The brominated pyrroloiminoquinone alkaloids discorhabdins B, L and G and 3-dihydro-7,8- dehydrodiscorhabdin C, isolated from methanol extracts of two specimens of Latrunculia sp. sponges collected ...near the Antarctic Peninsula, are here demonstrated for the first time to be reversible competitive inhibitors of cholinesterases. They showed Ki for electric eel acetylcholinesterase of 1.6–15.0 μM, for recombinant human acetylcholinesterase of 22.8–98.0 μM, and for horse serum butyrylcholinesterase of 5.0–76.0 μM. These values are promising when compared to the current cholinesterase inhibitors used for treatment of patients with Alzheimer's disease, to counteract the acetylcholine deficiency in the brain. Good correlation was obtained between IC50 data and results by molecular docking calculation on the binding interactions within the acetylcholinesterase active site, which also indicated the moieties in discorhabdin structures involved. To avoid unwanted peripheral side effects that can appear in patients using some acetylcholinesterase inhibitors, electrophysiological experiments were carried out on one of the most active of these compounds, discorhabdin G, which confirmed that it had no detectable undesirable effects on neuromuscular transmission and skeletal muscle function. These findings are promising for development of cholinesterase inhibitors based on the scaffold of discorhabdins, as potential new agents for treatment of patients with Alzheimer's disease.
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•Discorhabdins from Antarctic Latrunculia sponges show anticholinesterase activities.•Molecular docking studies show good correlation with experimental data.•Docking calculations revealed structural moiety involved in AChE interaction.•There are no side effects on neuromuscular transmission or skeletal muscle function.•Discorhabdin scaffold is promising in drug development for Alzheimer's treatment.
Aegerolysin proteins ostreolysin A6 (OlyA6), pleurotolysin A2 (PlyA2) and erylysin A (EryA) produced by the mushroom genus
bind strongly to an invertebrate-specific membrane sphingolipid, and ...together with a protein partner pleurotolysin B (PlyB), form transmembrane pore complexes. This pore formation is the basis for the selective insecticidal activity of aegerolysin/PlyB complexes against two economically important coleopteran pests: the Colorado potato beetle and the western corn rootworm. In this study, we evaluated the toxicities of these aegerolysin/PlyB complexes using feeding tests with two ecologically important non-target arthropod species: the woodlouse and the honey bee. The mammalian toxicity of the EryA/PlyB complex was also evaluated after intravenous administration to mice. None of the aegerolysin/PlyB complexes were toxic against woodlice, but OlyA6/PlyB and PlyA2/PlyB were toxic to honeybees, with 48 h mean lethal concentrations (LC
) of 0.22 and 0.39 mg/mL, respectively, in their food. EryA/PlyB was also tested intravenously in mice up to 3 mg/kg body mass, without showing toxicity. With no toxicity seen for EryA/PlyB for environmentally beneficial arthropods and mammals at the tested concentrations, these EryA/PlyB complexes are of particular interest for development of new bioinsecticides for control of selected coleopteran pests.
•nPoly-3-APS preferentially blocks the skeletal neuromuscular transmission.•nPoly-3-APS acts by a non-depolarizing mechanism.•In vivo toxicity of nPoly-3-APS mainly occurs through antagonist action ...on nAChRs.
The effects of natural polymeric alkylpyridinium salt (nPoly-3-APS), a potent acetylcholinesterase inhibitor isolated from the marine sponge Reniera sarai, were studied on isolated mouse phrenic nerve-hemidiaphragm muscle preparations using electrophysiological approaches. nPoly-3-APS inhibited nerve-evoked isometric muscle twitch and tetanic contraction in a concentration-dependent manner (IC50=29.4μM and 18.5μM, respectively) and produced a 30–44% decrease of directly muscle-elicited twitch and tetanus amplitudes at 54.4μM. Additionally, nPoly-3-APS (9.1–27.2μM) markedly decreased the amplitude of miniature endplate potentials, while their frequency was only affected at the highest concentration used. Endplate potentials were also inhibited by nPoly-3-APS in a concentration-dependent manner (IC50=20.1μM), without significant change in the resting membrane potential of muscle fibers (up to 54.4μM). In conclusion, our results show, for the first time, that nPoly-3-APS preferentially blocks the neuromuscular transmission, in vitro, by a non-depolarizing mechanism. This strongly suggests that the in vivo toxicity of nPoly-3-APS mainly occurs through an antagonist action of the compound on nicotinic acetylcholine receptors of skeletal muscles.
Alzheimerjeva bolezen je kronična progresivna nevrodegenerativna bolezen, za katero oboleva vse večje število ljudi po vsem svetu. Je najpogostejši vzrok za demenco (pri 50–80 % vseh primerov) in ...obenem tudi najhujša oblika demence, za katero so značilne kognitivne in vedenjske motnje pri ljudeh, večinoma starejših od 65 let. Patogeneza bolezni še ni popolnoma pojasnjena. Obstaja več hipotez, ki vključujejo kombinacijo genetskih in okoljskih dejavnikov ter načina življenja. Glede na začetek bolezni lahko bolezen opredelimo kot obliko z zgodnjim začetkom, običajno pred 65. letom starosti, in obliko s poznim začetkom, običajno po 65. letu starosti. Redka oblika bolezni z zgodnjim začetkom je povezana z mutacijami v genu za amiloidni prekurzorski protein ter v genih za presenilin 1 in 2. Genetski dejavnik tveganja za nastanek bolezni s poznim začetkom je prisotnost alela ε4 za apolipoprotein E. Za nastanek oblike s poznim začetkom obstaja več vzročnih hipotez: a) holinergična hipoteza, b) amiloidna hipoteza, c) hipoteza o hiperfosforilaciji proteina tau, č) hipoteza o mitohondrijski kaskadi, d) vnetna hipoteza, e) nevro-vaskularna hipoteza, f) hipoteza o kovinskih ionih ter g) hipoteza limfnega sistema. Poleg tega obstajajo tudi presnovni in drugi dejavniki tveganja za nastanek Alzheimerjeve bolezni, med katere sodijo: zvišana raven holesterola v krvi, debelost, zvišan krvni tlak, sladkorna bolezen tipa 2, motnje spanja idr. Kljub velikemu številu raziskav etiopatogeneza do danes še ni dokončno pojasnjena, prav tako ne poznamo zdravila, ki bi preprečilo nastanek bolezni ali ustavilo njeno napredovanje. Zdravljenje Alzheimerjeve bolezni trenutno temelji na farmakološkem zdravljenju, ki poteka na osnovi doslej znane etiologije bolezni. Veliko obeta tudi imunoterapija z antiamiloidnimi protitelesi.
Detailed shape analysis of cells is important to better understand the physiological mechanisms of toxins and determine their effects on cell morphology. This study aimed to develop a procedure for ...accurate morphological analysis of cell shape and use it as a tool to estimate toxin activity. With the aim of optimizing the method of cell morphology analysis, we determined the influence of ostreolysin A and pleurotolysin B complex (OlyA/PlyB) on the morphology of murine neuronal NG108-15 cells. A computational method was introduced and successfully applied to quantify morphological attributes of the NG108-15 cell line before and after 30 and 60 min exposure to OlyA/PlyB using confocal microscopy. The modified circularity measure
C
2
n
(
S
)
for shape analysis was applied, which defines the degree to which the shape of the neuron differs from a perfect circle. It enables better detection of small changes in the shape of cells, making the outcome easily detectable numerically. Additionally, we analyzed the influence of OlyA/PlyB on the cell area, allowing us to detect the cells with blebs. This is important because the formation of plasma membrane protrusions such as blebs often reflects cell injury that leads to necrotic cell death. In summary, we offer a novel analytical method of neuronal cell shape analysis and its correlation with the toxic effects of the pore-forming OlyA/PlyB toxin in situ.
The effects of Beauvericin (BEA) produced by the fungusBeauveria bassianaandFusariumsp. on neuromuscular transmission and contractility were determined in an isolated neuromuscular mouse ...hemidiaphragm preparation. BEA (5 µM) significantly inhibits indirectly elicited twitch amplitude. At higher concentrations (7.5 and 10 µM), BEA produces a significant reduction of directly elicited, or complete block of indirectly evoked, muscle contraction. BEA also appears to be myotoxic, as indicated by a slowly developing muscle contracture. Development of neuromuscular blockade and contracture is concentration dependent. BEA acted by presynaptically depressing spontaneous acetylcholine release as indicated by the reduction in the frequency of spontaneous miniature endplate potentials (MEPPs), while the membrane potential of muscle fibers remained unchanged. At higher concentrations (7.5 and 10 µM), BEA progressively reduces or completely blocks MEPPs and EPPs amplitudes. Changes in MEPPs and EPPs are associated with substantial depolarization of muscle fibers when exposed to 7.5 and 10 µM of BEA. These results indicate that BEA has neurotoxic and myotoxic effects, which overlap in a narrow range of concentrations.
Cholinesterases (ChEs) show increased activities in patients with Alzheimer’s disease, and remain one of the main therapeutic targets for treatment of this neurodegenerative disorder. A library of ...organoruthenium(II) complexes was prepared to investigate the influence of their structural elements on inhibition of ChEs, and on another pharmacologically important group of enzymes, glutathione S-transferases (GSTs). Two groups of organoruthenium(II) compounds were considered: (i) organoruthenium(II) complexes with p-cymene as an arene ligand, and (ii) organoruthenium(II) carbonyl complexes as CO-releasing molecules. Eight organoruthenium complexes were screened for inhibitory activities against ChEs and GSTs of human and animal origins. Some compounds inhibited all of these enzymes at low micromolar concentrations, while others selectively inhibited either ChEs or GSTs. This study demonstrates the importance of the different structural elements of organoruthenium complexes for their inhibitory activities against ChEs and GSTs, and also proposes some interesting compounds for further preclinical testing as ChE or GST inhibitory drugs.
The aim of this study was to detect cyanobacterial cytoskeletal elements and the cytoskeletal framework using immunostaining with anti-bovine α-tubulin mouse monoclonal antibodies. After strong ...permeabilization of axenic cyanobacterial cell lines, the cytoskeleton elements become visible in all cells. A mild cell permeabilization procedure allows the discrimination between healthy and senescent or otherwise stressed cyanobacteria whose cell integrity has been jeopardized. This technique can be useful to investigate cyanobacterial bloom lysis provoked by various natural or artificial factors. Viruses, among others, are important mortality agents of cyanobacteria. The presence of non-hepatotoxic cyclic cyanopeptides can provoke lysis of non-axenic Microcystis aeruginosa cell lines. This is presumably due to lytic cycle induction in lysogen cyanobacteria. A susceptible cyanobacterial cell line exposed to the depsipeptide planktopeptin BL1125 has been analysed with transmission electron microscopy to corroborate the involvement of virus like particles (VLP) in the process of lysis. VLP that correspond in shape and size to tailed cyanophages have been observed only in samples where the process of lysis has been triggered. The immunostaining of cytoskeletal elements by using epifluorescence and confocal microscopy has confirmed that the lysis expands from single infected cells or cell groups, the focal points, to their immediate environment. Our in vitro experiments demonstrate that lysogen focal point formation, which follows induction by endogenous cyanobacterial cyclic peptides, could constitute, also in the natural environment, the basis for an extremely rapid and extensive cyanobacterial bloom collapse.
Ostreolysin (Oly), a cytolytic and cardiotoxic protein from the oyster mushroom (
Pleurotus ostreatus), is lethal for mice with an LD
50 of 1170
μg/kg following intravenous application. Its ...cardiotoxicity is associated with hyperkalemia, which is probably a consequence of potassium released from the lysed cells. Moreover, sub-micromolar concentrations of Oly induce a concentration-dependent increase in rat aortic ring tension, suggesting that ischaemia, and consequent hypoxic injury of cardiomyocytes, could also derive from vasospasm induced by this toxic protein.
The purpose of the present study was to demonstrate histopathological lesions caused by Oly after parenteral application to rats, and to define the mechanisms of Oly-induced vasoconstriction using inhibitors verapamil, lanthanum chloride, and selective endothelin receptor antagonist TBC3214, which have different molecular targets,
in vitro on porcine coronary artery rings. We found that Oly causes endothelial injury with perivascular oedema in the heart and lungs, as well as myocardial haemorrhages in rats. Treatment of porcine coronary artery rings with Oly causes concentration-dependent vasoconstriction and prevents endothelium-mediated relaxation. Using TBC3214 as a selective blocker of the endothelin A receptor, we showed that vasoconstriction induced by Oly was independent of endothelin release and its effects. Verapamil (1
μM) greatly reduced Oly-evoked contractions of porcine coronary artery rings, while lanthanum abolished them completely. These results provide evidence that the contraction of coronary arteries by Oly is due mainly to the increased influx of Ca
2+ from the extracellular space through voltage-dependent L-type Ca
2+ channels and cation non-selective channels. Experiments suggest that Oly damages endothelial cells both
in vitro and
in vivo, and probably exhibits direct contractile effects on coronary smooth muscle cells.
Ostreolysin (Oly), an acidic, 15
kDa protein from the edible oyster mushroom (
Pleurotus ostreatus), is a toxic, pore-forming cytolysin. In this paper, its toxic properties have been studied in ...rodents and the LD
50 in mice shown to be 1170
μg/kg. Electrocardiogram, arterial blood pressure and respiratory activity were recorded under general anaesthesia, in intact, pharmacologically vagotomised and artificially respirated rats injected with one mouse LD
50. A few seconds after intravenous Oly injection, a transient increase in arterial blood pressure was recorded, followed by a progressive fall to mid-circulatory pressure accompanied by bradicardia, myocardial ischaemia and ventricular extrasystoles. Similar changes produced by Oly were observed in vagotomised and artificially respirated animals, indicating that vagotomy and hypoxia play no primary role in toxicity. Oly induced lysis of rat erythrocytes
in vitro, and probably also
in vivo as indicated by the increase in serum potassium. Although direct action of the protein on the cardiomyocytes or heart circulation cannot be excluded, the hyperkalaemia resulting from the haemolytic activity probably plays an important role in its toxicity. The lethality and cardiorespiratory toxic action of Oly are thus shown to be candidates for the cause of the recorded adverse effects of oyster mushroom.
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