Nicotinic acetylcholine receptors: Diversity and physiological importance for neurodegenerative disorders and development of organophosphate antidotes

The communication between the nervous and other systems in the organism is carried out by the transmission of nerve impulses. Diverse neurotransmitters are released into the synaptic cleft and bind to the specific receptors at the neighbouring cell to transmit the signal further. One of such receptors are nicotinic acetylcholine receptors (nAChR), integrated membrane proteins (ligand-gated ion channels) activated by the binding of a neurotransmitter acetylcholine. nAChR\u27s main characteristic is their diversity, as they consist of five of the same or mutually different subunits, which contribute to the specific receptors properties and biological activity. During the assembly of a pentameric protein structure, various combinations of subunits are linked together. After the discovery of nAChR’s involvement in various diseases, they became an important therapeutic target, for example in the treatment of neurodegenerative diseases (Alzheimer\u27s and Parkinson\u27s) and in the treatment of organophosphorus compound poisoning. This paper presents an overview of current knowledge on nicotinic receptors and an accompanying discussion on diseases, poisonings, potential drugs and treatments is given.</p

PNPLA porodica enzima – karakterizacija i biološka uloga

This paper brings a brief review of the human patatin-like phospholipase domain-containing protein (PNPLA) family. Even though it consists of only nine members, their physiological roles and mechanisms of their catalytic activity are not fully understood. However, the results of a number of knock-out and gain- or loss-of-function research models suggest that these enzymes have an important role in maintaining the homeostasis and integrity of organelle membranes, in cell growth, signalling, cell death, and the metabolism of lipids such as triacylglycerol, phospholipids, ceramides, and retinyl esters. Research has also revealed a connection between PNPLA family member mutations or irregular catalytic activity and the development of various diseases. Here we summarise important findings published so far and discuss their structure, localisation in the cell, distribution in the tissues, specificity for substrates, and their potential physiological role, especially in view of their potential as drug targets.Ovaj revijalni rad donosi pregled dosadašnjih spoznaja o porodici PNPLA (engl. patatin-like phospholipase domain-containing proteins) ljudskih enzima. Iako ovu porodicu čini samo 9 članova, najmanji sadrži 253 aminokiseline, a najveći više od 1360 aminokiselina, fiziološka uloga i mehanizam katalitičke aktivnosti nisu do sada potpuno razriješeni niti za jednoga. Međutim, rezultati brojnih tzv. knock-out, gain-of-function i loss-of-function modela istraživanja upućuju na važnu ulogu ovih enzima u mnogim biološkim procesima, uključujući održavanje homeostaze i integriteta stanične membrane, rast stanice, staničnu signalizaciju, staničnu smrt i metabolizam lipida kao triacilglicerola, fosfolipida, ceramida i retinil estera. Također, rezultati istraživanja upozoravaju na povezanost mutacija i nepravilne aktivnosti pojedinih članova s razvojem raznih bolesti. Radi boljeg razumijevanja PNPLA porodice enzima i naglaska na njihov potencijal kao mete razvoja novih lijekova, donosimo sveobuhvatni pregled do sada poznatih spoznaja koje uključuju strukturu, lokalizaciju u stanici, distribuciju u tkivima, specifičnost prema supstratima i potencijalnu fiziološku ulogu

Crossroads in Life Sciences

This is editorial, no abstract</p

Učinci imidazolijevih i kloriranih bispiridinijevih oksima povezani s njihovom toksičnosti na stanicama SH-SY5Y

Current research has shown that several imidazolium and chlorinated bispyridinium oximes are cytotoxic and activate different mechanisms or types of cell death. To investigate this further, we analysed interactions between these oximes and acetylcholine receptors (AChRs) and how they affect several signalling pathways to find a relation between the observed toxicities and their effects on these specific targets. Chlorinated bispyridinium oximes caused time-dependent cytotoxicity by inhibiting the phosphorylation of STAT3 and AMPK without decreasing ATP and activated ERK1/2 and p38 MAPK signal cascades. Imidazolium oximes induced a time-independent and significant decrease in ATP and inhibition of the ERK1/2 signalling pathway along with phosphorylation of p38 MAPK, AMPK, and ACC. These pathways are usually triggered by a change in cellular energy status or by external signals, which suggests that oximes interact with some membrane receptors. Interestingly, in silico analysis also indicated that the highest probability of interaction for all of our oximes is with the family of G-coupled membrane receptors (GPCR). Furthermore, our experimental results showed that the tested oximes acted as acetylcholine antagonists for membrane AChRs. Even though oxime interactions with membrane receptors need further research and clarification, our findings suggest that these oximes make promising candidates for the development of specific therapies not only in the field of cholinesterase research but in other fields too, such as anticancer therapy via altering the Ca2+ flux involved in cancer progression.Praćenjem učinka odabranih imidazolijevih i kloriranih bispiridinijevih oksima utvrđeno je da uzrokuju citotoksičnost i aktiviraju različite mehanizme ili tipove stanične smrti. Kako bismo to detaljnije istražili, analizirali smo aktivaciju nekoliko signalnih putova, kao i interakcije acetilkolinskih receptora (AChR) s navedenim oksimima te procijenili može li se opaženi toksični učinak objasniti njihovim utjecajem na ove specifične mete. Rezultati su pokazali da su klorirani bispiridinijevi oksimi prouzročili vremenski-ovisnu citotoksičnost, bez smanjenja razine ATP-a uz aktivaciju ERK1/2 i p38 MAPK-vezanih signalnih kaskada i inhibiciju fosforilacije STAT3 i AMPK proteina. Imidazolijevi oksimi djelovali su vremenski neovisno, uz značajno smanjenje razine ATP-a i inhibiciju ERK1/2 signalnog puta te fosforilaciju p38 MAPK, AMPK i ACC proteina. Navedeni signalni putovi obično se aktiviraju ili promjenom unutarnjega staničnog statusa, osobito energetskoga, ili vanjskim signalima, što upućuje na moguće interakcije oksima s nekim membranskim receptorima. Zanimljivo, in silico analizom procijenjeno je da je najvjerojatnija interakcija testiranih oksima s porodicom G-protein-spregnutih membranskih receptora (GPCR). K tomu, eksperimentalno je potvrđeno da testirani oksimi djeluju kao mogući antagonisti acetilkolina za vezanje na membranske AChR, potvrđujući tako i računalnu in silico procjenu. Iako interakcije ispitanih oksima s membranskim receptorima treba dodatno potvrditi, takve bi ih interakcije učinile kandidatima za razvoj specifičnih terapija u drugim područjima istraživanja, osim u istraživanjima povezanima s kolinesterazama, npr. kao moguće protutumorske lijekove, putem utjecaja na fluks iona Ca2+ uključenoga u progresiju tumora

Flavonoids as Inhibitors of Human Butyrylcholinesterase Variants

Inhibicijom butirilkolinesteraze (BChE, EC 3.1.1.8) moguće je liječiti bolesti kod kojih dolazi do smanjenja koncentracije neuroprijenosnika acetilkolina, poput Alzheimerove bolesti. Međutim, takav pristup terapiji ne smije zanemariti polimorfizam BCHE gena koji može utjecati na njezin konačan ishod. Nekolicina poznatih kolinergičnih lijekova (npr. galantamin, huperzin i rivastigmin) potječe iz biljaka ili su sintetski derivati biljnih spojeva, što opravdava daljnje istraživanje novih terapeutika iz biljaka. Flavonoidi pripadaju velikoj obitelji biološki aktivnih polifenolnih spojeva što se nalaze u mnogim biljkama, a poznata je činjenica da neki inhibiraju BChE. U radu smo se usredotočili na istraživanje flavonoida galangina, kvercetina, fisetina i luteolina kao inhibitora prirodnih inačica ljudske BChE: uobičajene, atipične i fluorid-rezistentne. Pokazano je da ispitani flavonoidi reverzibilno inhibiraju sve inačice BChE s konstantama disocijacije kompleksa enzim-inhibitor (Ki) u rasponu od 10 do 170 μmol/L. Inhibitorna moć ispitanih flavonoida spram svih triju inačica BChE povećavala se sljedećim redoslijedom: luteolin<fisetin<kvercetin<galangin, pri čemu je dokazano da inhibicija ne ovisi o polimorfizmu BChE. Rezultati upućuju na zaključak da flavonoidi mogu pomoći u daljnjem razvoju terapeutika, tj. inhibitora BChE za liječenje simptoma neurodegenerativnih bolesti i demencije.The inhibition of butyrylcholinesterase (BChE, EC 3.1.1.8) appears to be of interest in treating diseases with symptoms of reduced neurotransmitter levels, such as Alzheimer’s disease. However, BCHE gene polymorphism should not be neglected in research since it could have an effect on the expected outcome. Several well-known cholinergic drugs (e.g. galantamine, huperzine and rivastigmine) originating from plants, or synthesised as derivatives of plant compounds, have shown that herbs could serve as a source of novel target-directed compounds. We focused our research on flavonoids, biologically active polyphenolic compounds found in many plants and plant-derived products, as BChE inhibitors. All of the tested flavonoids: galangin, quercetin, fisetin and luteolin reversibly inhibited usual, atypical, and fluoride-resistant variants of human BChE. The inhibition potency increased in the following order, identically for all three BChE variants: luteolin<fisetin<quercetin<galangin. The determined enzyme-inhibitor dissociation constants (Ki) ranged from 10 to 170 mmol/L. We showed that no significant change in the inhibition potency of selected flavonoids exists in view of BChE polymorphism. Our results suggested that flavonoids could assist the further development of new BChE-targeted drugs for treating symptoms of neurodegenerative diseases and dementia

Translacija učinkovitosti piridinijevih oksima kod trovanja tabunom iz in vitro sustava u in vivo primjenu

Even if organophosphorus (OP) nerve agents were banned entirely, their presence would remain a problem as weapons of terror (like in Syria). Oxime antidotes currently used in medical practice still fall short of their therapeutic purpose, as they fail to fully restore the activity of cholinesterases, the main target for OPs. As orphan drugs, these antidotes are tested too seldom for anybody’s benefit. Over the last few decades, search for improved reactivators has reached new levels, but the translation of data obtained in vitro to in vivo application is still a problem that hinders efficient therapy. In this study, we tested the strengths and weaknesses of extrapolating pyridinium oxime antidotes reactivation efficiency from in vitro to in vivo application. Our results show that this extrapolation is possible with well-determined kinetic constants, but that it also largely depends on oxime circulation time and its tissue-specific distribution. This suggests that pharmacokinetic studies should be planned at the early stages of antidote development. Special attention should also be given to improving oxime distribution throughout the organism to overcome this major constraint in improving overall OP therapy.Iako su organofosforni živčani bojni otrovi potpuno zabranjeni za upotrebu, njihova je prisutnost i dalje velik problem, posebice kao kemijsko oružje u terorističkim napadima (poput nedavnih u Siriji). Oksimi koji se danas koriste kao protuotrovi u tretmanu nemaju dostatno djelovanje na reaktivaciju aktivnosti kolinesteraza, glavnih meta djelovanja organofosfornih spojeva. Valja napomenuti kako se klinička testiranja ovih protuotrova rijetko provode zbog svoje iznimne specifičnosti. Tijekom zadnjih desetljeća učinjen je napredak u istraživanju novih učinkovitijih protuotrova, međutim još je uvijek veliki nedostatak u poboljšavanju terapije translacija in vitro dobivenih rezultata u in vivo primjenu. Ovom studijom ispitali smo mogućnosti ekstrapolacije reaktivacijske učinkovitosti određene za oksimske protuotrove iz in vitro u in vivo sustav. Naši rezultati pokazuju kako je ova translacija moguća uz detaljno određene kinetičke parametre in vitro i uz poznavanje distribucije oksima i vremena cirkulacije u organizmu. Takav rezultat ističe važnost planiranja i farmakokinetičkih istraživanja već u samom početku razvoja protuotrova. Također, poseban naglasak u istraživanju trebalo bi staviti i na poboljšanje tkivo-specifične distribucije oksima u organizmu čime bi se poboljšala cjelokupna terapijska učinkovitost

Metabolic Insulin Resistance and Purine Metabolism

Metabolički sindrom ili sindrom X nekoliko je međusobno povezanih abnormalnosti koje povećavaju rizik od nastanka kardiovaskularnih i cerebrovaskularnih bolesti. To su dijabetes, hipertenzija, dislipidemija, hiperuricemija, debljina, odnosno bolesti u kojih se na početku patofi ziološkog slijeda nalazi inzulinska rezistencija. S kliničkoga gledišta, hiperuricemija treba upozoriti kliničara na povećani rizik od vaskularnih bolesti. Uloga urične kiseline i njezina povezanost s inzulinskom rezistencijom, kardiovaskularnim i cerebrovaskularnim bolestima, bubrežnim bolestima i hipertenzijom, svaki dan postaje sve važnija i jedan je od esencijalnih čimbenika u kliničkoj evaluaciji i gradiranju fenomena metaboličkog sindroma i dijabetesa tipa 2.Metabolic syndrome or syndrome X is a complex association of several interrelated abnormalities that increase the risk of cardiovascular and cerebrovascular diseases. These diseases include diabetes, hypertension, dyslipidaemia, hyperuricemia, obesity and other diseases resulting from insulin resistance. From a clinical standpoint, hyperuricemia should alert a clinician to an overall increased risk of vascular diseases. The topical role of uric acid and its relation to cardiovascular and cerebrovascular diseases and insulin resistance, renal disease and hypertension is rapidly evolving and it is one of essential factors in the clinical clustering phenomenon of the metabolic syndrome and type 2 diabetes mellitus

Mastitis u krava prouzročen brzorastućim mikobakterijama iz okoliša

Rapid-growth mycobacteria were isolated from two cases of cow mastitis with similar clinical appearance and within a narrow time frame. Mycobacteria were isolated on blood esculine agar. The isolated mycobacteria were Gram stained, Ziehl-Nielsen stained and tested for growth at 25°C, 37°C and 42°C, iron uptake, growth on Löwenstein-Jensen (LJ) agar with and without 5% NaCl, arylsulphatase (3 days), tween 80 hydrolysis, tellurite reduction, nitrate reductase and niacin synthesis. Molecular identification was performed using the Mycobacteria GenoType CM and AS tests (Hain Diagnostika, Nehren, Germany). One isolate was additionally sequenced for the hsp65, rpoB, 16S rRNA gene sequence and transcribed spacer sequence (ITS) DNA. Susceptibility testing of isolates was performed on the Sensititre Rapmycol plate (TREK Diagnostic Systems Ltd.) for trimethoprim/sulfamethoxasole, linezolid, ciprofloxacin, imipenem, moxifloxacin, cefepime, cefoxitin, amoxicillin / clavulanic acid, amikacin, ceftriaxone, doxycycline, minocycline, tigecycline, tobramycine and clarythromycine. Gram-positive acid-resistant rods were observed in stained smears. Both strains grew at 25°C, 37°C and 42°C on LJ medium, and on LJ medium containing 5 % NaCl. The conventional biochemical tests for iron uptake, arylsulphatase (3 days), Tween 80 hydrolysis, tellurite reduction and nitrate reductase were positive, while the niacin test was negative. Both isolates were identified by the GenoType Mycobacterium CM as Mycobacterium fortuitum II/ Mycobacterium mageritense, while application of the GenoType Mycobacterium AS kit identified both isolates as belonging to the species Mycobacterium smegmatis. Analysis of the isolate sequences (strain DS) for 16S ribosomal RNA confirmed a 100% identical result with Mycobacterium smegmatis strain INHR2. According to the CLSI criteria, both strains were sensitive to sulfametoxazole/trimethoprim, linezolid, doxicycline, amikacin and tobramycin. The strains differed in their sensitivity to cefoxitim, and both strains were resistant to clarithromycin. There was a strong difference between the isolates in sensitivity toward cefoxitime and tigecycline.Brzo-rastuće bakterije iz roda Mycobacterium izdvojene se iz dva klinički slična slučaja mastitisa krava u relativno kratkom vremenu. Mikobakterije su izdvojene na krvnom agaru s dodatkom eskulina, obojene po Grammu i Ziehl-Nielsenu te im je provjerena sposobnost rasta pri 25 °C, 37 °C i 42 °C, korištenje željeza, sposobnost rasta na Löwenstein-Jensen agaru bez i s dodatkom 5 % NaCl, tvorba arilsulfataze, sposobnost hidrolize tween 80, redukcije telurita i tvorbe nitrat reduktaze i niacina. Molekularna identifikacija izolata provedena je korištenjem testova Mycobacteria GenoType CM i AS. Odsječci gena hsp65, rpoB, 16S rRNK te tzv. (engl. transcribed spacer sequence (ITS)) jednog izolata su sekvencirani. Osjetljivost izolata provjerili smo pomoću SENSITITRE RAPMYCOL ploča (TREK Diagnostic Systems Ltd.) prema trimetoprim/sulfametoksazolu, linezolidu, ciprofloksacinu, imipenemu, moksifloksacinu, cefepimu, cefoksitinu, amoksicilinu s klavulanskom kiselinom, amikacinu, ceftriaksonu, doksiciklinu, minociklinu, tigeciklinu, tobramicinu i klaritromicinu. Gram-pozitivne acido-rezistentne štapićaste bakterije uočili smo u obojenim razmascima. Oba soja rasla su pri temperaturama 25 °C, 37 °C i 42 °C te na podlozi LJ s i bez dodatka 5  % NaCl. U oba soja utvrdili smo sposobnost korištenja željeza, tvorbe arilsulfataze, hidrolize Tween 80, redukcije telurita i tvorbe reduktaze nitrata. Sojevi nisu tvorili niacin. Oba izolata su testom GenoType Mycobacterium CM identificirana kao Mycobacterium fortuitum II/ Mycobacterium mageritense dok su GenoType Mycobacterium AS testom identificirana kao Mycobacterium smegmatis. Sekvencijskom analizom odsječka gena 16S ribosomske RNK soja DS i usporedbom s javno dostupnim izolatima, utvrdili smo potpunu podudarnost sa sojem Mycobacterium smegmatis soj INHR2. Prema CLSI kriterijima, oba izolata su osjetljiva prema sulfametoksazolu s trimetoprimom, linezolidu, doksiciklinu, amikacinu i tobramicinu, a rezistentna prema klaritromicinu. Razlike između sojeva očitovale su se u osjetljivosti prema cefoksitimu i tigeciklinu

Učinak ketamina na vijabilnost, primarna oštećenja DNA i parametre oksidacijskog stresa u stanicama HepG2 i SH-SY5Y

Ketamine is a dissociative anaesthetic used to induce general anaesthesia in humans and laboratory animals. Due to its hallucinogenic and dissociative effects, it is also used as a recreational drug. Anaesthetic agents can cause toxic effects at the cellular level and affect cell survival, induce DNA damage, and cause oxidant/antioxidant imbalance. The aim of this study was to explore these possible adverse effects of ketamine on hepatocellular HepG2 and neuroblastoma SH-SY5Y cells after 24-hour exposure to a concentration range covering concentrations used in analgesia, drug abuse, and anaesthesia (0.39, 1.56, and 6.25 μmol/L, respectively). At these concentrations ketamine had relatively low toxic outcomes, as it lowered HepG2 and SH-SY5Y cell viability up to 30 %, and low, potentially repairable DNA damage. Interestingly, the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) remained unchanged in both cell lines. On the other hand, oxidative stress markers [superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT)] pointed to ketamine-induced oxidant/antioxidant imbalance.Ketamin je disocijativni anestetik koji se koristi za izazivanje opće anestezije u određenim medicinskim postupcima kod ljudi, kao i u anesteziji laboratorijskih životinja. Zbog svojih halucinogenih i disocijativnih učinaka koristi se i kao rekreacijska droga. Anestetici također mogu prouzročiti toksične učinke na staničnoj razini i, utječući na preživljavanje stanica, izazvati oštećenje DNA te neravnotežu oksidansa i antioksidansa. Cilj ove studije bio je istražiti moguće štetne učinke ketamina na hepatocelularne HepG2 i neuroblastoma SH-SY5Y stanice nakon 24-satne izloženosti širokom rasponu koncentracija, uključujući koncentracije relevantne u slučajevima korištenja u analgeziji, zlouporabi droga i anesteziji (0,39, 1,56 odnosno 6,25 μmol/L). Naši rezultati pokazali su da je ketamin u ovim ispitivanim koncentracijama izazvao relativno nisku citotoksičnost, budući da je do 30 % smanjio preživljenje stanica HepG2 i SH-SY5Y, ali je uočen neznatan porast razine primarnih oštećenja DNA. Zanimljivo je da su razine reaktivnih kisikovih vrsta (ROS), malondialdehida (MDA) i glutationa (GSH) ostale nepromijenjene u objema staničnim linijama. S druge strane, markeri oksidacijskog stresa [suporeksid dismutaza (SOD), glutation peroksidaza (GPx), katalaza (CAT)] upućivali su na oksidacijsko-redukcijsku neravnotežu izazvanu ketaminom