Study of prostaglandins and thromboxanes B2 using mass-spectrometry of secondary ions

Prostaglandins E, F, I2 and thromboxane B2 have been studied by secondary ion mass spectrometry. It is shown that the method is suitable for direct identification of these compounds either as free acids or as their sodium salts. The spectra of the former reveal their structural features, while with the latter information on the molecular weight can be obtained. The limit of detection (about 1 microgram) allows the analysis of prostaglandin solutions of 1 microgram/microliter concentrations used in pharmacological tests.     

Synthesis and antiaggregation activity of prostacyclin analogs. II. Direct synthesis of 15-fluoro-13,14-didehydrocarbacyclin

E- and Z-isomers of 15-fluoro-13,14-dehydrocarbacyclin were synthesized starting from 2,3-epoxy-bicyclo[3.3.0]octan-6-one ethylene ketal with the use of 3-fluoro-1-octynydlithium.BF3 reagent and Wittig condensation. The ratio of isomeric the oxirane opening reaction and Wittig olefinization products was in each case 1:1. The synthesized compounds were identified by 13C NMR spectra. The antiaggregating activity of 5E-isomer was 2 x 10(-4) of the activity of corresponding 15-hydroxy compound, 5Z-isomer being even less active.     

Arachidonoyl amino acids and arachidonoyl peptides: Synthesis and properties

N-Arachidonoyl (AA) derivatives of amino acids (glycine, phenylalanine, proline, valine, γ-aminobutyric acid (GABA), dihydroxyphenylalanine, tyrosine, tryptophan, and alanine) and peptides (Semax, MEHFPGP, and PGP) were synthesized in order to study the biological properties of acylamino acids. The mass spectra of all the compounds at atmospheric pressure electrospray ionization display the most intense peaks of protonated molecular ions; the detection limits for these compounds are 10 fmol per sample. AA-Gly showed the highest inhibitory activity toward fatty acid amide hydrolase from rat brain (IC₅₀ 6.5 μM) among all the acylamino acids studied. AA-Phe, AA-Tyr, and AA-GABA exhibited a weak but detectable inhibitory effect (IC₅₀ 55, 60, and 50 μM, respectively). The acylated amino acids themselves, except for AA-Glu, were stable to the hydrolysis by this enzyme. All the arachidonoylamino acids inhibited cabbage phospholipase D to various degrees; AA-GABA and AA-Phe proved to be the most active (IC₅₀ 20 and 27 μM, respectively). Attempts to detect the biosynthesis of AA-Tyr in homogenates of rat liver and nerve tissue in vitro were unsuccessful; however, AA-dopamine and AA-Phe, the products of its metabolism, were found. The highest contents of these metabolites were detected in liver homogenate and in the brain homogenate, respectively. Acylamino acids exert no cytotoxic effect toward the glioma C6 cells. It was shown that N-acylation of Semax with arachidonic acid results in enhancement of its hydrolytic stability and increases its affinity for the sites of specific binding in rat cerebellum membranes.     

Prediction of the three-dimensional structure of aflatoxin of Aspergillus flavus by homology modelling

Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus spp. The toxic effects of aflatoxins have adverse consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a protein containing a zinc-finger DNA-binding motif. AFLR-Protein three-dimensional model was generated using Robetta server. The modeled AFLR-Protein was further optimization and validation using Rampage. In the simulations, we monitored the backbone atoms and the C-α-helix of the modeled protein. The low RMSD and the simulation time indicate that, as expected, the 3D structural model of AFLR-protein represents a stable folding conformation. This study paves the way for generating computer molecular models for proteins whose crystal structures are not available and which would aid in detailed molecular mechanism of inhibition of aflatoxin.     

Effects of arachidonoyl dopamine, haloperidol, and their mixtures on regeneration of freshwater Hydra attenuata

Arachidonoyl dopamine and haloperidol, both separately and in different combinations, inhibit regeneration of the gastral and basal regions of hydra. In addition, both substances induce stable anomalies of morphogenesis in the form of outgrowths and additional tentacles in gastral regenerates. In the presence of both substances at different combinations, anomalies either do not appear altogether, or exist for a short time, thus suggesting the normalization of morphogenesis. Possible mechanisms underlying the effects of these substances are discussed.     

Polyenic acid 5-hydroxytryptamides and 3-hydroxytyramides as tools for studying of the pre-nervous biogenic monoamine functions

Three main effects of the amides on embryos of opistobranch molluscs, sea urchins and starfish, were revealed. First, a rather independent and clear protective action of 5-HYDROXYTRYPTAMIDES AND 3-HYDROXYTYRAMIDES against cytostatic antagonists of serotonin and dopamine, resp. Second, prevention of developmental abnormalities induced by protein kinase C activators both by hydroxytryptamides and hydroxytyramides. Third, the cytostatic effect of 3-HYDROXYTRIPTAMIDES eliminated or prevented by 5-HYDROXYTRYPTAMIDES. These effects quantitatively depended on the structure of their fatty acids part. Some functionally active regulatory substances similar to 5-HYDROXYTRYPTAMIDES AND 3-HYDROXYTYRAMIDES may exist in the early embryos.     

The Pre-nervous Serotonergic System of Developing Sea Urchin Embryos and Larvae: Pharmacologic and Immunocytochemical Evidence

Forty serotonin-related neurochemicals were tested on embryos and larvae of Lytechinus variegatus and other sea urchin species. Some of these substances (agonists of 5-HT₁ receptors, antagonists of 5-HT₂, 5-HT₃ or 5-HT₄ receptors, and inhibitors of the serotonin transporter, SERT) perturbed post-blastulation development, eliciting changes in embryonic/larval phenotypes typical for each class of receptor ligand. These developmental malformations were prevented completely or partially by serotonin (5-HT) or 5-HT analogs (5-HTQ, AA-5-HT), providing evidence for the putative localization of cellular targets. Immunoreactive 5-HT, 5-HT receptors and SERT were found in pre-nervous embryos and larvae of both L. variegatus and Strongylocentrotus droebachiensis. During gastrulation, these components of the serotonergic system were localized to the archenteron (primary gut), mesenchyme-like cells, and often the apical ectoderm. These results provide evidence that pre-nervous 5-HT may regulate early events of sea urchin embryogenesis, mediated by 5-HT receptors or the 5-HT transporter.     

Arachidonoylcholine and N,N-Dimethylaminoethyl Arachidonate,New Cholinergic Compounds

Choline and N,N-dimethylaminoethyl esters of arachidonic and some other fatty acids were synthesized. Experiments on the embryos and larvae of sea urchins, sensitive to cholinergic compounds, showed that arachidonoylcholine exhibited cholinomimetic activity similar to that of nicotine while N,N-dimethylaminoethyl arachidonate acted as acetylcholine antagonist. The corresponding esters of docosahexaenoic acid were found to manifest similar biological properties.     

New aspects of biosynthesis and metabolism of N-acyldopamines in rat tissues

Possible biosynthetic pathways of N-acyldopamines in rat tissues were compared. It was shown that an insignificant amount of the conjugation products was formed during the incubation of arachidonic acid and dopamine, whereas the substitution of tyrosine for dopamine resulted in the productive biosynthesis of N-arachidonoyldopamine. The biosynthesis presumably involves several closely conjugated enzymatic stages, and free fatty acids rather than their CoA esters served as the starting substrates. The decarboxylation stage probably precedes the stage of catechol system formation, because N-acetyltyramine (a probable intermediate) was easily oxidized by monophenol monooxygenase to N-acyldopamine, whereas N-acyltyrosine is hydrolyzed under these conditions. Biosynthesis of N-acyldopamines in a cell-free medium was accompanied by their methylation. The possibility of oxidative metabolism of N-acyldopamines, which could serve as co-substrates or inhibitors of different oxidoreductases, was shown for the first time.