some way with Colcemid to nullify its activity.     

Podophyllotoxin,Colcemid and Cold Temperature Interfere with Cilia Regeneration in Stentor*

SYNOPSIS Stentor coeruleus, induced to shed their ciliary membranellar bands, regenerate these and associated oral structures within a few hours after treatment. In cells placed in media containing optimal concentrations of mitotic spindle inhibitors, regeneration of the ingestive organelles is reversibly inhibited. Inhibitory effects of Colcemid, podophyllotoxin, and cold temperature reported here are compared with previous results using colchicine, griseofulvin and isopropyl-n-phenyl carbamate on regenerating oral membranellar cilia and cell growth.     

Oral Regeneration in the Ciliate Stentor coeruleus: A Scanning and Transmission Electron Optical Study

SYNOPSIS. Elaboration of ciliated feeding organelles in the protozoon Stentor coeruleus was reinvestigated for the first time by scanning electron microscopy which gives the most realistic 3-dimensional images. Parallel transmission EM studies of synchronized regenerating stentors gave further ultrastructural details of stomatogenesis, while also confirming the expectation that in the structure of its kineties this now classical experimental object does not differ from other species of Stentor previously studied. Within 2 hr after the stimulus to regeneration, several generations of new kinetosomes for the oral primordium are produced, first in association with kinetosomes of kineties at the restricted primordium site. These kinetosomes rapidly sprout membranellar cilia as well as subpellicular microtubules but are still randomly oriented (anarchic field). The forming membranellar band increases from its center-line to both sides while it grows in length. Young cilia are blunt-ended. Recession of the early anlage occurs without rupture of the pellicle; soon apparent is the clear border stripe of unknown function along the right side of the membranellar band. Instantaneous fixation of beating cilia in early primordia revealed random beating, with coordination and presumably membranellar organization not yet attained. In late anlagen there are 2 types of metachronal rhythm: transversely from cilium to cilium across any given membranelle, as well as the easily observable serial beating of membranelles along the entire band. A single file of cilia leads the subsequent cytostomal invagination. The posterior end of the membranellar band then follows to line the cytopharynx.     

Central administration of growth hormone-releasing hormone triggers downstream movement and schooling behavior of chum salmon (Oncorhynchus keta) fry in an artificial stream

Anadromous salmonids migrate downstream to the ocean (downstream migration). The neuroendocrine mechanism of triggering the onset of downstream migration is not well known. We investigated the effects of 14 chemicals, including neuropeptides, pineal hormones, neurotransmitters, and neuromodulators (growth hormone-releasing hormone: GHRH, thyrotropin-releasing hormone, corticotropin-releasing hormone: CRH, gonadotropin-releasing hormone, melatonin, N-acetyl serotonin, serotonin, β-endorphin, enkephalin, dopamine, norepinephrine, epinephrine, acetylcholine, and histamine) on the onset of downstream migration in chum salmon (Oncorhynchus keta) fry. We defined downstream migration as a downstream movement (negative rheotaxis) with schooling behavior and counted the number of downstream movements and school size in experimental circulation tanks. An intracerebroventricular injection of GHRH, CRH, melatonin, N-acetyl serotonin, or serotonin stimulated the number of downstream movements. However, GHRH was the only chemical that also stimulated an increase in schooling behavior. These results suggest that CRH, melatonin, N-acetyl serotonin, and serotonin are involved in the stimulation of downstream movement in chum salmon, while GHRH stimulates both downstream movement and schooling behavior.     

The Effect of Colchicine on Regenerating Membranellar Cilia in Stentor coeruleus

SYNOPSIS. Stentors treated with toxic substances can be induced to shed their oral bands (19, Fig. 1), complex structures composed of many cilia organized into membranelles. Regenerating membranellar bands were observed in control stentors removed from toxic (urea-containing) medium at about 3.5 hours. At 8 hours regenerated control organisms were indistinguishable from normal unshed stentors. Experimental animals replaced into colchicine medium were inhibited from regeneration at low, nontoxic concentrations of this mitotic spindle inhibitor. Upon removal of the colchicine and replacement of the shed animals into normal medium or normal medium to which GTP had been added, complete and normal regeneration of the membranellar band ensued. Our observations are consistent with many suggesting that colchicine acts by reversibly binding with a protein during processes involving microtubule formation. Colchicine inhibition of membranellar band formation further indicates that oral membranelles are specialized evolutionary homologs to other centriole (= basal body, = kinetosome) derivatives such as mitotic spindle fibers, cilia and flagella, axopods, etc. (structures containing the ubiquitous microtubules of eukaryotic cells).     

Accumulation of Indole‐3‐Acetic Acid in Rice sl Mutant Leaves Infected with Bipolaris oryzae

Rice leaves accumulate serotonin in response to infection by Bipolaris oryzae. The leaves of the sl mutant, which is deficient in the gene encoding tryptamine 5‐hydroxylase, accumulate tryptamine instead of serotonin upon infection by B. oryzae. Because tryptamine is a possible precursor of indole‐3‐acetic acid (IAA), we investigated the accumulation of IAA in sl leaves infected with B. oryzae. Liquid chromatography coupled with tandem mass spectrometry analysis indicated that IAA accumulated at approximately 1.5 μmol/gFW in the leaves of sl mutant. This accumulation was suppressed by 95% by the treatment with the tryptamine decarboxylase inhibitor, (S)‐α‐(fluoromethyl)tryptophan, at 100 μm , indicating that tryptamine served as the precursor of IAA. The accumulation of IAA was not reproduced by treatment with CuCl₂ or by exogenous feeding of tryptamine. Furthermore, inoculation of Magnaporthe grisea induced only a lower level of IAA accumulation. On the other hand, B. oryzae produced IAA in culture media containing tryptamine. These findings strongly suggested that the metabolism of tryptamine by B. oryzae was responsible for IAA accumulation in the leaves of the sl mutant. Serotonin added to the culture media was also converted into 5‐hydroxyindole‐3‐acetic acid (5HIAA) at a rate similar to that of tryptamine. Considering that wild‐type rice leaves accumulate serotonin for defensive purposes, reducing the concentration of serotonin by conversion into 5HIAA may be significant as a detoxification process in the interaction between B. oryzae and rice.     

Con A Binds to the Membranellar and Somatic Cilia of Stentor and to the Developing Oral Primordium During Oral Regeneration1

Binding sites for Concanavalin A have been located in the ciliate Stentor coeruleus by utilizing FITC-Con A and fluorescence microscopy. When both nonregenerating and regenerating Stentor are fixed prior to FITC-Con A exposure, FITC-Con A binds intensely to the cilia of the membranellar band and to the somatic cilia that cover much of the cell surface. No binding is observed between the ciliary rows. The FITC-Con A also binds to the developing oral primordia of regenerating cells. Binding of FITC-Con A in the early stages of regeneration (prior to stage 4) appears to be less intense than that in the later stages. Additional FITC-Con A binding appeared as a granular fluorescence in the area of the developing buccal cavity beginning at about stage 4 and disappearing around stages 6–7. The presence of α-D-methyl mannoside prevented the binding of FITC-Con A to either regenerating or nonregenerating cells. If nonregenerating Stentor are exposed to FITC-Con A prior to fixation, the binding pattern is entirely different with the fluorescence primarily in the form of random, granular patches spread over much of the cell but with no binding to either type of cilia. These results demonstrate that membrane glycoproteins capable of binding Con A are located primarily in the membranellar and somatic cilia and in the developing oral primordia during oral regeneration in Stentor. Concanavalin A binding to these sites may be involved in the Con A-induced inhibition of oral regeneration observed in earlier studies.     

Metaphase arrest and delay in cell cycle kinetics of root apical meristems and mouse bone marrow cells treated with leaf aqueous extract of Clerodendrum viscosum Vent

Objectives

To study cell cycle delay and metaphase arresting activity of leaf aqueous extract of Clerodendrum viscosum Vent. (LAECV) in root apical meristems and mouse bone marrow cells.

Materials and methods

Cell cycle delay and metaphase arresting activities of LAECV were analysed, in root apical meristems of onion and wheat, and in mouse bone marrow cells, by scoring mitotic index, metaphase frequency and transition of cells from metaphase to anaphase. Colchicine was used as the standard metaphase arresting drug. Phytochemicals present in LAECV were detected and their phytotoxic activity was evaluated by analysing green‐gram (Vigna radiata) seedling's root growth retardation and branch root swelling phenomenon.

Results

LAECV treatment resulted in dose‐dependent root growth retardation of green‐gram seedling root length (P < 0.01) and half maximal growth inhibitory concentration (IC₅₀) of LAECV was 0.87 mg/ml at 144 h. In onion and wheat root meristem cells the mitotic index decreased, metaphase frequency increased and transition from metaphase to anaphase reduced. Experimentation with mouse bone marrow cells indicated that LAECV induced metaphase arrest (164.3% increase in arrested metaphases per 300 mg/kg body weight, over 2.5 h). Phytochemicals like carbohydrates, glycosides, saponins, terpenoids, triterpenoids, tannins and trace amounts of alkaloids were detected in LAECV.

Conclusion

It may be said that LAECV contains mitostatic and metaphase arresting components that are able to induce significant metaphase arrest in root apical meristems and also in mouse bone marrow cells.

     

Ciliary Proteins and Cytodifferentiation in Stentor coeruleus*

To elucidate further the molecular events required for cytodifferentiation in Stentor coeruleus, the effects of several chemical metabolic inhibitors were tested on the outgrowth in situ of the membranellar cilia of the oral feeding organelle. The chemicals used included several inhibitors of cytoplasmic and mitochondrial protein synthesis (cycloheximide, emetine, and chloramphenicol), and an antimitotic agent (colchicine). Ciliary growth was affected only by colchicine, suggesting that a pool of “ciliary proteins” exists in interphase Stentor of sufficient size to permit complete reformation of the membranellar cilia. The implication of these observations to an understanding of the more complicated process of oral regeneration is discussed.     

Melatonin metabolism,signaling and possible roles in plants

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 5-hydroxylase (T5H), which catalyzes the conversion of tryptamine into serotonin, has been proposed as a target to create a melatonin knockout mutant presenting a lesion-mimic phenotype in rice. With a reduced anabolic capacity for melatonin biosynthesis and an increased catabolic capacity for melatonin metabolism, all plants generally maintain low melatonin levels. Some plants, including Arabidopsis and Nicotiana tabacum (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 N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK), N-acetyl-5-methoxykynuramine (AMK) and 5-methoxytryptamine (5-MT) are also produced when melatonin is applied to Oryza sativa (rice). The signaling pathways of melatonin and its metabolites act via the mitogen-activated protein kinase (MAPK) cascade, possibly with Cand2 acting as a melatonin receptor, although the integrity of Cand2 remains controversial. Melatonin mediates many important functions in growth stimulation and stress tolerance through 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 N-acetylserotonin deacetylase (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.     

Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxlyase results in a dark brown phenotype and stunted growth

A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library. This mutant contained a copy of the T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxlyase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxlyase activities that converted tryptophan to tryptamine, which could be converted to serotonin by a constitutively expressed tryptamine 5′ hydroxylase (T5H) in rice plants. Over-expression of TDC-1 and TDC-3 in transgenic rice recapitulated the stunted growth, dark-brown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown color was proportional to the expression levels of TDC-1 and TDC-3. The levels of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the dark-brown leaves and hulls in the TDC-overexpressing transgenic rice were caused by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color.     

Morphogenetic Actions of 5-hydroxytryptamine and Some Analogous Substances on the Regeneration of the Planarian Worm Dugesia tigrina

The effects on regeneration of serotonin and analagous substances such as tryptamine, DMT, bufotenine, melatonin, auxin and gramine were studied on small pieces of D. tigrina. By comparison with results obtained in previous works with DOPA, α methyl DOPA and noradrenaline, and also with substances of the series of tetrahydrocannabinol it is shown that the normal morphogenetic action of the neurohormone is dependant of the presence of a free hydroxyl on a flat ring (phenol or indole) and of a free amine on the lateral chain of the molecule. Essays to detect the mechanism of the morphogenetic action did not allow to give any clear conclusion.     

Analysis of biogenic amines in plasma of hypertensive patients and a control group

Procedures were developed for the determination of 17 circulating amines using gas chromatography-negative ion chemical ionisation mass spectrometry. The amines were quantified against their appropriate deuterated isotopomers. The mean concentrations and ranges of catecholamines and trace amines were high compared with previous studies. In comparison with nonhypertensives, plasma from hypertensives had higher concentrations of the following amines: noradrenaline (t=4.0%); normetanephrine (t=6.1%) and metanephrine (t=1.9%). There were no significant differences between 5HT levels in plasma from hypertensives and controls. The following trace amine could be detected in variable amounts in plasma:p-tyramine,m-tyramine,p-octopamine,m-octopamine,p-synephrine,m-synephrine, and salsolinol. The trace amines melatonin,N-acetyl 5HT, tryptamine, 6-hydroxymelatonin and 5-methoxytryptamine could not be detected in plasma with limits of detection lying in the range 20–100 pg ml^–1.     

Salt stress‐induced seedling growth inhibition coincides with differential distribution of serotonin and melatonin in sunflower seedling roots and cotyledons

Indoleamines regulate a variety of physiological functions during the growth, morphogenesis and stress‐induced responses in plants. Present investigations report the effect of NaCl stress on endogenous serotonin and melatonin accumulation and their differential spatial distribution in sunflower (Helianthus annuus) seedling roots and cotyledons using HPLC and immunohistochemical techniques, respectively. Exogenous serotonin and melatonin treatments lead to variable effect on hypocotyl elongation and root growth under NaCl stress. NaCl stress for 48 h increases endogenous serotonin and melatonin content in roots and cotyledons, thus indicating their involvement in salt‐induced long distance signaling from roots to cotyledons. Salt stress‐induced accumulation of serotonin and melatonin exhibits differential distribution in the vascular bundles and cortex in the differentiating zones of the primary roots, suggesting their compartmentalization in the growing region of roots. Serotonin and melatonin accumulation in oil body rich cells of salt‐treated seedling cotyledons correlates with longer retention of oil bodies in the cotyledons. Present investigations indicate the possible role of serotonin and melatonin in regulating root growth during salt stress in sunflower. Effect of exogenous serotonin and melatonin treatments (15 μM) on sunflower seedlings grown in the absence or presence of 120 mM NaCl substantiates their role on seedling growth. Auxin and serotonin biosynthesis are coupled to the common precursor tryptophan. Salt stress‐induced root growth inhibition, thus pertains to partial impairment of auxin functions caused by increased serotonin biosynthesis. In seedling cotyledons, NaCl stress modulates the activity of N‐acetylserotonin O‐methyltransferase (HIOMT; EC 2.1.1.4), the enzyme responsible for melatonin biosynthesis from N‐acetylserotonin.     

Melatonin receptor pharmacology: toward subtype specificity

The recent cloning of three distinct melatonin receptor subtypes (Mel_1a, Mel_1b and Mel_1c) which are part of a new family of G-protein coupled receptors, and probably mediate the physiological actions of the hormone, has spurred interest in the design of analogues with subtype selectivity. The 5-methoxyl and N-acetyl groups of melatonin are important for binding to and activation of the receptor. The indole nucleus serves to hold these two groups at the correct distance from one another and allows them to adopt the required orientation for interaction with the receptor binding pocket. We have investigated the subtype selectivity of a number of analogues of melatonin in which the structure has systematically been modified in order to probe the similarities and differences in the interaction of ligand and receptor subtype. At all three subtypes 5-methoxyl and N-acetyl groups of melatonin are important for high affinity binding. However, replacing the 5-methoxyl group (eg with 5-H, 5-OH, 5-Me or 5-BzO) reduces affinity much less at the Mel_1b receptor subtype than at either Mel_1a or Mel_1c cloned subtypes. This suggests differences between the Mel_1b and Mel _1a/1c subtypes in the size and shape of the binding pocket or in the manner in which melatonin interacts with the receptor at this position. Further studies have revealed that analogues with longer N-acyl carbon chains behave similarly at each subtype. These observations suggest that the ‘pocket’ into which the N-acetyl group fits is very similar for each subtype. Substitutions at the 2-position on the indole ring improved affinity at each receptor subtype but did not give selective analogues. The systematic ‘mapping’ of the requirements for binding at each receptor subtype should allow the design of more selective agonists and antagonists, which will be valuable tools for the characterization and classification of functional melatonin receptors.     

Production of soluble,active acetyl serotonin methyl transferase in Leishmania tarentolae

N-acetyl serotonin methyl transferase (ASMT) is the last enzyme in the melatonin synthesis pathway. Evidence linking autism-related disorders with disorders of melatonin metabolism, and, more specifically, with mutations of the gene encoding ASMT, prompted us to investigate the properties and localization of this enzyme. As a first step, we undertook to overproduce the protein in a recombinant host. Early attempts to produce ASMT in recombinant Escherichia coli yielded only insoluble and heavily degraded material. However, recombinant ASMT (rASMT) could be produced in soluble, active form and purified in milligram amounts when the gene was cloned and expressed in Leishmania tarentolae.     

Effect of LED light spectra on exogenous prolactin-regulated circadian rhythm in goldfish,Carassius auratus

We investigated the effect of light spectra on circadian rhythm by exogenous prolactin (PRL) using light-emitting diodes (LEDs): red, green and purple. We injected PRL into live fish or treated cultured brain cells with PRL. We measured changes in the expressions of period 2 (Per2), cryptochrome 1 (Cry1), melatonin receptor 1 (MT1) mRNAs, and MT1 proteins, and in the plasma PRL, serotonin and melatonin levels. After PRL injection and exposure to green light, MT1 expression and plasma melatonin levels were significantly lower, but the expressions of Per2 and Cry1 were significantly higher than the others. Plasma serotonin after PRL injection and exposure to red light was significantly lower than others. These results indicate that injection of high concentration PRL inhibits melatonin, and inhibited melatonin regulates circadian rhythm via clock genes and serotonin. Thus, exogenous PRL regulates the circadian rhythm and light spectra influence the effect of PRL in goldfish.     

N-Acetyltransferase activity of the rat Harderian gland.

Harderian gland extracts from male rats catalyze the conversion of serotonin to N-acetylserotonin and of tryptamine to N-acetyltryptamine. The reaction is linear up to 14 mg tissue and departs from linearity after 10 min. The pH otpimum with tryptamine as substrate is between 8 and 9. Enzymic activity of the gland in vivo does not show diurnal variations. Enzymic activity of tissue in organ culture is not stimulated by 10 micrometer isoproterenol or 100 micrometer dibutyryl cyclic AMP. Harderian gland tissue in culture can acetylate tryptamine and serotonin and can O-methylate the N-acetylserotonin to form melatonin.     

Effects of tryptamine on active sodium and chloride transport in the isolated bullfrog cornea

The effects of the serotonin analogue, tryptamine, on the active transepithelial transport of Na⁺ and Cl⁻ in the in vitro bullfrog cornea were studied. Tryptamine, 1 mM, inhibited both the short-circuit current (I_sc) and potential difference (PD) of corneas transporting either Na⁺ alone or both Na⁺ and Cl⁻. The electrical resistance, R, increased in all cases. Both unidirectional Na⁺ and Cl⁻ fluxes were decreased by tryptamine and these changes accounted for the inhibitory effects on the I_sc. The effects of tryptamine were considered along with with those of 2 mM theophylline and 0.1 mM ouabain. Tryptamine inhibited the I_sc and both undirectional Cl⁻ fluxes which were previously stimulated by theophylline. Theophyline addition, after tryptamine preincubation, increased the Cl⁻ undirectional fluxes but did not restore the inhibited I_sc. The inhibitory effects of tryptamine on active Na⁺ and Cl⁻ transport were different from those of ouabain. While both drugs inhibited the forward Na⁺ and Cl⁻ fluxes, their backfluxes decreased with tryptamine and increased with ouabain. The addition to the bathing solution of tryptamine after ouabain preincubation reduced the ouabain-increased backward Cl⁻ flux and further increased the electrical resistance. These results are analyzed in terms of an electrical model from which it appears that tryptamine's mechanism of action was to decrease cellular permeability to the transepithelial movement of Na⁺ and Cl⁻.     

Melatonin: A potential regulator of plant growth and development?

Summary Recent research has reported the presence of melatonin (N-acetyl-5-methoxytryptamine), a mammalian indoleamine neurohormone, in higher plants, indicating that melatonin may be an important metabolic regulator that has been highly conserved across biological kingdoms. Melatonin is synthesized from tryptophan in the mammalian pineal gland and a similar biosynthetic pathway was recently described in St. John's wort shoot tissues, wherein radiolabel from tryptophan was recovered in serotonin and melatonin as well as indoleacetic acid. There is growing information describing melatonin control of physiological processes in mammals, yeast, and bacteria, including diurnal responses, detoxification of free radicals, and environmental adaptations. However, at the current time, there is no known specific role for melatonin in plant physiology. Alterations in melatonin concentrations in plant tissues have been shown to affect root development, mitosis, and mitotic spindle formation. The recent advancements in melatonin research in plants and some directions for important areas of future research are reviewed in this article.