Effect of N-acylethanolamines on adrenal cortex function

The possibility of NAE to take part in the regulation of the function of adrenal glands was studied. It was shown that two times NAE (18:0) injection in a dose 5 mg/kg of weight increased the content of 11-hydroxysteroids in blood of intact male rats. NAE caused the raise of the blood hormone content by 4 times under the immobilization stress. It is apparent that augment of stress response under the influence of NAE in vivo is explained by the activation of hypophysis-adrenal cortex system. In vitro NAE lowered steroidogenesis by near 40%. One can suggest that this decrease is caused by membranotropic properties of NAE.     

Basic directions of research of the department of lipid biochemistry

The review deals with the scientific activity of the Department of Biochemistry of Lipids of the Palladin Biochemistry Institute of the National Academy of Sciences of Ukraine. The estimation of the functional role of some major lipids and of the minor lipid components, namely, N-acylethanolamines (NAE), is the main problem of the scientific investigations of the Department. The role of some lipids in the pathogenesis of diseases accompanied by the oxidative stress was also studied. The Department was the first to find and identify NAE in neuroblastoma C1300 N18 cells. It was shown that NAE with long saturated acyl chains inhibited veratridine-activated fast sodium channels. NAE also activated uterine plasma membrane smooth muscle Ca2+, Mg(2+)-ATPase, and inhibited Fe(2+)-induced free radical oxidation in mitochondria. The results of these investigations served as a basis for development of pharmacological substances with membrane protective properties. It was shown that, at different diseases accompanied by the oxidative stress, a significant change in the lipid composition of cell membranes occurred. Sometimes these changes were adaptive in character, which favored the cell viability adaptation to pathological conditions. The new level of regulation of adaptive reactions can be accompanied by the development of additive injuries of cell viability, which may be caused by the altered level of biologically active cell lipid components. Based on the results of these investigations, the preparation intended for treatment of male infertility was developed. The pharmacological substances intended for treatment of morphine abuse and acute ischemia of myocardium were created.     

Effect of Pb on leaf antioxidant enzyme activities and ultrastructure of the two ecotypes of <Emphasis Type="Italic">Sedum alfredii</Emphasis> Hance

Hydroponic experiments were conducted to study the effect of Pb on growth, leaf antioxidant enzyme activities, and ultrastructure of the accumulating ecotype (AE) and non-accumulating ecotype (NAE) of Sedum alfredii Hance. AE was found to be more tolerant to excessive Pb levels in growth medium. Concentrations of Pb in the shoots of the AE were 1.98 times higher than those in the NAE when 0.2 mM Pb was supplied. Both chlorophyll a and b did not decrease significantly in AE plants after Pb treatment, while a significant decrease was noted in chlorophyll a and b of NAE plants treated with Pb concentrations greater than 0.05 mM. The results showed that activities of superoxide dismutase (SOD) and catalase (CAT) were elevated in the leaves of AE under Pb stress. However in NAE, Pb-caused enhancement was noticed only in the activity of SOD while activity of CAT was declined as compared to the control plants. With increased Pb level, malondialdehyde (MDA) content increased significantly in both ecotypes of S. alfredii, indicating that Pb toxicity led to lipid peroxidation and membrane damage, but MDA content in the leaves of NAE was always higher than in AE plants. The ultrastructural analysis of the spongy mesophyll cells revealed that excessive Pb concentrations obviously damaged the cell membrane, chloroplasts, and mitochondria of both the ecotypes but damage was more severe in NAE. Although growth, leaf physiology, and ultrastructure of both the ecotypes were affected by Pb treatment, deleterious effects were more pronounced in NAE. This text was submitted by the authors in English.     

Neuroprotective effect of N-acylethanolamines in chronic morphine dependence. II. Effect on the rat brain fatty acid composition

The effect of N-acylethanolamines mixture (NAE) with saturated and unsaturated acyl chains on the fatty acid composition of the rat brain under chronic morphine dependence was investigated. Long-term administration of NAE reduced morphine-induced decrease of mono- and polyunsaturated fatty acids in the rat brain crude lipid extract. Furthermore, NAE restored the acyl chain spectrum, especially the content of docosahexaenoic acid in essential phospholipids--phosphatidylcholine and phosphatidylethanolamine. Pharmacological activity of NAE depended on a dose.     

Lipidomic analysis of N-acylphosphatidylethanolamine molecular species in Arabidopsis suggests feedback regulation by N-acylethanolamines

N-Acylphosphatidylethanolamine (NAPE) and its hydrolysis product, N-acylethanolamine (NAE), are minor but ubiquitous lipids in multicellular eukaryotes. Various physiological processes are severely affected by altering the expression of fatty acid amide hydrolase (FAAH), an NAE-hydrolyzing enzyme. To determine the effect of altered FAAH activity on NAPE molecular species composition, NAE metabolism, and general membrane lipid metabolism, quantitative profiles of NAPEs, NAEs, galactolipids, and major and minor phospholipids for FAAH mutants of Arabidopsis were determined. The NAPE molecular species content was dramatically affected by reduced FAAH activity and elevated NAE content in faah knockouts, increasing by as much as 36-fold, far more than the NAE content, suggesting negative feedback regulation of phospholipase D-mediated NAPE hydrolysis by NAE. The N-acyl composition of NAPE remained similar to that of NAE, suggesting that the NAPE precursor pool largely determines NAE composition. Exogenous NAE 12:0 treatment elevated endogenous polyunsaturated NAE and NAPE levels in seedlings; NAE levels were increased more in faah knockouts than in wild-type or FAAH overexpressors. Treated seedlings with elevated NAE and NAPE levels showed impaired growth and reduced galactolipid synthesis by the "prokaryotic" (i.e., plastidic), but not the "eukaryotic" (i.e., extraplastidic), pathway. Overall, our data provide new insights into the regulation of NAPE-NAE metabolism and coordination of membrane lipid metabolism and seedling development.     

Delta sleep-inducing peptide in the blood and hypothalamus of rats with various resistance to emotional stress

Enzyme immunoassay was used to study delta-sleep peptide content in blood and hypothalamus in rats of Wistar lines under acute emotional stress. It was found that the content of delta-sleep peptide in blood and hypothalamus of stable rats was higher as compared with rats predisposed to emotional stress. After 1.5-hour emotional stress the content of delta-sleep peptide increased in blood and hypothalamus both in stable rats and predisposed ones. After 3-hour stress there was an increase in delta-sleep peptide content in hypothalamus, and contrary to its decrease in blood in both stable and predisposed animals. It is supposed that delta-sleep peptide along with other oligopeptides is one of the factors determining individual animal resistance to emotional stress, which is supported by significant delta-sleep peptide increase in hypothalamus in stable rats.     

Neuroprotective effect of N-acylethanolamines in chronic morphine dependence. I. Rat brain phospholipids as a target of their action

The influence of saturated and unsaturated N-acylethanolamines (NAEs) mixture on the rat brain lipid composition under the chronic morphine dependence was studied. It was shown that the long-term administration of NAE to rats with experimental morphine dependence restored the morphine-induced alterations of the brain phospholipid composition. This effect can probably be explained by a fatty acyl transfer from NAE to sn-1 position of some brain glycerophospholipids. Another reason of NAE beneficial effect could be due to its antioxidative property, thus providing the remodelling of phospholipid composition. The restoration of the brain essential phospholipids is associated with the decline in morphine consumption in rats with experimental morphine dependence. The mechanism of this phenomenon requires further investigations.     

Participation of cyclic nucleotides, protein kinase A and C in realization of n-acylethanolamine action in human adrenocortical cells

The messenger mechanisms mediating N-acylethanolamines (NAE) regulatory signals in the adrenal cortex were studied. An analysis of the mechanisms of realization of NAE effects in the post-operation human adrenal cortex was carried out in vitro. Influence of NAE mix on cAMP and cGMP level, protein kinase A and C activity in sub-cellular fraction of adrenocorticocytes and homogenates of conditionally normal adrenal cortex tissues was investigated. It was shown, that N-acylethanolamines treatment resulted in a decrease of cAMP level in adrenocortical cells. cGMP level is not changed in these conditions. The rise of protein kinase C activity was obtained in the membrane fraction after N-acylethanolamines in vitro treatment (3.3 microg/ml). Activity of cAMP-dependent protein kinase A significantly decreased in cytosol fraction of adrenocorticocytes. It was concluded, that steroid genesis activation is determined by protein kinase C activation, inhibition is determined by cAMP-dependent messenger system.     

Stability of plant vacuolar membranes under the conditions of osmotic stress and influence of redox agents

Stability of plant vacuolar membranes (tonoplast), which underwent two types of osmotic stress and also the effect of redox agents (glutathione in its oxidized (GSSG) and reduced (GSH) forms) and nitric oxide (NO) under various pH values, has been investigated. The fatty-acid content of tonoplast has also been determined. It has been shown that stability of vacuoles decreased under hyperosmotic stress but not under hypoosmotic stress. These effects are unlikely caused by the fatty-acid content of vacuolar membranes. Stability of vacuoles changed under various redox conditions; this process was more intensive under the conditions of hypoosmotic stress in the experiments with oxidized glutathione. The influence of nitrogen oxide was different for various kinds of stress: stability of vacuoles decreased substantially under hypoosmotic stress, while under the conditions of hyperosmotic stress NO elevated the stability level of the vacuolar membranes.     

The distribution of N-acetyl-beta-glucosaminidase, beta-glucuronidase, acid alpha-naphthyl acetate esterase and alpha-naphthyl acetate esterase in subpopulations of thymocytes, bone marrow cells and other lymphoid organs in mice

Thymocytes, bone marrow lymphocytes, as well as lymphocytes from spleen, lymphoid nodes and peripheral blood were obtained from BALB/c mice. Subpopulations of BALB/c bone marrow T-lymphocyte precursors and immature (small) and mature (large) thymocytes, as established by the percentage of terminal deoxynucleotidyl transferase (TdT) and peanut agglutinin (PNA) positive cells, were obtained by centrifugation on discontinuous density gradients. The activities of N-acetyl-beta-glucosaminidase (NAG), beta-glucuronidase (BG), acid alpha-naphthyl acetate esterase (ANAE) and alpha-naphthyl acetate esterase (NAE) were determined by enzymatic assays of cell extracts of the diverse T-lymphocyte subpopulations, in order to follow their evolution with the maturation of the T-lymphocytes in the thymus. These activities were compared with that determined in lymphocytes from spleen, lymphoid nodes and peripheral blood. The glucidases BG and NAG and the esterases ANAE and NAE present a high decrease in their activities from bone marrow T-lymphocyte progenitors to immature thymocytes. BG, NAG and ANAE activities undergo an about 3-fold increase with the evolution of the thymocytes from small to large cells. Whereas the level of the NAE activity decreases (2-fold) with that evolution of the thymocytes. Lymphocytes from spleen and lymphoid nodes exhibit activities of the glucidases and, specially, the esterases marked by higher than those of thymocyte populations. Peripheral blood lymphocytes also present NAG, ANAE and NAE activities higher than in thymocytes, but their BG activity is lower.     

Phospholipid composition of normal and transformed cells cultivated with N-acylethanolamines

Effects of N-stearoylethanolamine (NSE), N-oleoilethanolamine (OEA) and mixture of more than 20 saturated and unsaturated N-acylethanolamines on phospholipids composition of normal and transformed fibroblasts in the culture were compared in the work. It was shown that cultivation of cells NSE decreases percent content of phosphatidylinositol (PI), phosphatidylserine (PS) and sphingomyeline (SM) - important mediators of cell signaling. Under the influence of OEA the level of SM decreases as well, but at the same time PI and PS levels increase. NAEs mixture decreases PS and PI levels in cells and causes phosphatidylcholine (PC) amount increase. Moreover NSE and NAEs mixture decrease the content of main mitochondria membrane lipid - diphosphatidylglicerole (DPG). OEA increases DPG amount. In transformed fibroblasts (line L929) NAE modulate lipid composition as well: NSE decreases the level of phosphatidylethanolamine (PE), OEA and NAEs mixture increase sphingomyeline levels. It is shown that response of normal and transformed fibroblasts on NAE application is different, depending on substance structure and cell-target type.     

HSC70 protein of human blood mononuclears as a sign of adaptation under normobaric hypoxia training

We investigated two isoforms of heat shock protein 70 kDa: HSP70 and HSC70, in the human blood mononuclears under normobaric hypoxia training. It was shown that hypoxia regimen does not lead to manifestation of stress but exerts activation of the organism. The obtained organism adaptation is achieved with a little cost that is confirmed by absence of HSP70 content increase. HSC70 content in the blood mononuclears was increased in most case up to 1.5-2.0 fold. HSC70 displays itself as a sign of adaptation. We connect the increase of HSC70 with mitochondria biogenesis which is given a leading importance under adaptation of aerobic organism cells to hypoxia.     

Lead induced changes in the growth and antioxidant metabolism of the lead accumulating and non-accumulating ecotypes of Sedum alfredii

The phytotoxicity and antioxidative adaptations of lead （Pb） accumulating ecotype （AE） and non-accumulating ecotype （NAE） of Sedum alfredii Hance were investigated under different Pb treatments involving 0, 0.02 mmol/L Pb, 0.1 mmol/L Pb and 0.1 mmol/L Pb/0.1 mmol/L ethylenediaminetetraacetic acid （EDTA） for 6days. With the increasing Pb level, the Pb concentration in the shoots of AE plants enhanced accordingly, and EDTA supply helped 51% of Pb translocation to shoots of AE compared with those treated with 0.1 mmol/L Pb alone. Moreover, the presence of EDTA alleviated Pb phytotoxicity through changes in plant biomass, root morphology and chlorophyll contents. Lead toxicity induced hydrogen peroxide （H2O2） accumulation and lipid peroxidation in both ecotypes of S. alfredii. The activities of superoxide dismutase （SOD）, guaiacol peroxidase （G-POD）, ascorbate peroxidase, and dehydroascorbate reductase elevated in both leaves and roots of AE as well as in leaves of NAE with the increasing Pb levels, but SOD and G-POD declined in roots of NAE. Enhancement in glutathione reductase activity was only detected in roots of NAE while a depression in catalase activity was recorded in the leaves of NAE. A significant enhancement in glutathione and ascorbic acid （AsA） levels occurred in both ecotypes exposed to Pb and Pb/EDTA treatment compared with the control, however, the differences between these two treatments were insignificant. The dehydroascorbate （DHA） contents in roots of both ecotypes were 1.41 to 11.22-fold higher than those in leaves, whereas the ratios of AsA to DHA （1.38 to 6.84） in leaves altering more to the reduced AsA form were much higher than those in roots. These results suggested that antioxidative enzymes and antioxidants play an important role in counteracting Pb stress in S. alfredii.     

N-Acylated phospholipid metabolism and seedling growth: Insights from lipidomics studies in Arabidopsis

N-Acylphosphatidylethanolamines (NAPEs) are precursors of endogenous bioactive lipids, N-acylethanolamines (NAEs). NAPEs, which occur as a minor membrane lipid, are hydrolyzed in a single enzymatic step catalyzed by a type of phospholipase D (PLD) to generate fatty acid ethanolamides. Although, the occurrence of NAPE is widespread in the plant kingdom, the physiological roles remain under appreciated due to the lack of sensitive tools to quantify the pathway metabolites. In Kilaru et al. (2012, Planta, DOI 10.1007/s00425-012-1669-z), comprehensive mass spectrometry (MS)-based methods were developed to gain a clearer understanding of the complex network of metabolites that participate in NAE metabolic pathway. This targeted lipidomics approach allowed insights to be drawn into the implications of altered NAE levels on NAPE content and composition, and the overall regulation of PLD-mediated hydrolysis in Arabidopsis. Based on these results, we point out here the important need for the identification of the precise isoform(s) of PLD in plants that is (are) involved in the regulated hydrolysis of NAPE and formation of NAE lipid mediators in vivo.     

GC/MS analysis of anandamide and quantification of N-arachidonoylphosphatidylethanolamides in various brain regions, spinal cord, testis, and spleen of the rat

Anandamide [N-arachidonoylethanolamide (NAE)] was initially isolated from porcine brain and proposed as an endogenous ligand for cannabinoid receptors in 1992. Accumulating evidence has now suggested that, in the tissue, NAE is generated from N-arachidonoylphosphatidylethanolamides (N-ArPEs) by phosphodiesterase. In this study a sensitive and specific procedure was developed to quantify NAE and N-ArPE, including organic solvent extraction, reverse-phase C-18 cartridge separation, derivatization, and gas chromatography/mass spectrometry (GC/MS) analysis. NAE is converted by a two-step derivatization procedure to a pentafluorobenzoyl ester followed by pentafluoropropionyl acylation. Quantification was performed by isotope dilution GC/MS using deuterium-labeled NAE (NAE-2H8) as an internal standard. The same chemical derivatization was applicable to N-ArPE quantification. The separated N-ArPE fractions were converted by a two-step cleavage/derivatization procedure into the pentafluorobenzoyl ester of NAE and then to its pentafluoropropionyl amide. The derivative was quantified by GC/MS using deuterium-labeled 1,2-[2H8]dioleoyl-sn-glycero-3-phospho(arachidonoyl)ethanolamid e as an internal standard. Using these methods, we have found that endogenous NAE levels in rat brain, spleen, testis, liver, lung, and heart were below the level of quantification achievable (0.1 pmol/mg of protein) but that N-ArPE is readily quantifiable and is widely distributed in the rat CNS with the highest level in the spinal cord. The striatum, hippocampus, and accumbens contain intermediate concentrations of N-ArPE, whereas the value is lowest in the cerebellum.     

Changes in the energy resources of rat gastric mucosa exposed to dopamine agonists and antagonists in conditions of experimental stress

Changes in adenyl nucleotide and glycogen content in rat gastric tissue under the action of stressogenic factor and injection of the dopamine blocker metoclopramide and the dopamine precursor L-DOPA were investigated with the use of the "social stress" method devised by the authors. It was established that stress induced gastric mucosal lesions and reduced the content of adenyl nucleotides and glycogen in the gastric tissue. Preliminary injection of L-DOPA potentiated the effect of stress while metoclopramide inhibited it. The data show a role of energy balance disturbances in the gastric tissue in the pathogenesis of an acute ulcer. In the authors' opinion, the disturbances seen during immobilization psychogenic stress were caused by undue stimulation of central dopamine receptors.     

N-acylethanolamines are metabolized by lipoxygenase and amidohydrolase in competing pathways during cottonseed imbibition

Saturated and unsaturated N-acylethanolamines (NAEs) occur in desiccated seeds primarily as 16C and 18C species with N-palmitoylethanolamine and N-linoleoylethanolamine (NAE 18:2) being most abundant. Here, we examined the metabolic fate of NAEs in vitro and in vivo in imbibed cotton (Gossypium hirsutum) seeds. When synthetic [1-(14)C]N-palmitoylethanolamine was used as a substrate, free fatty acids (FFA) were produced by extracts of imbibed cottonseeds. When synthetic [1-(14)C]NAE 18:2 was used as a substrate, FFA and an additional lipid product(s) were formed. On the basis of polarity, we presumed that the unidentified lipid was a product of the lipoxygenase (LOX) pathway and that inclusion of the characteristic LOX inhibitors nordihydroguaiaretic acid and eicosatetraynoic acid reduced its formation in vitro and in vivo. The conversion of NAE 18:2 in imbibed cottonseed extracts to 12-oxo-13-hydroxy-N-(9Z)-octadecanoylethanolamine was confirmed by gas chromatography-mass spectrometry, indicating the presence of 13-LOX and 13-allene oxide synthase, which metabolized NAE 18:2. Cell fractionation studies showed that the NAE amidohydrolase, responsible for FFA production, was associated mostly with microsomes, whereas LOX, responsible for NAE 18:2-oxylipin production, was distributed in cytosol-enriched fractions and microsomes. The highest activity toward NAE by amidohydrolase was observed 4 to 8 h after imbibition and by LOX 8 h after imbibition. Our results collectively indicate that two pathways exist for NAE metabolism during seed imbibition: one to hydrolyze NAEs in a manner similar to the inactivation of endocannabinoid mediators in animal systems and the other to form novel NAE-derived oxylipins. The rapid depletion of NAEs by these pathways continues to point to a role for NAE metabolites in seed germination.     

Profiling the Cross Reactivity of Ubiquitin with the Nedd8 Activating Enzyme by Phage Display

The C-terminal peptides of ubiquitin (UB) and UB-like proteins (UBLs) play a key role in their recognition by the specific activating enzymes (E1s) to launch their transfer through the respective enzymatic cascades thus modifying cellular proteins. UB and Nedd8, a UBL regulating the activity of cullin-RING UB ligases, only differ by one residue at their C-termini; yet each has its specific E1 for the activation reaction. It has been reported recently that UAE can cross react with Nedd8 to enable its passage through the UB transfer cascade for protein neddylation. To elucidate differences in UB recognition by UAE and NAE, we carried out phage selection of a UB library with randomized C-terminal sequences based on the catalytic formation of UB∼NAE thioester conjugates. Our results confirmed the previous finding that residue 72 of UB plays a “gate-keeping” role in E1 selectivity. We also found that diverse sequences flanking residue 72 at the UB C-terminus can be accommodated by NAE for activation. Furthermore heptameric peptides derived from the C-terminal sequences of UB variants selected for NAE activation can function as mimics of Nedd8 to form thioester conjugates with NAE and the downstream E2 enzyme Ubc12 in the Nedd8 transfer cascade. Once the peptides are charged onto the cascade enzymes, the full-length Nedd8 protein is effectively blocked from passing through the cascade for the critical modification of cullin. We have thus identified a new class of inhibitors of protein neddylation based on the profiles of the UB C-terminal sequences recognized by NAE.     

Chk1 inhibitor SCH 900776 enhances the antitumor activity of MLN4924 on pancreatic cancer

MLN4924 inhibits the cullin-RING ligases mediated ubiquitin-proteasome system, and has showed antitumor activities in preclinical studies, but its effects and mechanisms on pancreatic cancer (PC) remains elusive. We found that MLN4924 inhibited the proliferation and clonogenicity of PC cells, caused DNA damage, particularly double-strand breaks, and leaded to Chk1 activation and cell-cycle arrest. Chk1 inhibitor SCH 900776 alone exhibited minimal cytotoxicity, and caused no DNA damage on PC cells. But in the combination therapy, SCH 900776 enhanced the cytotoxicity and DNA damage caused by MLN4924, likely by abrogating G2/M arrest and promoting DNA re-replication. In vivo study on a xenograft PC mouse model also showed that SCH 900776 increased the efficacy of MLN4924. We also evaluated the level of NEDD8-activating enzyme (NAE), the direct target of MLN4924, and found that NAE level was elevated in PC tissues compared with normal pancreas, but was irrelevant with prognosis. Our findings provide the preclinical evidence and the rationale of the combination therapy of MLN4924 with SCH 900776 or other Chk1 inhibitors to treat PC.     

Humoral factors of the spleen in the regulation of the calcium content in blood plasma of rats]

Introduction of protein-free peptides-enriched spleen extract increases the calcium content in blood plasma of rats. After the effect of stress factor (long swimming) it falls. The value of the index under study increases in the splenectomized rats and remains unchanged after stress and introduction of the spleen factors. The most pronounced increase of the calcium concentration is observed in the case of experimental hypersplenism induced by methylcellulose introduction. The calcium-regulating effects of introduction of the enriched protein-free spleen extract and pharmacopoeial splenin preparation are compared. It is supposed that spleen contains two humoral factors of different chemical nature which are able to influence somewhat differently the calcium content in blood plasma.