The Influence of the Cholesterol Level in Cells on Endovanilloid Cytotoxicity.

The influence of the cellular cholesterol content on the cytotoxicity of endovanilloids acyldopamines was studied in MDA-MB-231 and MCF 10A cells. The activity of acyldopamines depends on the cellular cholesterol content, and a decrease in cholesterol content increases the cytotoxicity of acyldopamines.

Neuroprotective and Antioxidant Activity of Arachidonoyl Choline, Its Bis-Quaternized Analogues and Other Acylcholines.

The antioxidant activity and protective effect in the toxicity model of H2O2 were studied for arachidonic (AA-CHOL), docosahexaenoic (DHA-CHOL), linoleic (Ln-CHOL), and oleic (Ol-CHOL) fatty acids, as well as arachidonoyl dicholine (AA-diCHOL) and O-arachidonoyl bistetramethylaminoisopropanol (ABTAP). AA-CHOL, DHA-CHOL and Ln-CHOL provided a 20% increase in cell survival. AA-CHOL, AA-diCHOL, Ol-CHOL, and ABTAP had a radical-scavenging effect in the ABTS test, approximately equal to the activity of a standard radical scavenger Trolox.

Selective Action of N-Arachidonoyl Dopamine on Viability and Proliferation of Stromal Cells from Eutopic and Ectopic Endometrium.

We performed a comparative study of the cytotoxic effect of endocannabinoid N-arachidonoyl dopamine (AA-DA) on cultured stromal cells of ectopic and eutopic endometrium. It was found that AA-DA in the concentration range of 1-20 µM produces more selective cytotoxic effect on the stromal cells of the ectopic endometrium due to interaction with cannabinoid type 1 receptor. In concentrations below 1 µM, AA-DA stimulated the proliferation of stromal cells of the eutopic endometrium and did not affect the division of ectopic endometrium cells. This effect was realized due to its interaction with cannabinoid type 2 receptor.

Neuroprotective Action of Amidic Neurolipins in Models of Neurotoxicity on the Culture of Human Neural-Like Cells SH-SY5Y.

It was established that in neurodegeneration models in the human neuron-like cell line SH-SY5Y, amide derivatives of arachidonic and docosahexaenoic acids were inactive in experiments with MPP+ and CoCl2 but protected from H2O2. The protective activity of neurolipins decreased in the series DHA-DA > AA-SER ≥ AA-GLY > AA-GABA ≥ AA-EA and was manifested starting from a concentration of 0.5 nM.

Neuroprotective Properties of Endocannabinoids N-Arachidonoyl Dopamine and N-Docosahexaenoyl Dopamine Examined in Neuronal Precursors Derived from Human Pluripotent Stem Cells.

Neuroprotective properties of endocannabinoids N-arachidonoyl dopamine (NADA) and N-docosahexaenoyl dopamine (DHDA) were examined in neuronal precursor cells differentiated from human induced pluripotent stem cells and subjected to oxidative stress. Both compounds exerted neuroprotective activity, which was enhanced by elevating the concentration of the endocannabinoids within the 0.1-10 µM range. However, both agents at 10 µM concentration showed a marked toxic effect resulting in death of ~30% of the cells. Finally, antagonists of cannabinoid receptors as well as the receptor of the TRPV1 endovanilloid system did not hamper the neuroprotective effects of these endocannabinoids.

Expression of Type I Cannabinoid Receptors at Different Stages of Neuronal Differentiation of Human Fibroblasts.

Differential expression of type 1 cannabinoid receptors (CR1) was evaluated at different stages of human skin fibroblast transformation into terminally differentiated neurons. Immunocytochemical staining detected no CR1 on fibroblasts, but their transformation into induced pluripotent stem cells was accompanied by marked stimulation of CR1 expression. In neuronal precursors, the receptors were located mainly on cell bodies and at the base of their processes. This distribution was retained at the terminal stage of differentiation of induced pluripotent stem cells into neurons.

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.

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 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.

[The optimization of the nitric oxide quantitative analysis for its determination in the cultural medium of mammalian cell culture].

The protocol for the quantitative analysis of nitric oxide as nitrite-ion suitable for determination of its production by a mammalian cell culture was developed. The optimal results were obtained using microvolume-adjusted Griess method after the preliminary reduction of NO3- to NO2- with non-activated cadmium. The protocol was verified on a rat glioma C6 cell culture. The developed method may be used for the nitric oxide determination in 96-well and 48-well microplates; the detection limit is 2.1 ± 0.1 µM for NO2- and 2.9 ± 0.1 µM for NO3-.

[GABA-ergic mechanism of cerebrovascular and antiischemic effects of docosahexaenoic acid].

In experiments on rats, measurements of the local blood flow in the cortex of cerebrum with the aid of a laser Doppler flow meter showed that docosahexaenoic acid (DHA) enhanced the local cerebral circulation in animals with global transient cerebral ischemia, while not influencing that in intact animals. This vasodilatory effect of DHA in ischemized rats is blocked by bicuculline (specific GABA(A) receptor blocker), which is indicative of a GABA-ergic mechanisms of the vascular tone regulation. The results of radioligand binding assay in vitro showed the possibility of direct DHA interaction with cerebrovascular GABA(A) receptors.

[STUDYING NEUROPROTECTIVE ACTIVITY OF GABA CONJUGATE WITH ARACHIDONIC ACID UNDER CONDITIONS OF LOCAL PERMANENT BRAIN ISCHEMIA IN RATS].

We have studied the effect of a GABA conjugate with arachidonic acid (AA) on the morphological state of rat brain tissues after left median cerebral artery occlusion. The results showed that a 6- and 12-day course administration of the GABA - AA conjugate at dose of 2 mg/kg (i.p.) in rats with this model of local permanent brain ischemia led to significant recovery processes in brain tissues. The tissue morphology pattern in the group of animals treated with the GABSA - AA conjugate for 12 days was almost identical to that in intact tissues.

[A new fluorescent analogue for the investigation of anandamide transport in cell cultures].

For the first time a new fluorescent analogue of anadamide incorporating BODIPY®-FL-fluorophore, attached to arachidonic acid via 2,2'-(ethylenedioxy)-bis(ethylenediamine), was prepared. Using rat glioma C6 cells it was demonstrated that the fluorescent analogue is a substrate of the cellular anandamide uptake system (Km 4.5 ± 0.9 µM, Vmax 20 ± 1 amol/(min x cell)).

Effect of N-arachidonoyl dopamine on activity of neuronal network in primary hippocampus culture upon hypoxia modelling.

We studied the effect of endocannabinoid N-arachidonoyl dopamine on spontaneous bioelectric activity of cultured hippocampal neurons in a model of hypoxia/reoxygenation. Incubation under hypoxic conditions induced irreversible decrease in spontaneous bioelectric activity of neurons and their death. Application of N-arachidonoyl dopamine during hypoxia and in the post-hypoxic period preserved bioelectric activity and viability of neurons. The protective effect of N-arachidonoyl dopamine was primarily mediated by type I cannabinoid receptors.

[Antiaggregation activity of arachidonic acid conjugates with neurotropic peptides proglyprol and semax].

The influence two original derivatives of a therapeutically important peptide, bearing arachidonic acid residue with semax and proglyprol, upon platelet aggregation have been studied in vitro. It is established that both derivatives, in contrast to the parent peptide, possess moderate anti-aggregant properties and produce a dose-dependent decrease in the interplatelet interaction induced by ADP, epinephrine, and arachidonic acid within the concentration range of 0.018 - 1.8 mM. This activity was more pronounced for arachidonoylsemax in comparison with arachidonoylproglyprol.

[Cerebrovascular effects of GABA-prostaglandin E2 conjugate in comparison with prostaglandin E2].

Experiments have shown that GABA conjugate with prostaglandin E2 enhances cerebral blood flow in rats after global transient ischemia, while not affecting the cerebral hemoperfusion in intact animals. It is established that cerebrovascular activity of the GABA conjugate with prostaglandin E2 under conditions of cerebral ischemia is based on GABAergic mechanisms of vascular tone regulation, since it is removed by GABA(A)-receptor blocker bicuculline. At the same time, cerebral blood flow of intact rats and rats after global transient ischemia of brain is equally enhanced by prostaglandin E2 alone. This effect is not neutralized by bicuculline.

[Peculiarities of cerebrovascular effects of GABA conjugate with docosahexaenoyl dopamine under conditions of separate and combined vascular pathology of brain and heart].

A GABA conjugate with docosahexaenoyl dophamine (DHED) enhanced local cerebral blood flow in rats under conditions of global transient cerebral ischemia, experimental myocardial infarction, and combined vascular pathology of brain and heart. At the same time, the GABA-DHED conjugate did not influence brain hemoperfusion in intact animals. The cerebrovascular effect of this conjugate is determined by its direct action on the vascular tone, since no changes in blood pressure have been observed. Under conditions of the combined vascular pathology of brain and heart, the cerebrovascular effect of GABA-DHED conjugate is inhibited by bicuculline, which is evidence for the involvement of GABAergic mechanisms in the drug action upon cerebrovascular tone.

[Isolation of expressed in E. coli human interferon beta1b (Ser17) by ion-exchange chromatography].

A method for isolation of interferon beta1b (Serl7) from inclusion bodies, comprising the steps of solution and reduction of protein from the inclusion bodies, refolding, chromatography on DEAE-Sepharose, chromatography on SP-Sepharose, concentrating, desalting and addition of stabilizers. The solution of reduced protein was diluted with pH 8.0 buffer of 50 mM Tris-HCl, 25 microM CuCl2 and 0.5% Twin 20 for refolding. We used gradient of pH (from 9.3 upto 11.3) for elution of interferon-beta from cation-exchange column. We concentrated of eluate and then desalted on the Sephadex G-50 column with 1 mM NaOH. Then the protein solution was neutralized with mannitol and Na-phosphate. Obtained preparation of interferon-beta was pure by gel-electrophoresis and by HPLC analysis, and had practically indentical level of antiproliferative activity with well-known preparation of Betaferone. Thus we show the possibility of isolation and obtaining of pure and active interferone-beta by ion-exchange chromatography in the presence of non-ion detergent Twin 20. We believe this method for interferon betalb preparation is perspective for scaling and using in the develop of industrial technology for production of this preparation.

In vitro effects of anandamide and prostamide e2 on normal and transformed nerve cells.

We studied the effects of endocannabinoid anandamide and its cyclooxygenase derivative prostamide E2 on cultured cerebellar granular cells and C6 glioma cells from rats. Prostamide E2 prevented apoptosis in cerebellar neurons induced by potassium deprivation of cultures, while anandamide had no neuroprotective properties. Prostamide E2 did not modulate the survival rate of glioma cells, while anandamide produced a cytotoxic effect. Our results indicate that cyclooxygenase transformation of anandamide is followed by the loss of antitumor activity of this agent. By contrast, prostamide E2 exhibited strong neuroprotective properties.

[Cerebrovascular effects of GABA conjugate with arachidonic acid under conditions of separate and combined vascular pathology of brain and heart].

Experiments on rats showed that, under conditions of global transient ischemia, a conjugate of GABA with arachidonic acid enhances the local cerebral blood flow due to a decrease in the vascular tone. In intact rats, the examined neurolipin did not show unidirectional changes in the cerebral perfusion. Under conditions of experimental myocardial infarction and combined vascular pathology of brain and heart, the GABA conjugate with arachidonic acid increased the blood flow in the parietal region of brain cortex in most experiments, while decreasing the level of blood pressure.