M. G. Akimov

Sulfation of N-acyl dopamines in rat tissues

Sulfation of N-acyl dopamines has been shown for the first time in cytosolic fractions of rat liver and nervous system. Sulfation of dopamine amides of docosahexaenoic and oleic acids occurred in all tissues studied, N-arachidonoyl dopamine was sulfated in the liver and spinal cord, and N-stearoyl dopamine was sulfated only in the liver. Depending on the substrate and tissue, the sulfation activity varied from 0.5 to 3.5 nmol/min per mg total protein. Kinetic parameters of N-docosahexaenoyl dopamine sulfation in the brain were determined. The findings characterize the sulfation system as the most productive metabolic pathway of N-acyl dopamines, but the role of this system in the body is unclear because of high Km value.

N-acyl dopamines induce cell death in PC12 cell line via induction of nitric oxide generation and oxidative stress.

It was shown that dopamine amides of arachidonic, oleic, and docosahexaenoic acids exhibit toxicity with respect to PC12 pheochromocytoma cell line. The mechanism of realization of the cytotoxic effect of acyl dopamines is the induction of oxidative stress. This event is preceded by triggering the synthesis of nitric oxide.

Methods of Protein Digestive Stability Assay - State of the Art

The evaluation of protein stability in digestive tract is an important topic in various fields of biomedical sciences. The most straightforward task within it is the evaluation of food quality, as during the food processing the digestibility of a protein could fall dramatically. Another task, tightly connected with the previous one, is the evaluation of protein allergenicity. This parameter is shown to be dependent on protein’s digestibility, the less digestible ones having the highest probability to induce immune response. Finally, the task of development of new peptide drugs also requires digestive stability check for per os drug forms that are the most convenient for a patient.

Docosahexaenoyl dopamine in freshwater hydra: Effects on regeneration and metabolic changes

The effects of docosahexaenoyl dopamine and docosahexaenoic acid on the regeneration of hydra gastric and basal fragments are studied. Docosahexaenoyl dopamine induced morphogenetic abnormalities such as single ectopic tentacles in the gastric region and projections in the gastric and basal regions. Docosahexaenoic acid had no effect on the morphogenesis except for a mild slowing of the regeneration rate. Since no hydrolysis of docosahexaenoyl dopamine was detected in hydra extract, it was assumed that the morphogenetic effect could be associated with the dopamine component of this complex.

Investigation of peptide stability upon hydrolysis by of fragments of the organs of the gastrointestinal tract of rats

The hydrolytic stability of a range of cyclic tripeptides, including the therapeutically important dalargin and stemokin, as well as peptides modified by ibuprofen and aspirin, has been studied. The first two experimental systems used utilized purified enzymes (pepsin, trypsin, and chymotrypsin), while the second one utilized fragments of the stomach and small intestine of rats. The linear peptides containing only L-amino acid residues were shown to be hydrolyzed by stomach and intestine fragments, although some of these peptides were resistant to hydrolysis by individual enzymes. The peptides containing D-amino acid residues and cyclic peptides were stable under all of the conditions used, but the peptides modified by aspirin lost the acetyl group of the aspirin moiety in acidic media, this process being accelerated in the presence of pepsin.

N-acyl dopamines induce apoptosis in PC12 cell line via the GPR55 receptor activation

Dopamine amides of arachidonic, docosahexaenoic, and oleic acids were found to induce apoptosis in PC12 cells, which was blocked exclusively by antagonists and preincubation agonists of the receptor GPR55, belonging to the group of non-CB1/CB2 receptors.

Optimization of the procedure of nitrogen oxide quantitation in mammalian cell culture media

A procedure for quantitative analysis of nitric oxide production by cultured mammalian cells based on nitrite ion quantitation has been optimized. The Griess assay adapted for microvolumes was found to yield optimal results when the NO3− ions formed from NO2− ions in the reaction medium were reduced by nonactivated cadmium prior to the assay to form the analyte NO2−. The assay was verified for the culture of C6 rat glioma cells. The assay developed is suitable for quantitative determination of nitrogen oxide in 96- and 48-well cell culture plates; the detection limit for NO2− was 2.1 ± 0.1 μM and that for NO3− was 2.9 ± 0.1 μM.

A new fluorescent analogue for the studies of anandamide transport in cell cultures

A new fluorescent analogue of anandamide bearing a BODIPY®-FL-fluorophore and linked to arachidonic acid via a 2,2′-(ethylenedioxy)-bis(ethylenediamine) residue was prepared. The fluorescent analogue was demonstrated to be a substrate of the cell anandamide uptake system (Km 4.5 ± 0.9 μM, Vmax 20 ± 1 amol/(min cell)) in rat glioma C6 cells.

Synthesis of N-acyl derivatives of Pro-Gly-Pro-Leu peptide: Proteolytic stability in vitro and effects on mouse macrophage cells RAW264.7

Acetyl, oleoyl, arachidonoyl, and docosahexaenoyl derivatives of the Pro-Gly-Pro-Leu peptide with a chemical purity of 99.8% were synthesized. The degradation kinetics of the Pro-Gly-Pro-Leu derivatives under the action of leucine aminopeptidase, nasal mucus, and microsomal fraction of the brain and blood of rats was studied. It was shown that the N-acyl derivatives of Pro-Gly-Pro-Leu proved to be more resistant to the action of leucine aminopeptidase and other enzyme systems. The study of the cytotoxic and anti-inflammatory activity of preparations on the mouse macrophage cell line RAW264.7 showed that acylation with oleic and arachidonic acid makes the peptide cytotoxic with LC50 in the range of 70–15 μM and gives it anti-inflammatory properties with EC50 of 32 and 36 μM, respectively.

N-Acyl Dopamines Induce Apoptosis in PC12 Cell Line via the O-1918-Sensitive non-CB1-non-CB2 Receptor

Dopamine amides of long chain fatty acids (NADA) are a family of endogenous mammalian lipids with an unknown function; they are anti-proliferative for many cancer cell lines. The aim of this study was to identify the NADA receptor responsible for cell death induction. Using PC12 cells treated with NADA in combination with various receptor blockers, or pre-treated with the known receptor agonists to down-regulate the proposed receptor, NADA were shown to induce apoptosis. This activity was blocked only by the non-CB1-non-CB2 receptor antagonist O-1918, and by the pre-treatment with the agonists of this receptor ethanolamides of palmitic and oleic acids.