Catabolism and disposition of exogenous 5-hydroxytryptamine in cockroach,Periplaneta americana,tissues

The concentrations of tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, N-acetyl 5-hydroxytryptamine and N-acetyl dopamine were determined in the cerebral ganglia, haemolymph and Malpighian tubules of the cockroach Periplaneta americana, using high performance liquid chromatography with electrochemical detection. Injected 5-hydroxytryptamine was rapidly removed from the haemolymph with a concomitant elevation of circulating N-acetyl 5-hydroxytryptamine and little accumulation of 5-hydroxytryptamine in the cerebral ganglia. N-acetyl 5-hydroxytryptamine and N-acetyl dopamine were also rapidly removed from the haemolymph. Incubation of haemolymph from 5-hydroxytryptamine-injected insects and glucosidase or phosphatase, indicated that most of the injected 5-hydroxytryptamine had been converted to a sugar conjugate of N-acetyl 5-hydroxytryptamine. Whole haemolymph did not catabolize 5-hydroxytryptamine or N-acetyl 5-hydroxytryptamine whereas Malpighian tubules N-acetylated both 5-hydroxytryptamine and dopamine and metabolized N-acetyl 5-hydroxytryptamine. Injection of p-chlorophenylalanine (200 and 500 μg/g) had no effect on 5-hydroxytryptamine concentrations in the cockroach cerebral ganglia.     

Influence of fasting and isolation on biogenic amine levels in the nervous system of the male cockroach Blaberus craniifer Burm. (Dictyoptera,Blaberidae)

1. The effects of long-term starvation and isolation on biogenic amine contents of the central nervous system of the male cockroach Blaberus craniifer Burm. were investigated.2. Levels of tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, a 5-hydroxyindolacetic acid-like substance, N-acetyl-5-hydroxytryptamine, dopamine, N-acetyldopamine and octopamine were measured by HPLC with electrochemical detection in fasted-isolated and fed-crowded insects from day 0 (imaginal molt) to day 30.3. Isolation and fasting resulted in general lower levels, except for serotoninergic pathway at day 10.4. From these data, by comparison with the isolation effect registered in females: (i) it is hypothesized that fasting exerts an inhibitory influence; and (ii) it is speculated that this effect is convergent with that of the loss of mechanical feeding stimuli on neurosecretory cells, as described in other male cockroaches.     

Further characterization and quantitative determination of 5-methoxy-N-methyltryptamine in Phalaris arundinacea

The 100 MHz PMR spectrum of 5-methoxy-N-methyltryptamine (5MMT) isolated from Phalaris arundinacea is elucidated, acid-induced fluorescence characteristics of 5-methoxy- and 5-hydroxytryptamine alkaloids on Avicel and silica gel TLC are described, and the concentration of 5MMT in P. arundinacea is determined by fluorometric TLC scanning.     

Selective destruction of 5-hydroxytryptamine receptors by neuraminidase

The responses of isolated frog skin to 5-hydroxytryptamine (increased active sodium transport and decreased passive chloride permeability) are diminished by incubation with the enzymes neuraminidase and N-acetylneuraminic acid aldolase but only in the absence of Ca²⁺ and presence of EDTA. The responses induced by oxytocin, adrenalin and aldosterone are unaffected by enzyme treatment.     

Phosphatidylcholine synthesis in adult Dirofilaria immitis females

Crude homogenates of adult Dirofilaria immitis females were able to incorporate choline into phosphatidylcholine (PC) and also were able to methylate phosphatidyl (N,N-dimethyl)-ethanolamine, using S-adenosylmethionine as the methyl donor, to form PC. The finding of choline phosphotransferase (EC 2·7·8·2) and phosphatidyl (N,N-dimethyl) ethanolamine methyltransferase activity in the paniculate (mainly microsomal) fraction of the homogenates provided further evidence that adult D. immitis females can synthesize PC by way of choline and cytidine 5'-diphosphocholine (Kennedy pathway) and also by way of S-adenosylmethionine-mediated sequential methylation of phosphatidylethanolamine (Bremer-Greenberg pathway).     

Spliceosome Sm proteins D1, D3, and B/B' are asymmetrically dimethylated at arginine residues in the nucleus

We report a novel modification of spliceosome proteins Sm D1, Sm D3, and Sm B/B′. L292 mouse fibroblasts were labeled in vivo with [³H]methionine. Sm D1, Sm D3, and Sm B/B′ were purified from either nuclear extracts, cytosolic extracts or a cytosolic 6S complex by immunoprecipitation of the Sm protein-containing complexes and then separation by electrophoresis on a polyacrylamide gel containing urea. The isolated Sm D1, Sm D3 or Sm B/B′ proteins were hydrolyzed to amino acids and the products were analyzed by high-resolution cation exchange chromatography. Sm D1, Sm D3, and Sm B/B′ isolated from nuclear fractions were all found to contain ω-N^G-monomethylarginine and symmetric ω-N^G,N^G′-dimethylarginine, modifications that have been previously described. In addition, Sm D1, Sm D3, and Sm B/B′ were also found to contain asymmetric ω-N^G,N^G-dimethylarginine in these nuclear fractions. Analysis of Sm B/B′ from cytosolic fractions and Sm B/B′ and Sm D1 from cytosolic 6S complexes showed only the presence of ω-N^G-monomethylarginine and symmetric ω-N^G,N^G′-dimethylarginine. These results indicate that Sm D1, Sm D3, and Sm B/B′ are asymmetrically dimethylated and that these modified proteins are located in the nucleus. In reactions in which Sm D1 or Sm D3 was methylated in vitro with a hemagglutinin-tagged PRMT5 purified from HeLa cells, we detected both symmetric ω-N^G,N^G′-dimethylarginine and asymmetric ω-N^G,N^G-dimethylarginine when reactions were done in a Tris/HCl buffer, but only detected symmetric ω-N^G,N^G′-dimethylarginine when a sodium phosphate buffer was used. These results suggest that the activity responsible for the formation of asymmetric dimethylated arginine residues in Sm proteins is either PRMT5 or a protein associated with it in the immunoprecipitated complex.     

Control of the melanophores of the crab,Pachygrapsus marmoratus: Release of pigment dispersing and pigment concentrating neurohormones by amines

1. The black pigment, in the melanophores, of Pachygrapsus marmoralus, a crab, disperses in specimens on a black background and concentrates in specimens on a white background.2. Bilateral eyestalk ablation results in black pigment concentration.3. These melanophores are regulated by pigment dispersing and concentrating hormones.4. In intact Pachygrapsus, 5-hydroxytryptamine produces black pigment dispersion whereas dopamine produces black pigment concentration.5. Neither 5-hydroxytryptamine nor dopamine affects melanophores in isolated legs. Presumably, therefore, these amines affect melanophores of intact Pachygrapsus indirectly only; 5-hydroxytryptamine by stimulating release of black pigment dispersing hormone and dopamine by stimulating release of black pigment concentrating hormone.     

Identification of N5,N10-methylene tetrahydrofolate reductase as the enzyme involved in the 5-methyl tetrahydrofolate-dependent formation of a beta-carboline derivative of 5-hydroxytryptamine in human platelets.

An enzyme in human platelets or rat brain incubated with 5-methyl tetrahydrofolate (5MeH₄folate) yields formaldehyde (4, 13), which will combine with biogenic amines to form β-carbolines (5) or tetrahydroisoquinolines. This activity was purified 500-fold from human platelets which are the main storage site for 5-hydroxytryptamine in man. This enzyme was identical to N⁵, N¹⁰-methylene tetrahydrofolate (N⁵,N¹⁰-methylene H₄folate) reductase by the following criteria: (i) co-purification, (ii) heat denaturation, (iii) pH response, (iv) molecular weight, (5) cofactor requirements. A mechanism involving the enzymatic generation of formaldehyde followed by adduct formation with a biogenic amine is proposed.     

Effect of polyphloretin phosphate and 7-oxa-13-prostynoic acid on the vasoactive actions of prostaglandin E2 and 5-hydroxytryptamine on isolated human umbilical arteries

Two prostaglandin antagonists, polyphloretin phosphate (PPP) and 7-oxa-13-prostynoic acid (EC-I-148) were examined for their ability and selectivity to block the vasoactive actions of prostaglandin E₂ and 5-hydroxytryptamine on isolated human umbilical arteries. Polyphloretin phosphate was found to be a weak antagonist of prostaglandin E₂ and exhibited a low degree of selectivity since responses to 5-hydroxytryptamine were also blocked. EC-I-148 (3.25 × 10^?5M) demonstrated a strong and selective antagonism to the contractions produced by prostaglandin E₂ because contractions to 5-hydroxytryptamine were not altered. EC-I-148 contracted umbilical artery strips in concentrations which antagonized responses to PGE₂.     

Synthesis and antimicrobial activities of N6-hydroxyagelasine analogs and revision of the structure of ageloximes

(+)-N⁶-Hydroxyagelasine D, the enantiomer of the proposed structure of (?)-ageloxime D, as well as N⁶-hydroxyagelasine analogs were synthesized by selective N-7 alkylation of N⁶-[tert-butyl(dimethyl)silyloxy]-9-methyl-9H-purin-6-amine in order to install the terpenoid side chain, followed by fluoride mediated removal of the TBDMS-protecting group. N⁶-Hydroxyagelasine D and the analog carrying a geranylgeranyl side chain displayed profound antimicrobial activities against several pathogenic bacteria and protozoa and inhibited bacterial biofilm formation. However these compounds were also toxic towards mammalian fibroblast cells (MRC-5). The spectral data of N⁶-hydroxyagelasine D did not match those reported for ageloxime D before. Hence, a revised structure of ageloxime D was proposed. Basic hydrolysis of agelasine D gave (+)-N-[4-amino-6-(methylamino)pyrimidin-5-yl]-N-copalylformamide, a compound with spectral data in full agreement with those reported for (?)-ageloxime D.     

Control of fluid secretion by isolated salivary glands of the lone star tick.

Adrenaline, noradrenaline, cyclic-AMP, and theophylline stimulated salivary fluid secretion in glands isolated from rapidly engorging females of the lone star tick, Amblyomma americanum. Cyclic-AMP and theophylline effectively initiated secretion only after the glands were pre-incubated and pre-stimulated to secrete with adrenaline. Pilocarpine nitrate, 5-hydroxytryptamine (5-HT), and glutamate did not stimulate glandular secretion, 2,4-Dinitrophenol (DNP) slowed adrenaline's ability to stimulate secretion. Results obtained after additions of harmaline and ouabain point to the possibility of a (Na⁺+K⁺)-ATPase system for helping to move fluid across the glandular epithelium. The significance of these findings to the mechanisms and control of tick salivary glandular function is discussed.     

Ascaris lumbricoides and Ascaridia galli: Biogenic amines in adults and developmental stages

Ascaris lumbricoides var. hominis and Ascaridia galli contain 5-hydroxytryptamine, histamine, dopamine, and norepinephrine. The chick parasite showed lower levels of monoamines compared to human ascaris. Amine concentrations in females were higher than in males. In all specimens, 5-hydroxytryptamine was the highest while norepinephrine was found to be uniformly low. The female reproductive organ contained the maximum amount of dopamine while intestine was rich in histamine. A progressive increase in the concentrations of biogenic amines was noticed during development.     

5-Hydroxytryptamine metabolism by the nuclear fraction of rat-liver homogenate

1. The metabolism of 5-hydroxy[1′-¹⁴C]tryptamine creatinine sulphate in the nuclear fraction of rat-liver homogenate was studied. In the incubation mixture five metabolites were found. 2. Two metabolites were not radioactive; one of them was identified as 5-hydroxyindole-3-carboxylic acid and the second tentatively as 5-hydroxyindole-3-aldehyde. 3. 5-Hydroxyindol-3-ylacetic acid, 1′-N-acetyl-5-hydroxytryptamine and 5-hydroxytryptophol were not precursors of 5-hydroxyindolealdehyde and 5-hydroxyindolecarboxylic acid. 4. It was shown that the metabolism of 5-hydroxytryptamine in the nuclear fraction involves monoamine oxidase, the precursor of 5-hydroxyindolealdehyde and 5-hydroxyindolecarboxylic acid being most probably 5-hydroxyindol-3-ylacetaldehyde.     

Microradioisotopic assay for indoleamine N-methyltransferase and analysis of products by liquid chromatography

A rapid method for the separation of tryptamine, 5-hydroxytryptamine, and their N-methylated derivatives is described. The method involves liquid chromatography using a cation exchange column with the eluant monitored either by ultraviolet or fluorescence spectroscopy. The latter technique permits the detection of picogram quantities of indoleamines. Using normal-phase liquid chromatography a complete separation of tryptamine, its N-methylated derivatives, and their β-carboline analogs was also achieved. A radioisotopic assay with the potential to detect indoleamine N-methyltransferase activity in milligram quantities of rabbit lung tissue was developed. The radioisotopically labeled products formed from a number of substrates in such assays were characterized by liquid chromatography.     

Alkaloids of Duboisia hopwoodii

Leaf and root collections of Duboisia hopwoodii were made from Alice Springs in central and Western Australia. From D. hopwoodii collected at Alice Springs were isolated nornicotine, nicotine, myosmine and N-formylnornicotine; cotinine, N-acetylnornicotine, anabasine, anatabine, anatalline and bipyridyl were detected by GC/MS. Root material contained hyoscyamine, scopolamine, nicotine and nornicotine; N-formylnornicotine was detected by GC/MS. D. hopwoodii from Western Australia yielded nicotine, nornicotine, hyoscyamine and metanicotine. Root material contained nornicotine, hyoseyamine, myosmine and N-formylnornicotine, GC/MS detected cotinine and N-acetylnornicotine.     

N-Substituted acetamide glycosides from the stems of Ephedra sinica

Five new N-mono-/bis-substituted acetamide glycosides, N-{4-O-[3-O-(4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (1), N-methyl-N-{4-O-[3-O-(4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (2), N-methyl-N-{4-O-[3-O-(6-O-benzoyl-4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (3), N-methyl-N-{4-O-[3-O-(6-O-benzoyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (4), and N-methyl-N-{4-O-[3-O-(6-O-trans-cinnamoyl-4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (5), along with one known acetamide derivative, N-methyl-N-(4-hydroxyphenethyl)-acetamide, the shared aglycone of 2–5, were isolated from the ethanol extract of the stems of Ephedra sinica. The structures of these new compounds were elucidated on the basis of extensive spectroscopic examination, mainly including multiple 1D and 2D NMR and HRESIMS examinations, and qualitative chemical tests. All N,N-bissubstituted acetamide glycosides were found to show the obvious rotamerism, as in the case of the isolated known N-methyl-N-(4-hydroxyphenethyl)-acetamide, under the experimental NMR conditions, with the ratios of integrated intensities between anti- and syn-rotamers always being found to be about 4 to 3.     

Alkaloids from Galanthus rizehensis

Two new Amaryllidaceae alkaloid N-oxides, incartine N-oxide (1) and lycorine N-oxide (2) together with one β-carboline alkaloid, 1-acetyl-β-carboline (3) and six known alkaloids namely, incartine (4), N-trans feruloyltyramine (5), lycorine (6), O-methylnorbelladine (7), vittatine (8) and 11-hydroxyvittatine (9) were isolated from Galanthus rizehensis Stern (Amaryllidaceae). The structures of the alkaloids were elucidated by spectroscopic analyses (UV, IR, MS, 1D and 2D NMR). Acetylcholinesterase inhibitory activity potentials of the compounds were also determined.     

The effects of 5-hydroxytryptamine on the isolated and intact circulatory system of the pinto abalone,Haliotis kamtschatkana,and its presence in cardiac and non-cardiac tissues

1. The in vivo and in vitro effects of 5-hydroxytryptamine (5-HT) on the circulatory system of the pinto abalone, Haliotis kamtschatkana, were examined.2. In the isolated heart, 5-HT caused a dose-dependent increase in beat frequency.3. In the intact animal, however, this effect was accompanied by increases in aortic systolic, diastolic, and pulse pressures and, at very high doses, efferent ctenidial pressure.4. The effects of 5-HT were inhibited by several antagonists.5. Furthermore, high levels of 5-HT were found in the tissue samples taken from the auricles, and ventricle and in the hemolymph, and pericardial fluid.6. Therefore, 5-HT may have a role as an excitatory neurotransmitter in the control of circulation in H. kamtschatkana.     

Purification and characterization of an ovulation stimulating substance from the male accessory glands of Drosophila suzukii

An ovulation stimulating substance (OSS) was isolated from males of the fruit fly Drosophila suzukii, and purified to a homogeneous state by a 5-step purification procedure: extraction with 80% methanol, chloroform wash, heat treatment, ion-exchange chromatography, and reverse phase high performance liquid chromatography. Approximately 100-fold purification was obtained thereby yielding 39 μg of OSS from 1000 males for an overall yield of 34%. The OSS is a single peptide consisting of at least 35 amino acid residues and having a molecular weight of 3990. The purified OSS not only initiated ovulation in unmated females but also suppressed their receptivity towards males. The peptide of D. suzukii was found to be effective in the females of D. melanogaster, a species that belong to a different subgroup, but was less effective in a more closely related species, D. pulchrella.     

Mucin of the rat submaxillary gland: influence of the animal's age and sex on the composition of its glucidic fraction

Mucin was isolated from submaxillary glands of young (30 and 45 days) and adult (100 days) male and female rats; it was purified first by fractional precipitation with ethanol and then by zone electrophoresis at pH 6.0 on Pevikon. The mucin contains N-acetylhexosamines (N-acetylglucosamine and N-acetylgalactosamine, 3:2), galactose and sialic acid. The total sugar content of the glycoprotein of adult rats is about twice that of the young animals, while the galactose content remains almost unchanged. Incorporation of [¹⁴C]glucosamine into the rat submaxillary gland mucin starts to increase considerably at the moment of sexual maturity and reaches a constant level (90 days); the increase in biosynthesis velocity is higher in males than in females.