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The relevance of studying the regulation of protein-ligand interactions is due to the emergence of new views on the role of metabolites and their key importance in vital processes. To study the protein-ligand interaction, the AB0 antigen-antibody blood system and the enzyme-substrate system of dehydrogenases were used as a test system, and ethanol was used as an influencing factor. In experiments performed with A and B blood erythrocyte antigens, natural AB0 system antibodies and monoclonal antibodies under the influence of ethanol performed change of the degree of agglutination and the time to onset of erythrocyte agglutination. It was found that ethanol can regulate the enzyme-substrate interactions of dehydrogenases: lactate dehydrogenase (EC 18.104.22.168), glyceraldehyde phosphate dehydrogenase (EC 22.214.171.124), and α-glycerol phosphate dehydrogenase (EC 126.96.36.199). The increase in the activity of studied enzymes under the influence of ethanol in the whole blood hemolysate was 2.5 - 3 times higher than in the isolated medium (with pure enzyme preparations).
Inferences on the development and morphology of extinct brachiopods must be informed by the ontogeny and shell ornamentation of extantbrachiopods. Although the adult shells of extant brachiopods are wellstudied,detailed descriptions of the embryonic and juvenile shells of extant lingulides are lacking. Here, we describe in detail the shells of juveniles of Lingula anatina Lamarck, 1801 from Vietnam and the Republic of the Philippines. The following previously unknown properties of the lingulide shell are described: (i) a distinct border between the protegulum and the brephic shell; (ii) drapes that develop on both the protegulum and brephic shell; and (iii) the notched anterior margin of the brephic shell. The drapes and cogs on the brephic shell may becaused by the formation of setal follicles during the planktonic stage. Specimens of L. anatina from the Philippines have larger brephic shells than those from Vietnam probably because the former have a longer planktonic stage. Based on comparisons of the first-formed shells of extant brachiopods with published data on fossil brachiopods, we suggest that the life cycle of extant lingulides, in which planktotrophic juveniles with a shell hatch from the egg envelopes, is the mostevolutionarily advanced brachiopod life cycle and appeared in the Early Silurian. We suggest criteria for determining the type of life cycle based on the structure of the first-formed shell of brachiopods. Finally, we consider hypothetical scenarios of life cycles of fossil brachiopods including true planktotrophic larvae in the Cambrian linguliforms.
Brachiopoda is a relict phylum of marine benthic animals that have not been adequately studied with modern microscopy methods. Microscopic study may provide useful information on the evolution of the brachiopod body plan and brachiopod phylogeny. Understanding the organisation of the coelomic system is important because of its role in body form and compartmentalisation. Most brachiopods are considered to have a bipartite coelomic system; the only known exception is Lingulida, which have a tripartite coelomic system. In the present study, we provide the first complete 3D reconstruction of the coelomic system in the craniide brachiopod Novocrania anomala (Müller, 1776). Its coelomic system consists of the following five main parts, which are entirely separated from each other: 1) a pair of large brachial canals; 2) a complex system of paired small brachial canals and a perioesophageal coelom; 3) frontal coelomic chambers; 4) a main trunk coelom, which includes several semi-detached muscular chambers and mantle sinuses; and 5) a pair of posterior adductors chambers. These results indicate that the coelomic system of N. anomala (and perhaps of other craniides) is complex and cannot be considered to be bipartite or tripartite. The frontmost part of the coelomic system is represented by a pair of frontal chambers, which are considered to be a part of the lophophore but which are derived from dorsal mantle fold extensions and thus may be a part of the trunk coelomic system. A number of similarities were discovered between craniiformean and rhynchonelliformean coelomic systems, including the prominent dorsal projections of the large brachial canals and the morphological features of the perioesophageal coelom. The complex subdivision of the N. anomala trunk coelom is explained by the location and function of muscles, and by the location of several mesenteries
Human leukocyte antigen (HLA) class I molecules play a crucial role in the development of a specific immune response to viral infections by presenting viral peptides at the cell surface where they will be further recognized by T cells. In the present manuscript, we explored whether HLA class I genotypes can be associated with the critical course of Coronavirus Disease-19 by searching possible connections between genotypes of deceased patients and their age at death. HLA-A, HLA-B, and HLA-C genotypes of n = 111 deceased patients with COVID-19 (Moscow, Russia) and n = 428 volunteers were identified with next-generation sequencing. Deceased patients were split into two groups according to age at the time of death: n = 26 adult patients aged below 60 and n = 85 elderly patients over 60. With the use of HLA class I genotypes, we developed a risk score (RS) which was associated with the ability to present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) peptides by the HLA class I molecule set of an individual. The resulting RS was significantly higher in the group of deceased adults compared to elderly adults [p = 0.00348, area under the receiver operating characteristic curve (AUC ROC = 0.68)]. In particular, presence of HLA-A*01:01 allele was associated with high risk, while HLA-A*02:01 and HLA-A*03:01 mainly contributed to low risk. The analysis of patients with homozygosity strongly highlighted these results: homozygosity by HLA-A*01:01 accompanied early deaths, while only one HLA-A*02:01 homozygote died before 60 years of age. Application of the constructed RS model to an independent Spanish patients cohort (n = 45) revealed that the score was also associated with the severity of the disease. The obtained results suggest the important role of HLA class I peptide presentation in the development of a specific immune response to COVID-19.
The Keap1-Nrf2 signaling axis is a validated and promising target for cellular defense and survival pathways. This minireview discusses the potential off-target effects and their impact on future drug development originating from Keap1-targeting small molecules that function as displacement activators of the redox-sensitive transcription factor Nrf2. We argue that small-molecule displacement activators, similarly to electrophiles, will release both Nrf2 and other Keap1 client proteins from the ubiquitin ligase complex. This non-specificity is likely unavoidable and may result in off-target effects during Nrf2 activation by targeting Keap1. The small molecule displacement activators may also target Kelch domains in proteins other than Keap1, causing additional off-target effects unless designed to ensure specificity for the Kelch domain only in Keap1. A potentially promising and alternative therapeutic approach to overcome this non-specificity emerging from targeting Keap1 is to inhibit the Nrf2 repressor Bach1 for constitutive activation of the Nrf2 pathway and bypass the Keap1-Nrf2 complex.
Previous studies have demonstrated the formation of stable complexes between inorganic pyrophosphatase (PPase) and three other Escherichia coli enzymes – cupin-type phosphoglucose isomerase (cPGI), class I fructose-1,6-bisphosphate aldolase (FbaB) and l-glutamate decarboxylase (GadA).
Here, we determined by activity measurements how complex formation between these enzymes affects their activities and oligomeric structure.
cPGI activity was modulated by all partner proteins, but none was reciprocally affected by cPGI. PPase activity was down-regulated upon complex formation, whereas all other enzymes were up-regulated. For cPGI, the activation was partially counteracted by a shift in dimer ⇆ hexamer equilibrium to inactive hexamer. Complex stoichiometry appeared to be 1:1 in most cases, but FbaB formed both 1:1 and 1:2 complexes with both GadA and PPase, FbaB activation was only observed in the 1:2 complexes. FbaB and GadA induced functional asymmetry (negative kinetic cooperativity) in hexameric PPase, presumably by favoring partial dissociation to trimers.
These four enzymes form all six possible binary complexes in vitro, resulting in modulated activity of at least one of the constituent enzymes. In five complexes, the effects on activity were unidirectional, and in one complex (FbaB⋅PPase), the effects were reciprocal. The effects of potential physiological significance include inhibition of PPase by FbaB and GadA and activation of FbaB and cPGI by PPase. Together, they provide a mechanism for feedback regulation of FbaB and GadA biosynthesis.
These findings indicate the complexity of functionally significant interactions between cellular enzymes, which classical enzymology treats as individual entities, and demonstrate their moonlighting activities as regulators.
We review the diversity and development of archegonia, the female reproductive organs of land-plant gametophytes. The archegonium is a uniquely land-plant structure, and studies of its evolution benefit from use of a comparative approach in a phylogenetic context. Archegonia of most land plants share a common developmental motif, here termed a T-shaped pattern. A primary axial cell produces a primary cover cell and a central cell by horizontal division. The upper cell usually divides vertically and the lower one horizontally. In mosses such as Atrichum, the T-shaped stage is shifted towards the end of archegonium development, whereas in vascular plants it appears at the beginning of development, but these stages are still probably homologous. The fully exposed archegonia are traditionally viewed as an ancestral (plesiomorphic) condition in land plants, but there is no direct support for this view. We speculate that the fully exposed condition is derived and synapomorphic for setaphytes (mosses and liverworts). The fully sunken hornwort archegonia may be similar to the ancestral type of land-plant archegonia. Developmental evidence suggests that archegonium necks of setaphytes and tracheophytes are not homologous to each other. The neck wall of pteridophytes is composed of four-celled tiers, and one such tier is present in gymnosperms with motile male gametes. Neck-cell arrangement is much more plastic in archegonia of gymnosperms with sperm cell delivery by pollen tube (siphonogamy), in which the neck plays a role similar to pollen-tube transmitting tissue of angiosperms. Angiosperm synergids are probably homologues of gymnosperm neck cells, and the angiosperm egg cell is probably homologous to the ventral canal cell of gymnosperms. Developmental genetic bases of archegonium diversity in land plants remain to be understood. Even descriptive developmental data are currently missing or controversial for some key lineages of land plants.
Breast cancer (BC) is the leading cause of death from malignant neoplasms among women worldwide, and metastatic BC presents the biggest problems for treatment. Previously, it was shown that lower expression of ELOVL5 and IGFBP6 genes is associated with a higher risk of the formation of distant metastases in BC. In this work, we studied the change in phenotypical traits, as well as in the transcriptomic and proteomic profiles of BC cells as a result of the stable knockdown of ELOVL5and IGFBP6 genes. The knockdown of ELOVL5 and IGFBP6 genes was found to lead to a strong increase in the expression of the matrix metalloproteinase (MMP) MMP1. These results were in good agreement with the correlation analysis of gene expression in tumor samples from patients and were additionally confirmed by zymography. The knockdown of ELOVL5 and IGFBP6 genes was also discovered to change the expression of a group of genes involved in the formation of intercellular contacts. In particular, the expression of the CDH11 gene was markedly reduced, which also complies with the correlation analysis. The spheroid formation assay showed that intercellular adhesion decreased as a result of the knockdown of the ELOVL5 and IGFBP6 genes. Thus, the obtained data indicate that malignant breast tumors with reduced expression of the ELOVL5 and IGFBP6 genes can metastasize with a higher probability due to a more efficient invasion of tumor cells.
The review addresses and highlights the main results of research on the physicohemical properties of single-phase and composite materials based on transition metal oxides in relation to their practical application as electrode materials for symmetrical solid oxide fuel cells. The electronic structures and thermodynamic stability of transition metal oxides with the perovskite structure are discussed. A detailed consideration is given to the thermal behaviour, chemical stability, electrical conductivity and electrochemical properties of a broad range of electrode materials based on iron-, chromium- and manganese-containing perovskite-like oxides and oxides that crystallize in other structure types. The analysis revealed the most promising compositions of electrode materials for symmetrical solid oxide fuel cells and effective approaches to the improvement of their functional characteristics.
MicroRNAs (miRNAs) are short RNA molecules regulating multiple cellular processes through posttranscriptional gene silencing. Over the last decade, miRNA have been found in extracellular space and have been consistently shown to mediate functional communication between cells. While it remains widely accepted that miRNA transfer between cells occurs via extracellular vesicles (EVs), multiple other carriers of cell-free miRNA have been described. In addition, some studies have demonstrated that both miRNA and their binding partners, Argonaute proteins, remain hardly detectable in common isolates of EVs. In this work, we summarize the state-of-the-art mechanisms of miRNA sorting and secretion, discuss methodological challenges associated with extracellular miRNA research, and suggest experimental steps to resolve current inconsistencies in the field of miRNA-mediated cell-cell communication.
Thismia is characterized by an exceptionally complicated floral morphology that is currently not understood properly. In the taxonomic literature, descriptive rather than morphological terms are often applied to parts of the flower in Thismia, relating to the general appearance of the floral organs instead of their precise homologies. Precise understanding of the floral structure is complicated by the rarity of Thismia spp. and the paucity of appropriate material. Here we provide a comprehensive study of reproductive organs of three Thismia spp. (T. annamensis, T. javanica and T. mucronata) including the first investigation of inflorescence architecture and early floral development in Thismiaceae. We found a hitherto unknown diversity of the reproductive shoots in the genus, manifested in the number of floral prophylls (two or three, in contrast to a single prophyll in the vast majority of monocots) and in the branching plane resulting in two distinct inflorescence types, a drepanium and a bostryx. We report the non-acropetal sequence of initiation of floral whorls (with stamens being the last elements to initiate), never previously described in monocots, and the gynoecium composed of completely plicate carpels, also a rare feature for monocots. Floral vasculature is relatively uniform in Thismia, but significant interspecific differences are found in tepal
innervation, including the number of tepal traces; some of these differences are not immediately related to the external tepal morphology. We argue that the annulus, which acts as a roof of the hypanthium, possesses an androecium nature and represents congenitally fused bases of stamen filaments. We describe the stamens as laminar structures, which are also shortly tubular in the distal part of the supraconnective with the adaxial tubular side forming a skirt-like appendage. Finally, the placentas, which are column-like when mature, are initially parietal, becoming secondarily similar to freecentral placentas through schizogenous separation from the ovary wall.
MiRNA isoforms (isomiRs) are single stranded small RNAs originating from the same pri-miRNA hairpin as a result of cleavage by Drosha and Dicer enzymes. Variations at the 5ʹ-end of a miRNA alter the seed region of the molecule, thus affecting the targetome of the miRNA. In this manuscript, we analysed the distribution of miRNA cleavage positions across 31 different cancers using miRNA sequencing data of TCGA project. As a result, we found that the processing positions are not tissue specific and that all miRNAs could be correctly classified as ones exhibiting homogeneous or heterogeneous cleavage at one of the four cleavage sites. In 42% of cases (42 out of 100 miRNAs), we observed imprecise 5ʹ-end Dicer cleavage, while this fraction was only 14% for Drosha (14 out of 99). To the contrary, almost all cleavage sites of 3ʹ-ends (either Drosha or Dicer) were heterogeneous. With the use of only four nucleotides surrounding a 5ʹ-end Dicer cleavage position we built a model which allowed us to distinguish between homogeneous and heterogeneous cleavage with the reliable quality (ROC AUC = 0.68). Finally, we showed the possible applications of the study by the analysis of two 5ʹ-end isoforms originating from the same exogeneous shRNA hairpin. It turned out that the less expressed shRNA variant was functionally active, which led to the increased off-targeting. Thus, the obtained results could be applied to the design of shRNAs whose processing will result in a single 5ʹ-variant.
The review analyzes the potential advantages and problems associated with using HIF
prolyl hydroxylase inhibitors as a treatment for COVID-19. HIF prolyl hydroxylase inhibitors
are known to boost endogenous erythropoietin (Epo) and activate erythropoiesis by
stabilizing and activating the hypoxia inducible factor (HIF). Recombinant Epo
treatment has anti-inflammatory and healing properties, and thus, very likely, will be
beneficial for moderate to severe cases of COVID-19. However, HIF PHD inhibition
may have a significantly broader effect, in addition to stimulating the endogenous Epo
production. The analysis of HIF target genes reveals that some HIF-targets, such as furin,
could play a negative role with respect to viral entry. On the other hand, HIF prolyl
hydroxylase inhibitors counteract ferroptosis, the process recently implicated in vessel
damage during the later stages of COVID-19. Therefore, HIF prolyl hydroxylase inhibitors
may serve as a promising treatment of COVID-19 complications, but they are unlikely to aid
in the prevention of the initial stages of infection.
Hypoxia is an extensively investigated condition due to its contribution to various pathophysiological processes including cancer progression and metastasis formation. MicroRNAs (miRNAs) are well-known post-transcriptional gene expression regulators. However, their contribution to molecular response to hypoxia is highly dependent on cell/tissue types and causes of hypoxia. One of the most important examples is colorectal cancer, where no consensus on hypoxia-regulated miRNAs has been reached so far. In this work, we applied integrated mRNA and small RNA sequencing, followed by bioinformatics analysis, to study the landscape of hypoxia-induced miRNA and mRNA expression alterations in human colorectal cancer cell lines (HT-29 and Caco-2). A hypoxic microenvironment was chemically modeled using two different treatments: cobalt(II) chloride and oxyquinoline. Only one miRNA, hsa-miR-210-3p, was upregulated in all experimental conditions, while there were nine differentially expressed miRNAs under both treatments within the same cell line. Further bioinformatics analysis revealed a complex hypoxia-induced regulatory network: hypoxic downregulation of hsa-miR-148a-3p led to the upregulation of its two target genes, ITGA5 and PRNP, which was shown to be a factor contributing to tumor progression and poor survival in colorectal cancer patients.
In humans, trophoblast hypoxia during placental development can be a cause of serious pregnancy complications, such as preeclampsia and fetal growth restriction. The pathogenesis of these conditions is not fully clear and may be associated with changed expression of some genes and regulatory molecules, including miRNA, in trophoblast cells. The aim of this study was to analyze miRNA profiles and measure the expression of their target genes in a model of trophoblast hypoxia. Human choriocarcinoma BeWo b30 cells were used as a trophoblast model. Hypoxia was induced by cobalt chloride (CoCl2) and an oxyquinoline derivative. MRNA and miRNA expression profiles were evaluated by means of next generation sequencing (NGS); the expression of individual genes was analyzed by PCR. We studied the secondary structure of mRNAs of target genes for those miRNAs whose expression had changed significantly and analyzed potential competition between these miRNAs for the binding site. The observed changes in the expression of the key genes involved in the response to hypoxia confirmed the feasibility of using CoCl2 and the oxyquinoline derivative as hypoxia inducers. The analysis revealed an increase in miR-374 levels following the activation of the hypoxia pathway in our trophoblast model. The changes were accompanied by a reduction in FOXM1 mRNA expression; this mRNA is a target for hsa-miR-374a-5p and hsa-miR374b-5p, which can compete with hsa-miR-21-5p for the binding sites on FOXM1 mRNA. The involvement of FOXM1 in the regulation of the invasive cell potential suggests the role of miR-374 and FOXM1 in the pathogenesis of disrupted trophoblast invasion during placental development as predisposing for fetal growth restriction and preeclampsia.
Analysis of regulatory networks is a powerful framework for identification and quantification of intracellular interactions. We introduce miRGTF-net, a novel tool for construction of miRNA-gene-TF networks. We consider multiple transcriptional and post-transcriptional interaction types, including regulation of gene and miRNA expression by transcription factors, gene silencing by miRNAs, and co-expression of host genes with their intronic miRNAs. The underlying algorithm uses information on experimentally validated interactions as well as integrative miRNA/mRNA expression profiles in a given set of samples. The latter ensures simultaneous tissue-specificity and biological validity of interactions. We applied miRGTF-net to paired miRNA/mRNA-sequencing data of breast cancer samples from The Cancer Genome Atlas (TCGA). Together with topological analysis of the constructed network we showed that considered players can form reliable prognostic gene signatures for ER-positive breast cancer. A number of signatures demonstrated remarkably high accuracy on transcriptomic data obtained by both microarrays and RNA sequencing from several independent patient cohorts. Furthermore, an essential part of prognostic genes were identified as direct targets of transcription factor E2F1. The putative interplay between estrogen receptor alpha and E2F1 was suggested as a potential recurrence factor in patients treated with tamoxifen. Source codes of miRGTF-net are available at GitHub (https://github.com/s-a-nersisyan/miRGTF-net).
Finding morphological differences between cytotypes that are stable throughout their geographical range is important for understanding evolution of polyploid complexes. The ancient monocot lineage Acorus includes two groups, of which A. calamus s.l., an important medicinal plant, is a polyploid complex with a centre of diversity in Asia. European plants are sterile triploids introduced by humans. An early study suggested that plants from temperate Asia are tetraploids, but subsequent work revealed diploids and triploids rather than tetraploids in Asiatic Russia; however, cytotype diversity in Western Siberia is insufficiently known. We document the occurrence of diploids and triploids in Western Siberia. Triploids that do not differ in genome size from European Acorus are abundant in the valley of the river Ob where the ability for extensive vegetative propagation provides ecological advantages. An isolated population of aneuploid triploids with 33 chromosomes is found outside the Ob valley. Flow cytometry provides an efficient tool for identification of aneuploid plants in Acorus. All triploids are sterile, but their flowers develop uniform parthenocarpic fruits. Fruits of diploids usually vary in size within a spadix depending on the number of developing seeds. In contrast to North America, where the native diploid plants differ from the introduced triploids by the absence of a secondary midrib of the ensiform leaf blade, Siberian diploids are similar to triploids in possessing a secondary midrib. We confirm that diploids differ from triploids in the size of air lacunae in leaves, which is determined by cell number rather than cell size in septa of aerenchyma. A combination of spathe width and spadix length measured after the male stage of anthesis shows different (slightly overlapping) patterns of variation between diploids and triploids in our material.
Echiura is small group of unsegmented marine worms that are sometimes abundant in the benthos of all areas of the World Ocean. The study of echiuran morphology and anatomy is important for understanding echiuran biology and the function of benthic communities where echiurans dominate. The current study used paraffin histology, scanning electron microscopy, and 3D reconstruction to describe the anatomy of organ systems in Lissomyema mellita, which is within the tribe Thalassematini of the subfamily Thalassematinae. The body of L. mellita consists of a trunk and a proboscis. The trunk terminates at the anal lobe, which contains many large glands, the secretions of which may be used for processing of the hard substratum where studied animals were found. The proboscis has lateral ciliated grooves that are < 7 µm wide and are probably used for collecting and separating food particles. The coelom is divided into the coelom of the proboscis and the coelom of trunk. The location of proboscis coelom may suggest that the proboscis is derived from the oral segment of the body of a hypothetical segmented echiuran ancestor. The shortness and histological uniformity of the foregut indicates that L. mellita feeds on soft food particles that do not require mechanical processing. The circulatory system has a complex organization and contains several vascular elements that have not been previously described in echiurans: voluminous plexus around the foregut, voluminous dorsal and ventral lacunes, and additional vessels between neurointestinal and circular blood vessels. The dorsal blood vessel is very short; the circumchaetael vessel is absent. Most of the anatomical peculiarities of L. mellita appear to be related to its biology. The newness of the data on the organization of the circulatory system reflects the general scarcity of knowledge on echiuran anatomy. The newly discovered features should be incorporated into general schemes of echiuran organization.
Herein, we present a novel electrode platform for H2O2 detection based on the immobilization of recombinant Tobacco Peroxidase (r-TOP) onto graphite electrodes (G) modified with p-phenylenediamine (p-PD) diazonium cation grafted multi-walled carbon nanotubes (MWCNTs). The employment of both p-phenylenediamine moieties and covalent cross-linking by using glutaraldehyde allowed to enhance the sensitivity, stability and selectivity toward H2O2 detection, as well as preventing the enzyme inactivation due to the electro-Fenton reaction. This reaction continuously produces hydroxyl radicals, whose high and unselective reactivity is likely to reduce drastically the operating life of the biosensor. The protection against the electro-Fenton reaction is through cross-linking the enzyme in combination with interaction between the uncoupled -NH2 groups (mainly uncharged at pH 7, considering a pKa of 4.6) available on the electrode surface and the enzyme. In particular, the electrode based on the r-TOP/p-PD/MWCNTs/G platform showed a lower limit of detection of 1.8 µM H2O2, an extended linear range between 6 and 900 µM H2O2, as well as a significant increase in sensitivity (63.1 ± 0.1 µA mM-1 cm-2) compared with previous work based on TOP. Finally, the r-TOP/p-PD/MWCNTs/G electrode was tested in several H2O2 spiked food samples as a screening analytical method for the detection of H2O2.
Changes in the parameters of miniature endplate potential (MEPP) of mouse diaphragm caused by exogenous application of two classical endocannabinoids, 2-arachidonoylglycerol (2-AG) (1 μM) and anandamide (AEA) (30 μM), were compared. A slowly developing stable increase in the MEPP amplitude by 50%, without affecting the frequency of MEPPs, was caused by 2-AG. This effect was prevented by AM-251, an inverse agonist of CB1-receptors, as well as by vesamicol, a blocker of the vesicular acetylcholine (ACh) transporter. On the contrary, AEA did not cause significant changes in the MEPP amplitude but induced a slowly developing (within 2 h) increase in MEPP frequency by 75% on average. The effect of AEA was prevented by AM-251 (1 μM) as well as by blocking of L-type Ca2+-channels with nitrendipine (1 μM) and inhibition of PKA activity by H89 (1 μM). It was concluded that both 2-AG and AEA are able to exert a noncanonical facilitating presynaptic effect on spontaneous ACh release. Even though these endocannabinoids activate the same type of CB-receptors, their facilitating effects do not overlap and are strictly aimed either at potentiating the size of ACh quanta (in case of 2-AG) or increasing the frequency of MEPP (in case of AEA). We assume that different intracellular targets and signaling pathways may be involved in the differentiated facilitating effects of 2-AG and AEA in mouse neuromuscular junctions.